The bioinorganic chemistry of copper-containing systems

The bioinorganic chemistry of copper-containing systems

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The bioinorganic chemistry of copper-containing systems from Type-3 systems pertinent to Alzheimer's disease to mononuclear hydrolysis involved in biological development
Da Silva, Giordano Faustini Zimmerer
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[Tampa, Fla.]
University of South Florida
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Reactive oxygen species
Sea urchin
Dissertations, Academic -- Chemistry -- Doctoral -- USF ( lcsh )
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theses ( marcgt )
non-fiction ( marcgt )


ABSTRACT: Although transition metals are essential for life, misregulation of redox-active metal uptake, delivery, storage, and excretion has been linked with a series of neurodegenerative disorders. Alzheimer's disease (AD) is considered an epidemic and is the most widespread of all forms of dementia. Copper ions found in large concentrations localized in amyloid-≈í‚⧠plaques in the brain of AD patients have been linked with the generation of reactive oxygen species which are suspected to be the culprits leading to neuronal cell death. Herein a series of mechanistic and spectroscopic studies elucidate the chemistry about the metal-centered oxidation of biomolecules, including catecholamine neurotransmitters and some analogues by copper-complexes of amyloid-≈í‚⧠peptide. Transition metals can also be useful tools for characterization of metalloproteins due to their unique chemical and spectroscopic features. Herein a series of studies of the native Zn¬¨â€šâ§+ and Cu¬¨â€šâ§+-derivative of recombinant Blastula Protease 10 (BP10) from the sea urchin Paracentrotus lividus are presented in order to elucidate its catalytic mechanism, with the use of enzymology, metal substitution, and electronic absorption spectroscopy.
Dissertation (Ph.D.)--University of South Florida, 2007.
Includes bibliographical references.
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by Giordano Faustini Zimmerer Da Silva.

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Da Silva, Giordano Faustini Zimmerer.
4 245
The bioinorganic chemistry of copper-containing systems :
b from Type-3 systems pertinent to Alzheimer's disease to mononuclear hydrolysis involved in biological development
h [electronic resource] /
by Giordano Faustini Zimmerer Da Silva.
[Tampa, Fla.] :
University of South Florida,
Dissertation (Ph.D.)--University of South Florida, 2007.
Includes bibliographical references.
Text (Electronic dissertation) in PDF format.
System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
Title from PDF of title page.
Document formatted into pages; contains 279 pages.
Includes vita.
Co-adviser: Li-June Ming, Ph.D.
Co-adviser: Brian T. Livingston, Ph.D.
3 520
ABSTRACT: Although transition metals are essential for life, misregulation of redox-active metal uptake, delivery, storage, and excretion has been linked with a series of neurodegenerative disorders. Alzheimer's disease (AD) is considered an epidemic and is the most widespread of all forms of dementia. Copper ions found in large concentrations localized in amyloid-§ plaques in the brain of AD patients have been linked with the generation of reactive oxygen species which are suspected to be the culprits leading to neuronal cell death. Herein a series of mechanistic and spectroscopic studies elucidate the chemistry about the metal-centered oxidation of biomolecules, including catecholamine neurotransmitters and some analogues by copper-complexes of amyloid-§ peptide. Transition metals can also be useful tools for characterization of metalloproteins due to their unique chemical and spectroscopic features. Herein a series of studies of the native Zn¨§+ and Cu¨§+-derivative of recombinant Blastula Protease 10 (BP10) from the sea urchin Paracentrotus lividus are presented in order to elucidate its catalytic mechanism, with the use of enzymology, metal substitution, and electronic absorption spectroscopy.
Reactive oxygen species.
Sea urchin.
0 690
Dissertations, Academic
x Chemistry
t USF Electronic Theses and Dissertations.


The Bioinorganic Chemistry Of Copper-Cont aining Systems: From Type-3 Systems Pertinent To AlzheimerÂ’s Disease To Mononuc lear Hydrolysis I nvolved In Biological Development by Giordano Faustini Zimmerer Da Silva A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Co-Major Professor: Li-June Ming, Ph.D. Co-Major Professor: Br ian T. Livingston, Ph.D. Steven H. Grossman, Ph.D. Randy L. Larsen, Ph.D. Date of Approval: May 9, 2007 Keywords: kinetics, reactive oxygen species, metallopeptide, amyloid, enzymology, sea urchin, metalloprotease Copyright 2007 Giordano F.Z. Da Silva


NOTE TO THE READER Note to Reader: The original of this docum ent contains color that is necessary for understanding the data. The orig inal dissertation is on file at the USF library in Tampa, FL.


DEDICATION Although the graduate school experi ence is highly a personal journey, it would be foolish to believe that I was solely responsible for my accomplishments. I then would like to dedicate this wo rk to all of those who have played a major role in this difficult yet rewarding part of my life. Firs t my grandmother Ermelinda who is the sole reason why I am in the US, my parents Mauric io and Marta for sacrificing the presence of their son, and my sister Marcella for be ing sibling, friend and pa rent through part of my formative years. Second I must dedicate this work to the Kelley family for being my parents, brother, and sisters; Moms, Pops, John, Tere, and Abbie, I would not be here today without your love and support. Last and not least I must also dedicate this work to Rachel for being a friend, always in my h eart, through difficult and joyous times, for her love and support without which I would have ne ver remained in the US I truly hope that the rest of my career will make the efforts of all of those herein mentioned worthwhile.


ACKNOWLDGEMENTS I must acknowledge my friends Bri and Br enda, who were my sisters through the laughter, tears, and often difficu lt times, and hope to remain life-long friends. I must also acknowledge William and Kash for being brothers in arm, and Vaso for inspiration. I would also like to acknowledge Altan for being a patient mentor during my undergraduate years and believing in my pot ential. I would like to acknowledge my collaborator Dr. Alexander Ange rhofer from UF for his avai lability and willingness to share his time and effort. Rae Reuille must be acknowledged for her patience and guidance while teaching molecular biology to a chemist. Dr. Randy Larsen and Dr. Steven Grossman must also be acknowledge d for their efforts as my committee members and for always being present to discuss matters of science, life, histor y, and politics. Last and not least I must acknowledge Dr. Li-June Ming and Dr. Brian T. Livingston. In an ever-changing academic world it is often difficu lt to find mentors who still have a pure scholarÂ’s mind. Through their efforts, patien ce, guidance, and disc ussions about science and life, I feel prepared to move on to the ne xt stage of the scientif ic experience. They were teachers, mentors, and friends and I am et ernally grateful for their time. I truly hope that the rest of my career will make the efforts of all of those herein mentioned worthwhile.


i TABLE OF CONTENTS List of Tables vi List of Figures vii List of Abbreviations xi Abstract xv Chapter I: Introduction 1 Background 1 Metabolic Generation of Reac tive Oxygen Species 7 Superoxide and Superoxide Dismutase 10 Metal-Binding to A and Structure of the Complex 19 Generation of ROS by Metallo-A 22 Type-3 Copper Oxidase Models and Me tallo-ROS 29 Concluding Remarks 52 List of References 53 Chapter II: Cu2+A Complexes as Redox-Active Catalysts Toward the Oxidation of 1,2,3 Tri-Hydroxy Benzene 87 Introduction 87 Experimental 90 DNA cleavage 90


ii THB Oxidation Assay 90 Metal Titration to A 91 1H NMR Co2+ Titration to A 92 Molecular Mechanics (MM3) 92 Results and Discussion 92 Oxidative Double-Stranded DNA Cleavage 92 Kinetics and Mechanism of Oxidative Catalysis by CuA 100 Metal Binding and Structure 112 Concluding Remarks 122 List of References 124 Chapter III: Catechol Oxidas e and Phenol Monooxygenase Activities of CuA 1-20 133 Introduction 133 Experimental 135 Phenol Monooxygenase MBTH Assay 135 Results and Discussion 137 Catechol Oxidase Activity 137 Zn2+ Dilution 139 Effect of H2O2 140 Phenol Hydroxylation 144 DCC Optical Titration 147 Concluding Remarks 149


iii List of References 150 Chapter IV: Metallo-ROS in Alzheimer Â’s Disease: Metal-Centered Oxidation of Neurotransmitters by Cu2+Amyloid Provides an Alternative Perspective for the Neuropathology of AlzheimerÂ’s Disease. 154 Introduction 154 Experimental 155 Neurotransmitter Oxidation Assay 156 Results and Discussion 156 Catecholamine oxidation 156 Effect of H2O2 162 Effect of SDS 163 Effect of NADH and NADPH 165 DCC Titration to A 1-40 167 Serotonin Oxidation 174 Concluding Remarks 177 List of References 179 Chapter V: Methionine 35 is not a Reducing Agent for the Metal-Centered Oxidation Chemistry of Cu2+-Amyloid: Kinetic and EPR Studies 187 Introduction 187 Experimental 189 Catechol Oxidase MBTH Assay 189 L-Met Optical Titration 190 Effect of Reducing Agents 190


iv EPR and ESEEM 190 Results 192 Effect of L-Met 192 Effect of Reducing Agents 196 Electronic Spectrum of L-Met CuA 1-20 196 CW-EPR of CuA 1-20 and L-Met + CuA 1-20 196 ESEEM Spectra CuA 1-20 and L-Met + CuA 1-20 200 Discussion 203 Concluding Remarks 209 List of References 211 Chapter VI: The Astacin Family of Endopep tidases and Embryogenesis 220 Introduction 220 List of References 236 Chapter VII: Overexpression and Ch aracterization of Recombinant Blastula Protease 10 from Paracentrotus lividus 237 Introduction 237 Experimental 241 Overexpression, Purification, and Refolding of Recombinant BP10 242 Circular Dichroism (CD) Studies 244 Preparation of the Copper Deriva tive of BP10 244 Gelatin Zymogram 245 Gelatinase Assay 245


v Hydrolysis of BAPNA by Zn-BP10 and Cu-BP10 246 Calcium Activation Assays 247 Inhibition Studies 247 pH Profiles 247 Electronic Spectrum of Cu-BP10 247 Homology Modeling and Substrate Docking 247 Results and Discussion 248 Overexpression and Refolding of Recombinant BP10 248 CD Spectra of ZnBP10 and Cu BP10 251 Kinetics of Gelatin Hydrolysis 251 Kinetics of BAPNA Hydrolysis 253 Mechanistic Studies of the Copper Derivative of BP10 (Cu-BP10) 258 Homology Modeling 271 Concluding Remarks 271 List of References 274 About the Author End Page


vi LIST OF TABLES Table 1: Standard Redox Potentials for Dioxygen in H2O. 15 Table 2: Kinetic Parameters for Dopamine Oxidation by CuA 158 Table 3: Kinetic Parameters for the Oxidation of Catecholamines to o -quinone by CuA 1–20. 164


vii LIST OF FIGURES Figure 1.1: Electronic stat es and standard reduction potentials for O2. 16 Figure 1.2: Types of enzymes that activate O2. 31 Figure 1.3: Structure of CuO inte rmediates identified in biomimetic complexes. 35 Figure 1.4: Early models of Type-3 oxidases by Karlin and Tolman 41 Figure 1.5: Type-3 model systems by Itoh and Stack. 47 Figure 2.1: Concentration and metal-dependent assay of dsDNA cleavage. 94 Figure 2.2: Time course reactivity assay of dsDNA cleavage. 97 Figure 2.3: Time course reactivity assay toward the cleavage of 150 ng dsDNA. 98 Figure 2.4: Effect of peroxide conc entrations on the rate of THB oxida tion in the presence of 7.5 M of CuA 1-20. 102 Figure 2.5: H2O2 saturation profile of CuA 1-16 and CuA 1-20. 104 Figure 2.6: Hanes analysis of the kinetic da ta. 109 Figure 2.7: Proposed mechanism for the oxidation of THB by CuA 111 Figure 2.8: Cu2+ titration to A 1-20 and A 1-16 monitored with their 113 oxidation activities. Figure 2.9: Electronic spectra of CuA 1-20. 116 Figure 2.10: 1H NMR spectra of A 1-20 and A 1-16 in DMSO. 119 Figure 2.11: Proposed metal coordinati on and solution structure of CuA 1-16. 121


viii Figure 3.1: Electronic spectrum of th e MBTH-o-quinone adduct. 136 Figure 3.2: Saturation kinetics with catechol, phenol and deut erated phenol. 138 Figure 3.3: Effect of Zn2+ on the oxidative activity of CuA 1-20 toward DTC 141 and phenol. Figure 3.4: The effect of H2O2 on the first order rate constant kcat toward the oxidation of phenol and catechol. 142 Figure 3.5: Hanes plot analysis of kinetic data from Figure 3.4. 143 Figure 3.6: Proposed mechanism for aer obic hydroxylation and oxidation of phenol. 146 Figure 3.7: Optical titrati on of DCC to 0.2 mM CuA 148 Figure 4.1: Oxidation of catechol and dopamine by 1.47 M CuA 1-40 in 100 mM HEPES at pH 7.0 and 25 C. 157 Figure 4.2: Catecholamine oxidation by 2.5 M CuA 1-20. 160 Figure 4.3: H2O2 effect on oxidation of catecholamines. 162 Figure 4.4: Effect of SDS on the oxidative activity of CuA 1-16,CuA 1-20, 166 and CuA 1-40 Figure 4.5: Influence of NAD(P)+ and NAD+ on the oxidative activity of CuA 1–20 toward dopamine oxidation. 168 Figure 4.6: Phosphate inhibition toward catechol oxidation by CuA 1–20. 169 Figure 4.7: Optical titrati on of DCC into 0.05 mM CuA 1–40. 171 Figure 4.8: Activity titration of Cu2+ into 2.5 M A 1–40 under 171 saturating conditions of catechol. Figure 4.9: Mechanism for the oxidation of catecholamine neurotransmitters and the cause of neurodegeneration by CuA 173 Figure 4.10: Aerobic oxidation of serotonin by 1.47 M CuA 1-40. 175


ix Figure 4.11: Saturation profile of phenol oxidation by 1.47 M CuA 1-40. 176 Figure 5.1: Effect of methionine toward dopamine oxidation by CuA 1–20. 193 Figure 5.2: Effect of methionine on kcat toward dopamine oxidation by CuA 1–20. 194 Figure 5.3: Lineweaver-Bur k plot of Met activation. 195 Figure 5.4: Rate of dopamine oxidation by CuA 1-20 in the presence of saturating amounts of L-Met and H2O2. 197 Figure 5.5: Glutathione and ascorbic acid inhibition towa rds dopamine 198 oxidation by Cu-A 1–20. Figure 5.6: Optical titration of L-methionine to 0.2 mM CuA 199 Figure 5.7: X-band EPR of CuA 1–20 in the presence and absence 201 of L-Met. Figure 5.8: ESEEM of CuA 1–20 in the presence and absence of L-Met 202 Figure 5.9: Proposed mechanism for the catech ol oxidase-like activity 204 of CuA toward the oxidation of dopamine. Figure 6.1: Active site motif co mmon to all astacins 221 Figure 6.2: Active site structur e of astacin with transition state analogue. 222 Figure 6.3: Phylogeny of the astacin family catalytic domains. 224 Figure 6.4: Domain organi zation of proteins from the astacin/CUB/EGF subfamily. 227 Figure 6.5: Proposed metallotriad mechanism. 231 Figure 7.1: SDS-PAGE gel (12.5 %) during purification of recombinant BP10. 249 Figure 7.2: SDS-PAGE showing intact BP10 afte r refolding; western blot 250 showing the time course of overexpression; ge latin zymogram showing concen tration-dependent substr ate hydrolysis by BP10.


x Figure 7.3: CD spectra of Zn-BP10 and Cu-BP10 in PBS pH 7.4. 254 Figure 7.4: Kinetics of gelatin hydrolysis by Zn2+ and Cu2+ derivatives of BP10. 255 Figure 7.5: Gelatin hydrolysis by ZnBP10 and Ca2+-dependent activation. 256 Figure 7.6: pH dependence of kcat and kcat/ Km for the hydrolysis of gelatin by 260 Zn-BP10 and BAPNA by Zn-BP10 and Cu-BP10. Figure 7.7: Hydrolysis of L-BAPNA by Zn-BP10. 261 Figure 7.8: Inhibition of Zn-BP10 toward L-BAPNA hydrolysis 263 by 1,10 phenanthroline and by Arg-NHOH; inhibition of Cu-BP10 by Arg-NHOH. Figure 7.9: Optical titrat ion of Arg-NHOH to Cu-BP10. 265 Figure 7.10: Inhibition of Cu-BP10 by Arg-NHOH. 266 Figure 7.11: pH profile of BAPNA hydrolys is by Cu-BP10. 267 Figure 7.12: The change in intensity of the LM CT transition of Cu-BP10 270 at 454 nm as a function of pH. Figure 7.13: Stereo view of the homology model of the BP10 active site. 272


xi LIST OF ABBREVIATIONS AD: AlzheimerÂ’s disease A : AmyloidCuO: Copper-oxygen intermediate Hcy: Hemocyanin Tyr: Tyrosinase ROS: Reactive Oxygen Species kDa: kilo Daltons APP: Amyloid Precursor Protein ADP: Adenosine 5Â’ diphosphate ATP: Adenosine 5Â’ triphosphate ET: Electron Transfer DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid NADP+: Nicotinamide adenine dinucleotide phosphate, oxidized form NADPH: Nicotinamide adenine dinucleotide phosphate, reduced form NAD+: Nicotinamide adenine dinucleotide, oxidized form


xii NADH: Nicotinamide adenine dinucleotide, reduced form PAGE: Polyacrylamide gel electrophoresis SDS: Sodium dodecyl sulfate Tris: Tris(hydroxymethyl)aminomethane DNA: Deoxyribonucleic acid mRNA: Messenger ribonucleic acid cDNA: Complementary DNA COX: Cytochrome oxidase activity CSF: Cerebral spinal fluid mtDNA: Mitochondrial DNA dsDNA: Double stranded deoxyribonucleic acid SOD: Superoxide dismutase MnSOD: Manganese dependent superoxide dismutase CuZnSOD: Copper-Zinc depende nt superoxide dismutase PHF: Paired helical fragments CNS: Central nervous system 1H NMR: Proton nuclear magnetic resonance CW-EPR: Continuous wave electron paramagnetic resonance ESEEM: Electron spin echo envelope modulation RAGE: Receptor for advanced glycation endproducts ERAB: Endoplasmic reticulum amyloidbinding protein PBN: N tert -butyl-a-phenylnitrone


xiii PBS: Phosphate buffered saline MNP: 2-methyl-2-nitrosopropane PrP: Prion protein DOPA: 3,4-dihydroxyphenylalanine XAS: X-ray absorption spectroscopy EXAFS: Extended X-ray absorption fine structure D M: Dopamine hydroxylating monooxygenase PHM: Peptidylglycine -hydroxylating monooxygenase PPh3: Triphenylphosphine THB: 1,2,3,-trihydroxybenzene DMSO: Dimethyl sulfoxide HEPES: N-(2-hydroxyethyl)-piper azine-N'-2-ethanesulfonic acid MES: 2-Morpholinoethanesulfonic acid TAPS: N-Tris(hydroxymethyl)m ethyl-3-aminopropanesulfonic acid CAPS: N-Cyclohexyl-3-aminopropanesulfonic acid MBTH: 3-methyl-2-benzothiazolinone hydrazone d6DMSO: Deuterated dimethyl sulfoxide UV-Vis: Ultraviolet-visibl e electronic spectroscopy LMCT: Ligand to metal charge transfer transition MALDI-TOF: Matrix assisted laser desorption mass spect rometry time of flight KIE: Kinetic isotope effect DTC: Di-tertbutyl catechol


xiv DCC: Dichlorocatechol CMC: Critical micelle concentration GSH: Reduced glutathione NQI: Nuclear quadrupole interactions BP10: Blastula Protease 10 CuBP10: Copper derivative of Blastula protease 10 ZnBP10: Zinc derivative of Blastula protease 10 BMP-1: Bone morphogenetic protein 1 EGF: Epidermal growth factor ECM: Extracellular matrix suBMP: Sea urchin bone morphogenetic protein 1 TGF: Transformation growth factor CUB: Complement-like domains Ni-NTA: Nickel nitrilotriacetic acid BAPNA: N-benzoyl-argininep -nitroanilide IPTG: Isopropyl-thiogalactopyronoside PMSF: Phenylmethyl sulfonyl fluoride PCR: Polymerase chain reaction OD: Optical density CD: Circular dichroism BCA: Bicinchoninic acid APS: Ammonium persulfate


xv TEMED: N,N,N',N'-Tetramethylethylenediamine OP: 1,10-phenanthroline Arg-NHOH: Arginine hydroxamate MM3: Molecular mechanics protocol


xvi The Bioinorganic Chemistry of Copper-Cont aining Systems: from Type-3 Systems Pertinent to AlzheimerÂ’s Disease to Mononuc lear Hydrolysis I nvolved in Biological Development Giordano F.Z. da Silva ABSTRACT Although transition metals are essential fo r life, misregulation of redox-active metal uptake, delivery, storage, and excre tion has been linked with a series of neurodegenerative disorders. AlzheimerÂ’s dis ease (AD) is considered an epidemic and is the most widespread of all forms of dementia Copper ions found in large concentrations localized in amyloidplaques in the brain of AD patie nts have been linked with the generation of reactive oxygen sp ecies which are suspected to be the culprits leading to neuronal cell death. Herein a series of mechanistic and sp ectroscopic studies elucidate the chemistry about the metal-centered oxidation of biomolecules, including catecholamine neurotransmitters and some analogues by copper-complexes of amyloidpeptide. Transition metals can also be useful tool s for characterization of metalloproteins due to their unique chemical a nd spectroscopic features. Herein a series of studies of the native Zn2+ and Cu2+-derivative of recombinant Blastula Protease 10 (BP10) from the sea urchin Paracentrotus lividus are presented in order to elucidate its catalytic mechanism, with the use of enzymology, metal substituti on, and electronic absorption spectroscopy.


1 CHAPTER I. COPPER-DIOXYGEN IN METABOLISM AND CATALYSIS I. BACKGROUND The interaction of transi tion metals with oxygen (O2, or dioxygen) is of paramount importance in biologi cal systems. The metal-O2 interaction is ubiquitous in metabolic pathways of aerobic organisms on th e earthÂ’s crust and it is fundamental to homeostasis by serving as catalysts in redox reactions as well as for the bidning and delivery of O2 in aerobes.1 For these purposes, iron and copper are the predominant, albeit not the only, transition metals utilized in metallopr oteins and metalloenzymes. Classic examples of ironcontaining O2 transport metalloproteins include hemoglobin,2 myoglobin,2 and hemerythrin,3 while redox-active non-heme iron metalloenzymes involved in O2 metabolism include methane monooxygenase,4 and protocatechuate 3,4 dioxygenase.5 Copper is also well re presented in the area of O2-binding with hemocyanin6 being the classic example, while catalytic copper-centers include tyrosinase,6 catechol oxidase,7 and galactose oxidase.8


2 Of particular interest in the recent years has been the nature of copper-O2 centered chemistry in metalloenzymes. Several re search groups have pur sued spectroscopic characterization, crystallography, and biomimetic studies of metalloproteins that contain copper and can interact with O2 through direct binding or for activation of O2 in chemical reactions like hydroxylation a nd oxygenation. These copper metalloenzymes are collectively termed Type-3 copper proteins attributed to the magnetically coupled dicopper center.6 Although the topic in this review is primarily from a health/disease or a more medicinal perspective, the lessons lear ned from the investigation of the chemistry of Type-3 copper proteins have some ove rlap with the area of neurodegeneration involving activated or reduced forms of O2. The focus of this chap ter will be the parallels of what is known about the chemistry of Type -3 copper oxidases and how it may relate to AlzheimerÂ’s disease. The ability of binuclear copper centers in proteins to bind O2 for transport and to use copper-O2 (CuO2) as the catalytic center for O2 activation has provided a wealth of understanding in both experimental and th eoretical chemistry. Understanding the interaction of O2 with copper has been an intense ar ea of study for bioinorganic chemists, both toward the native molecules and in biomimetic systems.9,10 Research in this area has utilized the spectroscopic pr operties of the Type-3 copper proteins, as well as crystal structures when available, to serve as te mplates toward the synthesis of small model systems that allow the inte ractions of copper with O2. Typically, the short-lived CuO2 intermediates cannot be well reso lved in the native systems either due to influence of the size of proteins in spectroscopic techniques that rely in magnetic resonance or because


3 crystal structures cannot offer a fast enough process in order to catch the transient species responsible for catalysis. Even in small biomimetic compounds, the unstable formation of CuO2 intermediates has forced the determination of the structure of reactive intermediates at very low temperatures.11 In fact, a significant effort has been made toward a systematic approach to synthesize biomimetic systems to approximate the chemical and spectroscopic prope rties of Type-3 copper protei ns. The choice of ligands, bridging ligands, solvent systems, and temper ature can play a significant role in the elucidation of both redox-activit y of model systems and thei r electronic properties that give rise to the now typical spectrosc opic fingerprints of Type-3 oxidases. In Type-3 metalloprotein biomimetic systems, the coordination environment around the metal ion is typically constituted of strong donor ligands such as the nitrogen atoms of pyridines that can mimic the hist idine residues which have been proposed to assist the stabilization of metal-oxygen interm ediates for electrophili c substitutions and 2electron oxidation chemistry. Th e stabilization of highly electrophilic/nuc leophilic CuO2 intermediates is also assist ed by a large hydrophobic enviro nment. However, several factors can influence the reactivity of th ese compounds; in essence, any alteration on solvent, and coordinated (and bridging) lig ands will change the reactivity of these biomimetic compounds.12 The now classic example of biomimetic O2-binding copper complexes that mimic the spectroscopic a nd chemical properties of their natural templates can be traced back to the efforts of Nobumasa Kitajima, Kenneth D. Karlin, and Edward I. Solomon from the late 1980Â’s to today.13-16


4 Although seemingly unrelated, the knowledge gained from investigating copperO2 interactions in metalloproteins and en zymes and their biomimetic analogues, may yield significant insight into the possible mechanism for metal-centered generation of reactive oxygen species (ROS). These ROS are generated at a metal center and may either diffuse away and cause damage to redox-sensitive biomolecules or remain bound to the metal and itself be an efficient redox catalyst. The common ground between these two areas of research then can be elucidated as the copper-O2 interaction. From the detailed spectroscopy of biomimetic compounds and their protein templates, we have a library of diagnostic patterns to elucidate the na ture of interactions of redox-active metals and O2. From the detailed mechanistic investig ation of the chemis try catalyzed by the metalloproteins and their model compounds we are now well aware of what reactivity can be expected from different conformations of reactive CuO2 intermediates. In recent years, a darker side of copper-O2 chemistry has surfaced in biological studies. As research in the areas of cancer, apoptosis, diabetes, heart disease, and neurodegenerative disorders becomes more focused and detaile d, the metal-centered generation of ROS has become a common theme. In fact, much of the focus has been placed on the nature of ROS rather than the interaction of the re dox-active metal center with the ROS. These ROS are the radical and reduced forms of oxygen that although present under normal metabolic conditions, could potentially damage oxidizable moieties in their environment if not properly regulated due to the much hi gher oxidation potential of ROS compared to O2. ROS alone are able to damage every majo r type of biological molecule, including membranes (by peroxidation of lipids),17 proteins (by oxidation of certain residues and


5 nitration by nitric oxide),18 DNA (by destruction of the structural stability of the double helix as ribose is oxidized),19 and smaller molecules such as neurotransmitters.20 According to recent studies, OH•, O2•–, and H2O2 have been assigned as the culprits in several types of neurological disorders, w ith a large number of research groups focusing on the role of ROS in the etiol ogy of Alzheimer’s disease (AD).21 However, as chemists further investigate the pathway of ROS genera tion and the fate of such molecules, it becomes more evident that a redox-active metal is necessary in order for the damaging and often lethal chemical imbalance that is caused by ROS (E = -300 mV for the one electron reduction of O2) to achieve its full neurodegenerative potential; in other words it is the metallo-ROS interaction that appears to be the culprit in oxidative stress that leads to neurodegeneration, not ROS alone. Recent observations demonstrate that although oxygen radicals and oxidation agents like H2O2 cannot achieve high enough concentration in order to be th e sole culprits in the neuropa thology of AD due to inherent ability of organisms to cope with this oxida tive stress, the presence of a metallo-ROS can form a very potent redox catalyst. Alzheimer’s disease is one of several neurodegenerative disorders affecting a large percentage of the population. An es timated 3 million individuals in the United States suffer from the slow, yet aggregate symptoms of AD. The number is expected to double by the year 2030.22 The progressive nature of AD that has even been called an epidemic,23 has also dire effects on the families of those diagnosed with the disease. The AD patients are “memory timers”, bringing reco gnition of events and people around them slowly to extinction with each passing day. W ith the gradual, yet always progressive and


6 degenerative nature of AD, families are placed under considerable stress while caring for their loved ones and coping with the loss of th eir existence in the minds of those afflicted by AD. To that end, numerous scientific st udies, amounting to tens of thousands, have been published, reflecting the interest in research funding and efforts toward understanding the neuropat hology of this disease. Largely, AD neuropathology concerns the interaction of a 40-42 amino acid peptide splice fragment from the ubiquitous apolipoprotein (or amyloid precursor protein, APP) with a number of intracellu lar components. These amyloid(A ) peptides are generated by the cleavage of APP by , and secretases.24 A in the form of insoluble plaques contains up to mM amounts of Zn2+, Cu2+, and Fe3+ in the neocortical region of the brain.25 However, the cause/effect connection of the metallo-A plaques with AD is still under debate.26 A has been the elusive culprit in AD studies with results ranging from free-radical generation to disruption of mitochondrial cell membrane potential, all hallmarks of oxidative stress. Formation of A plaques in the presence of metals has dominated a large percentage of investigatio ns in correlating th e physiology of AD with the metal chelating event (A serving as the ligand) a nd generation of ROS. The subsequent observations of ch emical events once metallo-A is formed have offered numerous hypotheses into the oxidation stress pr oposed as one of the main causes of AD. Herein we present a compendium of observations that bring together e fforts in the area of metal-O2 chemistry from a catalysis perspectiv e to better understand what role O2 and its reduced forms may have in disease. Rela ting the neuropathology of AD with possible


7 metal-centered pathways is a recent area of focus that help clarify the homeostatic generation of ROS and the possible eff ects of non-homeostasis ROS generation. II. METABOLIC GENERATION OF ROS Electron donors in the tri carboxylic acid cycle in m itochondria (i.e. NADH and succinate) to O2 are responsible for the generatio n of ATP for upkeep of cellular processes. Electron transfer (ET) processes rely on a series of molecular complexes to ensure that the transduction of oxidative en ergy and that the use of proton energy in ATP synthesis is carried out effi ciently. The complexes that are functionally connected to mitochondrial energy transduction in clude: Complex I (NADH:ubiquinone oxidoreductase), Complex II (succinate:ubi quinone oxido-reductase), Complex III (ubiquinol:ferricytochrome c oxido-reductase ), Complex IV(ferrocytochrome c:oxygen oxidoreductase), and Complex V (ATP-synthase).1, 27 In the inner mitochondrial membrane, with in Complexes I, III, and IV the energy transduced from ET is conserved by coupled pr oton translocation that is responsible for the generation of a membrane electrochemi cal potential of protons used in ATP synthesis. The whole ET system is reversib le and an electron flow can be generated against the current. However, cytochrome aa3 in Complex IV and O2, or the final step in ET is irreversible, shifting the equilibrium in the system toward ATP synthesis. Cytochrome aa3 retains all the partially reduced oxygen intermediates bound to its active sites until the O2 itself is completely reduced to water. However, through auto-oxidation


8 that can affect their reduced forms, other elements in the mitochondrial ET chain like ubiquinones and the cytochrome b family could transfer the electrons directly to O2, but do not retain the pa rtially reduced O2 intermediates in their active sites until the O2 is completely reduced to water. Because O2 accepts only one electron at a time, the superoxide radical (O2•–) is released. In the cytochrome b family, it is noteworthy that cytochrome b566 is closely involved with the proce sses of energy transduction in Complex III, wavering continually betw een a very low potential state of approximately –30 mV and a very high one of approximately 245 mV. The low potential of cytochrome b566 could play a primary role in the formation of mitochondrial O2•– because of an increase in its redox potential inhibiting the univalent transfer of electrons to oxygen. Therefore, electron leakage from the cytochrome b566 would appear to be a re al possibility which can thus bring about a continuous release of oxygen free radicals. During aging, the increasing amounts of these radicals that mana ge to escape the local defense mechanisms (e.g. scavengers, electron-trapping agents, et c.) may lead to multiple changes in the chemical and physical state of the membra nes. As a matter of fact, superoxide generation28,29 is significantly greater in the brain mitochondria of aged rats rather than young rats. This seems to be related to the f act that, with the exception of cytochrome aa3 in Complex IV, the metabolic levels of the electron carriers does not undergo significant change with aging. However, the decreas e in either the amount of cytochrome aa3 or its catalytic cytochrome oxidase activity (COA)30-34 in synaptic mitochondria from some cerebral regions (frontal co rtex, parieto-temporal cortex, hippocampus, cerebellum, etc.)35,36 may account for the finding that stoichio metric calculations show aging is


9 related to an increase in the per centage of ubiquinones and cytochrome b family. Although this increase is not dramatic, re latively it does explain how electrons can "escape" the ET sequence from electron donors to O2. The Km for cytochrome c is constant in aged rats but the Vmax decreases, suggesting that the COA activity is not related to the functional integrity of mitochondria accompanying senescence.35 Moreover, the COA activity drops significantly less in cor tical synaptic mitochondria from old rats fed a hypocaloric diet,37 and declines in the homogenate of old rat cerebral cortex38 and in insect mitochondria.39 Electrons can leak from the energytransduction sequences even in young animals, indicating that the formation of super oxide radicals could be associated with the normal process of mito chondrial respiration.40 The production of these radicals causes cell damage because of the dismutase reaction in which H2O2 is formed and which, with the involvement of low-molecular-weight ir on and copper complexes, leads to the highly dangerous hydroxyl radical. The catalytic ac tivity of Complex IV (COA activity) is low in three cortical areas but not in putamen and hippocampus of AD patients compared with age-matched controls.41,42 There is no correlation between the changes in COA and those of other marker mitochondrial enzymes, su ch as glutamate dehydrogenase and citrate synthase, leading to the conclu sion that the decrease in COA activity is not related to the loss of mitochondria.41 The decrease in COA activity in cortical areas and in the hippocampus from AD patients43,44 suggests a primary defect of Complex IV, resulting in more O2•– released. A specific decrease in COA in the platelets of AD patients has also been reported45 but not confirmed.46 COA is heterogeneously di stributed in the cerebral


10 spinal fluid (CSF).47 Expression of mRNA for COA in normal human and monkey brain is high in those regions which are most vul nerable to AD pathology and is particularly reduced in the same regions from AD patients.48, 49 In the mid-temporal gyrus, but not in the primary motor cortex, of AD patients ther e is a 50-65% decrease in the mRNA levels of the mitochondrial DNA (mtDNA)-encoded COA subunits I and III.50 However, the mitochondrial-encoded 12S ribosomal RNA (a mitochondrial transcript) does not change, suggesting that the observed reduction of COA I and III mRNA is not due to loss of mitochondria but to a specific alteration of transcriptional regulation. A behavioral study in rats treated with the selective sodi um azide COA inhibitor showed significant inhibition of a low-threshold form of hippo campal long-term potentiation and impaired spatial learning.51 This finding supports the theory that Complex IV alteration is somehow involved in the pathogenesis of AD and also raises the possibility of developing an animal model reflecting this aspect of AD provided it is specific. III. O2•– AND SOD Superoxide radicals are de scribed as having considerab le reactivity, short halflife, and limited diffusion through membranes. However, the latter property has been questioned, as these radicals can appear in the brain ex tracellular space.52,53 O2•– has a dual effect54 where it may help protect against inf ectious microorganisms, but they can also be harmful as it particip ates in the formation of th e very reactive hydroxyl radicals (OH•). Moreover, they can inactivate a numbe r of useful enzymes, such as antioxidat


11 enzymes (catalase55 and glutathione peroxidase56), enzymes involved in neurotransmission (glutamine synthase57), in signal transduction (adenylate cyclase58), and in energy transduc tion (creatine phosphokinase59 and NADH dehydrogenase and ATPase60). In AD brain and fibroblasts there is evidence of partial uncoupling of mitochondrial oxidation and phosphorylation.61,62 Apart from the neurodegeneration induced by the impairment of energy metabo lism, these oxidative abnormalities could contribute to the accumulation of cytoskeletal material. Addition of an uncoupler to cultured fibroblasts from normal subjects caus es the appearance of epitopes recognized by antibodies to paired helical filament s (PHF) and Alz-50 monoclonal antibodies,61 thus reproducing a pattern that is characteristic of fi broblasts from AD patients.63 When O2 accepts an electron from a reduci ng agent, the primary product is O2•– that, in aqueous environments, is in equilibrium with its protonated form (•O2H). When O2•– and •O2H approach equal molar concentratio ns, spontaneous dismutation occurs, and H2O2 plus 1O2 (singlet oxygen)64 are generated. 2 O2•– + 2H+ H2O2 + O2 (or 1O2) Scheme (l) In scheme 1, O2•– can be converted into H2O2 catalyzed by superoxide dismutase (SOD) that is present in varying concentrations in neural cells. Thus, the conversion removes O2•– and prevents its direct toxic action as we ll as its interaction with metal ions to increase the production of hydroxyl radicals (Scheme 9 below).


12 The rate constant for SOD-catalyzed dismut ation is approximatel y four orders of magnitude greater than that for the spontaneous dismutation of O2•– at physiological pH. For SOD protection/activity to work properly, it is absolutely vital for other enzymes (e.g., catalase, glutathione per oxidase, etc.) to convert H2O2 immediately into H2O, thus preventing the transformation of H2O2 by metal complexes into the highly toxic OH• (Scheme 9 below). In this last case, the intervention of SOD may be dangerous for neuronal cells in spite of what we expect as a beneficial biological scavenging of a highly reactive radical. The predominant two types of SOD are Mn-dependent and Cu-Zn dependent. The manganese-dependent (Mn-SOD) is locat ed in the mitochondria, where it interacts with the O2•– leaking from the ET chain. The copp erand zinc-dependent (Cu,Zn-SOD) is located in the neural cytosol where it ca rries out a more general catalytic function. Auto-oxidizable electron carriers located on the internal mitochondrial membrane can generate O2•– which is enzymatically dismutated to H2O2. However, a few reactions catalyzed by some enzymes (e.g., monoamine oxidase and L-aminoacid oxidase) can produce H2O2 directly. Thus, H2O2 may be generated either as a direct product or from each of the various sources of O2•– by auto-oxidation of a variety of low-molecular weight molecules, as byproducts of various enzyme catalyses such as between xanthine and xanthine oxidase, and by the mitochondrial ET system. The hydroxyl radical is one of the most potent reactive metabo lites produced in brain systems derived from O2. This radical, O2, and OH– are all products in the reaction shown in Scheme 2 when H2O2 is directly reduced by O2•–:


13 O2•– + H2O2 O2 (or 1O2) + OH• + OH– Scheme (2) H2O2 can cross cell membranes directly, whereas O2•– crosses cell membranes through anion channels. Although H2O2 cannot be classified as a radical because it does not contain unpaired electrons, it is still da ngerous because it easily permeates cell membranes and can migrate from where it is first generated to other organic compartments. It can interact with the redu ced forms of some meta l ions (generally, Fe2+ and Cu+) and decompose into the highly reactive OH• and the OH–, according to: H2O2 + Fe2+ Fe3+ + OH• + OH– Scheme (3) H2O2 + Cu+ Cu2+ + OH• + OH– Scheme (4) The formation of OH• requires reduced forms of metal ions, such as Fe2+ or Cu+. The superoxide radical O2•– can then give rise to Fe2+ or Cu+ by reducing Fe3+ or Cu2+ according to: O2•– + Fe3+ O2 + Fe2+ Scheme (5) O2•– + Cu2+ O2 + Cu+ Scheme (6) The reaction in Scheme (2) is slow at phys iological pH and would require steady-state concentrations of the reaction partners, which are much higher than those found in cerebral mitochondria to account for detectab le amounts of the highl y unstable radical.


14 As shown in Schemes 3-6, metal ions (Mn+) accelerate the reacti on in Scheme (2) by catalyzing two intermediate reactions: O2•–+ Mn+ O2 + M(n–l)+ Scheme (7) M(n-l)+ + H2O2 Mn+ + OH• + OH– Scheme (8) Overall: O2•– + H2O2 + O2 (or 1O2) + OH• + OH– Scheme (9) In Scheme 9, O2•– reduces redox-active transition metals (Fe3+, Cu2+), and generates oxygen or singlet oxygen. The reduced form of the metal subsequently reacts with H2O2 to produce the oxidized form of the metal, th e hydroxide ion, and the hydroxyl radical. In view of the evidence that in neural systems Scheme 2 proceeds very slowly, the hydroxyl radicals are possibly produced in the pres ence of a redox-active metal. An additional mechanism by which OH• may be generate d is supported by the observation that incubation of H2O2 with Fe2+ and iodide ions (I–) can generate a potent reactive molecule that is inhibited by scavengers of the hydroxyl radical (e.g., mannitol65 and ethanol66). Singlet oxygen63 is generated by the SOD in Scheme 2 or by the interaction with metal ions in Scheme 9 when one of the two unpaired electrons of O2 acquires sufficient energy to undergo spin inversion or both spin inve rsion and orbital tran sition. There are two distinct forms of singlet oxygen, and which are dependent respectively on whether the excited electron forms an el ectron pair in the same orb ital or remains unpaired in a different orbital. The form of singlet oxygen is more stable than the form. Singlet


15 Reaction E (V) O2 + e– O2•– O2•– + e– + 2 H+ H2O2 H2O2 + e– + H+ H2O + OH OH + e– + H+ H2O O2 + 2e– + 2 H+ H2O2 H2O2 + 2e– + 2 H+ 2 H2O O2 + 4 H+ + 4 e– 2 H2O –0.33 + 0.89 + 0.38 + 2.31 + 0.281 + 1.39 + 0.815 Table 1.1. Standard redox potentials fo r dioxygen species in water.


16 Figure 1.1. Electronic states and standard reduction potentials for O2. 67


17 oxygen is highly electrophilic and can react with electron-rich bi omolecules such as tryptophan, methionine, and molecules c ontaining unsaturated double bonds. The effect of physiological aging on br ain SOD is controversial, though most reports describe some age-related decline, mainly of the Cu-Zn form68,69,70,71,72 probably related to the decline of SOD mRNA. Howeve r, other studies report no change of this form and an increase in MnSOD.73,74 Thus, it would appear that higher levels of SOD are beneficial and lower levels of SOD detrimen tal. However, if the activity of SOD is increased without a concomitant enhancemen t of the activity of the enzymes which dispose of H2O2 (i.e. glutathione peroxidase) a nd the concentration of reduced glutathione, then H2O2 accumulates and reacts with O•– and Fe3+ and/or reduced metal ions to form the very reactive OH•. Thus the imbalance between SOD and H2O2converting enzymes results in a toxic e ffect of SOD by OH• generation, inducing DNA fragmentation, protein denatura tion, and activation of the auto catalytic process of lipid peroxidation. Down's syndrome (trisomy 21) has provide d some interesting clues on the balance among SOD, ROS, and antioxidants. Human Cu,Zn-SOD is encoded by a gene located on chromosome 21 and Down patients have a 50 % increase in the activity of this enzyme secondary to gene dosage effect.75 The increase in SOD, not accompanied by a concomitant adaptative rise in glutathione peroxidase,76 might induce oxidative damage to the CNS, including lipid peroxidati on and this might explain some of the neurobiological abnormalities found in Down's syndrome, such as accelerated aging and AD-type neuropathology. This is supported by the results in an an imal model of gene


18 dosage effect in transgenic mice carrying the human Cu,Zn-SOD gene.77,78 Cu,Zn-SOD and its mRNA are preferentially expressed in the large pyramidal neurons of Ammon's horn and granule cells of the dentate gyrus which are susceptible to degenerative processes in AD.79 Brain lipid peroxidation is also increased. In seeming contrast with this finding, increased Cu-Zn SOD in tran sgenic mice makes the hippocampus more resistant to the neurotox icity induced by amyloid.80 The levels of Cu,Zn-SOD protein an d mRNA in the vulnerable hippocampal neurons of AD patients,81 their association with ne urofibrillar dege neration (PHF),82 and the observation that the cell distribu tion of Cu,Zn-SOD mRNA in the human hippocampus is the same as amyloid mRNA83 suggest that high levels of enzymes for ROS decomposition are needed to remove excess superoxide ra dicals which indicate that ROS contribute to the degenerative processe s leading to neuropat hology in AD. Cu,ZnSOD activity is also upregulated in the te mporal cortex and nucleus basalis Meynert84,85 and in the fibroblasts of AD and Down patients.86 Cultured skin fibroblasts from both familial and sporadic AD patients are more susceptible to ROS-induced damage then from age-matched controls.87 Finally, high immunoreactivity for SOD and catalase in the AD brain is associated with some neur ofibrillar tangles and senile plaques.88 This immunoreactivity is absent in tangle-free neurons of AD and all neurons of normal control brains.


19 IV. METAL-BIDING TO A AND STRUCTURE OF THE COMPLEX The primary step in understanding metalcentered chemistry is the elucidation of the coordination of metal ions in its comp lexes. The geometry of metal complexes is often the determinant of reactivity. Once it was determined that transition metals could bind to A from analysis of isolated plaques,89 more detailed investigation of the nature of the metal-A interaction followed. Through the us e of competitive binding in a pH gradient column chromatography, Cu2+, Ni2+, and Zn2+ were determined to tightly bind to A 1-42; shorter fragments (A 1-16 and A 1-28) could also be retained in the metal-chelate column but did not bind as tightly.90 Based on the pKa of 6.1 determined from the pH gradient elution, His residues were suggest ed as the ligands responsible for metalbinding. Further pH dependent binding studi es using quantitative precipitation and turbidity revealed that Cu2+ can induce aggregation of A 1-40 and A 1-42. Chemical modification of His residues in the peptid e to N-carbethoxyhistidin e extinguishes the metal-induced aggregation.91 These results indicated that His residues were important in metal binding, but did not provide insight into geometry nor the affinity of A toward transition metals. Quantitative prec ipitation studies suggested that Cu2+ binds with an attomolar dissociation constant to A 1-42.92 This very low Kd, if correctly measured, would suggest that A could actually remove metal from hemes, some of the tightest binding ligands in biological systems. This result howev er, erroneously explained the metal-bidning because the combination of multiple molecular events in terms of euqilibrium can yield such a low metaldissociation constant. Since quantitative


20 precipitation measures both the metal affinity to A and also the affinity associated with peptide aggregation, the attomolar constant is thus a combination of different constants for the two processes that would not be quantified individually until chemical93 and spectroscopic94 methods were used to directly m easure metal-peptide interactions in solution. Both methods yielded Kd for metal-binding in the M range for A 1-16, A 120,93 and A 1-28.94 In order to further el ucidate the location of metal-binding in metal-A complexes resonance Raman was employed to directly observe metal-peptide interactions.97 The Raman spectra demonstrate that three His re sidues in the N-terminal hydrophilic region provide primary metal binding sites and the geometry of the metal-A complex is correlated with the metal binding mode. Zn2+ binds to the N atom of a His imidazole ring and the peptide was suggested to aggregate through intermolecular His(N )-Zn2+His(N ) bridges. The N -metal ligation also occurs in Cu2+-induced A aggregation at mildly acidic pH.91 At neutral pH, however, Cu2+ was shown to bind to N the other nitrogen of the His imidazole ring, and to a deprotonated amide nitrogen of the peptide main chain.95 The studies using electroni c absorption spectroscopy and 1H NMR techniques have also shown the nature of the metal-A interaction.93 The electronic spectrum of Cu2+-A 1-20 shows a typical tetragonally di storted octahedral environment that is consistent with many Cu2+ complexes in solution. Co2+ as a paramagnetic shift reagent in 1H NMR experiments revealed that th ree His resides coordinate all N nitrogens from the imidazole rings of His 6, His13, and His14. The results of the NMR


21 study also revealed that neith er Tyr10 nor the N-terminus bind to the metal as was previously suggested.95-97 Through the use of CW-EPR techniques and line broadeni ng observations in 1H NMR experiments, a model was suggested for the formation of A dimers that resembled the structure of the active site of Cu,Zn-SOD.96 The suggested structure yields the metal:peptide stoichiometry of 1:1 and that two metal metal centers can be bridged through a third His residue, suggested to be His6.96 These results were further confirmed by using chemically modified (methylated) His residues and CW-EPR.98 The broadening of the EPR spectrum was proposed to be due to antiferromagnetically couple Cu2+ that could only arise from a bridging ligand inter action or a close proxi mity of two metal centers. However, the distinct feat ures of Cu,Zn-SOD EPR are well known99 which do not resemble those of the CuA complexes. Indeed, the possibility of bridged metalcenters in CuA complexes is not preposterous whic h can be reasoned as an OH-bridged or even phosphate-bridged Cu cente rs (being that PBS is the pr eferred buffers in most of the experiments cited). The interpretation th at the broadening of EPR signals in CuA samples is likely due to the aggregation of the metallopeptides in solution and not because of magnetic coupling, since the metal-induced aggregation of A is a well known phenomenon.100-103 Although the metal-binding properties of A have been widely studied,25 questions about the correct stoichiometry were until recently not clarified. Recent studies using a number of different physical methods, ranging from 1H NMR to fluorescence and kinetics, have elucidated that each A monomer of varying lengths can only bind one


22 metal.93,94,104,105 Recent theoretical studies have s hown that the seeds for dimerization occur between residues 16-22, causing -sheet formation in smaller fragments that can range from dimmer to 16 peptides per fibril.106,107 It is thus possible for two individual metal centers to be close enough to each ot her due to dimerizati on and show binuclear metal-centered catalysis. Such structural motif may well explain the redox chemistry involved in etiology of AD. V. GENERATION OF ROS BY METALLO-A The cytotoxic effects of A which accumulates in the brain in AD, have been studied extensively.108 Early studies109 suggested that the aggregation state of A is related to its toxicity. It was shown that freshly solubilized A exhibited little toxicity, whereas A that had been aged for 7 days (formi ng aggregates in fibrillar states) was cytotoxic.109 However, the precise molecular mechanism by which A mediates cell death has remained a matter of considerable di spute. There is genera l agreement that the production of ROS (see section II) and th e influx of calcium ions into cells121 are both involved in toxicity, but it is still unclear how th e generation of ROS mi ght be related to A One possibility is that the aggregated peptide itself may be able to produce ROS directly in the forms of free radicals or H2O2.110,111 It is generally accepted that A needs to be in aggregated or partially aggregated states before it becomes toxic to cells. Ho wever, there is still no clear consensus on the precise nature of the toxic form of the pep tide. Dimers, soluble ol igomers, protofibrils


23 and annular protofibrils have all be en implicated in amyloid toxicity.112-117 Furthermore, it is becoming more evident that not all am yloid fibrils are toxi c to cultured cells. Recently a synthetic peptide correspondi ng to amino acid residues 1-15 of A protein (A 1-15) was shown to form amyloid fibrils in vitro that are completely non-toxic to cells, even at high concentrations.118 Some forms of synthetic A are also reported to be non-toxic,119 as is the A peptide mutant that contains a norleucine in the place of Met35.120 This leads to yet another area of re search that is ongoing and still trying to elucidate which forms of amyloid are toxic. What underlying molecular structures and mechanisms can explain why some amyloi ds are toxic, whereas others are not. Various other hypotheses have been put fo rward to explain the cytotoxic effects of A in addition to the direct production of ROS from the peptide, including the formation of ion calcium channels in cell membranes by A ,121 interactions between A and specific cell surface receptors, such as th e RAGE (receptor for advanced glycation endproducts) or scavenger receptors,122-124 interactions between A and intracellular target molecules such as ERA (endoplasmic reticulum A binding protein),125 and nonspecific intercalation of aggregated forms of A into membranes.126 These various interactions may not be related to the idea that the most import ant aspect of the toxicity of A is due to its ability to generate ROS directly once bound to a redox-active metal. The toxic process could be the re sult of a combination of bi nding and/or attachment of A aggregates to cell components, followed by the induction of oxidative damage. According to the oxidative stress hypot hesis, a spontaneous shower of A -derived free radicals is produced upon incubation of A in vitro and it is this phenomenon which


24 rendered the cytotoxic properties of A .111 Three main pathways can lead to the formation of free radicals from organic mol ecules: photolysis, thermal scission, and oneelectron redox reactions. Mechanical stress would be a fourth possible route that may apply in some special cases. Of these, photolys is is not relevant to neurodegenerative and amyloid diseases. Thermolysis of most chem ical bonds requires te mperatures above 450 C with the exception of peroxides and azo compounds. The temperature at which peroxides undergo unimolecular scission varies with the class of peroxide but can be between about 50 and 150 C.127,128 Likewise, nitrogen can be eliminated from certain azo compounds (general structure R-N= N-R) over a similar temperature range.129,130 However, neither of these classes of bond is present in the parent A peptide molecule. Perhaps the most important redox assisted bond scissions involve a metal ion that readily undergoes a one-electron transfer with one of the best known examples being the Fenton reaction: Mn+ + H2O2 M(n+1)+ OH• + OH– Metal assisted homolysis reactions can ta ke place at much lower temperatures than the corresponding unassisted reaction. Wh ile such reactions in peptide molecules cannot be totally discounted at 37 C, they do not provide an obvious route to the spontaneous generation of pep tidyl free radicals from A However, it has been suggested on purely theoretical studies th at peptidyl free radicals might be generated from A by a mechanism involving mechanical stress, i.e physical cont act among metallo-A monomers in the plaque.131


25 Electron paramagnetic resonance spectrosc opy (EPR) is the preferred technique for the detection of free radicals and geomet rical states around pa ramagnetic transition metal ions with unpaired electrons. EPR is highly sensitive and interpretation of the hyperfine structure associated with each spect rum allows the electr onic nature of the radical to be established. However, the hi gh reactivity and short li fe-time coupled with low steady-state concentrations of nearly all radicals prevent their direct detection in most cases. A simple experimental technique that is coupled with EPR is to employ spintrapping, in which a reactive ra dical reacts with a nitrone or a nitroso compound to form a much more stable nitroxyl radical whose concentration rises we ll above the detection limit of the spectrometer. The selection of the appropriate spin-trap is important and depends upon the nature of the initial radical and on the objectives behind the experiment. Choice of incorrect spin-tra ps may yield data that are ambiguous and misinterpreted (discussed below). The suggestion that A itself might spontaneously ge nerate free radicals was first made in 1994 when EPR spectra were observed during 6 hour incubation of A 1-40 at 37 C in phosphate buffered saline (PBS) in the presence of the spin-trap N tert -butylphenylnitrone (PBN).111 The observation therein and fu rther EPR spectra employing PBN as a spin-trap132,133 led to the “molecular shrapnel” hypot hesis. The main feature of these EPR spectra was a 4-line pattern, and such a pa ttern is not character istic of a true PBN adduct. In a similar expe riment, Tomiyama et al.134 observed a spectrum consisting of the superimposition of 3-line, 4-line, and 6line patterns during the incubation of A 1-40 in the presence of PBN in deionized water. Again, the 3-line pattern is no t characteristic of a


26 true PBN adduct. Under conditions of good spectral resolution, PB N adducts, without exception, consist of a 6-line pattern.135 Subsequently, Dikalov et al.136 reported that they were unable to detect any EPR spect ra following the 6 h incubation of A 1-40 and A 25-35 with PBN. When these same peptides we re incubated with a less pure sample of PBN they observed both 3-line a nd 4-line EPR spectra. These spectra were also observed in the absence of peptide, a nd were attributed to transiti on metal-catalyzed auto-oxidation of ditert -butylhydroxylamine and N tert -butylhydroxylamine, present as impurities within their sample of PBN. It was proposed that these impur ities were converted to their corresponding nitroxyls, ditert -butylnitroxide and tert -butylhydroaminoxyl. Further there is current evidence that A and -synuclein can generate ROS directly.118 The arguments presented herein could apply to pr oteins associated with some of the other protein conformational di seases mentioned above. In some recent publications Bush and co-workers have reported that A 1-40 forms H2O2 in the presence of both c opper and iron and can reduce Fe3+ to Fe2+ as well as Cu2+ to Cu+.110,137-139 The possible formation of H2O2 during the incubation of A in PBS could provide some interes ting insights into the ch emistry outlined. First, H2O2 is a strong oxidizing agent and, if pr esent, would certainly prom ote the hydrolysis of PBN. Secondly, the presence of both H2O2 and Fe2+ and/or Cu+ would lead to the formation of hydroxyl radicals by Fenton chemistry. It has been f ound that low concentrations of metals are present even in chelex buffers, a nd that significant amounts of metals are also bound to the peptide itself.140 If formed, the hydroxyl radical would be trapped by PBN, but the resulting adduct is unstable and is not directly observed in aqueous solution.


27 There is good evidence that at pH 7 and above PBN rapidly transforms into the tert butylhydroaminoxyl radical.141 This could also help to explain the origin of the characteristic 4-line spectrum of the tert -butylhydroaminoxyl radical obtained when A is incubated in the presence of PBN. Consequently, it may be that the very weak EPR signals of tert butylhydroaminoxyl observed by Butterfield and co-workers132,133,142-145 and by Monji et al146,147 are resulted from the formation of H2O2 in the presence of low concentrations of Fe and Cu rather than the spontaneous formation of peptide-derived radicals. A second scheme involving reactions of 2-methyl-2-nitro sopropane (MNP) and PBN explains the origins of the 3a nd 6-line spectra in the presence of A and ambient laboratory lighting. The possibility that H2O2 might be formed in the immediate vicinity of the peptide is interesting si nce, as noted above, in the pr esence of the appropriate metal ions it would be readily c onverted to hydroxyl radicals via Fenton chemistry. The hydroxyl radical is a strong oxi dant that can attract hydr ogen atoms from organic molecules with a rate constant close to the diffusion controlled limit.148 Because of its high reactivity the hydroxyl radical is very unselective and removes hydrogen atoms from primary, secondary and tertiary carbon atoms w ith almost equal facil ity. It also adds to C=C double bonds with a rate close to the diffusion controlled limit. Consequently, immediate oxidative damage woul d occur to any organic molecule in vicinity of the site of hydroxyl radical. Bush and co-workers110, 139 have reported results showing the production of H2O2 in systems involving A 1-40, A 1-42 and low concentrations of Fe3+ and Cu2+ in PBS. Quantitative measurement of the H2O2 levels was carried out using the standard


28 thiobarbituric acid reac tive species (TBARS) assay and not spin-trapping. Their results are reasonably convincing, however, not in agreement with other studies where H2O2 levels appear to be lower than those re ported by Bush, as double integration of DMPO hydroxyl radical adduct signals against calibra tion standards indicate s the concentration of adducts is not higher than about 0.5 mM from 100 mM CuA 1-40. A significant number of the aggregating polype ptides that have been implicated in neurodegenerative disease have been shown to bind to transition metal ions such as iron, zinc, copper or manganese. Another common emerging theme is that some of these polypeptides seem to function normally as an tioxidants through stru ctural or activity similarities to the active site of superoxide dismutase. This seems to apply to prion protein (PrP)149 and the A peptide.150 When these proteins are in an altered pathological configuration, they could actua lly become pro-oxidants. If we take the example of A there is strong evidence s upporting that binding of Cu2+, Zn2+, and possibly Fe3+ to the peptide involves coordination with the thre e histidine residues at positions 6, 13 and 14, along with the tyrosine residue at position 10.150 In the general case of biologically relevant redox-active transition metal ions, bi nding to peptide molecu les would still leave available coordination sites, allowing oxygen to bind, possibly in the form of a peroxo bridge. Such bridges are common in a variety of metal complexes,5,6,10,11,16 but in this case there is a further issue th at should be considered. As the peptide molecules change conformation and begin to aggregate this coul d have the effect of twisting the bridging oxygens, resulting in a straining of the complex. However, in the dimeric and small polymeric forms of the soluble peptide th e possibility of the formation of strong


29 electrophiles such as the Type-3 copper mode ls (discussed below) cannot be discounted. The conversion of superoxide to H2O2 is a well established reacti on. It is possible that the superoxide, and subsequently hydrogen pe roxide, is formed as a byproduct of the aggregation and fibril assembly process. In other words, the amyloid-induced toxicity could actually be associated with the ge neration of ROS during the process of fibril extension and growth. In this case, a search for one or more types of special toxic aggregate may yield several varian ts of toxic intermediates. It is assumed that Fenton chemistry can occur if peroxide is present in close proximity to Fe3+ resulting in the formation of hydr oxyl radicals which will react with organic molecules in situ including peptides. In this case a peptidyl radical may be detectable. Yanker and coworkers151 investigated the neurotoxic ity of a range of peptides spanning the entire A 1-40 sequence and concluded that 25-35 was the toxic domain. This report was supported by Pike et al109 who observed the formation of stable aggregates and neurotoxicity in synthetic peptides containing residues 29-35. These results focused attention on the possibility that a particular amino aci d residue within this sequence might play a key role. In particul ar the methionine 35 residue has attracted attention.145 Substitution of this methionine by aspartate, serine and cysteine all resulted in peptides which were neither aggregated nor neurotoxic.152 Conversely, substitution by leucine, norleucine, lysine and tyrosine re sidues resulted in peptides which neither aggregated nor were neurotoxi c. Significantly, substitution of the methionine residue by serine or cysteine led to peptides which were at least as neurotoxic as A 25-35 itself suggesting that methionine is not unique in promoting these properties.152


30 VI. TYPE-3 COPPER OXIDAS E MODELS AND METALLO-ROS Because the generation of activated forms of O2 through metal-centered reactions has been implicated in the etiology of neurodegenerative disorders, it warrants the question: Is the ROS generated and then di ffused or does it remain metal bound? Thus, even if the fields of Type-3 copper oxid ase biomimetics and neurodegeneration by ROS seem unrelated, at least in terms of goals, they are certainly connected at the chemical level by the drive to understand copper-O2 interactions. The oxidation of organic substrates w ith molecular oxygen under mild conditions is of great interest for industrial and synthetic processes from an economical and environmental point of view.153 Although the reaction of organic substances with dioxygen is thermodynamically favorable, it is ki netically unfavorable due to the triplet ground state of O2. In biological systems this problem is overcome by the use of copperor iron-containing metalloproteins which serve as highly efficient oxidation catalysts.154-157 Some examples of Cu-oxidases/ oxygenases are shown in Figure 1. The catechol oxidases are Type-3 copper enzymes containing a binuclear copper center.158,159 Well-known representa tives of these Type-3 copper proteins are hemocyanin,160-162 the O2 carrier for arthropods and mollusks, and tyrosinase.163 Catechol oxidase belongs, like tyrosinase, to the pol yphenol oxidases which catalyze the oxidation of phenolic compounds to qui none in the presence of O2. Tyrosinase catalyzes the hydroxylation of tyrosine to DOPA and th e oxidation of DOPA to dopaquinone with


31 Figure 1.2. Types of enzymes involved in O2 metabolism.16


32 electron transfer to O2, while catechol oxidase only catal yzes the oxidation of catechols to quinones.164 This reaction is of significance in me dicine and biology since catecholamine neurotransmitters like epinephrine, norepine phrine, and DOPA are part of the tyrosine metabolic pathway.165 Secondary reactions like melanin formation are downstream after oxidation of the catechol moiety of catec holamine substrates in the presence of polyphenol oxidases, which are also responsible for the brown color of injured plants.166 The copper in isolated catechol oxidases was determined to be EPR-silent due to an antiferromagnetically coupled Cu2+–Cu2+ pair.167 The electronic absorption spectrum of oxycatechol oxidase from Ipomoea batatas exhibits an intense absorption band at 343 nm and a weaker band at 580 nm, later found to be due to the peroxo complexes of hemocyanin and tyrosinase. These intense electronic transitions were assigned to a peroxo to Cu2+ charge transfer transitions168,169 with an O–O stretch vibrational band at 749 cm–1 indicating a possible 2: 2 bridging mode of the peroxo group. X-ray abosorption spectroscopy (XAS) i nvestigations on the native met forms of catechol oxidases from Lycopus europaeus and Ipomoeas batatas have revealed that the active site consists of a dicopper(II) center, in which the metal atoms are coordinated by four N:O donor ligands. Multiple scattering extended x-ray absorption fine structure (EXAFS) calculations have shown high significance for one or two coordinating histidine residues.170 The short metal–metal distance of 2.9 and the results of EPR investigations indicate that a -OH bridged dicopper(II) ce nter resides in the active site in the met forms of the proteins.171 Model studies of synthetic analo gues have furthered the understanding of the structural and chemical properties of these proteins.11,16 Current interest is focused


33 on the elucidation of the mechanism of c opper complexes that show catecholase and phenolase activities with different structural and electronic featur es around the copper ions. In these studies, monoor multinuclear complexes have been synthesized and the properties of the chelating ligands have been varied with respect to geometry, number, and nature of the donor atoms. There are seve ral structural constraints for proper Type-3 oxidase activity, such as square-planar mononuclear Cu2+ complexes exhibit only little catalytic activity while non-planar mononuclear Cu2+ complexes show a high activity.172,173 It was also found that binuclear co mplexes can also catalyze oxidation reactions if the CuCu distan ce is less than 5 . A steric match between substrate and complex is believed to be one of the determ inants for reactivity where two metal centers have to be located in close proximity to facilitate binding of the two hydroxyl oxygen atoms of catechol prior to the electron transfer.172 This conclusion wa s supported by the observation that binuclear copper complexes are generally more reactive towards the oxidation of catechols than are th e corresponding mononuclear species.174 It has been particularly challenging to s ynthesize structures where a substrate bridged compound can be resolved. Thus far only one case is know n in which the crystal structure of a catalytically active binuclear Cu2+ complex with a coordina ted catecholato ligand has been solved.175 Two other examples of mononuclear square-planar copper complexes176 were also effective catalysts, demonstrating that geometrica l effects are only one aspect of the complex activity. In mechanistic studi es, the same authors pointed out that a narrow range of redox potentials for effective catalysis exists betw een ease of reduction by the substrate and subsequent reoxidation by O2.177 Although some general structure-


34 reactivity patterns have been found, the oxidation chemistry of structurally wellcharacterized copper complexe s is still not fully underst ood, especially regarding the parameters affecting the catecholase activity. For example, there appears to be no direct correlations between the rates of reaction and the redox potential s of these complexes. Exploration of the oxidation chemistry of we ll-characterized copper complexes together with a detailed understanding of the function of O2 activating copper enzymes is expected to provide the basis for new catalytic oxi dation systems for synthetic and industrial processes. Current understanding of the types of c opper–dioxygen species or intermediates relevant to O2-binding to or activation by copper prot eins comes from the combination of biochemical/biophysical studies and coordination chemistry effo rts, with the latter having played a significant role. Figure 2 on the ne xt page provides a su mmary of structural types of nearly all now well established reactive intermediates of Cu-O2 species. These are the starting point for describing the possi ble conformation of reactive intermediates based on copper chemistry. As seen by the la rge number of examples (Figure 2), many structural types exist which are perhaps fa r more than are known for heme or nonheme iron enzymes or complexes. Synthetic bioinorganic copper(I)–O2 chemistry has flourished in the last 25 years.11,16,178-181 These efforts to elucidate fundame ntal chemical aspects of Type-3 oxidases have been inspired by understanding of the diverse nature of the active-site chemistry of copper-protein O2-carriers, monooxygenases, and oxidases.182-185 Enzymes with active sites having one, two, three, or f our copper ions are known (Figure 1), and the


35 Figure 1.3. Structure of CuO2 intermediates identified in biomimetic complexes.16


36 variations in ligand environment and reac tivity patterns are vast. The prototype for dioxygen binding to copper, and subsequent the primary model for ligand design has been the structure and reversible O2-binding of arthropodal and molluscan blood oxygen transporting protein, hemocyanin.185 The dicopper motif, also found in tyrosinase182and catechol oxidase,186 is of considerable interest with respect to the relationship of dioxygen binding and activation. Formation of the copper-dioxygen adducts leads to copper-mediated reduction of O2 to superoxo (O2 –), peroxo (O2 2–), or O–O cleaved products (copper-oxo, O2–), which constitute the active sp ecies responsible for substrate oxidation. While there is ample literatu re for oxidative reactions by copper complexes11,178,179,181,187,188 it is only within the past few y ears and recent advances in low temperature spectroscopy and reactivity that we have been able to assign a given organic reaction to specific copper-O2 derived species. In f act, a number of the CuO2 species can be placed into groups of reactivity types, for example as nucleophilic or electrophilic reactive intermediates. Following is a br ief summary of the known reactivity of biomimetic complexes of Type-3 copper oxida ses which is the basi s for categorizations of different CuO2 intermediates responsible for reactivity. The 2: 2 peroxo-dicopper(I I) (Figure 1.2 SP ) complex was first elucidated with x-ray crystallography in a synt hetic system by K. Fujisawa and the late N. Kitajima,189,190 and subsequently confirmed for oxy-hemocyanin.191 This moiety was also detected in tyrosinase182and catechol oxidase186 with spectroscopic methods It is likely, and now generally accepted, that the 2: 2 peroxo-dicopper(II) core is closely involved in aromatic hydroxylations, both in enzymes like ty rosinase and chemical systems. Notably,


37 through the research efforts of W.B. Tolman and co-workers there is an established highvalent bis--oxo-dicopper(III) species,16, 188 (Figure 1.2 O ) that is thus far not found in biochemistry but only exists as an interes ting example of how chemists can elucidate useful new chemical possibiliti es. A moiety such as this, or even containing more than two copper ions in a different overall redox stat e, is suggested to be resposnsible for the hydroxylation of methane in particul ate methane monooxygenase (p-MMO), a membrane-bound copper-dependent enzyme found in methanogenic bacteria.192 Mono and binuclear copper-hydroperoxo re active intermediate s are also known. Cu2+-OOH and more recently Cu2+-superoxide species, were s uggested to be present in the copper-containing enzymes dopamine -monooxygenase (D M) and peptidylglycine -hydroxylating monooxygenase (PHM).183,193,194 While D M and PHM have two activesite copper ions, they are separated by a large distance (~11 ) and the oxidative hydrogen-atom chemistry is though t to occur at the CuM site near the substrate binding pocket. The second metal center, dubbed CuH, has been suggested to assist the reaction by serving the role of an elec tron-transfer center to deliver the proper number of electrons to reduce O2. The chemistry of Cu-O2 species is being pursued more heavily as of late and interesting new results from Klinman a nd co-workers will offer a new insight into both ligand design and mechanistic approach es to studying Type-3 oxidase systems.195 Binuclear peroxo-dicopper(II) complexes (Figure 1.2 TP and SP) were the first to be well characterized.16,190 In synthetic chemical systems, it is difficult to stabilize the principal reaction product of Cu+ and O2 (Cu-O2), from coupling with another ligand-copper(I) complex to form a Cu2+-(O2 2–)-Cu2+ product.180 These compounds were originally


38 stabilized at low temperature (–80 C) in organic solvents. Early examples are [Cu2 2+(XYLO–)(O2)]+ which contained an asymmetrical end-on peroxo moiety,196 a 1,2-peroxo complex [{(TMPA)Cu2+}2(O2)]2+, (Figure 1.2 TP)197 and [Cu2 2+ (N4)(O2)]2+ Figure 1.3).198 Several analogues of [{(TMPA)Cu2+}2(O2)]2+ and [Cu2 2+ (N4)(O2)]2+ have been characterized, showing vari able stability and reactivity.16,199,200 These complexes were used 201 to generate a reactivity profile based on the type of reactive intermediate. It was determined that [Cu2 + (XYLO–)]+ reversibly binds O2 and that the resulting peroxo complex is basic. This means that a ca talyst that works through a nucleophilic substitution mechanism. It was also determined that the complex [Cu2 + (XYLO–)O2]+ does not oxygenate triphenylphosphi ne nor CO, but it releases O2 and binds PPh3 and CO. The peroxo complex however can be protonated to afford a -1,1-hydroperoxo complex, (Figure 1) or it can be acyla ted to form an analogous -1,1-acylperoxo complex. 202 Further, [Cu2 2+(XYLO–)(O2)]+ reacts as a nucleophile with CO2, with the formation of a percarbonato species that ther mally decomposes to the carbonato complex [Cu2 2+(XYLO–)(CO3)]+. Phenols are not hydroxylated no r oxidatively coupled (forming a dimer product) by [Cu2 2+(XYLO–)(O2)]+, but it was the first exam ple of a model system in the area of Type-3 oxidase biomimetics to show reactivity consistent with general acid-general base chemistry. The same nucleophil ic chemistry is observed in reactions of [{(TMPA)Cu2+}2(O2)]2+.201,203 With these preliminary resu lts it became evident that the nature of the intermediate would determine reactivity. By contrast, the 2: 2 side-on peroxo complex [Cu2 2+ (N4)(O2)]2+ reacts differently with the same substrates tested for the end-on peroxo complexes201 yielding


39 O=PPh3 in a reaction with PPh3. Perhaps one of the simplest and most elegant example of a model compound for Type-3 oxidases was synthesized by Stack and co-workers;204 they showed that a side-on peroxo complex formed from a simple binucleating ligand, namely [{(L)Cu}2(O)2]2+ (L=N,N-di-tert-butyl-N,N’-dim ethylethylenediamine Figure 1.4 B), also oxygenates PPh3. Further, [Cu2 2+(N4)(O2)]2+ does not protonate, acylate, nor react with CO2. The peroxo complex oxidizes phenols by H+ abstraction, leading to catalytic oxidative coupling chemistry under pre-equilibrium conditions (i.e. in the presence of excess phenol and dioxygen). T hus, a side-on peroxo complex is nonbasic or electrophilic in its reactivity toward these substrates. The electrophilic behavior of these model compounds has also been demonstrated in reactions of greater impact, as in hydroxylation of arenes (including phenols) ; this reactivity can have significant implications toward green chemistry, since phenols and chlorinate d phenols are rather stable pollutants which can contaminate soil and water ways. Activation of the peroxo complex [Cu2 2+(XYLO–)(O2)]+,201 was also determined to be electrophilic with H+ or CO2, affording [Cu2 2+(XYLO–)(O2H)]2+ 201 and the percarbonato species [Cu2 2+(XYLO–)(CO4)]+, respectively. These, in contra st to the original end-on peroxo complex [Cu2 2+(XYLO–)(O2)]+, readily convert PPh3 to O=PPh3. Thus, in biochemical systems, hydroperoxo-copper complexes are a po ssible reactive intermediate which may include percarbonato species, since CO2 as bicarbonate is certainly available in biological media. While carried out fo r a specific set of peroxo-di copper(II) compounds, the trends observed in these early reactivity studies of reactive intermediate types in copperdioxygen chemistry have generally been found to be consistent; however, there are some


40 exceptions.190,205,206 The end-on peroxo complexes with nucleophilic behavior have not yet shown oxidative chemistry comparable to the side-on complexes; however, Karlin and co-workers continue to pursue if ths alternative structure can perform useful chemistry, such as in the Baeyer-Villiger oxidation (oxidation of ketones to carboxylic acids in the presence of peracids). It is now known that binuclear copper en zymes such as tyrosinase and catechol oxidase adopt the side-on 2: 2-peroxo structure, as do model compounds like the Kitajima/Fujisawa complex, the Karlin complex [Cu2 2+(N4)(O2)]2+ and several other analogues.16 The great number of investigations in copper(I)–dioxygen reactivity have also revealed a novel binding motif in which the peroxo O–O bond is fully cleaved, after receiving 2 electrons from di-Cu2+, resulting in a high-vale nt dicopper(III) bis--oxo (Cu2 3+-O2) species (Figure 1.2 O ), first characterized by Tolman’s group,207 using tridentate substituted triazacyclononane as ligands, and also by Stack and coworkers,208,188 using ethylenediamine-based donors.16,188 The two isoel ectronic species have dinstinct spectros copic features and structural differences. The main structural difference is the shortened Cu–Cu distan ce of ~2.8 for the bis--oxo-dicopper(III) species when compared to the side-on pe roxo-dicopper(II) Cu–Cu distance of ~3.5 . Theoretical calculations haved predicted that there is a small difference in the free energies of the side-on peroxo Cu2 2+-O2 and the Cu2 3+-O2 species and a low barrier for interconversion,209-211 meaning that under experimental conditions (ambient temperature) the two forms are under equilibrium. This is experimentally observed in several systems


41 Figure 1.4. Early examples of Type-3 oxidase models by Karlin.196198 and Tolman.207


42 where the side-on peroxo-dicopper(II) and bis--oxo-dicopper(III) sp ecies are found in a dynamic equilibrium, strongly influenced by the na ture of the ligand such as denticity, Nalkyl vs. N-pyridyl donor, and steric effect, as well as reaction conditio ns, including concentration, counter ion eff ect, temperature, and solvent.16,188 These well-characterized Cu2O2-containing synthetic complexes are quite reactive, with oxidative capabilities ranging from alcohol/catechol oxidation, oxo transfer to phosphines and sulfides, to aromatic/aliphatic hydroxyla tions, and also oxidati ve N-dealkylations.11 The existence of two oxidative species coexisting in equilibrium complicates the determination of which form is the true reactive intermediate responsible for the reactivity. A brief summary and comparison of Ka rlin’s and Tolman’s effors is discussed below, highlighting preferenti al reactivity patterns of side-on peroxodicopper(II) and bis--oxo-dicopper(III) species. Karlin’s group212,213 elucidated several examples of copper-promoted dioxygen activation. While stoichiometric in nature this monooxygenase model system is a rare chemical system where an unactivated C–H bond substrate is rapi dly hydroxylated under “very mild” conditions utilizing dioxygen (–80 C and 1 atm O2).213,214 The dicopper(I) complex [Cu2 +(XYL-H)]2+ reacts with O2 to form [Cu2 2+(XYLO–)(OH)]2+ stoichiometrically. Isotope labeling experiments (using 18O) demonstrate the product phenol O-atom is derived from dioxygen, and the observed O2-consumption stoichiometry (Cu:O2 = 2:1) is consistent with monooxygenases. Detailed low temperature stopped-flow kinetic studies180,215 revealed the intermediate


43 [Cu2 2+(XYL-H)(O2)]2+. Its peroxo structure was confir med by utilizing the electron withdrawing R=NO2 derivative, which results in a decreased kon but even more diminished first order rate constant fo r the hydroxylation step, thus allowing for spectroscopic investigation.179,215 In resonance Raman studies, characteristics of [Cu2 2+ (NO2-XYL-H)(O2)]2+ are conistent with a 2: 2-peroxo moiety, with the O–O vibrational stretch at 747 cm–1.216 Analysis of spectrosc opic and kinetic studies indicates that the side-on peroxo moiety is the reactive species responsible for oxygentation, supported by the observation that the O–O stretching disappears at the same rate as the phenolic C–O bond stretch at 1320 cm–1 in [Cu2 2+(NO2-XYLO–)(OH)]2+.216 A number of results support the m -xylyl hydroxylation reaction where [Cu2 2+(R-XYL-H)(O2)]2+ acts as an electroph ile, attacking the -system of the arene substrate. Side-on peroxo complexes [Cu2 2+(R-XYL-H)(O2)]2+ are very similar to [Cu2 2+(N4)(O2)]2+, shown to be electrophilic. Stopped-flow kinetic studies showed that rate constant for hydroxylation in creases as the R substituent becomes more electron donating (Hammett plot = –2.1),179 with deuterium substitution into the 2-H position of the substrate not affecting the firs t order rate constant for hydroxylation. The lack of a deuterium kinetic isot ope effect is consistent with an electrophilic attack on the arene substrate -system, precluding C–H bond cleavag e in the rate-determining step.179 Methyl substitution into the xylyl 2-positi on where hydroxylation occurs for the parent compound [Cu2 +(XYL-CH3)]2+ in a reaction with O2, results in aromatic hydroxylation followed by a 1,2-migration of the methyl gr oup. The process is similar to the ‘‘NIH shift’’, observed previously in iron hydroxyl ases via isotope labeling, where a carbonium


44 ion intermediate is obtained as a result of an electrophilic iron-oxo reactive intermediate, resulting in R migration.217 Computational studie s indicate that the side-on peroxo moiety is indeed electrophilic; the xylyl hydroxyla tion reaction can be seen as derived from interactions of the empty */ orbitals of peroxo O–O with the filled p orbitals in the arene substrate.216,218 The favorable proximity of the side-on peroxo moiety to the m xylyl substrate, due to the ligand design and resulting intramolecular O2 reaction with two copper(I) centers, is certainly important in promoting the aromatic hydroxylation reaction. In this sense, the system resembles reactions at enzyme active sites, where preorganization of the substrate and reactive species occur. Since the discovery and characterizati on of the model monooxygenase system [Cu2 +(XYL-H)]2++O2 [Cu2 2+(XYLO–)(OH)]2+, a good number of analogous systems have been described.11,179 Tolman and co-workers,206 however, have shown that a bis-oxo-dicopper(III) complex is also capable of aromatic hydroxylation. This observation is opposite in trend to the general reactivity patterns establishedthus far, serving to demonstrate the general points that placing a reactive intermediate species in close proximity to a substrate by ligand design grea tly enhances the proba bility of turnover, and that different types of intermedieates will promote certain placements thus promoting different reactivities. Itoh and co-workers219 have demonstrated the importance of the side-on peroxo-dicopper(II) spec ies as relevant to phenol o -hydroxylation both in synthetic systems and in the enzyme tyrosinase.220 Cresolase activity ( o -hydroxylation of phenols, i.e cresol) was succe ssfully mimicked with a Cu+ complex possessing a tridentate ligand N N -bis[2-(2-pyridyl)-


45 ethyl]-R,R-dideuteriobenzylamine (LPy2Bz, Figure 1.4 A) that binds Cu+ and reacts with O2 to generate a low-temperature stab le side-on peroxo-dicopper(II) species.219,220 Addition of lithium salts of p -substituted phenols gi ves the corresponding o -hydroxylated catechols in high yields. Ne ither oxidation to the o -quinone nor C–C/C–O coupled dimerization was observed. With the use of 18O isotope labeling experiments, it was confirmed that the incorporated cat echol oxygen atom is derived from O2.220 Mechanistic studies revealed substrate sa turation kinetics, consisent of formation of a phenolate/Cu2O2 complex prior to the rate-determining oxygenation step. An observed increase in reaction rate with increasing electron dona ting ability of the X substituent (p-XC6H4O-Li) coupled with the lack of a deuterium kineti c isotope effect, suggests the side-on peroxo species reacts via an electrophilic aromatic substitution mechanism,219 similar to that described for the Karlin system,179 the dicopper-mediated m -xylyl aromatic hydroxylation. To compare and contrast, Itoh and co-workers220 studied the analogous chemistry on tyrosinase. As it was also s een in the model reaction, oxygenation rates increased with increasing p -substituted phenol electron don ating properties, clearly demonstrating that the enzyme acts as an electrophilic reactive intermediate. Hammett plots of the enzymatic reaction give a value of –2.4, similar to the model reaction ( = – 1.8) and Karlin’s XYL hydroxylation ( = –2.1),179 supporting the hypothesis that the phenolase activity of tyrosinase occu rs by an electrophilic attack by a 2: 2-peroxodicopper(II) species. Casella and co-workers221,222 and Sayre and coworkers223,224 have also contributed in the area of o -phenol hydroxylation chemistry and their papers should be noted.


46 Consistent with the conclusions described a bove, CasellaÂ’s group has also concluded that the side-on 2: 2-peroxo species is critical in this chemistry.221 Stack and co-workers205 showed that a side-on peroxo-dicopper(II) complex with a secondary amine ligand, N,Ndi-tert-butylethylenediamine (Figure 1.4 B), also exhibits some phenolase and catecholase activity, producing mixtures of catechol and o -benzoquinone from 2,4-ditert-butylphenolate as substrate. With addition of the neutral phenol, no oxidatively coupled dimer is produced, contra ry to KarlinÂ’s complex, [Cu2 2+(N4)(O2)]2+. StackÂ’s side-on peroxo complex is also able to pe rform other electrophilic types of reactions, converting PPh3 to O=PPh3, catechols to quinones, and benz yl alcohol and benzylamine to benzaldehyde and benzonitrile, respectively.205 Bis--oxo-dicopper(III) species have been implicated in a number of different reactivities, including al cohol oxidation, phenol coupli ng, oxidative N-dealkylation, aliphatic hydroxylation, and oxygena tion of phosphines or sulfides.188 Detailed mechanistic studies and theoretical calcula tions suggested that the bis--oxo core facilitates hydrogen-atom abstraction reactions.11,210,211,225 Tolman and co-workers11,226 studied intramolecular N-dealkylation reacti ons by bis--oxo-dicopper(III) complexes. The electrophilic nature of the bis--oxo core was elucidat ed by its lack of reactivity towards acids, while it can only act as an outer-sphere reactive intermediate in the presence of acid. The bis--oxo complex d ecomposes by oxidative N-deakylation, with one of the arms of the tri-substituted tri azacyclononane ligand being cleaved, resulting in production of the primary amine and the alde hyde/ketone. Double crossover experiments using labeled 18O2 confirm the carbonyl oxygen deri ves from the high-valent Cu2 3+-O2


47 Figure 1.5. Type-3 model systems by Itoh219 (A) and Stack204 (B). A B


48 core and that the reaction is intramolecular. Primary kinetic isotope effects and Eyring activation parameters revealed that C–H bond cleavage is the rate-determining step. A Hammett study with derivatives of triben zyl-substituted triazacyclononane ligands demonstrates that reaction rates increase with electron-donating substituents. A small observed negative value of –0.4 suggests the Cu2 3+-O2 core behaves as an electrophilic radical;11 similar values were reported for benzylic hydrogen-atom abst raction reactions using free radical reactive in termediates, as well as cytochrome P450 and porphyrinderived high-valent metal-oxo species. Bis--oxo-dicopper(III) complexes with pyridylalkylamine ligands have been used by Itoh et al.227 to model benzylic hydroxylation chemistry similar to dopamine-monooxygenase (D M) activity. Their experiments using 18O2 confirmed oxygen atom incor poration into the oxygenated alcohol, which in the experiment is trapped by coordinating to copper( II). Deuteration at benzylic positions revealed a la rge kinetic isotope effect of 35.4 at –80 C. The activation parameters are consistent with intr amolecular rate-determining hydrogen atom abstraction followed by rebound of a coppe r-bound OH group, analogous to cytochrome P450 monooxygenase chemistry. A Hammett pl ot based on experiments with varying substituents at the para position gives = –1.48, consistent with benzylic hydrogen atom abstraction reactions. With the tridentate ligand LPy2Bz, benzylic hydroxylation was also observed, but spectroscopic evidence, reveals the low-temperature dioxygen adduct formed is a side-on 2: 2-peroxo-dicopper(II) species. For this system, the deuterium kinetic isotope effects are much smaller th an expected for a rate-determining hydrogen atom abstraction step, and the rate of hydroxylation is not affected by the p -substituent of


49 the ligand. Instead, the rate of reaction was found to be solvent dependent. From the combined observations, it was proposed that cleavage of the O–O bond to form the highvalent copper(III) bis--oxo core was rate-limiting, which is then responsible for the hydrogen atom abstraction.228 Karlin and co-workers229,230 showed that copper(I)/O2 reactivity with tridentate liga nds give rise to mixtures of side-on peroxodicopper(II) and bis--oxo-dicopper(III) complexes, in rapi d equilibrium at –80 C. These showed oxidative chemistry towards different subs trates. With respect to dimethylaniline oxidative N-dealkylation, mechanistic studies using p -substituted and deuterated dimethylanilines suggest the reactive intermediate can operate via either a rate-limiting hydrogen-atom abstraction or electron-trans fer pathway, depending on the R group in the complex [{Cu(R-Me-PY2)}2(O2)]2+ and the ease of the substrate oxidation.231 Itoh and co-workers232 have been successful in distinguishing the active intermediate for the radical coupling of neutra l phenols. According to their spectroscopic features, the Cu2O2 adducts are the bis--oxodicoppe r(III) and the side-on peroxodicopper(II) species (Figure 1.2) respectively. Both isomeric species react with neutral phenols to produce solely the C–C coupled dimers. Both Cu2 3+-(O)2 and Cu2 2+-(O2) species exhibit this same behavior. However, the rate constant for the reaction of the bis-oxo species was two orders of magnitude gr eater than that for the peroxo complex. This could be due to two reasons: either th e bis--oxo is intrinsi cally a better reactive intermediate than the side-on peroxo because of a difference in redox potentials, or the (LPY1)2Cu2 3+-(O)2 bis--oxo complex is the true reac tive intermediate, which would be in much higher concentration than a small amount of bis--oxo isomer present in


50 equilibrium with the side-on complex (LPy2Bz)2Cu2 2+-(O2). The hypothesis that the bis-oxo is the true reactive species is favored b ecause if the side-on peroxo complex was able to couple phenols via the proton-coupled elect ron transfer mechanism, then the reaction with phenolates should also occur through an outer-sphere el ectron transfer mechanism to give the coupled phenol. However, this c ontradicts previous obs ervations that the 2: 2-peroxo-dicopper(II) complex reacts wi th phenolates to yield solely o -catechol products. In the chemistry described for hydroperoxodicopper(II) (Figure 1.2 -1,1 hydroperoxo), enhanced electrophi lic reactivity occurs due to protonation of less reactive and nucleophilic peroxo-dicopper( II) complex, at least with respect to triphenylphosphine oxygenation. While the latter re action is not of great impor tance due to the ease of PPh3 oxygenation, protonation of peroxo-copper or other O2-derived species may be important as a means of peroxo-copper activation.233-235 In heme enzymes such as cytochrome P450 monooxygenase, protonation of a heme-peroxo l eads to O–O heterolytic cleavage and generation of the high-valent ferryl speci es responsible for el ectrophilic oxidation reactions like C–H hydroxyl ation or epoxidation.236 Activation to an alternate reactive intermediate appears to be pres ent in heme oxygenase, where an O2-derived hemehydroperoxo species attacks the porphyrin meso-carbon substrate.237,238 More unique examples of reactivity for binuclear hydroperoxo complexe s which do not have the ArO– bridging ligands have been reported.239 Although structural descri ptions are not as readily available, kinetic and/or sp ectroscopic probing shows such Cu2+-(–O2H)-Cu2+ intermediates are capable of arene hydroxylation. Thus, Karlin, Zuberbhler, and co-


51 workers239 report that reaction of a dicoppe r(II) complex with ligand XYL-H and hydrogen peroxide also leads to efficient aren e hydroxylation. It is likely, according to reactivity, that a 1: 1-OOH hydroperoxo-dicopper(II) sp ecies facilitates arene hydroxylation. Casella and co-workers240 reported a compound capable of performing double arene hydroxylation, again in a dicopper(II) plus hydroge n peroxide reaction; not only were kinetic parameters determined fo r the kinetics of s ubstrate attack by an 1: 1hydroperoxo-dicopper(II) intermed iate, but the latter was al so characterized by UV-Vis and EPR spectroscopies. Thus, dicopper(II) -hydroperoxo species, with or without an additional phenoxo bridging ligand, which c ould, be replaced by a water or hydroxo ligand in solution,240 can effect oxidation reactions, a nd further exploration of their chemistry should be considered. The work involved in ligand design a nd low-temperature techniques leads to stable enough species to be characterized spectroscopically, but may prevent efficient reactivity, especially for hydro carbon-based substrates. Thus, there is a general caveat in concluding that an observed or ganic substrate oxidative reac tion actually occurs from a well-defined and fairly stable Cun-O2(H) complex. The true reactive intermediate may be something other than what can be observed spectroscopically. It is possible that a transient mononuclear Cu3+-oxide Cu2+-O•– species is the most likely candidate for the reaction of Type-3 oxidases both in the nati ve proteins and their biomimetic models.188


52 VII. CONCLUDING REMARKS Considerable effort in trying to el ucidate the nature of the cause of neurodegeneration in AD and the catalyti c mechanism through which Type-3 copper oxidases catalyze their chemical transforma tions has yielded some common threads. First, it is known that oxyge n radicals and reduced forms of oxygen collectively termed ROS are generated during normal metabolism. It is also know that the amyloidfibrils and plaques can generate those same ROS via a metal-centered mechanism. These activated forms of O2 are no different than the metal-bound forms associated with Type-3 copper oxidases. It is expected that furt her analysis of metal complexes of amyloidcan show that the chemistry proposed as the culp rit in the etiology of AD is very much consistent with the reactivity of Type-3 c opper oxidases and their biomimetic systems.


53 VI. LIST OF REFERENCES 1) R.J.P. Williams, J.J.R. Frausto da Silva, The Biological Chemistry of the Elements: The Inorganic Chemistry of Life Oxford University Press, New York, 2001. 2) K. Shikama, Nature of the FeO2 bonding in myoglobin a nd hemoglobin: A new molecular paradigm. Prog. Biophys. & Mol. Biol. (2006), 91, 83-162. 3) R.E. Stenkamp, Dioxygen and Hemerythrin. Chem. Rev. (1994), 94, 715-26. 4) M.-H. Baik, M. Newcomb, R.A. Friesner, S. J. Lippard, Mechanistic Studies on the Hydroxylation of Methane by Methane Monooxygenase. Chem. Rev. (2003), 103, 2385-2419. 5) S.J. Lange, L. Que Jr., Oxygen activating nonheme iron enzymes. Curr. Opinion Chem. Biol. (1998), 2, 159-172. 6) E.I. Solomon, P. Chen, M.Metz, S.-K L ee, A.E. Palmer, Oxygen binding, activation, and reduction to water by copper proteins. Angew. Chem. Int. Ed. (2001), 40, 45704590. 7) C. Gerdemann, C. Eicken, B. Krebs, The cr ystal structure of cat echol oxidase : New insight into the function of type-3 copper proteins. Acc. Chem. Res. (2002), 35, 183191. 8) J.W. Whittaker, The radical chemistry of galactose oxidase. Arch. Biochem. Biophys. (2005), 433, 227-239.


54 9) I.A. Koval, P. Gamez, C. Belle, K. Se lmeczi, J. Reedijk, Synthetic models of the active site of catechol oxida se: Mechanistic studies. Chem. Soc. Rev. (2006), 35, 814840. 10) A. Decker, E.I. Solomon, Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electroni c structures and reactivities. Curr. Opinion Chem. Biol. (2005), 9, 152-163. 11) E.A. Lewis, W.B. Tolman, R eactivity of Dioxygen-Copper Systems. Chem. Rev. (2004), 104, 1047-1076. 12) J. Anekwe, A. Hammerschmidt, A. Romp el, B. Krebs, Altering the activity of catechol oxidase model compounds by electronic influence on the copper core. Zeitschrift fuer Anorganische und Allgemeine Chemie (2006), 632, 1057-1066. 13) K.D. Karlin, M.S. Haka, R.W. Cruse, G.J. Meyer, A. Farooq, Y. Gultneh, J.C. Hayes, J. Zubieta, Dioxygen-copper reactivity. M odels for hemocyanin: Reversible O2 and CO binding to structurally characteri zed dicopper(I) complexes containing hydrocarbon-linked bis[2-(2-pyridyl)ethyl]amine units. J. Am. Chem. Soc. (1988), 110, 1196-1207. 14) N. Kitajima, K. Fujisawa, C. Fujimoto, Y. Morooka, S. Hashimoto, T. Kitagawa, K. Toriumi, K. Tatsumi, A. Nakamura, A new model for dioxygen binding in hemocyanin. Synthesis, characteri zation, and molecular structure of the 2: 2 peroxo binuclear copper(II) complexes, [Cu(HB(3,5-R2pz)3)]2(O2) (R = isopropyl and Ph). J. Am. Chem. Soc. (1992), 114, 1277-1291.


55 15) M.J. Baldwin, D.E. Root, J.E. Pate, K. Fujisawa, N. Kitajima, E.I. Solomon, Spectroscopic studies of side-on peroxide-bridged bi nuclear copper (II) model complexes of relevance to oxyhemocyanin and oxytyrosinase. J. Am. Chem. Soc. (1992), 114, 10421-10431. 16) L.M. Mirica, X. Ottenwaelder, T.D.P. Stack, Structure and Spectroscopy of CopperDioxygen Complexes. Chem. Rev. 2004, 104, 1013-1045. 17) G. Stark, Functional Consequences of Oxidative Membrane Damage. J. Membrane Biol. (2005), 205, 1-16. 18) M. Valko, H. Morris, M.T.D Cronin, Me tals, toxicity and oxidative stress. Curr. Med. Chem. (2005), 12, 1161-1208. 19) L.R. Ferguson, M. Philpott, N. Karuna singhe, Oxidative DNA damage and repair: significance and biomarkers. J. Nutrition. (2006), 136, 2687S-2689S. 20) M.E. Goetz, M. Gerlach, Formation of radicals. Brain Damage and Repair (2004), 135-164. 21) E. Gaggelli, H. Kozlowski, D. Valens in, G. Valensin, Copper Homeostasis and Neurodegenerative Disorders (Alzheimer's Prion, and Parkinson's Diseases and Amyotrophic Lateral Sclerosis) Chem. Rev (2006), 106, 1995-2044. 22) page=newsdetail&id=5 21133&ap=68&brand=30 23) ticle.cfm/ID/1352/T/Alzheimers/searchtext/ 24) Y. Ling, K. Morgan, N. Kalsheker, Int. J. Biochem. Cell. Biol (2003), 35, 15051535.


56 25) A. I. Bush, The metallobiology of Alzheimer's disease. Trends Neurosci (2003), 26, 207-214. 26) A. Kontush, C. Berndt, W. Weber, V. A kopian, S. Arlt, S. Schippling, U. Beisiegel, Free Rad. Biol. Med (2001), 30, 119-128. 27) R.A. Capaldi, Structural features of the mitochondrial electron-transfer chain. Curr. Op. Struct. Biol. (1991), 1, 562-568. 28) M. Sawada, U. Sester, J.C. Carlson, S uperoxide radical form ation and associated biochemical alterations in the plasma memb rane of brain, heart, and liver during the lifetime of the Rat. J. Cell. Biochem. (1992), 48, 296-304. 29) R.S. Sohal, L.A. Arnold, B.H. Soha l, Age-related changes in antireactive intermediate enzymes and proreactive intermediate generation in tissues of the rat with special reference to parameters in two insect species. Free Rad. Biol. Med. (1990), 10, 495-500. 30) G.M. Abu-Erreish, D.R. Sanadi, Age-relate d changes in cytochro me concentration of myocardial mitochondria. Mech. Ageing Dev. (1978), 7, 425-432. 31) G. Benzi, Enzymatic activities related to energy transduction in the mature rat brain. Interdiscip. Topics Geront. (1979), 15, 104-120. 32) G. Benzi, Peroxidation, energy tran sduction and mitochondria during aging. London, Paris: John Libbey Eurotext, 1990. 33) G. Benzi, O. Pastoris, F. Marzatico, R.F. Villa, F. Dagani, D. Curti, The mitochondrial electron transfer alteration as a factor i nvolved in the brain aging. Neurobiol. Aging (1992), 13, 361-368.


57 34) P.H. Gold, M.W. Gee, B.L. Strehler, Effect of age on oxidative phosphorylation in rat. J. Gerontol. (1968), 23, 509-512. 35) D. Curti, G. Benzi, Cytochrome c oxida se activity in discrete brain regions: agerelated changes. Arch. Gerontol. Geriatr. Suppl. (1991), 2, 549-556. 36) D. Curti, M.C. Giangar, M.E. Redol fi, I. Fugaccia, G. Benzi, Age-related modifications of cytochrome c oxidase activity in discrete brain regions. Mech. Ageing Dev. (1990), 55, 171-180. 37) S. Algeri, C. Cafa, A. Cagnotto, F. Marz atico, T. Mennini, L. Raimondi, G. Sacchetti, Calory restriction counteract s some of the neurochemical changes in the cortical neurons of senescent rats. Neurobiol. Aging (1992), 31, S134. 38) G. Barja de Quiroga, R. Perez-Campo, M. Lfipez Torres, Antirea ctive intermediate defences and peroxidation in liv er and brain of aged rats. Biochem. J. (1990), 272, 247250. 39) R.S. Sohal, Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria. Free Rad. Biol. Med (1993), 14, 583-588. 40) Q.L. Wang, W. Luo, W. Zheng, Y. Liu, H. Xu, G. Zheng, Z. Dai, W. Zhang, Y. Chen, J. Chen, Iron supplement prevents le ad-induced disruption of the blood-brain barrier during rat development. Toxicol. App. Pharmacol. (2007), 219, 33-41. 41) S.J. Kish, C. Bergeron, A. Rajput, S. Dozic, F. Mastrogiacomo, L.-J. Chang, J.M. Wilson, L.M. DiStefano, J.N. Nobrega, Br ain cytochrome oxidase in Alzheimer's disease. J. Neurochem. (1992), 59, 776-779.


58 42) S.J. Kish, L.M. DiStefano, S. Dozic, J.N. Nobrega, Cerebral cortical but not hippocampal cytochrome oxidase activity is reduced in Alzheimer's disease. Neurobiol. Aging (1992),13, suppl. 106. 43) H. Reichmann, S. Flerke, G. Hebenstreit, H. Schrubar, P. Riederer, Analyses of energy metabolism and mitochondrial genome in postmortem brain from patients with Alzheimer's disease. J. Neurol. (1993), 240, 377-380. 44) K.J. Reinikainen, L. Paljarvi, T. Halonen, O. Malminen, V.-M. Kosma, M. Laakso, P.J. Riekkinen, Dopaminergic system and monoamine oxidaseactivity in Alzheimer's disease. Neurobiol. Aging (1988), 9, 245-252. 45) W.D. Parker Jr., C.M. Filley, Parks, J. K. Cytochrome oxidase deficiency in Alzheimer's disease. Neurology (1990), 40, 1302-1303. 46) A.J. Van Zuylen, G.J.C.G.M. Bosman, W. Ruitenbeek, P.J.C. Van Kalmthout, W.J. De Grip, No evidence for reduced thrombocyte cytochrome oxidase activity in Alzheimer's disease. Neurology (1992), 42, 1246-1247. 47) M.T.T. Wong-Riley, Cytochrome oxida se: An endogenous metabolic marker for neuronal activity. Trends Neurosci. (1989), 12, 94-101. 48) K. Chaudrasekaran, J. Stoll, D.R. Bra dy, S.I. Rapoport, Distribution of cytochrome oxidase (COX) activity and mRNA in monkey and human brain. COX mRNA distribution correlates with neurons vulnerable to Alzheimer pathology. Soc. Neurosci. Abstr. (1992), 18, 557 49) K. Chandrasekaran, J. Stoll, D.R. Bra dy, S.I. Rapoport, Localization of cytochrome oxidase (COX) activity and COX mRNA in th e hippocampus and ento rhinal cortex of


59 the monkey brain: correlation wi th specific neuronal pathways. Brain. Res (1992), 579, 333-336. 50) K. Chandrasekaran, T. Giordano, D.R. Br ady, J. Stoll, L.J. Martin, S.I. Rapoport, Impairments in mitochondrial cytochrome oxidase gene expression in Alzheimer disease. Mol. Brain Res. (1994), 24, 336-340. 51) M.C. Bennet, D.M. Diamond, S.L. Stryker, J.K. Parks, W.D. Parker Jr., Cytochrome oxidase inhibition: A novel animal model of Alzheimer's disease. J. Geriat. Psych. Neurol. (1992), 5, 93-101. 52) H.A. Kontos, E.P. Wei, E.F. Ellis, L.W. Jenkins, J.T. Povlishock, G.T. Rowe, M.L. Hess, Appearance of superoxide anion radi cal in cerebral extracellular space during increased prostaglandin synthesis in cats. Circ. Res. (1985), 57, 142-151. 53) S. Nakamura, T. Kawamata, I. Akiguchi M. Kameyama, N. Nakamura, H. Kimura, Expression of monoamine oxidase B activit y in astrocytes of senile plaques. Acta Neuropathol. (1990), 80, 419-425. 54) M. Valko, D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazu r, J. Telser, Free radicals and antioxidants in normal physiologi cal functions and human disease. Intern. J. Biochem. Cell Biol. (2006), 39, 44-84. 55) Y. Kono, I. Fridovich, Superoxi de radical inhibits catalase. J. Biol. Chem. (1982), 257, 5751-5754. 56) J. Blum, I. Fridovich, Inac tivation of glutathione peroxi dase by superoxide radical. Arch. Biochem. Biophys. (1985), 240, 500-508.


60 57) N.F. Schor, Inactivation of mammalia n brain glutamine synthetase by oxygen radicals. Brain Res. (1988), 456, 17-21. 58) G.C. Palmer, Free radicals generated by xanthine oxidasehypoxanthine damage adenylate cyclase and ATPase in gerbil cerebral cortex. Metabol. Brain Dis. (1987), 2, 243-256. 59) J.M. McCord, W.J. Russell, Superoxi de inactivate creatine phosphokinase during reperfusion of ischemic heart. In: P.A. Ce rutti, I. Fridovich, J.M. McCord, eds. Oxyradicals in molecular biol ogy and pathology. New York: Alan R. Liss; (1988), 27-35. 60) Y. Zhang, Marcillat, C. Giulivi, L. Erns ter, K.J.A. Davies, The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J. Biol. Chem. (1990), 265, 16330-16336. 61) J.P. Blass, A.C. Baker, L.-W. Ko, R.S. Black, Induction of Alzheimer antigens by an uncoupler of oxidative phosphorylation. Arch. Neurol. (1990), 47, 864-869. 62) N.R. Sims, J.M. Finegan, J.P. Blass, D. M. Bowen, D. Neary, Mitochondrial function in brain tissue in primary degenerative dementia. Brain Res (1987), 436, 30-38. 63) A.C. Baker, L.-W. Ko, J.P. Blass, System ic manifestations of Alzheimer's disease. Age (1988), 11, 60-65. 64) J.N. Kanfer, J.W. Pettegrew, J. Mooss y, D.G. McCartney, Alterations in selected enzymes of phospholipids metabolism in Alzhei mer's disease brain tissue as compared to nonAlzheimer's demented controls. Neurochem. Res. (1993), 18, 331-334. 65) P.J. Benke, H. Levcovitz, J. Paupe, E. To zman, Scavengers of free radical oxygen affect the generation of low molecular wei ght DNA in stimulated lymphocytes from


61 patients with systemic lupus erythematosus. Metabol. clin. experiment. (1990), 39, 1278-1284. 66) M. Krenz M.V. Cohen Michael, J,D. Downey, The protective and anti-protective effects of ethanol in a myocardial infarct model. Annals N.Y. Acad. Sci. (2002), 957 103-114. 67) 68) G. Benzi, O. Pastoris, F. Marzatico, R.F. Villa, Cerebral enzyme antireactive intermediate system. Influence of aging and phosphatidylcholine. J. Cereb. Blood Flow Metabol. (1989), 9, 373-380. 69) A. Gupta, M. Hasan, R. Chander, N. K. Kapoor, Age-related elevation of lipid peroxidation products: diminution of super oxide dismutase activity in the central nervous system of rats. Gerontol. (1991), 37, 305-309. 70) G. Rao, E. Xia, A. Richardson, Effect of age on the expre ssion of antireactive intermediate enzymes in male Fischer F344 rats. Mech. Ageing Dev. (1990), 53, 49-60. 71) I. Semsei, G. Rao, A. Richardson, Expressi on of superoxide dismutase and catalase in rat brain as a function of age. Mech. Ageing Dev. (1991), 58, 13-19. 72) A. Vanella, E. Geremia, G. D'Urso, P. Tiriolo, I. Di Silvestr o, R. Grimaldi, R. Pinturo, Superoxide dismutase activities in aging rat brain. Gerontol. (1982), 28, 108113.


62 73) M.C. Carrillo, S. Kanai, Y. Sato, K. Kitani, Age-related ch anges in antireactive intermediate enzyme activities are region and organ, as well as sex, selective in the rat. Mech. Ageing Dev. (1992), 65, 187-198. 74) H. C. Danh, M. Strolin Be nedetti, P. Dostert, Differe ntial changes in superoxide dismutase activity in brain and liver of old rats and mice. J. Neurochem. (1983), 40, 1003-1007. 75) G. Forloni, -amyloid neurotoxicity. Funct. Neurol. (1993), 8, 211-225. 76) B.W.L. Brooksbank, R. Balazs, Superoxide dismutase, glutathioneperoxidase and lipoperoxidation in Down's syndrome fetal brain. Dev. Brain Res. (1984), 16, 37-44. 77) Y. Groner, K.B. Avraham, M. Schickler, R. Yarom, H. Knobler, Down syndrome: Gene dosage and clinical symptoms in transgenic-copper-zinc-SOD mice. Adv. Mol. Gen. (1991), 4, 181-211. 78) C.J. Epstein, K.B. Avraham, M. Lovett, S. Smith, O. Elroy-Stein, G. Rotman, C. Bry, Y. Groner, Transgenic mice with increased co pper/zinc-superoxide dismutase activity: animal model of dosage effects in Down syndrome. Proc. Nat. Acad. Sci. U S A (1987), 84, 8044-8048. 79) I. Ceballos-Picot, A. Nicole, P. Briand, G. Grimber, A. Delacourte, A. Defossez, F. Javoy-Agid, M. Lafon, J.L. Blouin, P.M. Sine t, Neuronal-specific expression of human copper-zinc superoxide dismut ase gene in transgenic mice: Animal model of gene dosage effects in Down's syndrome. Brain Res. (1991), 552, 198-214.


63 80) A. L. Friedlich, T.W. Farris, E. Carlson, C.J. Epstein, L.L. Butcher, -Amyloid protein-induced neurotoxicity in wild type mice and in transgenic mice with elevated CuZn superoxide dismutase activity. Soc. Neurosci. Abstr. (1993), 19, 1036. 81) I. Ceballos, F. Javoy-Agid, A. Delacour te, A. Defossez, M. Lafon, E. Hirsch, A. Nicole, P.M. Sinet, Y. Agid, Neuronal loca lization of copper-zinc superoxide dismutase protein and mRNA within the human hi ppocampus from control and Alzheimer's disease Brains. Free Rad. Res. Comm. (1991), 12-13, 571-580. 82) D. Games, K.M. Khan, F.G. Soriano, I. Lieberburg, S. Sinha, Evidence for altered APP processing following acute head trauma in the rodent. Soc. Neurosci. Abstr. (1992), 18, 1437. 83) S. Bahmanyar, G.A. Higgins, D. Goldgaber, D.A. Lewis, J.H. Morrison, M.C. Wilson, S.K. Shankar, D.C. Gajdusek, Lo calizationof amyloid/3 protein messenger RNA in brains from patients with Alzheimer's disease. Science (1987), 237, 77-80. 84) G. Moll, Neurotransmittermetabolismus, Energiestoffwechsel und Radikale in der Pathogenese der Demenz vom Alzheimer-Typ. Z Gerontol. (1991), 24, 212-213. 85) S.S. Panter, M. Scott, Elevated temporal cortex superoxide di smutase in Alzheimer's Disease. Soc. Neurosci. Abstr. (1991), 17, 1072. 86) F. P. Zemlan, O.J. Thienhaus, H.B. Bosmann, Superoxide dismutase activity in Alzheimer's disease: Possible mechanism for paired helical filament formation. Brain Res. (1989), 476, 160-162.


64 87) G. Tesco, S. Latorraca, P. Piersanti, S. Piacentini, L. Amaducci, S. Sorbi, Alzheimer skin fibroblasts show increased susceptibility to free radicals. Mech. Ageing Dev. (1992), 66, 117-120. 88) M.A. Pappolla, R.A. Omar, K.S. Kim, N. K. Robakis, lmmunohist ochemical evidence of antireactive intermediate st ress in Alzheimer's disease. Am. J. Pathol. (1992), 140, 621-628. 89) R.A.Cherny, J.T. Legg, C.A. McLean, D.P. Fairlie, X. Huang, C.S. Atwood, K. Beyreuther, R.E. Tanzi, C.L. Masters, A.I. Bush, Aqueous dissolution of Alzheimer's disease A amyloid deposits by biometal depletion. J. Biol. Chem. (1999), 274, 23223-23228. 90) R. Balakrishnan, R. Parthasarat hy, E. Sulkowski, Alzheimer s -amyloid peptide: affinity for metal chelates. J. Peptide Res. (1998), 51, 91-95. 91) C.S. Atwood, R.D. Moir, X. Huang, R. C. Scarpa, N.M.E. Bacarra, D.M. Romano, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, Dramatic aggregation of Alzheimer A by Cu(II) is induced by conditions representing physiological acidosis. J. Biol. Chem. (1998), 273, 12817-12826. 92) C.S. Atwood, R.C. Scarpa, X. Huang, R.D. Moir, W.D. Jones, D.P Fairlie, R.E. Tanzi, A.I. Bush, Characterization of c opper interactions with Alzheimer amyloid peptides: identification of an attomola r-affinity copper bind ing site on amyloid 142. J. Neurochem. (2000), 75, 1219-1233. 93) G.F.Z. da Silva, W.M. Tay, and L.-J Ming, Catechol Oxidase-like Oxidation Chemistry of the 1–20 and 1–16 Fragments of Alzheimer's Disease-related -


65 Amyloid Peptide: THEIR STRUCT URE-ACTIVITY CORRELATION AND THE FATE OF HYDROGEN PEROXIDE. J. Biol. Chem. (2005), 280, 16601-16609. 94) C.D. Syme, R.C. Nadal, S.E.J. Rigby, J.H. Viles, Copper Binding to the Amyloid(A ) Peptide Associated with Alzheime r's Disease: FOLDING, COORDINATION GEOMETRY, pH DEPENDENCE, STOICH IOMETRY, AND AFFINITY OF A (1–28): INSIGHTS FROM A RANGE OF COMPLEMENTARY SPECTROSCOPIC TECHNIQUES J. Biol. Chem. (2004), 279, 18169-18177. 95) T. Miura, K. Suzuki, N. Kohata, H. Takeuchi, Metal Binding Modes of Alzheimer s Amyloid -Peptide in Insoluble Aggreg ates and Soluble Complexes. Biochemistry (2000), 39, 7024-7031. 96) C. Curtain, F. Ali, I. Volitakis, R.A. Cherny, R.S. Norton, K. Beyreuther, C.J. Barrow, C.L. Masters, A.I. Bush, Alzheimer's Disease AmyloidBinds Copper and Zinc to Generate an Allosterically Ordered Membrane-pen etrating Structure Containing Superoxide Di smutase-like Subunits. J. Biol. Chem. (2001), 276, 2046620473. 97) T. Miura, K. Suzuki, H. Takeuchi, Bindi ng of iron(III) to the si ngle tyrosine residue of amyloid -peptide probed by Raman spectroscopy. J. Mol. Struct. (2001), 598, 79–84. 98) D.P. Smith, D.G. Smith, C.C. Curtain, J.F. Boas, J.R. Pilbrow, G.D. Ciccotosto, T.L. Lau, D.J. Tew, K. Perez, J.D. Wade, A.I. Bush, S.C. Drew, F. Separovic, C.L. Masters, R. Cappai, K.J. Barnham, Copper-mediated AmyloidToxicity Is


66 Associated with an Intermolecular Histidine Bridge. J. Biol. Chem. (2006), 281, 15145-15154. 99) R.A. Lieberman, R.H. Sands, J.A. F ee, A study of the electron paramagnetic resonance properties of single monoclinic cr ystals of bovine superoxide dismutase. J. Biol. Chem. (1982), 257, 336-44. 100) C.S. Atwood, R.D. Moir, X. Huang, R. C. Scarpa, N.M.E. Bacarra, D.M. Romano, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, Dramatic aggregation of Alzheimer A by Cu(II) is induced by conditions representing physiological acidosis. J. Biol. Chem. (1998), 273, 12817-12826. 101) X. Huang, C.S. Atwood, R.D. Moir, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, Trace metal contamination initiates the appare nt autoaggregation amyloidosis, and oligomerization of Alzheimer s A peptides. J. Biol. Inorg. Chem. (2004), 9, 954960. 102) F.E.A. Ali, K.J. Barnham, C.J. Barrow, F. Separovic, Metal -Catalyzed Oxidative Damage and Oligomerization of the AmyloidPeptide of Alzheimer's disease. Australian J. Chem. (2004), 57, 511-518. 103) D. Drago, M. Folin, S. Baiguera, G. Tognon, F. Ricchelli, P. Zatta, Comparative Effects of A (1-42)-Al Complex from Rat and Human Amyloid on Rat Endothelial Cell Cultures. J. Alzheimer's Disease (2007), 11, 33-44. 104) L. Hou, M.G. Zagorski, NMR Reveals Anomalous Copper(II) Binding to the Amyloid A Peptide of Alzheimer's Disease. J. Am. Chem. Soc. (2006), 128, 9260-9261.


67 105) Y. Mekmouche, Y. Coppel, K. Hochgrafe, L. Guilloreau, C. Talmard, H. Mazarguil, P. Faller, Characterization of the Zn (II) binding to th e peptide amyloid1-16 linked to Alzheimer's disease. ChemBioChem. (2005), 6, 1663-1671. 106) L.O. Tjernberg, A. Tjernber g, N. Bark, Y. Shi, B.P. Ruzsicska, Z. Bu, J. Thyberg, D.J.E. Callaway, Assembling amyloid fibrils from designed stru ctures containing a significant amyloid -peptide fragment. Biochem. J. (2002), 366, 343-351. 107) U.F. Rohrig, A. Laio, N. Tantalo, M. Pa rrinello, R. Petronzio, Stability and structure of oligomers of the Alzheimer Peptide A 16-22: from the dimer to the 32-mer. Biophys. J. (2006), 91, 3217-3229. 108) L.L. Iversen, R.J. Mortishire-Smith, S.J. Pollack, M.S. Shearman, The Toxicity invitro of -Amyloid Protein. Biochem J. (1995), 311, 1-16. 109) C.J. Pike, D. Burdick, A.J. Wale ncewicz, C.G. Glabe, C.W. Cotman, Neurodegeneration Induced by -Amyloid Peptides in vitro: The Role of Peptide Assembly State. J. Neurosci. (1993), 13, 1676-1687. 110) X. Huang, C.S. Atwood, M.A. Harts horn, G. Multhaup, L.E. Goldstein, R.C. Scarpa, M.P. Cuajungco, D.N. Gray, J. Lim, R.D. Moir, R.E. Tanzi, A.I. Bush, The A Peptide of AlzheimerÂ’s Disease Directly Produces Hydrogen Peroxide through Metal Ion Reduction. Biochemistry (1999), 38, 7609-7616. 111) K. Hensley, J.M. Carney, M.P. Mattson, M. Aksenova, M. Harris, J.F. Wu, R.A. Floyd, D.A.A. Butterfield, A Model for -Amyloid Aggregation and Neurotoxicity Based on Free Radical Generation by the Pep tide: Relevance to Alzheimer Disease. Proc. Natl. Acad. Sci.USA (1994), 91, 3270-3274.


68 112) A.E. Roher, M.O. Chaney, Y.M. Kuo, S.D. Webster, W.B. Stine, L.J. Haverkamp, A.S Woods, R.J. Cotter, J.M. Tuohy, G.A. Krafft, B.S. Bonnell, M. R. Emmerling, Morphology and Toxicity of A -(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of AlzheimerÂ’s Disease. J. Biol. Chem. (1996), 271, 2063120635. 113) W.L. Klein, G.A. Krafft, C.E. Finch, Targeting Small A Oligomers: The Solution to an AlzheimerÂ’s Disease Conundrum? Trends Neurosci. (2001), 24, 219-224. 114) D.M. Hartley, D.M. Walsh, C.P. Ye, T. Diehl, S. Vasquez, P.M. Vassilev, D.B. Teplow, D.J. Selkoe, Protofibri llar Intermediates of Amyloid -Protein Induce Acute Electrophysiological Changes and Pr ogressive Neurotoxicity in Cortical Neurons. J. Neurosci. (1999), 19, 8876-8884. 115) J.D. Harper, S.S. Wong, C.M. Li eber, P.T. Lansbury, Assembly of A Amyloid Protofibrils: An in vitro Model for a Possible Early Event in AlzheimerÂ’s Disease. Biochemistry (1999), 38, 8972-8980. 116) M.S. Goldberg, P.T. Lansbury, Is Ther e a Cause-and effect Relationship Between synuclein Fibrillization a nd ParkinsonÂ’s Disease? Nature Cell Biol. (2000), 2, 115119. 117) D.R. Howlett, R.V. Ward, L. Bresciani, K.H. Jennings, G. Christie, D. Allsop, C.W. Gray, E.H. Karran, Biological Activity Associated with a High Molecular Weight Form of -Amyloid. AlzheimerÂ’s Rep. (1999), 2, 171-177.


69 118) S. Turnbull, B.J. Tabner, O.M.A. El -Agnaf, S. Moore, Y. Davies, D. Allsop, synuclein Implicated in ParkinsonÂ’s Dis ease Catalyses the Formation of Hydrogen Peroxide in Vitro. Free Radical Biol. Med. (2001), 30, 1163-1170. 119) Y. Fezoui, D.M. Hartley, D.M. Walsh, D.J. Selkoe, J.J. Osterhout, D.B.A. Teplow, A de novo Designed Helix-turn-helix Peptide Forms Nontoxic Amyloid Fibrils. Nature Structural Biol. (2000), 7, 1095-1099. 120) S. Varadarajan, S. Yatin, D.A. Butterfield, AlzheimerÂ’s Amyloid Peptide (1-42) Fibrils Are Not Always Neurotoxic. AlzheimerÂ’s Rep. (2000), 3, 71-76. 121) H. Lin, Y.J. Zhu, R. Lal, Amyloid Protein (1-40) Forms Calcium-permeable, Zn (II)-sensitive Channel in R econstituted Lipid Vesicles. Biochemistry (1999), 38, 11189-11196. 122) S.D. Yan, X.F.J Chen, M. Chen, H. Z hu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Na wroth, D. Stern, A.M. Schmidt, RAGE and AmyloidPeptide Neurotoxicity in AlzheimerÂ’s Disease. Nature (1996), 382, 685691. 123) J. El-Khoury, S.E. Hickman, C.A. Thomas L. Cao, S.C. Silverstein, J.D. Loike, Scavenger Receptor mediated Adhesion of Microglia to -Amyloid Fibrils. Nature (1996), 382, 716-719. 124) D.M. Paresce, R.N. Ghosh, F.R. Maxfie ld, Microglial Cells In ternalize Aggregates of the AlzheimerÂ’s Disease Amyloid -Protein via a Scavenger Receptor. Neuron (1996), 17, 553-565.


70 125) S.D. Yan, J. Fu, C. Soto, X. Chen, H. Zhu, F. Al-Mohanna, K. Collison, A. Zhu, E. Stern, T. Saido, M. Tohyama, S. Ogawa, A. Roher, D. Stern, An Intracellular Protein That Binds AmyloidPeptide and Mediates Neurotoxicity in AlzheimerÂ’s Disease. Nature (1997), 389, 689-695. 126) C.M. Yip, J. McLaurin, AmyloidPeptide Assembly: A Critical Step in Fibrillogenesis and Membrane Disruption. Biophys. J. (2001), 80, 1359-1371. 127) C. Kosa, I. Lukac, R.G. Weiss, Phot ochemical Transformation of Benzil Pendant Groups of Polystyrene Copolymers into Benzoyl Peroxide Mo ieties and Their Subsequent Thermal Decomposition. CrossLinking or Chain Scission ? Macromolecules (2000), 33, 4015-4022. 128) V. Verney, M.A. Vincent. Study of the thermal decomposition of polypropylene peroxides by means of melt rheology. Poly. Proc. Eng. (1986), 3, 321-335. 129) T.H. Peterson, B.K. Carpenter, Estima tion of dynamic effects on product ratios by vectorial decomposition of a reaction coordi nate. Application to thermal nitrogen loss from bicyclic azo compounds. J. Am. Chem. Soc. (1992), 114, 766-767. 130) H. Meier, K.P. Zeller, Thermal and photochemical nitrogen cycloelimination. Angew. Chem. (1977), 89, 876-890. 131) C.J. Kay, Mechanochemical Mechanism for Peptidyl Free Radical Generation by Amyloid Fibrils. FEBS Lett. (1997), 403, 230-235. 132) K. Hensley, M. Aksenova, J.M. Carney, D.A. Butterfield, Amyloid -Peptide Spintrapping. 1. Peptide Enzyme Toxicity is Related to Free-radical Spin Trap Reactivity. NeuroReport (1995), 6, 489-493.


71 133) K. Hensley, M. Aksenova, J.M. Carney, D.A. Butterfield, Amyloid -Peptide Spintrapping. 2. Evidence for Decompos ition of the PBN Spin Adduct. NeuroReport (1995), 6, 493-496. 134) T. Tomiyama, A. Shoji, K. Kataoka, Y. Suwa, S. Asano, H. Kaneko, N. Endo, Inhibition of Amyloid Protein Aggregation and Ne urotoxicity by Rifampicin. J. Biol. Chem. (1996), 271, 6839-6844. 135) A. Forrester, Nitroxides. In Magnetic Properties of FreeRadicals ; Fischer, H., Ed.; Springer: Berlin, (1989), 17, 8-71. 136) S.I. Dikalov, M.P. Vitek, K.R. Maples, R.P. Mason, Amyloid Peptides Do Not Form Peptide-derived Free Radicals Spontaneously, but Can Enhance Metal catalysed Oxidation of Hydroxylamines to Nitroxides. J.Biol. Chem. (1999), 274, 9392-9399. 137) A.I. Bush, Metals and Neuroscience. Curr. Opinion Chem. Biol. (2000), 4, 184-191. 138) A.I. Bush, X. Huang, D.P. Fairlie, Th e Possible Origin of Free Radicals from Amyloid -Peptides in AlzheimerÂ’s Disease. Neurobiol. Aging (1999), 20, 335-337. 139) X. Huang, M.P. Cuajungco, C.S. Atwood, M.A. Hartshorn, J.D.A. Tyndall, G.R. Hanson, K.C. Stokes, M. Leopold, G. Multhaup, L.E. Goldstein, R.C. Scarpa, A.J. Saunders, J. Lim, R.D. Moir, C. Glabe, E. F. Bowden, C.L. Masters, D.P. Fairlie, R.E. Tanzi, A.I. Bush, Cu(II) Potentiation of Alzheimer A Neurotoxicity Correlation with Cell-fr ee Hydrogen Peroxide Production and Metal Reduction. J. Biol.Chem. (1999), 274, 37111-37116.


72 140) S. Turnbull, B.J. Tabner, O.M.A. El-Agnaf, L.J. Twyman, D. Allsop, New Evidence that the Alzheimer Amyloid Peptide Does Not Spontaneously Form Free Radicals: An ESR Study Usi ng a Series of Spin-traps. Free Radical Biol. Med. (2001), 30, 1154-1162. 141) Y. Kotake, E.G. Janzen, Decay and Fate of the Hydroxyl Radical Adduct of Phenyl-N-tert-butylnitrone in Aqueous Media. J. Am. Chem. Soc. (1991), 113, 9503-9506. 142) S.M. Yatin, S. Varadarajan, C.D. Link, D.A. Butterfield, In Vitro and in Vivo Oxidative Stress Associated with AlzheimerÂ’s Amyloid -Peptide (1-42). Neurobiol. Aging (1999), 20, 325-330. 143) D.A. Butterfield, S. Varadarajan, M. Ak senova, C. Link, S.M. Yatin, On Methionine and AlzheimerÂ’s Amyloid -Peptide (1-42)-induced Oxidative Stress. Neurobiol. Aging (1999), 20, 339-342. 144) S. Varadarajan, S. Yatin, M. Aksenova, D.A. Butterfield, A Review: AlzheimerÂ’s Amyloid -Peptide-associated Free Radical Ox idative Stress and.Neurotoxicity. J. Struct. Biol. (2000), 130, 184-208. 145) S. Varadarajan, S. Yatin. J. Kanski, F. Jahanshahi, D.A. Butterfield, Methionine Residue 35 is Important in Amyloid -Peptide-associated Free Radical Oxidative Stress. Brain Res. Bull. (1999), 50, 133-141. 146) A. Monji, H. Utsumi, I. Yoshida, S. Hashioka, K. Tashiro, N. Tashiro, The Relationship Between A -associated Free Radical Generation and A Fibril


73 Formation Revealed by Negative Stain Electron Microscopy and Thioflavine-T Fluorometric Assay. Neurosci. Lett. (2001), 304, 65-68. 147) A. Monji, H. Utsumi, T. Uedta, T. Imot o, I. Yoshida, S. Hashioka, K. Tashiro, N. Tashiro, The Relationship Between the Aggregated State of the AmyloidPeptides and Free Radical Generation by the Peptides. J. Neurochem (2001), 77, 1425-1432. 148) M. Anbar, P.A. Neta, A Compilation of Specific Bimolecular Rate Constants for the Reactions of Hydrated Electrons, Hydroge n Atoms, and Hydroxyl Radicals with Inorganic and Organic Compounds in Aqueous Solution. Int. J. Appl. Radiat. Isot. (1967), 18, 493-523. 149) D.R. Brown, B.S. Wong, F. Hafiz, C. Cliv e, S.J. Haswell, I.M. Jones, Normal Prion Protein Has an Activity Like Th at of Superoxide Dismutase. Biochem. J. (1999), 344, 1-5. 150) L.M. Sayre, G. Perry, M.A. Smith, A. Redox Metals and Disease. Neurodegenerative Disease. Curr. Opinion Chem. Biol. (1999), 3, 220-225. 151) B.A. Yanker, L.K. Duffy, D.A. Kirshne r, Neurotrophic and Ne urotoxic Effects of Amyloid Protein: Reversal by Tachykinin Neuropeptides. Science (1990), 250, 279-282. 152) C.J. Pike, A.J. Walencewicz-Wasserman, J. Kosmoski, D. H. Cribbs, C.G. Glabe, C.W. Cotman, Structure reactivity Analyses of -Amyloid Peptides: Contributions of the A 25-35 Region to Aggregation and Neurotoxicity. J. Neurochem. (1995), 64, 253-265.


74 153) L.I. Simandi, Catalytic Activation of Dioxygen by Metal Complexes Kluwer Academic, Dordrecht, (1992). 154) S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry University Science, Mill Valley, CA, (1994). 155) W. Kaim, B. Schwederski, Bioinorganic Chemistry Wiley, Chichester, UK (1994). 156) R.H. Holm, P. Kennepohl, E.I. Solomon, St ructural and Functiona l Aspects of Metal Sites in Biology. Chem. Rev (1996), 96, 2239-2562. 157) E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Multicopper Oxidases and Oxygenases. Chem. Rev. (1996), 96, 2563. 158) K.D. Karlin, Z. Tyeklar (Eds.), Bioi norganic Chemistry of Copper, Chapman & Hall, New York, (1993). 159) E.I. Solomon, K.W. Penfield, D.E. WilC OA, Active sites in copper proteins. An electronic structure overview. Struct. Bond. (1983), 53, 1-57. 160) K.A. Magnus, B. Hazes, H. Ton-That, C. B onaventura, J. Bonaventura, W.G.J. Hol, Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences. Proteins Struct. Funct. Genet (1994), 19, 302-309. 161) K.A. Magnus, H. Ton-That, J.E. Carpenter, Recent Structural Work on the Oxygen Transport Protein Hemocyanin. Chem. Rev. (1994), 94, 727-735. 162) R.S. Himmelwright, N.C. Eickman, C. D. LuBien, E.I. Solomon, Chemical and spectroscopic comparison of the binuclea r copper active site of mollusc and arthropod hemocyanins J. Am. Chem. Soc. (1980), 102, 5378-5388.


75 163) H.S. Mason, W.L. Fowlks, E. Peterson, Oxygen transfer and electron transport by the phenolase complex. J. Am. Chem. Soc. (1955), 77, 2914-2915. 164) M. Tremolieres, J.B. Bieth, Isolation and characterization of the polyphenol oxidase from senescent leaves of black poplar. Phytochemistry (1984), 23, 501-505. 165) D. Meiwes, B. Ross, M. Kiesshauer, K. Cammann, H. Witzel, M. Knoll, M. Borchardt, C. Sandermaier Lab. Med. (1992), 15, 24. 166) K. Lerch, Tyrosinase: molecu lar and active-site structure. Enzym. Brown. Prev. (1995), 600, 64-80. 167) A. Rompel, H. Fischer, K. Bldt-Ka rentzopoulos, D. Meiwes, F. Zippel, H.-F. Nolting, C. Hermes, B. Krebs, H. Witz el, Structure and magnetism of novel tetranuclear -4-oxo-bridge d copper(II) complexes. J. Inorg. Biochem. (1995), 59, 715-720. 168) N.C. Eickman, R.S. Himmelwright, E.I. Solomon, Geometric and electronic structure of oxyhemocyanin: Spectral and chemical correlations to met apo, half met, met, and dimer active sites. Proc. Acad. Sci. USA (1979), 76, 2094-2098. 169) E.I. Solomon, M.J. Baldwin, M.D. Lowery, Electronic structures of active sites in copper proteins: contri butions to reactivity. Chem. Rev. (1992), 92, 521-542. 170) F. Zippel, F. Ahlers, B. Krebs, S. Be hning, K. Bldt-Karentzopoulos, H. Witzel, M. Oversluizen, X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exch anged form). Daresbury Annual Report, Daresbury Laboratory, Daresbury, UK, (1994:95), 102.


76 171) B. Krebs, K. Bldt-Karentzopoulos, C. Eicken, A. Rompel, H. Witzel, A. Feldmann, R. Kruth, J. Reim, W. Steinforth, S. Teip el, F. Zippel, S. Schindler, F. Wiesemann, in: DFG Deutsche Forschungsgemeinschaft (Ed.), Bioinorganic Chemistry: Transition Metals in Biology and their Coordination Chemistry D.14, VCH, Weinheim, (1997), p. 616. 172) N. Oishi, Y. Nishida, K. Ida, S. Kida, Reaction between various copper(II) complexes and ascorbic acid or 3,5-di-tert-butylcatechol. Bull. Chem. Soc. Jpn (1980), 53, 2847-2850. 173) S. Kida, H. Okawa, Y. Nishida, in: K.D. Karlin, J. Zubieta (Eds.), Copper Coordination Chemistry: Biochemical and Inorganic Perspectives Adenine, Guilderland, NY, (1983), p. 425. 174) S. Casellato, S. Tamburini, P.A. Vigato, A. de Stefani, M. Vi dali, D.E. Fenton, The preparation of binuclear complexes and thei r catalytic behavior in the oxidation of 3,5-di-tert-butylcatechol. Inorg. Chim. Acta (1983), 69, 45-51. 175) K.D. Karlin, Y. Gultneh, T. Nicholson, J. Zubieta, Catecholate coordination to copper: structural char acterization of a tetrachl oro-o-catecholate-bridged dicopper(II) complex as a model for interm ediates in copper-catalyzed oxidation of catechols. Inorg. Chem. (1985), 24, 3725-3727. 176) M.R. Malachowski, J. Carden, M.G. Davidson, W.L. Driessen, J. Reedijk, The preparation and catalytic properties of copper(II) complexes derived from a pyrazole containing ligand. X-ray crys tal structure of [Cu(pzmhp)(BF4)](BF4). Inorg. Chim. Acta (1997), 257, 59-67.


77 177) M.R. Malachowski, H.B. Huynh, L.J. Tomlinson, R.S. Kelly, J.W. Furbee Jr., Comparative study of the catalytic oxidat ion of catechols by copper(II) complexes of tripodal ligands. J. Chem. Soc. Dalton Trans. (1995), 31-36. 178) C.X. Zhang, H.-C. Liang, K.J. Humphr eys, K.D. Karlin In: Simandi L (ed) Catalytic activation of dioxygen by metal complexes Kluwer, Dordrecht, The Netherlands, (2003), 79–121. 179) K.D. Karlin, A.D. Zuberbuhler In: R eedijk J, Bouwman E (eds) Bioinorganic catalysis, 2nd edn. Marcel De kker, New York, (1999), pp 469–534. 180) K.D. Karlin, S. Kaderli, A.D. Z uberbuhler, Kinetics and Thermodynamics of Copper(I)/Dioxygen Interaction. Accs. Chem. Res (1997), 30, 139–147. 181) K.D. Karlin, Y. Gultneh, Binding and activation of molecular oxygen by copper complexes. Prog. Inorg. Chem (1987), 35, 219–327. 182) E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Multicopper Oxidases and Oxygenases. Chem. Rev (1996), 96, 2563–2605. 183) J.P. Klinman, Mechanisms Whereby Mononuclear Copper Proteins Functionalize Organic Substrates. Chem. Rev. (1996), 96, 2541–2561. 184) E.I. Solomon, P. Chen, M. Metz, S.-K. Lee, A.E. Palmer, Oxygen binding, activation, and reduction to water by copper proteins. Angew. Chem. Int. Ed. (2001), 40, 4570–4590. 185) K.A. Magnus, H. Ton-That, J.E. Carpenter, Recent Structural Work on the Oxygen Transport Protein Hemocyanin. Chem. Rev (1994), 94, 727–735.


78 186) C. Gerdemann, C. Eicken, B. Krebs, Th e crystal structure of catechol oxidase: New insight into the function of type-3 copper proteins. Acc. Chem. Res. (2002), 35, 183–191. 187) H. Gampp, A.D. Zuberbuhler, Co pper-catalyzed oxi dation and oxygenation. Metal Ions Biol. Syst (1981), 12, 133–189. 188) L. Que Jr., W.B. Tolman, Bis(-oxo)dim etal "diamond" cores in copper and iron complexes relevant to biocatalysis. Angew. Chem. Int. Ed. (2002), 41, 1114–1137. 189) N. Kitajima, K. Fujisawa, Y. Moro-oka, K. Toriumi, 2: 2-Peroxo binuclear copper complex, [Cu(HB(3,5-(Me2CH)2pz)3)]2(O2). J. Am. Chem. Soc. (1989), 111, 8975–8976. 190) N. Kitajima, Y. Moro-oka, Coppe r-Dioxygen Complexes. Inorganic and Bioinorganic Perspectives. Chem. Rev. (1994), 94, 737–757. 191) K.A. Magnus, B. Hazes, H. Ton-That, C. B onaventura, J. Bonaventura, W.G.J. Hol, Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences. Proteins Struct. Funct. Genet (1994), 19, 302–309. 192) S.I. Chan, K.H.C. Chen, S.S.F. Yu, C. L. Chen, S.S.J. Kuo, Polarized ATR-FTIR Spectroscopy of the Membrane-Embedded Domains of the Particulate Methane Monooxygenase. Biochemistry (2004), 43, 4421–4430. 193) J.P. Evans, K. Ahn, J.P. Klinman, Ev idence that dioxygen and substrate activation are tightly coupled in dopamine -monooxygenase: Implications for the reactive oxygen species. J. Biol. Chem. (2003), 278, 49691–49698.


79 194) S.T. Prigge, R.E. Mains, B.A. Eippe r, L.M. Amzel, New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol. Life Sci (2000), 57, 1236–1259. 195) J.P. Klinman, G.F.Z. da Silva, unpub lished results and personnal communication. 196) K.D. Karlin, R.W. Cruse, Y. Gultneh, J.C. Hayes, J. Zubieta, Peroxide coordination to a dicopper(II) cen ter. Dioxygen binding to a st ructurally characterized phenoxide-bridged binuclear copper(I) complex. J. Am. Chem. Soc. (1984), 106, 3372–3374. 197) R.R. Jacobson, Z. Tyeklar, K.D. Karli n, S. Liu, J. Zubieta, A copper-oxygen (Cu2O2) complex. Crystal structure and char acterization of a reversible dioxygen binding system. J. Am. Chem. Soc. (1988), 110, 3690–3692. 198) K.D. Karlin, M.S. Haka, R.W. Cr use,Y. Gultneh, Dioxygen-copper reactivity. Reversible oxygen and carbon monoxide binding by a new series of binuclear copper(I) complexes. J. Am. Chem. Soc. (1985), 107, 5828–5829. 199) Z. Hu, R.D.Williams, D. Tran, T.G. Spiro, S.M. Gorun, Re-engineering EnzymeModel Active Sites: Reversible Bindi ng of Dioxygen at Ambient Conditions by a Bioinspired Copper Complex. J. Am. Chem. Soc. (2000), 122, 3556–3557. 200) M. Kodera M, Kajita Y, Tachi Y, Katayama K, Kano K, Hirota S, Fujinami S, Suzuki M Synthesis, structure, and greatly improved reversible O2 binding in a structurally modulated 2: 2-peroxodicopper(II) complex with room-temperature stability. Angew. Chem. Int. Ed. (2004), 43, 334–337.

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80 201) P.P. Paul, Z. Tyeklar, R.R. Jac obson, K.D. Karlin, Reactivity patterns and comparisons in three classes of synthetic copper-dioxygen {Cu2-O2} complexes: implication for structure a nd biological relevance. J. Am. Chem. Soc. (1991), 113, 5322–5332. 202) K.D. Karlin, P. Ghosh, R.W. Cruse, A. Farooq, Y. Gultneh, R.R. Jacobson, N.J. Blackburn, R.W. Strange, J. Zubieta, Dioxygen-copper reactivity: generation, characterization, and reactivity of a hydroperoxodicopper(II) complex. J. Am. Chem. Soc. (1988), 110, 6769–6780. 203) I. Sanyal, P. Ghosh, K.D. Karlin, M ononuclear Copper(II)-Acylperoxo Complexes. Inorg. Chem. (1995), 34, 3050–3056. 204) V. Mahadevan, M.J. Henson, E.I. Solo mon, T.D.P. Stack, Differential Reactivity between Interconvertible Side-On Per oxo and Bis--oxodicopper Isomers Using Peralkylated Diamine Ligands. J. Am. Chem. Soc. (2000), 122, 10249–10250. 205) L.M. Mirica, M. Vance, D.J. Rudd, B. Hedman, K.O. Hodgson, E.I. Solomon, T.D.P. Stack, A Stabilized 2: 2 Peroxodicopper(II) Complex with a Secondary Diamine Ligand and Its Ty rosinase-like Reactivity. J. Am. Chem. Soc. (2002), 124, 9332–9333. 206) P.L. Holland, K.R. Rodgers, W.B. Tolma n, Is the bis(-oxo)d icopper core capable of hydroxylating an arene? Angew. Chem. Int. Ed. (1999), 38, 1139–1142. 207) J.A. Halfen, S. Mahapatra, E.C. Wilk inson, S. Kaderli, V.G. Young Jr, L.Que Jr., A.D. Zuberbhler, W.B. Tolman, Revers ible cleavage and formation of the dioxygen O-O bond within a dicopper complex. Science (1996), 271, 1397–1400.

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81 208) V. Mahadevan, Z. Hou, A.P. Cole, D.E. R oot, T.K. Lal, E.I. Solomon, T.D.P. Stack Irreversible Reduction of Dioxygen by Simp le Peralkylated Diamine-Copper(I) Complexes: Characterization a nd Thermal Stability of a [Cu2(-O)2]2+ Core. J. Am. Chem. Soc. (1997), 119, 11996–11997. 209) C.J. Cramer, B.A. Smith, W.B. Tolman, Ab Initio Characterization of the Isomerism between the 2: 2-Peroxoand Bis(-oxo)dicopper Cores. J. Am. Chem. Soc. (1996), 118, 11283–11287. 210) P. Spuhler, M.C. Holthausen, Mechanism of the aliphatic hy droxylation mediated by a bis(-oxo)dicopper(III) complex. Angew. Chem. Int. Ed. (2003), 42, 5961– 5965. 211) M.J. Henson, P. Mukherjee, D.E. Root, T.D.P. Stack, E.I Solomon, Spectroscopic and Electronic Structural Studies of the Cu(III)2 Bis--oxo Core a nd Its Relation to the Side-On Peroxo-Bridged Dimer. J. Am. Chem. Soc. (1999), 121, 10332–10345. 212) K.D. Karlin, P.L. Dahlstrom, S.N. Cozzette P.M. Scensny, J. Zubieta, Activation of oxygen by a binuclear copper(I) compound. Hydroxylation of a new xylyl binucleating ligand to produce a phenoxy-br idged binuclear copper(II) complex; xray crystal structure of [Cu2{OC6H3[CH2N(CH2CH2py)2]2-2,6}(OMe)] (py = 2pyridyl). J. Chem. Soc. Chem. Commun. (1981), 881-882. 213) K.D. Karlin, Y. Gultneh, J.C. Hayes, R.W. Cruse, J.W. McKown, J.P. Hutchinson, J. Zubieta, Copper-mediated hydroxylation of an arene: model system for the action of copper monooxygenases. Structur es of a binuclear copper(I) complex and its oxygenated product. J. Am. Chem. Soc. (1984), 106, 2121–2128.

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82 214) M.S. Nasir, B.I. Cohen, K.D. Karli n, Mechanism of aroma tic hydroxylation in a copper monooxygenase model system. 1,2-Meth yl migrations and the NIH shift in copper chemistry. J. Am. Chem. Soc. (1992), 114, 2482–2494. 215) K.D. Karlin, M.S. Nasir, B.I. Cohen, R. W. Cruse, S. Kaderli, A.D. Zuberbhler, Reversible Dioxygen Binding and Aromatic Hydroxylation in O2-Reactions with Substituted Xylyl Binuclear Copper( I) Complexes: Syntheses and LowTemperature Kinetic/Thermodynamic and Spect roscopic Investiga tions of a Copper Monooxygenase Model System. J. Am. Chem. Soc. (1994), 116, 1324–1336. 216) E. Pidcock, H.V. Obias, C.X. Zhang, S.D. Karlin, E.I. Solomon, Investigation of the reactive oxygen intermediate in an ar ene hydroxylation reaction performed by xylyl-bridged binuclear copper complexes. J. Am. Chem. Soc. (1998), 120, 7841– 7847. 217) M.S. Nasir, B.I. Cohen, K.D. Karli n, Mechanism of aroma tic hydroxylation in a copper monooxygenase model system. 1,2-Meth yl migrations and the NIH shift in copper chemistry. J. Am. Chem. Soc. (1992), 114, 2482–2494. 218) E. Pidcock, H.V. Obias, M. Abe, H.-C. Liang, K.D. Karlin, E.I. Solomon, Spectroscopic and Theoretical Studies of Oxygenated Dicopper(I) Complexes Containing Hydrocarbon-Linked Bis[2-(2-pyr idyl)ethyl]amine Units: Investigation of a Butterfly [Cu2(2: 2)(O2)]2+ Core. J. Am. Chem. Soc. (1999), 121, 1299– 1308. 219) S. Itoh, H. Kumei, M. Taki, S. Nagato mo, T. Kitagawa, S. Fukuzumi, Oxygenation of phenols to catechols by a (2: 2-peroxo)dicopper(II) complex: mechanistic

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83 insight into the phenolase activity of tyrosinase. J. Am. Chem. Soc. (2001), 123, 6708–6709. 220) S.-I. Yamazaki, S. Itoh, Kinetic evalua tion of phenolase activity of tyrosinase using simplified catalytic reaction system. J. Am. Chem. Soc. (2003), 125, 13034–13035. 221) G. Battaini, M. De Carolis, E. Monzan i, F. Tuczek, L. Casella, The phenol orthooxygenation by mononuclear copper(I) co mplexes requires a binuclear 2: 2peroxodicopper(II) complex rather than mononuclear CuO2 species. Chem. Commun. (2003), 726–727. 222) L. Santagostini, M. Gullotti, E. Monzan i, L. Casella, R. Dillinger, F. Tuczek, Reversible dioxygen binding and phenol oxyge nation in a tyrosinase model system. Chem. Eur. J (2000), 6, 519–522. 223) S. Mandal, D. Macikenas, J.D. Prot asiewicz, L.M. Sayre, Novel tert-Butyl Migration in Copper-Mediate d Phenol Ortho-Oxygenation Implicates a Mechanism Involving Conversion of a 6-Hydroperoxy-2,4cyclohexadienone Directly to an oQuinone. J. Org. Chem. (2000), 65, 4804–4809. 224) L.M. Sayre, D. Nadkarni, Direct Conve rsion of Phenols to o-Quinones by Copper(I) Dioxygen. Questions Regarding the Monophe nolase Activity of Tyrosinase Mimics. J. Am. Chem. Soc. (1994), 116, 3157–3158. 225) S. Mahapatra, J.A. Halfen, E.C. Wilkin son, G. Pan, X. Wang, V.G. Young Jr., C.J. Cramer, L. Que Jr., W.B. Tolman, Stru ctural, Spectroscopic, and Theoretical Characterization of Bis(-oxo)dicopper Complexes, Novel Intermediates in

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84 Copper-Mediated Dioxygen Activation. J. Am. Chem. Soc. (1996), 118, 11555– 11574. 226) S. Mahapatra, J.A. Halfen, W.B. Tolm an, Mechanistic Study of the Oxidative NDealkylation Reactions of Bi s(-oxo)dicopper Complexes. J. Am. Chem. Soc. (1996), 118, 11575–11586. 227) S. Itoh, M. Taki, H. Nakao, P.L. Holland, W.B. Tolman, L. Que Jr., S. Fukuzumi, Aliphatic hydroxylation by a bi s( -oxo)dicopper(III) complex. Angew. Chem. Int. Ed. (2000), 39, 398–400. 228) S. Itoh, H. Nakao, L.M. Berreau, T. K ondo, M. Komatsu, S. Fukuzumi, Mechanistic Studies of Aliphatic Ligand Hydroxylati on of a Copper Complex by Dioxygen: A Model Reaction for Copper Monooxygenases. J. Am. Chem. Soc. (1998), 120, 2890–2899. 229) C.X. Zhang, H.-C. Liang, E.-I. Kim, J. Shearer, M.E. Helton, E. Kim, S. Kaderli, C.D. Incarvito, A.D. Zuberbuhler, A.L. Rheingold, K.D. Karlin, Tuning CopperDioxygen Reactivity and Exogenous Substrate Ox idations via Alterations in Ligand Electronics. J. Am. Chem. Soc. (2003), 125:634–635. 230) M.J. Henson, M.A. Vance, C.X. Zhang, H.-C. Liang, K.D. Karlin, E.I. Solomon, Resonance Raman Investigation of Equa torial Ligand Donor Effects on the Cu2O2 2+ Core in End-On and Side-On -PeroxoDicopper(II) and Bi s--oxo-Dicopper(III) Complexes. J. Am. Chem. Soc. (2003), 125, 5186–5192. 231) J. Shearer, C.X. Zhang, L.Q. Hatcher, K.D. Karlin, Distinguishing Rate-Limiting Electron versus H-Atom Transfers in Cu2(O2)-Mediated Oxidative N-Dealkylations:

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85 Application of Interversus Intram olecular Kinetic Isotope Effects. J. Am. Chem. Soc. (2003), 125, 12670–12671. 232) T. Osako, K. Ohkubo, M. Taki, Y. Tachi, S. Fukuzumi, S. Itoh, Oxidation Mechanism of Phenols by Dicopper-Dioxygen (Cu2/O2) Complexes. J. Am. Chem. Soc. (2003), 125, 11027–11033. 233) D.E. Root, M. Mahroof-Tahir, K.D. Kar lin, E.I. Solomon, Effect of Protonation on Peroxo-Copper Bonding: Spectroscopic and Electronic Structure Study of [Cu2(UN-O-)(OOH)]2+. Inorg. Chem. (1998), 37, 4838–4848. 234) P. Chen, K. Fujisawa, E.I. Solomon, Spectroscopic and Theoretical Studies of Mononuclear Copper(II) Alkyland Hydroperoxo Complexes: Electronic Structure Contributions to Reactivity. J. Am. Chem. Soc. (2000), 122, 10177–10193. 235) P. Chen, E.I. Solomon, Frontier molecular orbital analysis of Cun-O2 reactivity. J. Inorg. Biochem. (2002), 88, 368–374. 236) M. Sono, M.P. Roach, E.D. Coulter, J.H. Dawson, Heme-Containing Oxygenases. Chem. Rev. (1996), 96, 2841–2887. 237) R. Davydov, V. Kofman, H. Fujii, T. Yoshida, M. Ikeda-Saito, B.M. Hoffman, Catalytic Mechanism of Heme Oxyg enase through EPR and ENDOR of Cryoreduced Oxy-Heme Oxygenase and Its Asp 140 Mutants. J. Am. Chem. Soc. (2002), 124, 1798–1808. 238) P.R. Ortiz de Montellano, EPR Spin -Trapping of a Myeloperoxidase Protein Radical. Curr. Opin. Chem. Biol. (2000), 4, 221–227.

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86 239) R.W. Cruse, S. Kaderli, C.J. Meyer, A.D. Zuberbhler, K.D. Karlin, Coppermediated hydroxylation of an arene: kinetics and mech anism of the reaction of a dicopper(II) m-xylyl-containing complex with H2O2 to yield a phenoxodicopper(II) complex. J. Am. Chem. Soc. (1988), 110, 5020–5024. 240) G. Battaini, E. Monzani, A. Perotti, C. Para, L. Casella, L. Santagostini, M.M. Gullotti, R. Dillinger, C. Nather, F.A. Tuczek, A Double Arene Hydroxylation Mediated by Dicopper(II )-Hydroperoxide Species. J. Am. Chem. Soc. (2003), 125, 4185–4198.

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87 CHAPTER II. Cu2+-AMYLOIDCOMPLEXES AS A REDOX-ACTIVE CATALYST TOWARD THE OXIDAT ION OF 1,2,3-TRIHYDROXY BENZENE‡ I. INTRODUCTION Abnormal metal-ion homeostasis has been closely associated with several neurodegenerative diseases, including Park inson’s, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease (i.e. mad cow di sease), and Alzheimer’s disease (AD).1–3 Because high cytoplasmic concentrations of free metal ions are toxic and potentially lethal, intricate physiological pathways have evolved to transport and distribute metal ions to their targets which include enzymes and proteins.4 With aging, physiological processes responsible for accurate delivery of metal ions break down and “leakage” of free metal ions can cause toxic effects to cells.5,6 Divalent ions of redox-active transition metals have often been associated with oxi dative stress and clos ely involved in the This work has been published: ‡ G.F.Z. da Silva, W.M. Tay, L.-J. Ming, Catechol Oxidase-like Oxidation Chemistry of the 1–20 and 1–16 Fragme nts of Alzheimer's Disease-related -Amyloid Peptide: THEIR STRUCTURE-ACTIVITY CORRELATION AND THE FATE OF HYDROGEN PEROXIDE, J. Biol. Chem. (2005), 280, 16601-16609.

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88 chemistry of reactive oxyge n species (ROS), including hydrogen peroxide and superoxide and hydroxyl radicals.7 Because increases in intr acellular concentrations of metal ions is closely related to the effects of aging, oxidative stress, and AD, there is considerable interest in i nvestigating the connection betw een malfunction of regulatory processes such as metal transport and the pr esence of ROS with the pathology of AD. The chemistry of redox-active metal complexes of -amyloid peptide (A ) has been an area of intense focus in the st udy of AD. The aggregation of A within the neocortex is closely related to the pa thology of AD and has been shown to be induced by metal binding.8,9 The A peptides are generated by the cleavage of the ubiquitous amyloid precursor protein (APP) by , and secretases.10 A in the form of insoluble plaques contains up to mM amounts of Zn2+, Cu2+, and Fe3+ in the neocortical region of the brain;7 however, the cause/effect connection of the metallo-A plaques with AD is still under debate.11 The metal coordination environment of the 1–40 and 1–42 peptides has been previously studie d and their pH dependent aggregation reported.9,12 The results showed the metal binding seemed to be nonstoichiometric with approximately 3.5 metal ions per pair of aggregated peptides and a cooperative bindi ng pattern as the amount of aggregates increases.7 Since A 1-42 and A 1-40 have been shown to bind Zn2+ and Fe3+ with nM and Cu2+ with aM apparent dissociation c onstants by means of quantitative determination of the metal-complex precipitates,7 understanding of the metal-binding domain and its structure may shed light on th e chemistry related to the neuropathology of AD.

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89 Though the coagulation of the pe ptide plaques leaves little doubt that interaction with cytoplasmic molecules is unlikely, smaller fr agments of the amyloid peptide are soluble and A fibrils extend across membranes, exposing them to the cytoplasm. Recently, insulin degrading enzyme (IDE) has been shown to digest the longer A peptides (40–42 amino acids) into smaller soluble fragments.13 Moreover, the cleavage of APP by and secretases produces the A 1-16 fragment of APP.14 These soluble fragments and intramembrane spanning fibrils still possess possibl e metal binding sites such as histidines, glutamate, aspartate, and tyrosine within the 1-20 fragment of A in the sequence DAEFR5 HDSGY10 EVHHN15 KLVFF20. Redox chemistry of A has been previously reported, wherein Met35 was suggested as a “built-in” reducing agent required for the redox cycling hypothesis.15 The lack of sufficient data on the redox chemistry of and the oxidative stress caused by metallo-A and the discrepancies in previous studies such as the presence or absence of free-radi cals and the nature of the metal-A interaction seem often to be the shortcoming in A research. Understanding of the chemical processes associated with metallo-A may provide insight into th e upstream and/or downstream regulatory processes that lead to AD. Here in, we describe the oxidation chemistry of CuA in the presence and absence of H2O2, showing conclusive metal-centered preequilibrium kinetics toward the oxidation of a simple substrate and the oxidative cleavage of double-stranded plasmid DNA.

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90 II. EXPERIMENTAL The 1-20 and 1-16 fragments of A were purchased from Aldrich (St. Louis, MO) or synthesized at the Peptide Center of the Univers ity of South Florida. The identities of the peptides have been confirmed with a Bruker MALDI-TOF mass spectrometer. The substrate 1,2,3-trihydroxylben zene (THB) was obtained from Sigma -ldrich, 3-methyl-2benzothiazolinone hydrazone hydrochloride mon ohydrate from Acros (Fairlawn, NJ), the plasmid pQE30Xa from Quiagen (Valen cia, CA), EDTA, DMSO, mannitol, H2O2, Cu(NO3)2, ZnCl2, and Ni(SO4) from Fisher (Swanee, GA), and CoCl2 from Mallickrodt (Paris, KY). All plastic ware was demetallized with EDTA and exte nsively rinsed with 18.0-M water to remove the chelator. The wa ter used for the studies of DNA cleavage was autoclaved to remove ubiquitous nucleases. DNA cleavage assay : The metal derivatives of A were prepared by dissolving the peptide in 18.0-M water and separated into aliquots followed by addition of corresponding metal ions at 1:1 stoichiometr ic ratio, which was further diluted into aliquots of working concentrations. The metal-complexes were freshly prepared in all experiments. A typical reaction toward DNA cleavage contained 150 ng of plasmid DNA, 4.0%, 2.0%, or 0.2% H2O2, and 5.0 M of metallo-A derivatives in 100 mM HEPES at pH 7.00 in a volume of 15.0 L. A time-course experiment was performed and analyzed in a 1.0 % agarose gel stai ned with ethydium bromide, and then photographed on a transilluminator. THB oxidation assay : Several concentrations of THB ranging from 0.10 to 5.0 mM were incubated with 7.5 M of CuA and 1.60, 3.20, 16.20, 32.3 or 70.0 mM H2O2 and

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91 buffered with 100 mM HEPES at pH 7.00 in a fi nal volume of 1.0 ml. The formation of product was monitored at 420 nm ( = 4,583 M–1 cm–1) on a Varian CARY50 Bio-UVVis spectrophotometer for 5 minutes, and the rates determined by the change in absorbance over time. The background oxida tion of THB was conducted in the same manner without CuA in the assay solution. Rates were fitted to appropr iate rate laws and rate constants determined by the use of SigmaPlot 8.0. The dependence of H2O2 on THB oxidation by CuA was determined by measuring the oxidation rate at several different concen trations of hydrogen peroxide with saturating concentration of THB (6.0 mM). The initial rates were determined and then fitted as a function of [H2O2] to an appropriate rate law to reveal the rate constant. Alternatively, the catechol oxi dase assay was performed as previously reported with minor changes to fit current studies.16 Same molar concentrations of THB and 3-methyl2-benzothiazolinone hydrazone (which serves as an ortho -quinone indicator) were mixed in 100 mM HEPES at pH 7.00 in the presence of 3.5 M CuA The red-adduct of the ortho -quinone product was monitored at 500 nm ( = 32,500 M–1cm–1) and rates calculated. Auto-oxidation of THB was dete rmined under the same conditions without Cu-A Metal Titration : Apo-A was dissolved in 100 mM HEPES at pH 7.00 to a final concentration of 1.0 mM. Cu2+ binding was monitored by titra ting the metal into the apoA solution and the electronic spectra collect ed after each addition of the metal. Cu2+ binding was also determined through the activity of CuA complex toward THB. In this case, Cu2+ was titrated into a fixed amount of the peptide in 100 mM HEPES at pH 7.00

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92 in the presence of 10.0 mM of THB and 70.0 mM H2O2. The oxidation rates were determined as a function of [Cu2+], then fitted to a simple equilibrium of metal:peptide = 1:1 or a cooperative binding pattern using the Hill equation. NMR Spectroscopy: All the NMR spectra were acquired on a Bruker DPX250 spectrometer at 1H resonance of 250 MHz. The metal-binding was monitored through the changes in the NMR spectra. The peptides A 1-16 and A 1-20 and the paramagnetic shift reagent Co2+ were prepared in d6-DMSO. The metal ion was gradually titrated into the peptide and the paramagnetically shifted 1H NMR signals detecte d. A typical spectrum showing the paramagnetically shifted 1H NMR signals consists of ~80,000 transients from accumulation of several spectra of 10,000–20,000 transients with a recycle time of ~50 ms and a spectral window of ~250 ppm. Solvent exchangeable signals were determined by adding a drop of D2O into the sample which di sappear after the addition. Molecular mechanics : The primary sequence of A 1-16 peptide was entered into BioCAChe 6.0 (Fujitsu, Beaverton, Oregon) and the energy of its structure under solvation using a simulated water droplet was minimized with the MM3 molecular mechanics method. Histidine side chains were considered the ligands in the calculations on the basis of the NMR data. III. RESULTS AND DISCUSSION Oxidative Double-Stranded DNA Cleavage: Though the bulk of the amyloid plaques in AD brain is membrane bound, proteolytic pr ocessing of amyloid has been shown to yield soluble fragments.12 It has also been demonstrat ed that the neuropathology of AD

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93 may directly affect DNA, even tually leading to apoptosis.17 As metal ions are involved in the formation of amyloid plaque s, the oxidative activity of metallo-A complexes against plasmid DNA was probed in vitro with gel electrophoresis. The oxidative activities of the Zn2+, Ni2+, and Cu2+ complexes of A 1-20 (ZnA 1-20, NiA 1-20, and CuA 1-20) toward the cleavage of plasmid DNA were determined by incubating several different concentrations of the complexes with plasmid DNA in the presence of 4.0 % H2O2 at room temperature for 30 minutes (Figure 2.1). Here, ZnA 1-20 serves as the control since Zn2+ is oxidative inactive. The plas mid in the presence of metallo A 1-20 at lower concentrations shows a middle band that is not present in the reference (Lane R, Figure 2.1). Comparing the middle band with the DNA ladder gives an approximate size of 3.5 kbp, consistent with the size of lineari zed plasmid from the manufacturer. The activities of the derivatives follow the trend CuA 1-20 > NiA 1-20 > ZnA 1-20, demonstrating the involvement of metal in the oxidative cleavage of dsDNA. One interesting result is shown in the H2O2 + DNA and the H2O2 + DNA + A 1-20 control experiments. The 4.0 % H2O2 shows a significant damage toward plasmid dsDNA (Lanes 1, Figure 2.1), whereas A 1-20 decreases the H2O2 damage of plasmid dsDNA and perhaps acts as a scavenger of ROS speci es (Lane 2, Figure 2.1). The role of A as an antioxidant has been previously reported,18 wherein the presence of Met35 was proposed to prevent lipid peroxidation while the M 35L mutant showed reduced antioxidant activity. In a similar study, A 1-40 was found to prevent the oxi dation of the lipoproteins from cerebral spinal fluid and plasma.11 Moreover, A 1-42 was shown to exhibit an

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94 Figure 2.1 Concentration and metal-dependent assay of dsDNA cleavage. R is the reference plasmid DNA which shows superco iled band (bottom) and nicked circular band (top); lane 1, DNA + 4% H2O2; lane 2, DNA + apo-A 1-20 (200 M) + 4% H2O2; lanes 3–6, ZnA 1-20 (40, 80, 100, and 200 M, re spectively) + 4% H2O2; lanes 7–13, NiA 1-20 (5, 10, 20, 40, 80, 100, and 200 M respectively) + 4% H2O2; CuA 1-20 (at 5, 10, 20, 40, 80, 100, and 200 M) + 4% H2O2 shows complete DNA cleavage (“blank” gel, not shown). All assays were incuba ted for 30 minutes. The middle band is the linearized plasmid detected at 3.5 kbp position, consistent with that obtained from the sequence of the plasmid pQE30Xa (Qiagen). Lanes 1’–6’ show the plasmid cleavage by Cu2+ ions (5 M) + 3.6% H2O2 at 10, 20, 40, 60, 90, 120 min. The standard DNA ladder starts with 1 kbp from the bottom with 1 kbp increment upward.

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95 antioxidant activity more effective than ascorbate in cerebral spinal fluid.19 The antioxidant activity of A was also demonstrated in the decrease of cytoplasmic amounts of 8-hydroxyguanosine, a major product of nucle ic acid oxidation present in elevated amounts in the brains of AD patients.20 These observations implied that the production of A could be related to prevention of oxidative stress. We have demonstrated here that even shorter fragments of A without a Met can serve as a protective agent against oxidative damage of DNA, corrobora ting with some previous reports11,19,20 and supporting the hypothesis that apo-A might be an effect of the oxidative stress in AD brains and might serve a specific purpose to scavenge ROS. This antioxidant activity is also observed in all concentrations of ZnA 1-20 (Lanes 3–6, Figure 2.1), consistent with the lack of redox chemistry of Zn2+ and a protection role against dsDNA cleavage as in the case of apo-A 1-20. Although Ni2+ is redox active and some of its comp lexes have been shown to exhibit oxidative damage toward DNA,21 NiA 1-20 does not show such “chemical nuclease” activity, probably attributed to its lo w redox potential. Conversely, like apo-A and ZnA 1-20 discussed above, NiA 1-20 shows a concentration-dependent protection against oxidative damage of dsDNA by H2O2, with better protection at higher concentrations while no significant protection at [NiA 1-20] < 80 M (Lanes 7–12, Figure 2.1). NiA has not been shown to be associated with AD pathology; however, it may serve as a structural and mechanistic probe in future studies of metallo-A or similar systems. The activity of 5.0 M CuA 1-20 (Lane 14, Figure 2.1) is exce edingly higher than that of ZnA 1-20 at all concentrations test ed in the presence of 4.0% H2O2 (Lanes 3-6, Figure

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96 2.1) which effectively oxidizes the entire dsDNA plasmid sample into fragments that are too small to be resolved with the agarose gel electrophoresis (empty lanes not shown). Cu2+ ion has been demonstrated in the literature to be active toward DNA cleavage in the presence of H2O2.22 The use of Cu2+ (5.0 M) in the presence of 3.6% H2O2 as control shows a much slower cleavage rate as the pl asmid is not completely digested after 2 hours of incubation. In order to m onitor the catalyti c activity of CuA 1-20 toward plasmid dsDNA, the concentration of H2O2 was reduced to 2.0% and a time course experiment conducted (Figure 2.2). CuA 1-20 completely oxidizes plasmid DNA within 5.0 minutes in the presence of 2.0% H2O2, leaving only a faint streak in the gel (Lane 7, Figure 2.2). The ability of apo-A 1-20 to act as a protector against oxi dation of dsDNA in the presence of H2O2 is once again demonstrated here. Fu rther reducing the concentration of H2O2 to 0.2% allows a clearer monitoring of plasmid cl eavage patterns (Figure 2.3). Within 10 minutes of incubation, 5.0 M of CuA 1-20 shows double-strande d DNA cleavage as evident in the appearance of a middle ba nd approximately 3.5 kbp (Lane 6, Figure 2.3). Within 20-30 minutes, complete conversion of the supercoiled plasmid into linear and nick-circular conformations is observed, eviden t in the changes in the intensity of the different forms of the plasmid compared to the reference (Lanes 3 and 4, respectively, Figure 2.3). After 30 minutes, plasmid is cleav ed into small pieces leaving a streak of oligonucleotide products (Lanes 1, 2, and 3). The different and quite oppos ite activities between apo-A 1-20 and CuA 1-20 toward dsDNA damage may hi nt a physiological role of small fragments of apo-A

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97 Figure 2.2 Time course reactivity assay of ds DNA cleavage. R is the reference plasmid DNA; lanes 1–7, DNA + 5.0 M CuA 1-20 + 2% H2O2 (60, 50, 40, 30, 20, 10, 5 min, respectively); lanes 8-11, DNA + A 1-20 (metal free) + 2% H2O2 (60, 40, 20, 10 min, respectively); lanes 12–15, DNA + 2% H2O2 (60, 40, 20, 10 min, respectively); and lanes 16–18, DNA + A 1-20 (metal free, peroxide free). The standard DNA ladder starts with 1 kbp from the bottom with 1 kbp increment upward.

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98 Figure 2.3. Time course reactivity assay toward the cleavage of 150 ng dsDNA. R is the reference DNA; lanes 1–6, DNA + 5.0 M CuA 1-20 + 0.2% H2O2 (60, 40, 30, 20, 15, 10 min, respectively); lanes 7-10, DNA + 5.0 M CuA 1-20 (peroxide free 60, 40, 20, 10 min, respectively); and lanes 11-15, DNA + 0.2% H2O2 (40, 30, 20, 15, 10 min, respectively). The st andard DNA ladder starts with 1 kbp from the bottom with 1 kbp increment upward. The cleavage of plasmid by 5.0 M Cu2+ ions and 3.6% H2O2 is shown in Fig. 1, which exhibits a much lower activity.

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99 To determine the role of the oxidizing agent in these reactions, the same concentration of CuA 1-20 was incubated with the plasmid in the absence of H2O2 up to 60 minutes, which shows neglig ible cleavage (Lane 7, Figure 2.3). The low activity of CuA 1-20 without H2O2 indicates a metal-centered activa tion of peroxide, such as the formation of a Cu2-peroxo center found in many Cu complexes23,24 which subsequently results in oxidative damage to dsDNA. To distinguish the reaction pathways of oxidative DNA cleavage by H2O2 in the presence and absence of CuA 1-20, a time-course experiment was established (Lanes 11-15, Fi gure 2.3). The reacti on patterns of dsDNA cleavage in these two cases are clearl y different. In the absence of A 1-20, dsDNA is cleaved into small fragments without formati on of a linear intermediate as evident by the faint band at 2.0 kbp. The nature of the band is not clear at this stage and is not associated with A The dsDNA cleavage by CuA 1-20 in the presence of H2O2 is conformation-dependent, most active towa rd supercoiled dsDNA as evident by the accumulation of nicked-circular and linear form s with time in the reaction, likely due to the structural constraints of the superco iled form. The accumulation of the linearized form (middle bands) is indicative of doubl e-stranded DNA cleavage, rather than a random single-stranded cleavage, which is a ke y trigger that can re sult in cell apoptosis.25 The linearization of the plasmid via cleav age of double-stranded (ds) DNA is also characteristic of the cleavage patter n by DNA-recognizing agents such as Cubleomycin.26 False regulation of metal homeostasis and RO S physiology is closel y related to aging and oxidative stress,6 wherein apo-A 1-20 seems to serve as a scav enger of metal ions due

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100 Obackgrond cat appv k CuATHB KTHB [][] '[] (1) to its large affinity constant with metal ions and a protective ag ent against oxidative damage of biological macromolecules by H2O2 based on the observations in this and other studies.27, 28 However, the presence of H2O2 can result in severe damage toward dsDNA and presumably other redox-sensit ive biomolecules as well by metallo-A when the metal ions are redox activ e as demonstrated herein. Kinetics and Mechanism of Ox idative Catalysis by CuA : To gain further insight into the mechanism for the oxidation activity of CuA 1-20 and its interaction with H2O2, the catechol analogue 1,2,3-trihydroxylbenzene (THB ) was utilized to provide detailed kinetic information owing to its easily acces sible oxidation state which also has been utilized for investigation of oxida tive activities of metal complexes.29 The oxidation rate of THB by 7.5 M CuA 1-20 was determined at different va lues of [THB] in the presence of H2O2 at various concentrations (Figure 2.4) which reached saturation at high THB concentrations. This saturation pattern suggest s a possibility of preequilibrium kinetics. The rate law for this reaction mechanism can be expressed as in Eq. (1), assuming that the concentration of the intermediate THB-CuA 1-20 complex is much lower than that of the unbound THB in which K’app = ( k–1 + kcat)/ k1 is the virtual disso ciation constant and k1 and k–1 are the rate constants for the formati on and dissociation, respectively, of the THBCuA complex. The data can be well fitted to Eq. (1), yielding kcat = 0.00767 s–1 and K’app = 1.67 mM, and a second-order rate constant kcat/ K’app of 4.59 M–1 s–1 for the

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101 reaction in the presen ce of 16.0 mM (0.0544%) H2O2. This represents a 724 fold increase in terms of the first-order rate constant when compared to the auto-oxidation of THB under the same reaction conditions in the absence of CuA 1-20 (determined to be k0 = 1.06 10–5 s–1). A plot of kcat as a function of [H2O2] from Figure 2.4 shows that kcat reaches a plateau at high H2O2 concentrations (inset, Fig. 1.4). However, the kcat value does not reach zero at 0% H2O2 that is higher than k0 of the auto-oxidation of the substrate. The oxidation reaction in the absence of H2O2 was further explored a nd discussed in a later section below. The plot seems slightly sigm oidal which indicates a possible presence of either a consecutive or a cooperative binding of H2O2 to the active center. Since catechol oxidation involves 2-electron tran sfer which matches with th e two-electron reduction of H2O2 to yield two oxides, a consecutive mechanism is not fundamentally necessary for the reaction to take place. The data were f itted to the Hill equati on to extract the Hill coefficient of 2.09 and kcat value of 0.00731 s–1 at 0% H2O2 (close to the value of 0.0065 s–1 directly measured in the absence of exogenous H2O2 discussed later), indicative of the presence of weak cooperativity and H2O2-independent oxidative catalysis. Interestingly, the smaller fragment CuA 1-16 showed more than 4-folds higher kcat of 0.0340 s–1 for the reaction with the same concentration of H2O2. However, its catalytic efficiency is only twice higher than the larger fragment in terms of the second order rate constant kcat/ K’app (10.5 M–1 s–1), which suggests a particip ation of the last four Cterminal hydrophobic residues (LVFF) in the reaction pathway. The hydrophobic Cterminus may influence substrate binding and product release as re flected by the higher

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102 Figure 2.4. Effect of peroxide concentrati ons on the rate of THB oxidation in the presence of 7.5 M of CuA 1-20 in the presence of 1.6 ( ), 3.2 ( ), 16.2 ( ), 32.3 ( ), 64.6 ( ), and 70.0 ( ) mM H2O2 (HEPES buffer of 100 mM at pH 7.0 and 25.0 C). The dotted line is CuA 1-16 in the presence of 16.2 mM H2O2. The inset shows the first order rate constant kcat as a function of hydrogen peroxide, wherein the solid trace is a fitting of the data to the Hill equation by taking into considera tion of an activity at 0% H2O2.

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103 K’app and kcat values for CuA 1-16 ( K’app = 3.23 mM). This observation suggests that the C-terminus is close enough to the metal center to influence THB binding or a transitionstate conformational change that affects both the binding of THB and the turnover of the reaction. It has previously been documented that H2O2 and other ROS generated by metallo-A may play a role in the pathology of AD.27,28,30 Since the local concentration of metalloA in an AD brain can reach sub-mM range,7 the above observation implies that a significant rate acceleration in redox reactions can be expected at a location where H2O2 is produced. This rate enhancement in the brains of AD patients can be metabolically catastrophic. In the studies shown here, I have further specified the fate of H2O2 in metallo-A -associated redox reactions. To further analyze the role of H2O2 in the reaction pathway, a saturation profile was constructed with a fixed amount of the substr ate THB at 6.0 mM. Under such conditions, the reaction reaches plateau at [H2O2] near 70.0 mM or 0.238% (Figure 2.5). The results can be well fitted to a pre-equilibrium kinetics (Eq. 2). This kinetics further corroborates a metal-centered mechanism as described above. The rate of acceleration against the ba ckground oxidation of THB at saturating [H2O2] is approximately 6,000 folds (background rate constant ko = 1.14 10–5 s–1) with kcat = Obackground cat app v kCuAHO K H O [][] '[]22 2 2 (2)

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104 Figure 2.5 H2O2 saturation profile of CuA 1-16 ( o ) and CuA 1-20 ( ) activity at a fixed [THB] of 6.0 mM (HEPES buf fer of 100 mM at pH 7.0 and 25.0 C). The inset shows that DMSO ( o ) and mannitol ( ) do not inhibit the oxi dation of THB by CuA

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105 0.066 s–1 and kcat/K’app = 37.2 M–1 s–1. The hydrophobicity at the C-terminus does not affect the binding of H2O2 as reflected by the similar apparent virtual dissociation constant K’ pp between CuA 1-20 and CuA 1-16 (17.3 and 16.6 mM, respectively). Qualitatively, since the kcat value is small, the K’ app value is expected to be closer to the dissociation of the CuA -THB complex. The binding of each H2O2 molecule in this case may not yield a complete turnover, suggesti ng that formation of the intermediate Cuperoxo active center is not favorable under th e experimental conditions which may well be the rate-determining step. This result co rroborates with chemi cal model studies of catechol oxidase and tyrosinase in that a di nuclear Cu-peroxo intermed iate is often shortlived and thermodynamically unstable.24,31 Because the involvement of free radical s in the redox chemis try of metallo-A had been implicated in previous reports,7 different amounts of DMSO and mannitol, two common scavengers for superoxide free radical and hydroxyl free radical,37,32 were added to the reaction solution separately wi th saturating concentrations of H2O2 and THB. No noticeable effect on the reaction rates was observed under the experimental conditions (Figure 2.5, inset). However, this does not discount possible free ra dical generation since the free radicals may well be metal-centered and free-radical oxida tion in solution may not be the predominant pathway in the oxidation of THB catalyzed by CuA 1-20. We have established here a metal-cen tered oxidative catalysis by CuA 1-20 and CuA 1-16 which can not only generate H2O2 as noted previously30 but also activate H2O2 for possible oxidation of biomolecules.33

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106 Since both H2O2 and THB can interact with the metal center and are considered “substrates” for CuA it is imperative to further na rrow down the mechanism about how these two substrates interact individually wi th the metal center. For this purpose, the redox indicator 3-methyl-2-benzothiazolinone hydrazone (MBTH) was used to probe the oxidation product of THB in the absence of H2O2. MBTH is a common indicator used in catechol oxidase assay which forms a red adduct with the o -quinone products instantaneously.16 The rate for the oxidation of a catechol into its corresponding o quinone can thus be easily monitored colorimetr ically as the oxidation is the rate-limiting step. The rate for the oxidation of THB by CuA 1-20 in the absence of H2O2 as a function of [THB] is not linear which can be well f itted to pre-equilibrium kinetics to give kcat = 0.0065 s–1 and K’app = 2.0 mM. The first order rate acc eleration of THB oxidation here is 650 folds with respect to the auto-oxidation (i.e. kcat/ ko; ko = 1.06 10–5 s–1). This oxidative reaction is much less significant in te rms of rate acceleration than that in the presence of a saturating amount of H2O2 described above. Here catechol is possibly oxidized by CuA in form of a dinuclear Cu2+ center via 2-electron transfer to afford 2Cu+ and o -quinone product. The reduced 2Cu+ in turn can bind O2 to form a dinuclear Cu2+-peroxo center and follow the catalytic pathway as the CuA /H2O2 system discussed above. Because a bi-substrate mechanism wa s implied from our results (i.e. both THB and H2O2 show saturation), further analysis of the data was performed. The Hanes analysis was used to minimize the error across the concentration range (Figure 2.6A).34 The virtual dissociation constant K’app for both substrates cannot be resolved only from the primary nonlinear fit ting without analyzing their combined effects.35 It is thus

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107 important to determine the rates at varying amounts of H2O2 when holding THB constant and vice versa. The data in Figure 2.4 were fitted to a two-substrate random-binding mechanism according to Equation 3 (Figure 2.6A),34 wherein the binding of THB and H2O2 to CuA 1-20 was assumed to be random and in rapid-equilibrium with a subsequent ordered product release. Unde r these conditions, a simple c onversion to a secondary plot of the slope (slope = (1+ K’ /[H2O2])/ Vmax) and the y-intercept (y intercept = ( K’app/ Vmax)*(1+ Ki/[H2O2] ) in Figure 2.6B versus 1/[H2O2] yields K ’ and K ’, the true values for the virtual dissociation of H2O2 and THB, respectively, and the intrinsic affinity constant of H2O2 Ki Moreover, if any cooperativity is present in this bi-substrate reaction mechanism, it would be revealed by the ratio of K ’app/ K ’. For a random equilibrium mechanism a ratio of K ’app/ K ’ between 1 and 5 would suggest little cooperativity.35 In the oxidative catalysis by CuA 1-20, the K’app/ K ’ ratio is 2.85 for THB oxidation and K’ app/ K ’ is 1.62 for H2O2 which indicates little cooperativity. It is important to note that based on the data alone it is difficult to distinguish between an ordered sequential-binding mechanism and the mechanism herein proposed.34 However, a random equilibrium phase is a sound assumption since both THB and H2O2 can interact with the metal centers separately. In our proposed reaction mechanism, only when both THB and H2O2 substrates bind to the metal-center can productiv e turnover be observed with se cond-order rate constants of 8.66 and 15.6 M–1s–1 for the oxidation of THB by CuA 1-20 and CuA 1-16, respectively, in [] [] [] []maxmaxTHB K HO V THB K V K HOO i 1 122 22 (3)

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108 the presence of H2O2 (whereas molecular O2 serves as the second substrate in the absence of H2O2). This pathway differs from the pr eviously proposed mechanism in the redox cycling of metallo-A wherein the presence of the thioether group of Met35 was accounted for the reduction of the metal center.7 The results presented here indicate substrate-mediated reduction of the metal cente r (since Met is absent in the studies) as well as oxidation of the substrate by metal-activated H2O2. However, our data do not discount the possibility of the involvement of Met in the re ductive pathway in A 1-40/42. Regardless, the redox chemistry of CuA presented here shows an important mechanism for possible destructive acti ons in Alzheimer’s disease. Taken together, the metal-centered redox cycle of CuA action in this study seems to match the mechanism of the dinucle ar Cu-containing catechol oxidase, wherein the oxidation of the substrate takes place both in the presence and absence of H2O2.38,39 Since the oxidation of catechols is a two-elec tron transfer process, the involvement of a dinuclear Cu center is thus a preferred path way as in the case of the enzyme. In the presence of THB and H2O2, the dinuclear 2, 2-peroxo-Cu2+ 2-THB transition state is eventually formed by assembling two metal cente rs together via the bridging peroxo as in the case of many mononuclear Cu2+ complexes23,24 (Figure 2.7, steps A–C ), which is followed by 2-electron transfer from the bound peroxide to the bound catechol (likely through the metal center) to yield Cu2+ 2--OH and o -quinone to complete a catalytic cycle (step D ). Here, the complexes oxy-Cu+ 2, Cu2+ 2--( 2, 2-peroxo), Cu2+ 2--( 1, 1.

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109 Figure 2.6. Hanes analysis of the kinetic da ta from Figure 4. The plots in A yield the apparent virtual dissociati on constant for THB. The re-plot of the slope ( )and yintercept ( ) from A reveals dissociation constants for THB ( KÂ’ = 0.41 mM) and H2O2 ( Ki = 17.3 mM) in a bi-substrate reaction.

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110 peroxo), and Cu3+ 2-(-oxo)2 ( B ) are isoelectronic24 which are not distinguishable in our study. In the absence of H2O2, the oxidation of the bound THB is achieved by 2-electron transfer to the dinuclear Cu2+ 2 center to yield Cu+ 2 (steps F–G ) which is followed by O2 and THB binding to regenerate the THB-Cu2+ 22-peroxo transition state (steps H B and C ). The binary and the ternary complexes then follow the same pathway as the case in the presence of H2O2 for another turnover. H2O2 is also generated according to this mechanism under reducing conditions (steps E and I ), which has been previously observed30 and can serve as a competing reac tion pathway toward the oxidation of catechols (steps C D ). The mechanism of oxidative “chemical nucleases” has been thoroughly studied and reviewed.36 According to the studies of some simple chemical nucleases such as Cu1,10-phenanthroline, a reduced state of the meta l center (by a reducing agent) is required for catalysis in the presence of O2. In our experiments, however, the absence of a reducing agent to convert Cu2+ to Cu+ and the use of H2O2 as the oxidation agent suggest a different oxidative pathway. Moreover, the free radical scavenger37 dimethyl sulfoxide (DMSO) did not inhibit the r eaction, suggesting the absence of free radicals to induce the oxidative damage. On the basis of the results shown here, we propose a 2-peroxobridged dinuclear Cu2+ active center for CuA 1-20 as observed in a number of chemical model systems23 and in catechol oxidase, tyrosinase, and hemocyanin.38,39 The nature of this transient 2-peroxo species and its attack on the substrates, although thoroughly

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111 Cu3+Cu3+ O2-O2-Cu2+O O Cu2+ Cu2+Cu2+ O O Cu2+Cu2+ O H Cu2+Cu2+ O O O O H H O O O H Cu+Cu+ Cu2+Cu2+ O O O H O O O H + HO2 O O O H H2O H2O2 H2O2 2H+2H+2H+THB 2H+THB O2, H+3H+_ _ _A F D C E B G H 2e-, I2H+H2O2 Figure 2.7. Proposed mechanism for the oxidation of THB by CuA in the presence (steps A–D ) and absence of H2O2 (steps F–H and B–D ). The production of H2O2 under reduction conditions (steps E ) is also consistent with this mechanism.

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112 studied, still has key mechanistic questions to be answered, such as the true structure of reaction intermediates, the role of substrate in the reaction mechanism, rate determining steps in catalysis, and specificity of meta l cofactor for the function of enzymes like catechol oxidase, tyrosinase, peptidylglycine monooxygengase, methane monooxygenase, fatty acid desaturase, and ribonucleotide reductase.40–42 It has been proposed that substrate ac cessibility in the active site after O2 binding plays a key role in the action of these proteins.40,43 Consequently, a reversible O2 binding has been demonstrated in hemocyanin because of the lack of substrate accessibility, wherein bulky substrates such as aromatic systems and the ribose moiety of DNA may not easily gain access to the O2-binding active center of the pr oteins. However, studies of catechol oxidase and tyrosinase have s hown the production of hydroxylated phenols and ortho -quinones, reflecting that substrates bind directly to the dinuclear Cu22-O2 active center which enables a direct attack on the substrates by the peroxo unit.44,24 Metal Binding and Structure: Detailed information about the metal-binding ligands and geometry of the metal site is needed to gain further insight into the metal-centered redox chemistry and to elucidate any struct ure-function correlation important for the action of metallo-A Since activity is an excell ent probe for monitoring reaction mechanism, it is thus chosen as a probe for the determination of the metal-binding stoichiometry of metallo-A Upon introduction of Cu2+ to A oxidative activity can be measured as described above. It is eviden t from the data that metal binding reaches saturation at slightly above 1:1 ligand-to-me tal ratio (Figure 2.8). Despite a previous

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113 Figure 2.8 Cu2+ titration toward A 1-20 ( ) and A 1-16 ( ) monitored with their oxidation activities (100 mM HEPES buffe r at pH 7.0 and 25.0 C). The dotted curves show the best fit tings to a noncooperative M+L ML equilibrium. Solid curves are the nonlinear fittings to the Hill Equation which yield Hill coefficients of 1.94 and 3.27 for Cu2+ binding to A 1-16 and A 1-20, respectively.

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114 EPR study which indicate the binding of two Cu2+ ions to A ,45 our result indicates the active species is a 1:1 CuA complex. Both a noncooperative binding equilibrium (a quadratic pattern) and a cooperative equilibrium (a sigmoida l pattern) were used to fit the data. It is evident from the fitting that the shorter CuA 1-16 fits equally well to both binding patterns with a me tal-to-ligand stoichiometr y of 1 to 1, whereas CuA 1-20 seems to fit better the cooperative binding pattern. The binding of Cu2+ to A 1-16 gives a Hill coefficient of 1.94, while the binding to A 1-20 shows a higher cooperativity with of 3.27. This result is consistent with previous reports of cooperativ e metal binding to the entire A determined on the basis of quantitative precipitation.46 The results presented here further indicate the pres ence of cooperativity in the oxidative activity as well as metal-binding. The higher C-terminal hydrophobicity of A 1-20 may influence intermolecular interactions, resulting in a mo re apparent cooperativ ity. The data were also analyzed to determine whether or not there were possible inactive dimer conformations of this metallope ptide by plotting activity as func tion of the square root of metal ion concentration as previously described.47 However, the data do not reflect the existence of such equilibrium in this r eaction pathway, adding supporting evidence to the dinuclear metal-centered redox mechanism he rein proposed. Dissociation constants ( Kd) for metal binding to A can be extrapolated from both fits with values of 3.96 and 4.30 M for CuA 1-16 and CuA 1-20, respectively. Since activity se rves as the probe here, the values obtained above are thus the intrinsi c dissociation constants attributable to the active CuA complexes and are not affected by coagulation equilibrium for A The intrinsic dissociation constant for metal binding in CuA 1-40 is likely to be in the same

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115 range of ~4 M for CuA 1-20 and CuA 1-16 owing to their probably similar metal-binding configuration. Indeed, dissociation cons tants in the range of ~0.1 M for CuA 1-28 and ~2 M for CuA 1-40 and CuA 1-42 were determined with ligand-competition45 and direct fluorescence measurement.48 An apparent dissociation constant Kd app of 0.50 pM for Cu2+ binding toA 1-40 was determined based on the formation of CuA 1-40 coagulates,46 which can be dissected into the intrinsic metal dissociation constant KCu of ~4 M and the dissociation constant of CuA 1-40 coagulates Kco ~0.13 M (i.e., Kd app = KCu Kco). The much smaller dissociation constant of 6.3 aM for Cu2+ binding toA 1-42 would thus afford an apparent dissociat ion constant of CuA 1-40 coagulates in the range of 1.6 pM. A recent report indicates that trace amounts of metal ions can significantly affect A coagulation, it is thus suspected that th e dissociation constants may be under estimated based on the coagulation.49 I report in this dissertation a direct and reliable means for the determination of meta l binding to soluble A fragments which is not complicated by the formation of the coagulation as previously observed46 that can be influenced by other factors, such as tra ce amount of metal ions.49 To further investigate the metal-coordinati on environment, the el ectronic spectrum of CuA 1-20 was obtained (Figure 2.9). The spectrum reveals a typical type-2 copper center with d-d transitions showing max at 610 nm (107 M–1 cm–1), clearly distinguishable from the near IR absorption at 820 nm for aqueous Cu2+ solutions. This absorption is consistent with that of the “CuH2L” species of acetyl-A 1–16 with three coordinated His side chains in a potentiometric study50 (617 nm and 117 M–1 cm–1) and another study45 (610 nm and ~50 M–1 cm–1 which seems to be too low an absorptivity51). The result

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116 Figure 2.9 Electronic spectra of A 1-20 with 0.5 equivalents (solid trace) and 1.0 equivalent Cu2+ (dashed trace) referenced against apo A 1-20 (100 mM HEPES buffer at pH 7.0). The dotted trace is the difference spectra of 1.4 and 1.0 equivalents of Cu+2 in A 1-20, showing no further increase in the d-d transition after the binding of one equivalent of Cu2+.

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117 agrees well with a tetragonally di storted octahedral environment51,52 for the Cu2+ in CuA 1-20. Upon addition of more than one equiva lent of metal, the spectrum does not change. This is consistent with the results when activity is used as the probe to monitor metal binding (Fig. 2.8), wh erein one equivalent Cu2+ is determined to bind to one peptide. It is also worth no ting that there are no intense tr ansitions in the near-UV range that can be possibly assigned to Tyr-to-Cu2+ charge-transfer transiti ons as observed in the Cu2+-substituted proteases astacin and serralysin.53 The metal coordination chemistry was also investigated by the use of Co2+ as an NMR probe. Co2+ has been well demonstrated to be an excellent probe for the investigation of metal-binding si tes in a number of metalloprot eins, including Zn and Cu proteins.54,55 Although A 1-20 has four additional hydrophobic amino acids on the Cterminus, the conformations of the two peptides in d6-DMSO are similar as they show nearly identical 1H NMR spectra (Figure 2.10). The signals due to L16VFF20 side chains in A 1-20 are clearly observed when comp ared with the spectrum of A 1-16, wherein LV are seen at ~0.6 ppm and FF ~7.2 ppm. Th is similarity reflects their similar configuration. There are two solvent ex changeable signals in the 14–16 ppm range (imidazole N-H signature chemical shifts56) with a 1:2 ratio in in tensity, corresponding to the three His side chains (ins ets, Figure 2.10 A and B). Upon Co2+ titration, the intensities of these solvent exchangeable His-imidazole signals gradually decreased which was acco mpanied by the appearance of three fardownfield paramagnetically shif ted signals in the region of 40–80 ppm as shown here for A 1-20 (Figure 2.10C). These far-shifted signa ls are also solvent exchangeable and

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118 correspond to the chemical shif t of the solvent ex changeable signal of a paramagnetic Co2+-bound imidazole group of a hist idine residue (which cannot be due to dipolar shift of unbound His residues si nce the octahedral Co2+ center is expected not to possess magnetic anisotropy) as observed in many Co2+-substituted metalloproteins.54,55 These three solvent exchangeable NH signals furthe r confirms the involvement of all three histidine residues in A 1-20 for metal binding, consistent with previous Raman spectroscopic studies,9 and is indicative of the absence of a bridging his tidyl imidazole (in contrast to previously suggested9,12) which would result in the loss of an imidazole NH signal. Tyr10 was suggested to be a possible ligand for Cu2+ and Fe3+ binding,9,12,57 but was suggested not to be a ligand in other studies.50 The 1H NMR signals of Tyr10 (the two asterisked doublets centere d at ~6.7 ppm in Figure 2.10 do not show any noticeable change upon the addition of the paramagnetic Co2+ ion. The binding of a Tyr-phenol group to Co2+ is expected to exhibit paramagnetically shifted 1H NMR signals of the bound phenol group outside the diamagnetic region as previously observed.58 This result indicates that this Tyr is not a metal-binding ligand, consistent with the lack of chargetransfer transitions for a possible Tyr-Cu2+ binding as described above. Our results also do not support the binding of the N-terminal amino group to the metal as previously suggested.45 This binding mode for paramagnetic Co2+ would show far upfield-shifted NH2 signal(s), a downfield-sifted CH proton, and slightly upfield-shifted CH2 protons owing to spin polarization, which were not observed.55

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119 C Figure 2.10 1H NMR spectra of A 1-20 (A) and A 1-16 (B) in DMSO. The insets show the imidazole N-H solvent exchangeable signals of His which disappear upon addition of a drop of D2O (remain intact with same amount of H2O). These signals disappear upon addition of paramagnetic Co2+, with concomitant appearance of three solv ent exchangeable hyperfine-shifted signals in the far-downfield region as shown here for Co-A 1-20 (C). The asterisked signals in A and B are due to Tyr ring protons which remain the same upon Co2+ binding.

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120 Molecular mechanical calculations have been applied to determine the structure of A 1-16 and its metal-binding domain. The energi es for different metal-binding modes have been calculated by the use of the MM3 fo rce field and a simulated water droplet to solvate the peptide. Binding of Cu2+ to His13 and His14 yields the lowest energy of –385 kcal/mol as compared to all other possible binding modes in the peptide. The binding to all three His side chains yiel ds a distorted octahedral geom etry (with 3 open coordination sites presumably occupied by water molecu les) and a slightly higher energy at –363 kcal/mol (Figure 2.11). The energy differen ce between these two metal-binding modes may be low enough to be easily overcome at r oom temperature. Extensive H-bonding are observed in this calculated structur e, particularly Gl u3-Arg5-Asp7 H-bonding interactions may stabilize the structure to a great extend (dotted lines in Fig. 2.11). The energies for Cu2+ binding to His6/13 and His6/14 are much higher at –125 and –210 kcal/mol, respectively, and are not likely to be the metal binding modes for A A histidine-bridged dimer form of the peptide previously proposed12 was also calculated which gave an unacceptably high overall energy of 52,500 kcal/mol. The binding of Tyr10 along with the histidine residues is al so highly unfavorable which puts undue stress on the phenol ring causing it to pu cker and the peptide backbone to distort, with a high overall energy of 570 kcal/mol. The recently suggested N-terminal binding mode (along with the binding of the 3 histidines) has al so been calculated to give an unfavorable overall energy of 147 kcal/mol. Since Cu+ can easily adopt a trigonal coordination sphere,52 a calculation with a fixed trigonal coor dination was performe d which yielded an energy of –318 kcal/mol. The low energy diffe rence between octahedr al and trigonal

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121 Figure 2.11 Proposed metal coordination a nd solution structure of CuA 1-16 in a relaxed-eye stereo view based on NMR study of Co2+ binding and molecular mechanics calculations. According to the molecular mechanical calculations both the two-histidine (H13/H14) and three-histidine bind ing patterns are stable, whereas NMR study suggests the latter bindi ng pattern. The dotted lines are H-bonds, which may prevent further bending of the peptide to al low the binding of the N-terminus to the metal as recently reported.45

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122 geometries rationalizes the redox cycle of th e Cu center in the catalysis of catechol oxidation. The binding of the 3 Hi s side chains to the metal renders one side of the metal center to have an open coordinati on sphere which can possibly bind H2O2 or O2 to form the dinuclear bridging 2-peroxo center described above. IV. CONCLUDING REMARKS The results presented here have added furthe r insight and support to the structure and chemistry of metallo-A which may assist better unders tanding of the neuropathology of AlzheimerÂ’s disease. A complete redox cycle for the action of CuA has been proposed from the kinetic studies which is consis tent with the mechanism proposed for the dinuclear Cu catechol oxidase. The results in this report, howeve r, do not resolve the cause/effect debate about the role of A in AD, but add more information to the chemistry of metallo-A As a cause for AD, I have shown and quantified redox chemistry of CuA in this dissertation which can serv e as a catalyst both in the absence and presence of H2O2 to cause severe oxidative damages in the brains of AD patients. As an effect of AD, A can be reasoned to be present as a regulator toward metal ion homeostasis due to its considerable metal a ffinities and its protective property toward oxidative DNA damage in the absence of Cu2+. In the latter case, abnormal homeostasis of redox-active metal ions can leach the metal io ns to yield metallo-A that can undergo redox destruction of biomolecules. I have presented data herein to revise the redox chemistry of the methionine-centered hypothe sis by showing a metalcentered catalysis

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123 as a significant contributi on to the oxidative damage in the pathology of the neurodegenerative AD. The fate of H2O2 generated by CuA in the presence of a reducing agent previously obser ved or an electron-donating subs trate shown here has also been established and quantified with exogenous addition of this oxidant. Further studies currently under way that focus on the stru cture-activity relati onship of metallo-A are expected to shed light on th e roles of metal ions and A in AD and with hope to provide useful information for treatment and prevention of AlzheimerÂ’s disease.

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124 IV. LIST OF REFERENCES 1) (a) S.R. Paik, H.J. Shin, J.H. Lee, C.S. Chang, J. Kim, Co pper(II)-induced selfoligomerization of alpha-synuclein. Biochem. J (1999), 340, 821-828. (b) S.R. Paik, H.J. Shin, J.H Lee, Metal-catalyzed oxida tion of alpha-synuclein in the presence of Copper(II) and hydrogen peroxide. Arch. Biochem. Biophys. (2000), 378, 269-277. 2) A.G. Estevez, J.P. Crow, J.B. Sampson, C. Reiter, Y. Zhuang, G.J. Richardson, M.M. Tarpey, L. Barbeito, J.S. Beckman, Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficie nt superoxide dismutase. Science (1999), 286, 24982500. 3) D. Mckenzie, J. Bartz, J. Mirwald, D. Olander, R. Marsh, J. Aiken, J. Reversibility of scrapie inactivation is enhanced by copper. J. Biol. Chem (1998), 273, 25545-25547. 4) V.C. Culotta, L.W.J. Komp, J. Strain, R. L.B. Casareno, B. Krems, J.D. Gitlin, The copper chaperone for superoxide dismutase. J. Biol. Chem. (1997), 272(38), 2346923472. 5) E. Roche, D. Romero-Alvira, Oxida tive stress in some dementia types. Med. Hypot. (1993), 40, 342-50. 6) L.A. Shinobu, M.F. Beal, in J.R. Conner, J. R. Ed. Metals in Oxidative Damage in Neurological Disorders ; Plenum, New York (1997). 7) A.I. Bush, The metallobiology of Alzheimer's disease. Trends Neurosci (2003), 26, 207-214.

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125 8) A.I. Bush, W.H. Pettingell, G. Multhaup, M. Paradis, J. Vonsattel, J.F. Gusella, K. Beyreuther, C.L. Masters, R.E. Tanz i, Rapid induction of Alzheimer A amyloid formation by zinc. Science (1994), 265, 1464-1467. 9) T. Miura, K. Suzuki, N. Kohata, H. Ta keuchi, Metal binding modes of Alzheimer's amyloid beta-peptide in insoluble aggregates and soluble complexes. Biochemistry (2000), 39, 7024-7031. 10) Y. Ling, K. Morgan, N. Kalsheker, Angiotensinogen: molecular biology, biochemistry and physiology. Int. J. Biochem. Cell. Biol (2003), 35, 1505-1535. 11) A. Kontush, C. Berndt, W. Weber, V. A kopian, S. Arlt, S. Schippling, U. Beisiegel, Amyloidis an antioxidant for lipoprotein s in cerebrospinal fluid and plasma. Free Rad. Biol. Med (2001), 30, 119-128. 12) C. Curtain, F. Ali, I.Volitakis, R.A. Ch erny, R.S. Norton, K. Beyreuther, C.J. Barrow, C.L. Masters, A.I. Bush, Alzheimer's disease amyloidbinds copper and zinc to generate an allosterically ordered me mbrane-penetrating structure containing superoxide dismutase-like subunits. J. Biol. Chem (2001), 276, 20466-2073. 13) G. Evin, A. Weidemann, Biogenesis a nd metabolism of Alzheimer's disease A amyloid peptides. Peptides (2001), 23, 1285-1297. 14) G. Evin, A. Zhu, D. Holsinger, C.L. Mast ers, Q. Li, Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets. J.Neurosci. Res. (2003), 74, 386-392.

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126 15) C. Sch neich, D. Pogocki, G.L. Hug, K. Bobrowski, Free Radical Reactions of Methionine in Peptides: Mechanisms Relevant to -Amyloid Oxidation and Alzheimer's Disease. J. Am. Chem. Soc (2003 ) 125, 13700-13713. 16) S.G. Srivatsan, P. Nigam, M.S. Rao, S. Verma, Phenol oxidation by coppermetallated 9-allyladenine-DVB polymer: r eaction catalysis and polymer recycling. Applied Catal. A: General (2001), 209, 327-334. 17) J.H. Jang, Y.J. Surh, beta-Amyloid i nduces oxidative DNA damage and cell death through activation of c-Jun N terminal kinase. Ann. NY Acad. Sci (2002), 973, 228236. 18) M.F. Walter, P.E. Mason, R.P. Mason, Alzheimer's disease amyloid peptide 25-35 inhibits lipid peroxidation as a resu lt of its membrane interactions. Biochem. Biophys. Res. Comm (1997), 233, 760-764. 19) K. Lonrot, K.T. Metsa, G. Molnar, J.P. A honen, M. Latvala, J. Peltola, T. Pietila, H. Alho, The effect of ascorbate and ubiqui none supplementation on plasma and CSF total antioxidant capacity. Free Rad. Med. Biol (1996), 21, 211-217. 20) A. Nunomura, G. Perry, J. Zhang, T. Montine, A. Takeda, S. Chiba, M.A. Smith, RNA oxidation in Alzheimer's and Parkinson's diseases. J. Anti-Aging Med (1999), 2, 227-230. 21) C.J. Burrows, V. Lepentsiotis, J. Domaga la, I. Grgic, R. van Eldik, J.G. Muller, Mechanistic information on the redox cyc ling of nickel(II/III) complexes in the

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127 presence of sulfur oxides and oxygen. Corre lation with DNA damage experiments. Inorg. Chem. (1999), 38, 3500-3505 22) (a) O.I. Aruoma, B. Halliwell, E. Gajewski, M. Dizdarogl u, Copper-ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem. J. (1991), 273, 601–604; (b) J. Sagripanti, K. H. Kraemer, Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide. J. Biol. Chem. (1989), 264 1729–1734; (c) S.-M. Chiu, L.-Y. Xue, L.R. Friedman, N.L. Oleinick, Differential Dependence on Chro matin Structure for Copper and Iron Ion Induction of DNA Double-Strand Breaks. Biochemistry (1995), 34, 2653-2661; (d) K. Yamamoto, S. Kawanishi, Site-specifi c DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom. J. Biol. Chem. (1991), 266, 1509–1515; (e) K. Yamamoto, S. Kawanishi, Hydroxyl free radical is not the main ac tive species in site-specific DNA damage induced by copper(II) ion and hydrogen peroxide. J. Biol. Chem. (1989), 264, 15435– 15440. 23) N. Kitajima, T. Koda, Y. Iwata, Y. Morooka, Reaction aspects of a -peroxo binuclear copper(II) complex. J. Am. Chem. Soc. (1990), 112, 8833-8839. 24) (a) E.A. Lewis, W.B. Tolman, Reactivity of Dioxygen-Copper Systems. Chem. Rev. (2004), 104, 1047-1076. (b) W.B. Tolman, Making and Breaking the Dioxygen O-O Bond: New Insights from Studies of Synthetic Copper Complexes. Acc. Chem. Res. (1997) 30, 227–237.

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128 25) M. Stanulla, J. Wang, D.S. Chervins ky, S. Thandla, P.D. Aplan, DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation duri ng the initial stages of apoptosis. Mol. Cell. Biol. (1997), 17, 4070–4079. 26) G.M. Ehrenfeld, L.O. Rodriguez, S.M. Hecht, Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission. Biochemistry (1985), 24, 81-92. 27) K. Zou, J.S. Gong., K. Yanagisawa, M. Michikawa, A novel f unction of monomeric amyloid -protein serving as an antioxida nt molecule against metal-induced oxidative damage. The J. Neurosci. (2002), 22, 4833-4841 28) A. Kontush, Amyloid: an antioxidant that become s a pro-oxidant and critically contributes to Alzheimer's disease. Free Rad. Med. Biol. (2001), 31, 1120-1131. 29) K. J. Humphreys, A. E. Johnson, K. D. Karlin, S. E. Rokita, Oxidative strand scission of nucleic acids by a multinuclear copper(II) complex. J. Biol. Inorg. Chem (2002), 7, 835-842. 30) C. Opazo, X. Huang, R.A. Cherny, R.D. Moir, A.E. Roher, A.R. White, R. Cappai, C.L. Masters, R.E. Tanzi, N.C. Inestrosa, A.I. Bush, Metalloenzyme-like activity of Alzheimer's disease -amyloid. J. Biol. Chem (2002), 277, 40302-40308. 31) V. Mahadevan, M.J. Henson, E.I. Solom on, T.D.P. Stack, Irreversible Reduction of Dioxygen by Simple Peralkylated DiamineCopper(I) Complexes: Characterization

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129 and Thermal Stability of a [Cu2( -O)2]2+ Core. J. Am. Chem. Soc. (2000), 122, 10249-10250. 32) D. Bagchi, A. Garg, R.L. Krohn, M. Ba gchi, M.X. Tran, S.J. Stohs, Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res. Comm. Mol. Pathol. Pharmacol. (1997), 95, 179-89. 33) See Chapter IV. 34) V. Leskovac, Comprehensive Enzyme Ki netics, Kluwer/Plenum, Boston, MA (2002). 35) J.R. Florini, C.S. Vestling, Graphical de termination of the dissociation constants for two-substrate enzyme systems. Biochim. Biophys. Acta (1957), 25, 575-578. 36) D.S. Sigman, A. Mazumder, D. Perrin, Chemical nucleases. Chem. Rev (1993), 93, 2295-2316. 37) P.S. Rao, J.M. Luber, J. Milinowicz, P. Lalezari, H.S. Mueller, Specificity of oxygen radical scavengers and assessment of free ra dical scavenger efficiency using luminol enhanced chemiluminescence. Biochem. Biophys. Res. Comm (1988), 150, 39-44. 38) I. Bertini, S.J. Lippard, H. B. Gray, J.S. Valentine, Eds. Bioinorganic Chemistry University Science Books, Saus alito, CA. (1994) Chapters 4 & 5. 39) C. Gerdemann, C. Eicken, B. Krebs, Th e crystal structure of catechol oxidase: New insight into the function of type-3 copper proteins. Acc. Chem. Res. (2002), 35, 183191. 40) N.J. Blackburn, F.C. Rhames, M. Ralle S. Jaron, Major changes in copper coordination accompany reduction of peptid ylglycine monooxygenase: implications

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130 for electron transfer and the catalytic mechanism. J. Biol. Inorg. Chem. (2000), 5, 341-353. 41) L. Que Jr., W.B. Tolman, Bis( -oxo)dimetal "diamond" cores in copper and iron complexes relevant to biocatalysis. Angew. Chem. (2002), 41, 1114-1137. 42) S.Y. Seo, V.K. Sharma, N. Sharma, Mushroom tyrosinase: Recent prospects. J. Agric. Food Chem. (2003), 51, 2837-2853. 43) P. Chen, E.I. Solomon, Oxygen Activati on by the Noncoupled Binuclear Copper Site in Peptidylglycine -Hydroxylating Monooxygenase. Reaction Mechanism and Role of the Noncoupled Nature of the Active Site. J. Am. Chem. Soc. (2004), 15, 49915000. 44) S. Yamazaki, S. Itoh, Kinetic evaluation of phenolase activity of tyrosinase using simplified catalytic reaction system. J. Am. Chem. Soc. (2003), 125, 13034-13035. 45) C.D. Syme, R.C. Nadal, S.E.J. Rigby, J.H. Viles, Copper Binding to the Amyloid(A ) Peptide Associated with Alzheimer's Disease: Folding, coordination Geometry, pH Dependence, Stoichiome try, and Affinity of A -(1-28): Insights from a Range of Complementary Spectroscopic Techniques. J. Biol. Chem. (2004), 279, 18169– 18177. 46) C.S. Atwood, R.C. Scarpa, X. Huang, R.D. Moir, W.D. Jones, D.P. Fairlie, R.E. Tanzi, A.I. Bush, Characterization of c opper interactions with Alzheimer amyloid peptides: identification of an attomola r-affinity copper bind ing site on amyloid 142. J. Neurochem (2000), 75, 1219-33.

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131 47) E.L. Hegg, S.H. Mortimore, C.L. Cheung, J.E. Huyett, D.R. Powell, J.N. Burstyn, Structure-Reactivity Studies in Copper(I I)-Catalyzed Phosphodiester Hydrolysis. Inorg. Chem. (1999), 38, 2961-2968. 48) W. Garzon-Rodriguez, A.K. Yatsimirs ky, C.G. Glabe, Binding of Zn(II), Cu(II), and Fe(II) ions to Alzheimer's A peptide studied by fluorescence. Bioorg. Med. Chem. Lett. (1999), 9, 2243-2248 49) X. Huang, C.S. Atwood, R.D. Moir, M.A. Hartshorn, R.E. Tanzi, A.I. Bush, Trace metal contamination initiates the appare nt auto-aggregation, amyloidosis, and oligomerization of Alzheimer's A peptides. J. Biol. Inorg. Chem. (2004), 9, 954960. 50) T. Kowalik-Jankowska, M. Ruta, K. Wi niewska, L. ankiewicz, Coordination abilities of the 1-16 and 1-28 fragments of -amyloid peptide towards copper(II) ions: a combined potentiom etric and spectroscopic study. J. Inorg. Biochem. (2003), 95, 270–282. 51) A.B.P. Lever, Inorganic Electronic Spectroscopy 2nd ed. Elsevier, (1986). 52) F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, 5th Ed., Wiley, New York, NY (1988). 53) H.I. Park, L.-J. Ming, Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism. J. Biol. Inorg. Chem (2002), 7, 600-610.

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132 54) I. Bertini, C. Luchinat, High spin coba lt(II) as a probe for the investigation of metalloproteins. Adv. Inorg. Biochem. (1984), 6, 71-111. 55) L.-J. Ming, Nuclear Magnetic Resonance of Paramagnetic Metal Centers in Proteins and Synthetic Complexes In Physical Methods in Bioinorganic Chemistry, Spectroscopy and Magnetism Que, L., Jr., Ed.; University Science Books, (2000). 56) A.R. Burger, S. J. Lippard, M.W. Pantoliano, J.S. Valentine, Nuclear magnetic resonance study of the exchangeable his tidine protons in bovine and wheat germ superoxide dismutases. Biochemistry (1980), 19, 4139-4143. 57) T. Miura, K. Suzuki, H. Takeuchi, Bindi ng of iron(III) to the si ngle tyrosine residue of amyloid -peptide probed by Raman spectroscopy. J. Mol. Struct (2001), 598, 79–84. 58) H.I. Park, L.-J. Ming, The mechanistic role of the coordinated tyrosine in astacin. J. Inorg. Biochem. (1998), 72, 57-62.

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133 CHAPTER III. CATECHOL OXIDASE AND PHENOL MONOOXYGNEASE ACTIVITIES OF CuA 1-20 ‡ I. INTRODUCTION Over the past few years an enormous effort has been directed towa rd the investigation of the metal-dependent mechanisms that lead to the neuropathology of Alzheimer’s disease (AD).1 The self-assembled metallo-amyloid (A ) peptide fibrils are the hallmark of this disease2 and have been attributed to FeIII and CuII-centered generation of H2O2 under reducing conditions which has been postulated to be of significant importance in connection with neuropathy in AD.3,4 However, an area of oversight has been the detailed chemical processes associ ated with the neuropathology of AD, besides the general acclaimed “ROS” (r eactive oxygen species including H2O2) assault.5 Hence, ‡ This work has been published: G.F.Z. da Silva, L.-J. Ming, Alzheimer’s Disease Related Copper(II)Amyloid Peptide Exhibits Phenol Monooxygenase and Catechol OxidaseActivities. Angew. Chem. Int. Ed. (2005), 44, 5501-5504.

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134 a better understanding of metal-centered redox chemistry and the mechanism for the generation of ROS and their fa te can provide insight into potential strategies for prevention and treatment of AD. Several examples of redox chemistry in biological systems are known to be associated with dior multi-nuclear “Type-3” Cu oxidases,6 which might be related to the redox activity of CuII-A .1–4 A number of chemical model systems targeting Type-3 copper centers have successfully been demonstr ated to contain highly active isoelectronic copper-dioxygen species (i.e. CuII 21: 1-peroxo, CuII 22: 2-peroxo, and CuIII 2-bis-oxo) responsible for copper-dependent oxidation and hydroxylation reactions.6–9 Despite the extensive modeling studies, peptid e mimics of these enzymes have apparently been excluded from the studies. CuII-A seems to fill the gap since it is a naturaloccurring Cu-peptide complex demonstr ated to exhibit oxygen-associated redox chemistry,1–4 although details about its oxygen binding and activation mechanisms is lacking. I present herein resu lts which bring together two di stinct fields of research, AD and Type-3 copper centers. The results elucidate that the CuII complex (CuA 1-20) of the icosapeptidyl metal-binding domain of A (DAEFR5 HDSGY10 EVHHN15 KLVFF20) exhibits metal-centered redox chemistry consis tent with the mechanisms of the Type-3 copper enzymes phenol monooxygenase (e.g., tyrosinase) and catechol oxidase.

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135 II. EXPERIMENTAL Phenol Monooxygenase assay: Phenol hydroxylation and ox idation and catechol oxidation were performed as previously reported10 with minor changes to fit current studies. Same molar concentrations of phenol or catechol and 3-methyl-2benzothiazolinone hydrazone (MBTH, which serves as an ortho -quinone indicator) were mixed in 100 mM HEPES at pH 7.00 in the presence of 5.0 M (phenol hydroxylation/oxidation) or 0.50 M CuA 1-20 (catechol oxidation). The red-adduct of the ortho -quinone product was monitored at 500 nm ( = 32,500 M–1cm–1; cf. Figure 3.1) Auto-oxidation of phenol and catechol were determined under the same conditions without CuA 1-20. The 1-20 fragment of A was synthesized at the Peptide Center of the University of South Florida. The identity of the peptide has been confirmed with a Bruker MALDI-TOF mass spectrometer. The rates were determined on a Varian CARY50 spectrophotome ter equipped with a temperature controller. The rate law for pre-equilibrium kinetics (Eq. 1), which CuIIA follows, is shown in Eq. 2, where kcat and K’app are the first-order rate constant and virtual dissociation constant, respectively, S is the substrate and vbackground is the autooxidation rate. CuII-A + S A -CuII-S CuII-A + Prod. k1k1 kcat (1) background cat appkCuAS KS[][] '[] (2)

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136 nm 400500600Abs 0.0 0.1 0.2 0.3 0.4 Figure 3.1. The production of o -quinone from phenol in the absence of H2O2 is monitored by the increase in the absorption due to its adduct with MBTH.

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137 The Hill equation can serve as a general model for cooperativity as reflected by the Hill coefficient which deviates from one in the presence of cooperativity (Eq. 3), wherein Kx is the intrinsic dissociation constant. ] CuA [ ] CuA [max x oK V V (3) In case of a bi-substrate cat alysis, such as phenol hydroxyl ation/oxidation and catechol oxidation in the presence of H2O2, both substrates can interact with the metal center independently. The data are fitted to the Ha nes plot (Eq. 4) to yi eld true values of substrate dissociation constants K’ .19 ] [ 1 ] [ ] [ 1 ] [2 2 max max 2 2 0O H K V K S V O H K v Si (4) III. RESULTS AND DISCUSSION Catechol Oxidase activity: The metal-centered redox chemistry of CuA 1-20 was probed using catechol and the much more inert phenol as substrates.10 The oxidation of catechol under aerobic conditions reaches a plat eau at low-mM concentrations (Fig. 3.2). This saturation profile fits nicely to preequilibrium kinetics (Eqs 1 and 2), affording kcat = 0.154 s–1, K’app = 0.35 mM and a significant se cond-order rate constant kcat/K’app= 440 M–1 s–1 ( Fig. 3.2). Since the formation of quinone from catechol is a two-electron oxidative process, the reaction is expected to follow the two-electron dinuclear reaction pathway for catechol oxidase,11 wherein the binding of catech ol to the active-site di-CuII

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138 [Substrate] mM mM/s 0 2e-5 4e-5 6e-5 8e-5 Figure 3.2. Saturation profile for the oxidati on of phenol (solid square), deuterated phenol (square), and catec hol (solid circle) in the absence of H2O2.

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139 center results in the reduction of the center to yield di-CuI with concomitant production of o -quinone. The reduced di-CuI center can bind dioxygen to afford the active peroxobridged di-CuII center, which can further oxidize a subsequently bound substrate. H2O2 can also be generated in this reactio n pathway from the peroxo-bridged di-CuII center in the presence of a reducing agent, such as the substrate itself. This H2O2 production pathway under reduction conditions is consis tent with previous observations in AD studies.3 This catechol oxidase-like mechanism ha s also been observed in kinetic studies in several chemical model systems12 and in polyphenol oxidation by CuA 1-20.13 It is noteworthy that recent density functional th eory (DFT) results reported mixed valence CuII-CuI transition states,14 corroborating with the reducti on pathway for the Cu center. Zn2+ dilution : I show in Chapter II that the CuII:A 1-20 stoichiometry to be in 1:1 ratio for oxidative activity with three N-coordinated imidazole hi stidine rings as metalbinding ligands.13 Since activity is an excellent probe for determining stoichiometry, gradual replacement of CuII in CuA 1-20 with redox-inactive ZnII can serve as a practical method for addressing the nature of the meta llo-active center by virtually “silencing” the active sites through the dilution with ZnII. A linear correlation between the activity and the extent of of CuII in the ZnII dilution should be observed for simple 1:1 metal binding if there is no cooperativity and/or in teractions between the active site CuII from different A strands. Conversely, a sigmoidal activity pr ofile was observed as a function of mole fraction of CuII in A 1-20 toward the oxidation of th e catechol derivative 3,5-ditertbutyl catechol (DTC, kcat = 0.411 s–1 and K’app = 0.781 mM) which can be nicely fitted to the Hill equation (Eq. 3) with a Hill coefficient of 2.40 and r2 = 0.99 (Figure 3.3A, solid

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140 trace). The data clearly cannot be fitted well to a quadratic equation for 1:1 noncooperative binding mode (r2 = 0.91; dashed trace, Figure 3.3 A). These results imply a possible presence of a cooperative dinuclear active CuII center during the catalytic oxidation of catechol by CuA 1-20, consistent with the catalytic cycle of catechol oxidase.11 Effect of H2O2: The presence of the ROS H2O2 (25 mM) significantly enhances the turnover and catalytic efficiency of CuA 1-20 toward catechol oxidation (Figure 3.4), yielding kcat = 0.531 s–1 and K’app = 0.342 mM and a significant second-order rate constant kcat/K’ = 1.51 103 M–1s–1 from the Hanes plot (to give K’ ; Eq. 4, Figure 3.5A)15 for a random bi-substrate reaction, wherein the bindings of the two substrates H2O2 and catechol are independent of each other. It is worth noting that the second-order rate constant for catechol oxidation is only about 20 times lower than that of the catechol oxidase from gypsywort.16 This pathway is consistent with the “peroxide shunt” in the action of catechol oxidase in the presence of H2O2, wherein a CuII 22: 2-peroxo intermediate is proposed to be th e active species (cf. Fig. 2.7 A-D).6 The oxidation of catechol to form o -quinone in the absence and presence of H2O2 exhibits remarkable 3.25 105 and 1.12 106 folds, respectively, rate accelerati on in terms of the first-order rate constant kcat compared to that of aerobic au to-oxidation of catechol without CuA 1-20 determined to be ko = 4.74 10–7 s–1.

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141 nCu/(nCu+nZn) (mM/s) 0 5e-5 1e-4 0.0 2.0e-6 4.0e-6 6.0e-6 A B Figure 3.3. Oxidative activity of CuA 1-20 toward the oxidation of DTC ( A ) and phenol ( B ) in the absence of H2O2 as a function of CuII mole fraction with a constant CuII + ZnII at pH 7.0 and 25 C. The solid traces are fittings to the Hill equation and the dotted traces are fittings to a quadratic binding pattern with metal:ligand = 1:1.

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142 [H2O2] mM 0102030kcat s1 0.0 0.2 0.4 0.6 Figure 3.4. The effect of H2O2 on the first order rate constant kcat toward the oxidation of phenol (solid square ) and catechol (solid circle).

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143 [phenol] mM -[phenol]/rate 4.0e+4 8.0e+4 1.2e+5 [catechol] mM -[catechol]/rate 4.0e+3 8.0e+3 1.2e+4 A B Figure 3.5. Hanes plot analysis of kinetic data from Figure 3.4.

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144 Phenol hydroxylation: Owing to their inertness, metal-centered hydroxylation of phenol and derivatives, particul arly polychlorophenols, poses some challenging tasks in chemical synthesis and ensvironmen tal detoxification and remediation17 and may provide further insight into the action of thos e metalloenzymes for arene monooxygenation.8,18 In addition to catechol oxidation described above, phenol was observed to be hydroxylated and oxidized by CuA 1-20 in the presence of saturating amount of H2O2 (> 50.0 mM), wherein the formation of o -quinone exhibits rate constants of kcat = 0.213 s–1 and K’app = 1.31 mM, and kcat/K’ = 457 M–1s–1 from the Hanes plot (Fig s. 3.1and 3.2). This result represents a remarkable ra te acceleration of 4.6 106 folds for the ydroxylation/oxidation of phenol to form o -quinone in terms of kcat compared to that of aerobic auto-oxidation of phenol (measured to be ko = 4.6 10–8 s–1). This reaction is expected to take place following a similar dinuclear mechanism as in the action of the di-Cu enzyme tyrosinase toward hydroxylation and oxidation of tyrosine, wherein the active center is believed to contain dinuclear 2: 2-peroxo-CuII 2 on the basis of spectroscopic studies.8,14 Cooperativity for the oxidation of bot h catechol and phenol in terms of kcat is observed upon titration of H2O2 as reflected in the sigmoida l activity profile with respect to H2O2 (Figure 3.4). The data from the oxidation of catec hol and phenol by H2O2 fit nicely to the Hill equation (Eq. 3), yielding Hill coefficients = 2.23 and 1.78, respectively. Moreover, fitting of the rate s to a random bi-substrate reaction mechanism yields corrected K’ values and a cooperativit y index based on the ratio K’app/K’ (Fig. 3.5 B), wherein a ratio of 1.70 for H2O2 in both phenol and catechol oxidations and 2.07 and

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145 2.10 for phenol and catechol, respectively, also suggest a small cooperativity and their independent binding to the active center. Of particular interest is th e observation of scarcely reported19 CuII-centered hydroxylation and oxidation of phenol aerobically without H2O2. The production of o quinone from phenol catalyzed by CuA 1-20 follows pre-equilibrium kinetics, yielding kcat = 3.90 10–3 s–1 and K’app = 1.23 mM (Figure 3.2) which represents a first-order rate acceleration of 8.67 104 folds with respect to aerobic auto-oxidation of phenol. The kcat value is lower than that for catechol oxidati on, indicating that the hydroxylation step here must be the rate-limiting step. Otherwise, these two reactions would have similar kcat values attributed to the oxidation of a bound catechol upon hydroxylation of a bound phenol. Moreover, the use of deuterated phe nol as substrate show s significant kinetic isotope effect (KIE), wherein kcat values of 1.12 10–3 and 0.0442 s–1 are obtained in the absence ( Fig. 3.2) and presence of 100 mM H2O2 which represent KIE of 3.46 and 4.77, respectively. The results indicate that hydroxylation and breakage of the o -C–H bond of phenol is the rate-limiting step, whic h is followed by a faster step to form o quinone. The different KIE values for phenol hydroxylation in the presence and absence of H2O2 suggest that the rate-deter mining step in these two case s may be different and/or possibly involve additional pathways. The K’app values are not significantly different between phenol and deuterated phenol (1.31 a nd 1.23 mM for the latte r in the presence and absence of H2O2, respectively), suggesting that kcat does not significantly contribute to the magnitude of K’app and that they may have a similar binding mode. The mechanistic reasoning (Figure 3.6) for the conversion of phenol to o -quinone may be

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146 O H CuII.O O ._ O2 O O H CuIICuI. A C DCuIIOH CuIIO O BCuIICuIICuIIO O O H Figure 3.6. Proposed mechanism for aerobic hydroxyl ation and oxidation of phenol in the absence of H2O2, wherein the binding of phenol and re duction of the meta l center is a key step ( A ). The binding of dioxygen and formation of superoxide ( B ) is proposed to be assisted b y a dinuclear center.

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147 attributed to that the CuII/I redox equilibrium can be achieved upon phenol binding, followed by electron transfer to afford CuI and phenol radical whic h was suggested to be stabilized through resonance structure w ith the free radical situated at the ortho and para positions19a (step A ). This intermediate is then attacked by dioxygen followed by electron transfer to possibly form a CuII-superoxide center which may be further stabilized by a di nuclear center (step B ). The free radical was pr oposed previously to be attached directly by triplet dioxygen,19a which however is not sy mmetrically favorable. Coupling between the bound phenol radica l and superoxide radical at an ortho position can then be expected to be a favorable step (step C ), which is followed by transfer of electrons and an oxygen atom to affo rd the final quinone product (step D ). An involvement of a dinuclear center for the cataly sis is possible as disc ussed below. Since the hydroxylation and oxidation of phenol is a multi-electron transfer process, the involvement of two metal centers is suspected. An activity profile is obtained for phenol oxidation similar to the case of the “ZnII dilution” experiment for catechol oxidation. The data can be fitted equally well to the Hill equation (Eq. 3) to afford value of 1.80 (r2 = 0.98) and a quadratic equati on for single metal-binding (r2 = 0.98), consisting with either a mononuclear oxidation19 or a cooperative mechanism involving a dinuclear center,14 or a combination of both pathways. DCC optical titration: To monitor substrate bind ing, a slow substrate 4,5dichlorocatechol (DCC which is approxima tely 200 times slower in terms of kcat than catechol) was titrated to 0.2 mM CuA 1–20 in the presence or absence of 100 mM H2O2 and the electronic spectrum collected at 25 C and pH 7.0 (Figure 3.4). Similar spectra

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148 wavelength (nm) 400500600700Absorption 0.0 0.1 0.2 0.3 0.4 [DCC] equiv Abs 0.0 0.1 0.2 0.3 Figure 3.7 Optical titration of DCC to 0.2 mM CuA in the presence of 100.0 mM H2O2 in 100 mM HEPES at pH 7.0. Analogous spectra were obtained in the absence of H2O2. The inset shows the change in ab sorbance at 437 nm as a function of equivalents of DCC added, consisting with the formation of a 1:1 adduct.

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149 were obtained, indicating that H2O2 was not involved in DCC binding to CuIIA under the experimental conditions. The absorpti on at 437 nm increases upon addition of DCC and reaches saturation at >1.2 e quivalents of its addition, which can be nicely fitted to single-substrate binding mode to yield a di ssociation constant of 0.24 mM. The result provides a direct evidence for catechol binding to the metal center, consistent with the observation in a chemical model20 and the mechanism proposed above. IV. CONCLUDING REMARKS In conclusion, we have establishe d the catalytic activities of CuA 1-20 toward the relatively inactive (according to their ko values) catechol and phenol in the presence and absence of H2O2 in aqueous solutions near physiological conditions. The reaction patterns are consistent with the mechanisms carried out by Type-3 copper centers as observed in catechol oxidase and tyrosina se and their dinuclear model systems.8,18 These results are unique thus far in metal-center ed redox chemistry related to AlzheimerÂ’s disease, and expected to offer further insight into the neuropathology of this disease since it has been suspected to connect with, in addi tion to many other factors, the oxidation of monoand diphenol-containing neurotransm itters such as dopamine, epinephrine, norepinephrine, and serotonin.21,22 Furthermore, the connect ion of this highly reactive Cu-oxygen chemistry with AlzheimerÂ’s disease can better define the role of metallo-A in the neuropathology of this disease and possi bly lead to different treatment strategies toward this disease.

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150 V. LIST OF REFERENCES 1) A. I. Bush, The metallobiology of Alzheimer's disease Trends Neurosci. (2003), 26, 207-214. 2) B. Tseng, M. Kitazawa, F.M. LaFerla, Amyloid -peptide: The inside story. Curr. Alzheimer Res. (2004), 1, 231-239 3) X. Huang, C. S. Atwood, M. A. Harts horn, G. Multhaup, L. E. Goldstein, R. C. Scarpa, M. P. Cuajungco, D. N. Gray, J. Lim, R. D. Moir, R. E. Tanzi, A. I. Bush, Characterization of copper intera ctions with Alzheimer amyloid peptides: identification of an attomolar-affini ty copper binding site on amyloid 1-42. Biochemistry (1999), 38, 7609-7616. 4) C. Opazo, X. Huang, R.A. Cherny, R.D. Moir, A. E. Roher, A. R. White, R. Cappai, C. L. Masters, R. E. Tanzi, N.C. Inestrosa, A. I. Bush, Metalloenzyme-like activity of Alzheimer's disease -amyloid. J. Biol. Chem (2002), 277, 40302-40308. 5) K. G. Manton, S. Volovik, A. Kulmin ski, ROS effects on neurodegeneration in Alzheimer's disease and related disorders: On environmental stresses of ionizing radiation. Curr. Alzheimer Res (2004), 1, 277-293. 6) T. E. Machonkin, U. M. Sundaram, E. I. Solomon, Multicopper Oxidases and Oxygenases. Chem. Rev. (1996), 96, 2563-2605. 7) W. B. Tolman, L. Que Jr., Bis( -oxo)dimetal "diamond" cores in copper and iron complexes relevant to biocatalysis. Angew. Chem. Int. Ed. (2002), 41, 1114-1137. 8) a)E. A. Lewis, W. B. Tolman, Reactivity of Dioxygen-Copper Systems. Chem. Rev. (2004), 104, 1047-1076; b) W. B. Tolman, Making and Breaking the Dioxygen O-O

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151 Bond: New Insights from Studies of Synthetic Copper Complexes Acc. Chem. Res. (1997), 30 227–237. 9) L. M. Mirica, X. Ottenwaelder, D. P. Stack, Structure and Spectroscopy of CopperDioxygen Complexes. Chem. Rev. (2004), 104, 1013-1045. 10) S. G. Srivatsan, P. Nigamb, M. S. Raob, S. Verma, Phenol oxidation by coppermetallated 9-allyladenine-DVB polymer: reac tion catalysis and polymer recycling. Appl. Catal. A: General (2001), 209, 327–334. 11) A. Rompel, H. Fischer, D. Meiwes, K. Bldt-Karentzopoulos, R. Dillinger, F. Tuczek, H. Witzel, B. Krebs, Structur e and magnetism of novel tetranuclear -4-oxobridged copper(II) complexes. J. Biol. Inorg. Chem. (1999), 4, 56–63. 12) a) C. T. Yang, M Vetrichelvan, X. Yang, B Moubaraki, K. S. Murray, J. J. Vittal, Syntheses, structural properties and cat echolase activity of copper(II) complexes with reduced Schiff base N-(2-hydroxybenzyl)-amino acids. J. Chem. Soc. Dalton Trans (2004), 113–121; b) A. Granata, E. Monzani, L. Casella, Mechanistic insight into the catechol oxidase activity by a biomim etic dinuclear copper complex. J. Biol. Inorg. Chem. (2004), 9, 903-913; c) R .Than, A. A. Feldmann, B. Krebs, Structural and functional studies on model compounds of purple acid phosphatases and catechol oxidases. Coord. Chem. Rev. (1999), 182, 211–241. 13) G. F. Z da Silva, W. T. Tay, L. -J. Ming, Catechol Oxidase-like Oxidation Chemistry of the 1–20 and 1–16 Fragments of Alzheimer's Disease-related -Amyloid Peptide: Their Structure-Activity Correlation and the Fate of Hydrogen Peroxide J. Biol. Chem (2005), 280, 16601-16609.

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152 14) P. E. M. Siegbahn, The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O-O bond cleavage. J. Biol. Inorg. Chem. (2003), 8, 567-576. 15) V. Leskovac, Comprehensive Enzyme Kinetics Kluwer/Plenum, Boston, 2002, p.119. 16) A. Rompel, H. Fischer, D. Meiwes, K. Buldt-Karentzopoulos, A. Magrini, C. Eicken, C. Gerdemann, B. Krebs, Substr ate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation. FEBS Lett. (1999), 445, 103–110. 17) M. Maumy, P. Capdevielle, Coppercatalyzed ortho-oxidation of phenols by dioxygen (tyrosinase mimics) do yields catechols as primary products. J. Mol. Catal., A: Chem. (1996), 113, 159-166. 18) K. D. Karlin, S. Kaderli, A. D. Zuberbhler, Kinetics and Thermodynamics of Copper(I)/Dioxygen Interaction. Acc. Chem. Res. (1997), 30, 139-147. 19) a) K. -Q. Ling, Y. Lee, D. Macikenas, J. D. Protasiewicz, L. M. Sayre, Copper(II)Mediated Autoxidation of tert-Butylresorcinols. J. Org. Chem. (2003), 68, 13581366; b) S. R. Starck, J. -Z. Deng, S. M. H echt, Naturally Occurring Alkylresorcinols That Mediate DNA Damage and Inhibit Its Repair. Biochemistry (2000), 39, 24132419. 20) S. Torelli, C. Belle, S. Hamman, J. P. Pierre, Substrate Binding in Catechol Oxidase Activity: Biomimetic Approach. Inorg. Chem. (2002), 41, 3983-3989. 21) H. Umegaki, N. Tamaya, T. Shinkai, A. Iguchi, The metabolism of plasma glucose and catecholamines in Alzheimer's disease. Gerontology (2000), 35, 1373-1382.

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153 22) W. Fu, H. Luo, S. Parthasarathy, M. P. Mattson, Catecholamines potentiate amyloid -peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis. Neurobiol. Disease (1998), 5, 229-243.

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154 CHAPTER IV. METALLO-ROS IN ALZHEIMER’S DISEASE: METALCENTERED OXIDATION OF NEUROTRANSMITTERS BY Cu2+AMYLOID PROVIDES AN ALTERNATIVE PERSPECTIVE FOR THE NEUROPATHOLOGY OF ALZHEIMER’S DISEASE‡ I. INTRODUCTION The generation of reactive oxygen species (R OS), including superoxide, peroxide, and free radicals, is associated with norma l redox metabolic pathways as side-tracks which can be regulated through the action of superoxide dismutase, catalase, and some reducing agents under homeostasis.1,2 However, long-term effects of such oxidative chemical imbalance in normal and disease states can be expected. ROS are often considered the culprits responsible for the devastating effects of oxidative stress,3 concerning cancer, aging, heart diseases, arthritis, diabetes, and the etiology of neurodegenerative disorders such as Park inson’s disease and Alzheimer’s disease (AD).4 AD affects primarily the elderly whic h causes considerable distress of the patients and emotional sufferings of their fa milies and close friends. One mechanism ‡ This work has been published: G.F.Z. da Silva, Ming L.-J, “Metallo-ROS” in Alzheimer’s Disease : Metal-Centered Oxidation of Neurotransmitters by CuII-Amyloid Provides an Alternative Perspective for the Neuropathology of Alzheimer’s Disease. Angew. Chem. Int. Ed. (2007), 46, in press.

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155 proposed for the neurodegeneration in AD focuses on amyloidpeptides (A ) and their metal complexes through formation of pl aques and fibrils and generation of the ROS H2O2 and free-radicals.5–9 Aggregation of A of 40 or 42 amino acids (DAEFR HDSGY10 EVHHQ KLVFF20 AEDVG SNKGA30 IIGLM VGGVV40 IA) in the brain is the hallmark in AD neuropathology induced by metal binding10–12 and is usually found as metalcontaining plaques and insoluble fibrils. Si milar pathological effects are also found in transgenic mouse models with human A .13,14 Moreover, soluble fragments of A can be generated in vivo by insulin degrading enzyme as well as and -secretases.15,16 Nevertheless, the cause or effect connection of the metallo-A plaques with AD is still under debate.17–19 Despite immense endeavor in A research, the potential risk of metal-centered oxidative cat alyses by metallo-A has been overlooked.20 The CuII complexes of metal-binding domains of A (CuA ) have recently been demonstrated to exhibit metal-centered oxidative catalys is, consistent with Type-3 copper oxidases.21,22 To verify the bio-relevance of th is metal-centered catalysis, we have determined the oxidation of several catech olamine and indoleamine neurotransmitters catalyzed by CuA 1–40 and two metal-binding N-terminal fragments CuA 1–16 and CuA 1–20 under various biomimetic conditions. The studies described herein are expected to provide chemical basis fo r better understanding the etiology of AD. II. EXPERIMENTAL The peptides A 1–16(20) were synthesized at the Peptide Center of the University of South Florida, and confirmed with a Bruker MALDI-TOF mass spectrometer. A 1–40 was purchased from Biopeptide Co., LLC (San Diego, CA). The CuII complex

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156 of A 1–40 was prepared according to literature procedures23 and concentration determined with a standard BCA assay. The CuA stock solutions were quickly aliquoted to prevent concentration devi ations caused by aggregation. Kinetic assays: Kinetic measurements were performed and analyzed as previously described 21,22 by the use of MBTH as a o-quinone indicator ( product = 32,500 M–1 cm– 1 for phenol and catechol24 and 28,900, 27,200, and 27,500 M–1cm–1 for Dopa, dopamine, and epinephrine/norepinephrine, respectively,25 and 35,200 M–1cm–1 for serotonin and 5-hydroxytriptophan). The da ta are analyzed with the bi-substrate Hanes plot for experiment s in the presence of H2O2 to yield the apparent KS and intrinsic KSi dissociation constants for the neurotransmitters and H2O2.37 III. RESULTS AND DISCUSSION Dopamine has been directly linked to the neurodegenerative Parkinson’s disease.26 Moreover, symptoms of Parkinson’s disease have also been recognized in AD patients.27 Thus, disturbance of dopamine me tabolism may be closely associated with AD. Dopamine is effectively oxid ized aerobically to dopaquinone by CuA 1–40 at pH 7.0, reaching saturation at high dopa mine concentrations (Fig. 4.1; Table 1) which is consistent with pre-equilibrium-like kinetics. There is an apparent cooperativity in the catalysis that is not usually expected in simple monomeric systems, which may be due to the tendency of the peptide to coagulate and/or the formation of a dinuclear center during cat alysis. Fitting the results to the Hill equation gives kinetic constants kcat = 7.48 10–4 s–1 and kcat/ K ’ = 2.77 M–1 s–1 and a Hill coefficient = 1.48. This reaction shows 85-fo ld rate enhancement relative to

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157 Figure 4.1 Oxidation of catechol (o) and dopamine ( ) by 1.47 M CuA 1-40 in 100 mM HEPES at pH 7.0 and 25 C. Dashed traces are fittings to simple pre-equilibrium kinetics while the solid traces are the fitting to the Hill equation. The inset shows the effect of H2O2 on kcat which also show the presence of cooperativity.

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158 CuA kcat (10–3 s– 1) K ’ (mM) kcat/ K ’ (M–1s– 1) krel 1–40 0.748 0.27 2.77 85 1–20 11.6 0.90 12.9 1,320 1–16 28.0 0.31 90.3 3,180 1–40b 5.61 0.27 21 312 1–20b 99.0 0.52 190 5,530 1–16b 230 0.68 339 12,700 Table 2 Kinetic parameters for dopamine oxidation by A a in 100 mM HEPES buffer at pH 7.0 and 25 C. b in the presence of 20 mM H2O2

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159 auto-oxidation of dopamine. The shorter CuA 1–20 and CuA 1–16 do not exhibit the apparent cooperativity and exhibit 16 and 37-fold, respectively, higher activity than CuA 1–40 toward dopamine oxidation in terms of kcat. Catechol oxidation shows the same catalytic trend (Fig. 4.1; Table 1), with CuA 1–40 showing cooperativity ( = 2.7) and CuA 1–16 exhibiting the highest activity. Th e results herein indicate that the soluble CuA fragments may be more severe in ca using oxidative stress in the brain of AD patients than th e coagulation-prone CuA 1–40. Recent studies also suggested pathological signi ficance of soluble forms of A .28 Collectively termed catecholamines, dopami ne, epinephrine, norepinephrine, and Dopa are catechol-cont aining neurotransmitters (whereas serotonin and its precursor 5-hydroxytryptophan are indole-containing) which are involved in cognitive, behavioral, physical, physiological and psychological functions.29 Oxidation of these molecules may cause severe alteration in bi oactivity, eventually leading to neuronal death.30 Metabolic malfunctions of neurot ransmitters are known phenomena in the physiology of AD31,32 and have also been suggested to be related to the neuropathology of this disease.31–34 However, a chemical mechanism for the neurotransmitter malfunction in AD is still unknown. These catecholamine neurotransmitters are found to be effectiv ely oxidized aerobically to their respective o -quinone products by CuA 1–20 (Fig. 4.2, Table 1). Herein, CuA 1-20 is shown to significantly accelerate aerobic oxidation ra te of these neurotransmitters by 333–2,420 times in terms of kcat relative to auto-oxidation rate constant ko.

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160 Figure 4.2 a) Catecholamine oxidation by 2.5 M CuA 1-20 in the absence of H2O2 toward dopamine ( ), (+/–)epinephrine ( ) and (–)norepinephrine ( ), L-DOPA ( ) and D-DOPA ( ). The solid lines are the best fi t to an enzyme-like pre-equilibrium kinetics). b) The dependence of th e first-order rate constant on H2O2 toward the oxidation of dopamine ( ), (+/–)epinephrine ( ), and (–) norepinephrine ( ).

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161 Compared to that of catechol, the auto-oxi dation rates of these neurotransmitters are faster by nearly 10 times in terms of ko. However, their oxidation rates by CuA are ~130–980-fold slower than that of catec hol, reflecting their higher resistance to oxidation by CuA by ~103–104-fold relative to catechol oxidation. The relatively higher stability against oxidative damage entitles these molecules better suited for the purpose of neurotransmission. Dopa shows a lower reactivity than other catecholamines, likely due to the inductiv e effect of the carboxyl group on the side chain. Chirality of these neurotransmitter s does not appear to play a role in this oxidation chemistry (Table 2). The ROS H2O2 has been commonly suggested to be a culprit causing the oxidative stress in AD.4,6 However, this ROS alone at 50.0 mM does not significantly affect the oxidations of neurotransmitters (footnote b, Table 2). Conversely, oxidations of these neurotransmitters by CuA in the presence of >15 mM (>0.051%)35 H2O2 under the same conditions exhib it significant rate enhancement ( krel, Table 2; Fig. 4.3), e.g., 312-fold rela tive to auto-oxidation rate constant ko for dopamine oxidation by CuA 1–40 with kcat = 0.0056 s–1 and kcat/ K’ = 21 M–1 s–1. The oxidation of dopamine is dependent on H2O2 and reaches a plateau at H2O2 > 15 mM with = 3.57 (Fig. 1, inset), which indicates that the ROS H2O2 can bind to CuA to afford a "metallo-ROS" in a cooperative ma nner. Oxidation of catechol without the side chain by Cu-A 1–40 exhibits a similar kinetic pattern (Fig. 4.1; Table 2), confirming the cooperative nature of this oxidative catalysis. The results conclude that "metallo-ROS" is much more reactive than free ROS alone as far as H2O2 is concerned. Once again, the shorter CuA 1–16(20) fragments exhibit higher activity than CuA 1–40 toward dopamine oxidation in the presence of

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162 Figure 4.3. H2O2 effect on oxidation of dopamine (a), (+/–)epinephrine (b), and (– )norepinephrine (c) catalyzed by 3.15 M CuA 1-20 in the presence of H2O2 (from bottom, 0, 1.0, 2.0, 4.0, 8.0, 16.0, and 20.0 mM). (d–f) Hanes plot analysis of catecholamine oxidation in the presence of H2O2 (from plots a–c). The lines are the best fits to a bi-substrate mechanism (g–i ) Secondary plots of the Hanes plots from d–f which reveal apparent and in trinsic dissociation constants K ’ values, by plotting of the slope ( ) and y-intercept ( ) from plots d–f as a function of 1/H2O2.

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163 H2O2 (Table 1). This observation once agai n suggests pathological significance of soluble A fragments in AD. Herein, CuA 1-20 is able to significantly accelerate oxidation rates of catechol and catecholamine neurotrans mitters by 1,350 to 2.24 105 times in terms of kcat relative to ko (Table 2). It is worth noting that kcat/ K ’ of 1,690 M–1 s–1 for catechol oxidation by CuA 1–16 approaches enzyme-like catalytic efficiency which is 5.3% of the activ ity of the catechol oxidase (32,000 M–1 s–1) from gypsywort ( Lycopus europaeus ).36 Since both H2O2 and the neurotransmitters can bind to the metal active-center, the data are analyzed with a two-substrate random-binding mechanism according to the Hanes equation (Fig. 4.3)37 to afford the apparent and intrinsic dissociation constants KS and Ki(S) (0.34 and 0.23 for catechol and 0.52 and 0.22 for dopamine, respectively). The ratios of KS/ Ki(S) are greater than one, indicating that the neurotransmitters and H2O2 are affecting the binding of each other in this bi-substrate reaction mechanism.37,38 Influence of micelles: A 1–40 and fragments are found in various cellular environments, including soluble forms in the cytosol and insoluble forms as membrane-bound plaques. Herein, the dete rgent sodium dodecyl sulfate (SDS) is used to approximate the amphiphilic nature of cell membrane. Structures of short A fragments in the presence of SDS differ considerably from that in the absence of this micelle-forming surfactant.39 The activities of CuA s in the presence of micelles may offer insight into the nature of the structure and activity of A The rate constant kcat of CuA 1–20 for the oxidation of dopamine is larg ely affected by the soluble form of SDS (4.5 times) and noticeably influenced (80%) by micelles with the critical micelle concentration (C MC) of SDS

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164 Substrate kcat(10–3 s–1) K’ (mM)a kcat/ K’ (M–1s–1) krel b kcat (10–3 s–1) sat. H2O2 kcat/ K’ (M–1s– 1)c sat. H2O2 k’rel d sat. H2O2 Catechole (by A 1–16) (by A 1–40)f dopamine (by A 1–16) (by A 1–40)f () Epinephrine (–) Epinephrine (–) Norepi. (+) Norepi. (–) Dopa (+) Dopa Phenole (by A 1–16) (by A 1–40)f 5-hydroxy-Trp serotonin (by A 1–16) (by A 1–40)f 154 280 0.87 11.6 28 0.75 3.1 2.9 2.7 2.8 1.1 1.2 3.9 6.4 0.44 6.4 6.7 26 0.28 0.35 0.31 0.19 0.90 0.31 0.27 0.60 0.61 0.52 0.57 0.34 0.32 1.23 0.59 1.25 0.45 1.47 0.63 0.33 440 900 4.6 13 90 2.8 5.2 4.8 5.2 4.9 3.2 3.8 3.2 11 0.35 14 4.5 41 0.84 3.25 105 5.92 105 1,850 1,320 3,180 85 2,420 2,270 1,330 1,380 333 364 8.67 104 1.41 105 9.54 103 8.25 103 7.75 103 3.03 104 324 531 1,150 11.2 99 230 5.61 24 22 17 18 9.11 9.03 213 340 1.43 43.9 30.4 250 7.8 1,510 1,690 28 190 340 11 34 54 32 39 27 28 170 58 1.14 97.5 67.6 380 24.4 2.24 105 7.64 105 1.96 104 5,530 1.27 104 312 9,420 8,630 6,940 7,350 1,360 1,350 4.16 106 6.64 106 2.79 104 5.64 104 3.28 104 2.69 105 8.41 103 Table 3 Kinetic parameters for the oxidation of catecholamines to o -quinone by A 1–20, except those indicated. Intrinsic dissociation constant for the CuA -S complex. b The background self-oxidation rate constant ko is calculated based on the MBTH detection of o quinone formation in the absence of CuA at 25 C in 100.0 mM HEPES pH 7.0 under aerobic conditions.

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165 around 8 mM (Fig. 4.4);xl whereas that of CuA 1–40 is slightly influenced by soluble SDS (2-fold) and greatly aff ected by micellar SDS (8-fold). Under our experimental conditions, SD S micelles do not influence the selfoxidation rates of neurotrans mitters. The shortest CuA 1–16 is only slightly affected (85% enhancement) in the presence of sa turating amount of SDS and shows only 14% enhancement in correspondence with CMC. The results indicate that the plaqueforming CuA 1-40 is expected to exhibit more significant oxidation chemistry when it is “solubilized” and incorporated into a hydrophobic environment; whereas the soluble CuA 1–16 and CuA 1–20 are already more powerful oxidation agents than CuA 1–40 in aqueous environments (Table 1). The results seem to also corroborate the “opposing activities” proposal for neuroprotection via proteolysis and aggregation,xli wherein the highly active small A 1–16(20) fragments are supposed to be eliminated by the former process whereas the activity of A 1–40 is much decreased by the latter process by forming aggregates. The local concentrations of metallo-A plaques can reach mM range,4 which can cause significant oxidative damages of proximal areas on the brain. Consequently the devastating metallo-ROS chemistry due to CuA 1–40 and fragments in the brain of AD patients can have a widespread effect in different cellular environments, particularly in hydrophobic membranes that significantly enhance the metal-centered oxida tive activities as demonstrated herein. Influence by reducing agents: The NAD(P)+/NAD(P)H ratios vary according to changes in metabolism and is species/tissue-dependent,xlii thus are expected to affect the redox property of CuA in vivo. Changes in the homeostatic levels in terms of NAD(P)+/NAD(P)H may reflect the neuroche mical status under oxidative stress.xliv Metabolic changes have been

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166 [SDS]/mM 01020% activation 0 200 400 600 800 Figure 4.4. Effect of SDS on the oxidative activity of CuA 1-16 ( ), CuA 120 (o), and CuA 1-40 ( ) in 100.0 mM HEPES pH 7.0 and 25 C. The data show influence of the activity by bot h the monomeric and the micellar forms of SDS with CMC of 8 mM.

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167 noted to be associated with several age-associated diseases, including neurodegenerative diseases.xliii It has been previously reported that these ratios are managed according to spatial and temporal constraints in the brain under oxidative stress.xliv The oxidation activity of CuA 1-20 is lowered by NAD(P)H. As the ratios of NAD(P)+/NAD(P)H decrease, the activity in terms of kcat toward dopamine oxidation decreases by 2.4 and 1.9 times, respectively (Fig. 4.5). Based on the proposed mechanism for catechol oxidation by Cu-A 1–20,21 the inhibitory effect of NAD(P)H is due to shifting in equilibrium toward H2O2 generation under reducing conditions.21,xlii The more pronounced inhibition caused by NADPH as compared to NADH might be attributed to the phosphate group in NADPH. Indeed, phosphate has been observed to be a competitive inhibitor toward the oxidation of dopamine, showing Ki = 4.7 mM (Fig. 4.6). The ratio of free NAD+/NADH has been under debate which nevertheless ha s recently been suggestedxlv to be around 600, consistent with the value based on potentiometric measurement.xlvi The relatively smaller availability of free form of NADH suggests that this bi ological reducing agent may not significantly influence the metalcentered oxidative catalysis of CuA under physiological conditions. To reveal the mechanism of the full-length A 1–40, the slow substrate 4,5dichlorocatechol (DCC) was titrated into a solution of CuA 1–40. The observation of a charge transfer transition at 438 nm (Fig. 4.7) is indicative of DCC binding to the CuII center (with an affinity constant of 6.4 105 M–1, dotted trace; Fig. 4.6) analogous to its binding to the soluble CuA 1–20.22 Cooperativity is apparent in DCC binding (solid

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168 XNAD(P)+ 0.000 0.004 0.008 Figure 4.5. Influence of NAD(P)+ (o) and NAD+ ( ) on the oxidative activity of CuA 1–20 toward dopamine oxidation with a fixed total concentration of NAD(P)+ + NAD(P)H = 10.0 mM.

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169 Figure 4.6 Phosphate inhibition towa rd catechol oxidation by CuA 1–20 in 100 mM HEPES at pH. 7.0. Phosphate concen trations are 0, 10, 20, and 40 mM (from bottom).

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170 trace; Fig. 4.7), probably due to the coagulation nature of A 1–40. The stoichiometry of DCC:CuA 1–40 = 1:2 verifies the dinuclear nature of the catalysis. The activation profile during CuII titration to A 1–40 is sigmoidal with a Hill coefficient =2.68 (Fig. 4.8) which further support a dinuclear catalys is. Cooperativity is not usually expected in mononuclear catalysis, wherein a linear correlation of the activity with CuII is expected until one equivalent is reached. The oxidation of catecholamine neurotransmitters by CuA 1–20 and the stoichiometry for DCC binding are consistent with the action of dinuclear catechol oxidase.21 The mechanism of this enzyme thus serves as a working model for the metal-centered oxidation of catecholamines by CuA Under aerobic conditions, the catechol moiety binds to a di-CuII center (Fig. 4.9) and is oxidized via 2-electron transfer to afford di-CuI and o -quinone product ( B ). Di-Cu+ then binds O2 to form the metallo-ROS -peroxo-Cu2 center ( C and D ) after 2-electron transfer from di-Cu2+ to O2, which may bind ( E ) and oxidize ( F ) another substrate. In the presence of an electron donor such as NAD(P)H ( H ), the oxidation of neurotra nsmitters is inhibited. Herein, H2O2 is formed to certain extents whic h can produce other types of ROS and may exacerbate the oxidative destructio n by going through the peroxide shunt ( G ) upon forming the highly reactive metallo-ROS -peroxo-di-CuII intermediate. The metal-bound H2O2 as a metallo-ROS in equilibrium of three possible iso-electronic speciesxlvii ( D ) has been demonstrated herein to be a more potent oxidative agent than H2O2 alone to cause neurodegeneration. The full-length plaque-forming CuA 1-40 also shows significant metal-centered oxidative activity toward the relatively mo re inert phenol (Fig. 4.10; Table 2),

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171 Figure 4.7. a) Optical titration of DCC into 0.05 mM CuA 1–40. Inset shows change of intensity at 438 nm upon DCC binding to a di-CuII center. Both were in 100.0 mM HEPES at pH 7.0.

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172 Figure 4.8. Activity titration of CuII into 2.5 micro-M A 1–40 under saturating conditions of catechol.

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173 Figure 4.9. Mechanism for the oxidation of catecholamine neurotransmitters and the cause of neurodegeneration by CuA The metal-bound H2O2 as a metallo-ROS is shown in D

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174 showing kcat = 0.439 and 1.43 s–1 (with a small cooperativity = 1.6) and kcat/ K ’ = 0.351 and 1.14 M–1 s–1, respectively, in the absence and presence of 50.0 mM H2O2. The activity reaches saturation at high H2O2 (Fig. 4.10), reflecting binding of H2O2 to the metal to afford metallo-ROS. Phenol hydroxylation and oxidation exhibits the same trend of reactivity with the shortest CuA 1–16 showing the highest activity (Table 2). Herein, oxidation of phenol is dramatically enhanced by 6.64 106 and 1.41 105 times by CuA 1–16 with and without 50.0 mM H2O2, respectively. Serotonin can be hydroxylated and oxidized by CuA 1–(16,20,40) into its quinone form with significant rate enhancem ents ranging from 324 to 3.03 104 and 8.41 103 to 2.69 105-fold, respectively, relative to ko in the absence and presence of a saturating amount of H2O2 (Fig. 4.11; Table 2). As in the case of catecholamine oxidation, the oxidation of serotonin by CuA 1–40 is slower than that by CuA 1–(16,20). Moreover, the precursor of serotonin 5-hydroxy-Trp can also be effectively hydroxylated and oxidized by CuA 1–20 (Table 2). The apparent and intrinsic dissociation constants KS and Ki(S) for phenol are determined to be 1.23 and 0.54 mM, respectively, from the Hanes plots, indicating H2O2 binding to the metal center decreases the binding of serotonin. Th e reactions may follow the tyrosinase mechanismxlviii for the hydroxylation and oxidation of phenol, serotonin, and 5hydroxy-Trp. However, CuII-A is still highly active without H2O2 in hydroxylation reaction, whereas the di-CuII met-form of tyrosinase is not. The large oxidation enhancements of the two indoleamines by CuA suggest that their oxidation possibly taking place in the brain of AD patients may alter serotonin-me diated physiological functions, including sleep disorder, mood cha nge, and anxiety often associated with AD patients.xlix

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175 Figure 4.10. (a) Aerobic oxidation of serotonin by 1.47 M CuA 1-40 in 100.0 mM HEPES at pH 7.0 and 25 C. Dashed traces are fittings to a pre-equilibrium kinetics while the solid traces are fittings to the Hill equation, showing the presence of cooperativity. (b) Oxidation of serotonin by CuA 1–40 in the presence of H2O2 as in (a).

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176 Figure 4.11. (a) Saturation profile of phenol oxidation by 1.47 M CuA 1-40 in 100.0 mM HEPES at pH 7.0 and 25 C. Dashed line is the fitting to a pre-equilibrium kinetics while the solid line is the fitting to the Hill equation ( kcat = 4.39 10–4, Km = 1.25 mM, kcat/ Km = 0.35 M–1s–1, = 1.60). (b) The effect of H2O2 on the first order rate constant kcat. Cooperativity is not very appa rent in this case, showing = 1.33.

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177 Cell culture experiments reveal that catecholamines can exacerbate the oxidative stress caused by A .l However, the metal-centere d oxidative catalysis has been overlooked.20 Oxidation of catecholamines is known to generate neurotoxic quinone productsli that are involved in pr otein modification (e.g., c ovalent modification of dopamine transporterlii) and polymerizing tau protein into fibrils.liii Age-related deficits of both dopamine and norepine phrine have been implicated in the vulnerability of noradrenergic neurons in the hippocampus,liv which suffers significant damage in AD. Moreover, loss of noradrene rgic neurons is linked to degradation of the locus ceruleus, which is rich in dopami nergic neurons that shows severe lesion in Hence, a possible mechanism for reduc tion in neurotransmitter-regulated alertness response, delay-period activity, sl eep cycle, mood stabilization, short-term memory, cognition, attention and problem solv ing capability, satisf action feeling, and coordination of physical movement experienced by AD patients31–34 may be due to excessive oxidation of neurotransmitters, hi nting at a possible neuropathological role of metallo-ROS associated with CuA IV. CONCLUDING REMARKS In conclusion, the full-length plaque-forming CuA 1–40 found in the brain of AD patients has been demonstrated to exhibit si gnificant activities toward the oxidation of neurotransmitters with or without H2O2 which can be further enhanced by interacting with membranes. The results suggest that imbalance of neurotransmitter metabolism

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178 can be created near A 1–40 plaques in AD. Moreover, the smaller fragments CuA 1– (16,20) show higher activities than CuA 1–40 toward the oxidation of neurotransmitters. This observation suggests that small fragments of A due to their soluble nature, can significantly disturb neurotransmission in a more systematic manner in the brain of AD patients and thus may play an importa nt role in neuropathology of this devastating disease.

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179 IV. LIST OF REFERENCES 1) K. Schuessel, S. Le utner, N.J. Cairns, W.E. Mueller, A. Eckert, Impact of gender on upregulation of antioxidant defense mech anisms in Alzheimer's disease brain. J. Neural Trans. (2004), 111, 1167-1182. 2) J. Apelt, M. Bigl, P. Wunderlich, R. Schliebs, Aging-related increase in oxidative stress correlates with developmental patte rn of beta-secretase activity and betaamyloid plaque formation in transgenic T g2576 mice with Alzheimer-like pathology. Inter. J. Develop. Neurosci. (2004), 22, 475-484. 3) (a) A.J. Nez-Sells, Antioxida nt therapy: Myth or reality? J. Braz. Chem. Soc. (2005), 16, 699-710. (b) M. Valko, M. Iza kovic, M. Mazur, C.J. Rhodes, J. Telser, Role of oxygen radicals in DNA damage and cancer incidence. Mol. Cell. Biochem. (2004), 266, 37-56. (c) A.C. Maritim, R.A. Sanders, J.B. Watkins, nfluence of treatment of diabetic rats wi th combinations of pycnogenol, -carotene, and -lipoic acid on parameters of oxidative stress. J. Biochem. Mol. Toxicol. (2003), 17 24-38. (d) A. Spector, Review: oxida tive stress and disease. J. Ocul. Pharmacol. Ther. (2000), 16, 193-201. 4) (a) A.I. Bush, The metallobi ology of Alzheimer's disease Trends Neurosci (2003), 26, 207-214. (b) D.A. Butterfield, Amyloid -peptide (1-42)-induced oxidative stress and neurotoxi city: implications for neurodegeneration in Alzheimer's disease brain. A review. Free Rad. Res. (2002), 36, 1307-1313.

PAGE 200

180 5) M.J. Del Rio, C. Velez-Pardo, Th e hydrogen peroxide and its importance in Alzheimer's and Parkinson's disease. Curr. Med. Chem. Cent. Nerv. Syst. Agen. (2004), 4, 279-285. 6) C. Opazo, X. Huang, R.A. Cherny, R.D. Moir, A.E. Roher, A.R. White, R. Cappai, C.L. Masters, R.E. Tanzi, N.C. Inestrosa, A.I. Bush, Metalloenzyme-like activity of Alzheimer's disease -amyloid. J.Biol. Chem. (2002), 277, 40302-40308. 7) R. Sultana, S. Newman, H. Mohmmad-Abdul, J.N. Keller, D.A Butterfield, Protective effect of the xanthate, D609, on Alzheimer's amyloid -peptide (1-42)induced oxidative stress in primary neuronal cells. Free Rad. Res. (2004), 38, 449458. 8) H. Engelberg, Pathogenic Factors in Va scular Dementia and Alzheimer's Disease. Demen. Geriat. Cogn. Disord. (2004), 18, 278-298. 9) D.A Butterfield, Amyloid -peptide 1-42-associated free radical-induced oxidative stress and neurodege neration in Alzheimer's dise ase brain: Mechanisms and consequences. Curr. Med. Chem. (2003), 10, 2651-2659. 10) A.I. Bush, W.H. Pettingell, G. Multhaup, M. Paradis, J. Vonsattel, J.F. Gusella, K. Beyreuther, C.L. Masters, R.E. Tanzi, Rapid induction of Alzheimer A amyloid formation by zinc. Science (1994), 265, 1464-1467. 11) T. Miura, K. Suzuki, N. Kohata, H. Takeuchi, Alzheimer's amyloid beta-peptide in insoluble aggregates and soluble complexes. Biochemistry 2000, 39 7024-7031. 12) R.E. Tanzi, L. Bertram, Twenty ye ars of the Alzheimer's disease amyloid hypothesis: a geneti c perspective. Cell (2005), 120, 545-555.

PAGE 201

181 13) I.H. Cheng, J.J. Palop, L.A. Esposito, N. Bien-Ly, F. Yan, L. Mucke, Aggressive amyloidosis in mice expressing human amyl oid peptides with the Arctic mutation. Nat. Med. 2004, 10 1190-1192. 14) K. Jin, V. Galvan, L. Xie, X. M. Ou, O.F. Gorostiza, D.E. Bredesen, D.A. Greenberg, Proc. Natl. Acad. Sci. USA Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw,Ind) mice. (2004), 101, 13363-13367. 15) G. Evin, A. Weidemann, Biogenesis and metabolism of Alzheimer's disease A amyloid peptides. Peptides (2002), 23, 1285-1297. 16) G. Evin, A. Zhu, D. Holsinger, C.L. Masters, Q. Li, Proteolytic processing of the Alzheimer's disease amyloid precur sor protein in brain and platelets. J.Neurosci. Res. (2003), 74, 386-392. 17) A. Kontush, C. Berndt, W. Weber, V. Akopian, S. Arlt, S. Schippling, U. Beisiegel, Amyloidis an antioxidant for lipoproteins in cerebrospinal fluid and plasma. Free Rad. Biol. Med (2001), 30, 119-128. 18) Y. Luo, P. Butko, Amyloidpeptide, a therapeutic target for Alzheimer's disease? Oxid. Stre. Dis. (2006), 22, 395-408. 19) J. Hardy, Has the amyloid cascad e hypothesis for Alzheimer's disease been proved? Curr. Alzhei. Res. (2006), 3, 71-73. 20) (a) D.P. Smith, D.G. Smith, C.C. Curtain, J.F. Boas, J.R. Pilbrow, G.D. Ciccotosto, T.-L. Lau, D.J.Tew, K. Perez, J.D. Wade, A.I. Bush, S.C. Drew, F. Separovic, C.L Masters, R. Cappai, K.J. Barnham, Copper-mediated AmyloidToxicity Is Associated with an Intermolecular Histidine Bridge. J. Biol. Chem. (2006), 281, 15145-15154; (b) F.E. Ali, F.B. Separovic, C.J. Barrow, R.A. Cherny, F. Fraser, A.I. Bush, C.L. Masters, K.J. Barnham Methionine regulates copper/hydrogen

PAGE 202

182 peroxide oxidation products of A J. Peptide Sci. (2005), 11, 353-360. (c) P.I. Moreira, K.Honda, Q. Liu, M.S. Santos, C. R. Oliveira, G. Aliev, A. Nunomura, Z. Akihiko, X. Zhu, M.A. Smith, G.Perry, Oxidative stress mechanisms and potential therapeutics in Alzheimer disease. J. Neur. Trans. (2005), 112, 921-932. 21) G.F.Z. da Silva, W.M. Tay, L.-J. Ming, Catechol Oxidase-like Oxidation Chemistry of the 1–20 and 1–16 Fragment s of Alzheimer's Disease-related -Amyloid Peptide: Their Structure-Activity Correlat ion and the Fate of Hydrogen Peroxide. J. Biol. Chem. (2005), 280, 16601-16609. 22) G.F.Z. da Silva, L.-J. Ming, Alzheimer s Disease Related Copper(II)-Amyloid Peptide Exhibits Phenol Monooxygenase and Catechol Oxidase Activities Angew. Chem. Int. Ed. (2005), 44, 5501-5504. 23) X. Huang, C.S. Atwood, R.D. Moir, M.A. Hartshorn, J.P. Vons attel, R.E. Tanzi, A.I. Bush, Zinc-induced Alzheimer's A 1-40 aggregation is mediated by conformational factors. J. Biol. Chem. (1997), 272, 26464-26470. 24) S. G. Srivatsan, P. Nigamb, M. S. Ra ob, S. Verma, Phenol oxidation by coppermetallated 9-allyladenine-DVB polymer: r eaction catalysis and polymer recycling. Appl. Catal. A (2001), 209, 327–334. 25) J.N. Rodrguez-Lpez, J. Escrib ano, F. Garca-Cnovas, A continuous spectrophotometric method for the determ ination of monophenolase activity of tyrosinase using 3-methyl-2 -benzothiazolinone hydrazone. Anal. Biochem. (1994), 216, 205-212. 26) D.B. Calne, J.W. Langston, Ae tiology of Parkinson's disease. Lancet (1983), 322, 1457-1459.

PAGE 203

183 27) (a) Ditter, S. M.; Mirra, S. S. Neuropath ologic and clinical features of Parkinson's disease in Alzheimer's disease patients. Neurology (1987), 37, 754-760. (b) Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M, Hashimoto M, Mucke L amyloid peptides enhance -synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheime r's disease and Parkinson's disease. Proc. Nat. Acad. Sci. USA (2001), 98, 12245-12250. 28) (a) K. Heinitz, M. Beck, R. Schliebs J.R. Perez-Polo Toxicity mediated by soluble oligomers of -amyloid(1-42) on cholinergic SN56.B5.G4 cells. J. Neurochem. (2006), 98, 1930-1945. (b) R. Kayed, E. Head, J.L. Thompson, T.M. McIntire, S.C. Milton, C.W. Cotman, C.G. Glabe, Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis. Science (2003), 300, 486–489. 29) (a) A.F. Arnsten, B.M. Li, Neurobiol ogy of Executive Functions: Catecholamine Influences on Prefrontal Cortical Functions. Biol. Psych. (2005), 57, 1377-1384. (b) S.F. Leibowitz, G.F. Weiss, G. Shor-P osner, pharmacologica l, biochemical, and behavioral analyses of its feeding-suppressive action. Clin. Neuropharm. (1988), 11, S51-S71. 30) E.B. Larson, W.A. Kukull, R.L. Katzman, R.L. Cognitive impairment: dementia and Alzheimer's disease. Ann. Rev. Pub. Heal. (1992), 13, 431-49. 31) J.B. Friedman, D.N. Adler, K.L. Davis, Diffusion tensor imaging in schizophrenia. Biol. Psychiatry (1999), 46, 1243-1252. 32) H. Umegaki, N. Tamaya, T. Shinkai, A. Iguchi, The metabolism of plasma glucose and catecholamines in Alzheimer's disease. Exp. Gerontol. (2000), 35, 13731382.

PAGE 204

184 33) R.J. Gruen, J. Ehrlich, R. Silva, J. W. Schweitzer, A.J. Friedhoff, Cognitive factors and stress-induced changes in catecholamine biochemistry. Psychia. Res. (2000), 93, 55-61. 34) J. Li, M. Zhu, A.B. Manning-Bog, D.A. Di Monte, A.L. Fink, Dopamine and Ldopa disaggregate amyloid fibrils: implic ations for Parkinson's and Alzheimer's disease. FASEB (2004), 18, 962-964 35) The off-the-shelf H2O2 solution in the U.S. is 3%. 36) A. Rompel, H. Fischer, D. Meiwes, K. Buldt-Karentzopoulos, A. Magrini, C. Eicken, C. Gerdemann, B. Krebs, Substr ate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation. FEBS Lett. (1999), 445, 103–110. 37) V. Leskovac, Comprehensive Enzyme Kinetics Kluwer, Boston, (2002), p.119. 38) J.R. Florini, C.S. Vestling, Graphical determination of the dissociation constants for two-substrate enzyme systems. Biochim. Biophys. Acta (1957), 25, 575-578. 39) C. Morgan, M. Colombres, M.T. Nunez, N.C. Inestrosa, Structure and function of amyloid in Alzheimer's disease. Prog. Neurobiol. (2004), 74, 323-349. xl) (a) G.W. Buchko, A. Rozek, D.W. Hoyt, R.J. Cushley, M.A. Kennedy, The use of sodium dodecyl sulfate to model the a polipoprotein environment. Evidence for peptide-SDS complexes using pulsedfield-gradient NMR spectroscopy. Biochim. Biophys. Acta. (1998), 1392, 101-108. (b) P. C. Griffiths, J. A. Roe, B. L. Bales, A. R. Pitt, A. M. Howe, Paramagnetic Resona nce and Small-Angle Neutron Scattering Studies of Mixed Sodium Dodecyl Su lfate and (Tetrade cylmalono)bis(Nmethylglucamide) Surfactant Micelles. Langmuir (2000), 16 8248-8254.

PAGE 205

185 xli) E. Cohen, J. Bieschke, R. M. Perc iavalle, J. W. Kelly, A. Dillin, Opposing Activities Protect Against Age-Onset Proteotoxicity. Science (2006), 313, 1604-1610. xlii) S.-J. Lin, L. Guarente, Longevity de terminant genes: what is the evidence? What's the importance? Panel discussion. Curr. Opin. Cell Biol. (2003), 15, 241–246. xliii) (a) F. Garcia Soriano, L. Virag, P. Jagtap, E. Szabo, J.G. Mabley, L. Liaudet, A. Marton, D.G. Hoyt, K.G. Murthy, A.L. Salzman, G.J. Southan, C. Szab, Diabetic endothelial dysfunction: the role of poly(ADP-ribose) pol ymerase activation. Nature Med. (2001), 7, 108-113. (b) Q. Zhang, D.W. Piston, R.H. Goodman, Regulation of corepressor function by nuclear NADH. Science (2002), 295, 18951897. xliv) L.K. Klaidman, S.K. Mukrherjee, J. D. Adams Jr., Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide. Biochim. Biophys. Acta (2001), 1525, 136-148. xlv) Q. Zhang, D.W. Piston, R.H. Goodman, Science (2002), 295, 1895-1897. xlvi) H. Sies, Metabolic Compartmentation Academic Press, London, (1982). xlvii) L.M Mirica, X. Ottenwaelder, T.D. P. Stack, Structure and Spectroscopy of Copper-Dioxygen Complexes. Chem. Rev. (2004), 104, 1013-45. xlviii) A. Snchez-Ferrer, J.N. Rodrigu ez-Lpez, F. Garca-Cnovas, F. GarcaCarmona, Tyrosinase: a comprehensiv e review of its mechanism. Biochim. Biophys. Acta (1995), 1247, 1-11. xlix) L. Volicer, M.Z. Wrona, W. Mats on, G. Dryhurst, Neur otoxic oxidative metabolite of serotonin: possible role in Alzheimer's disease. Bioimag. Neurodegen. (2005), 85-93.

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186 l) W. Fu, H. Luo, S. Parthasarathy, M.P. Mattson, Catecholamines potentiate amyloid -peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis. Neurobiol. Dis. (1998), 5, 229-243. li) (a) D.G. Graham, Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol. Pharmacol. (1978), 14, 633-643. (b) D.G. Graham, S.M. Tiffany, W.R Bell Jr. W.F. Gutknecht, Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Mol. Pharmacol. 1978, 14 644-653. (c) T.G. Hastings, J. Neurochem. (1995), 64, 919-924. lii) S.B. Berman, M.J. Zigmond, T.G. Ha stings, Enzymatic oxidation of dopamine: the role of prostaglandin H synthase. J. Neurochem. (1996), 67, 593-600. liii) (a) M.J. LaVoie, T.G. Hastings, Pe roxynitriteand nitrite-i nduced oxidation of dopamine: implications for nitric oxide in dopaminergic cell loss. J. Neurosci. (1999), 19, 1484-1491. (b) I. Santa-Maria, F. Hernandez, M.A. Smith, G. Perry, J. Avila, F.J. Moreno, eurotoxic dopamine quinone facilita tes the assembly of tau into fibrillar polymers. Mol. Cell. Biochem. (2005), 278, 203-212. liv) A.J. Nazarali, G.P. Reynolds, Monoamine neurotransmitters and their metabolites in brain regions in Alzheimer's disease: a postmortem study. Cell. Mol. Neurobiol. (1992), 12, 581-587. lv) M.A. Miller, P.E. Klob, J.B Levernz, E. R. Peskind, M.A. Raskind Preservation of noradrenergic neurons in the locus ceruleus that coexpress galanin mRNA in Alzheimer's disease. J. Neurochem. (1999), 73, 2028-2036.

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1 CHAPTER V. METHIONINE-35 IS NOT A REDUCING AGENT FOR THE METAL-CENTERED OXIDATION CHEMISTRY OF Cu2+-AMYLOID– KINETIC AND EPR STUDIES I. INTRODUCTION Oxidative stress1 has been a key topic of resear ch concerning cancer, aging, heart diseases, arthritis, diabetes, and neurodegene rative disorders such as Parkinson’s and Alzheimer’s diseases2 (AD). Mechanisms proposed for the neurodegeneration in AD brains generally focus on the amyloidpeptide (A ),2 a proteolytic product of 40–42 amino acids of the ubiquitously distributed amyloid precursor protein (APP), and its interaction with redox-active metal ions. The recent “A cascade hypothesis” suggested that A aggregates trigger a complex pa thological cascade which leads to neurodegeneration in AD,3 including generation of H2O2,4,5 free-radical induced oxidation,6–8 and the involvement of Met35 as a reducing agent9 in the redox chemistry of metallo-A A central focus of the neuropatholog y of AD thus has been the effects of

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2 redox-active transition metal ions and reactive oxygen species (ROS), such as superoxide, hydroxyl free radical, and H2O2.2 Although physiological processes responsible for dealing with ROS can be up-re gulated to tackle va riations in oxidative stress,10,11 long-term negative effects of such oxida tive chemical imbalance such as that taking place in the brains of AD patients can be expected. Some AD treatment strategies have targeted the metal center in A to prevent peptide aggregation and ROS generation.12–14 However, comparatively little e ffort has been focused on the metalcentered chemistry associated with th e bound metal ions, such as the detailed coordination chemistry and the reac tivity of the metal–bound ROS in CuA Transgenic mouse models with human A show similar effects as AD patients, including A aggregation and loss of memory.15,16 Rodent A has been shown to exhibit redox activity in vitro that was attributed to ROS generation via Met35,17 even though the metal-binding domain was mutated in rodent A (i.e., His13 Arg). Since A activity and aggregation in AD brains is se quence-specific and metal-dependent,2 it is a priority to establish the targets of re dox activity that can contribut e to the physiological and cognitive effects of AD. I have shown in Chapters II-IV that the Cu2+ complexes of A and its soluble fragments (A 1-16 and A 1-20) showed considerable activities toward the oxidation of phenol, polyphenol, catechol, and neurotransmitters to form o -quinones which challenges the redox role of Met35 that is not present in the fragments.18,19,20 The capability of A to bind copper21 with subsequent H2O2 generation under reduction conditions5 and catechol oxidation18–20 hint at the possible fo rmation of a reactive 2: 2-peroxo-di-Cu2+ species (or its isoelectr onic counterparts, oxy-di-Cu+, 1: 1-

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3 peroxo-di-Cu2+, and bis--oxo-di-Cu3+)22 which is the active species in action of the type3 dinuclear copper oxidases23 such as catechol oxidase. In this chapter I present kinetic and sp ectroscopic investigations of the oxidation chemistry of CuA 1-20 and the influence of methioni ne and reducing agents on the oxidation chemistry. The results support meta l-centered oxidative stress and shed light on the mechanistic role of Met35 and reduc ing agents in the redox chemistry of CuA II. EXPERIMENTAL The 1-20 fragment of A was synthesized by the use of the Fmoc chemistry at the Peptide Center of the University of South Florida, and the identity of the peptide confirmed with a Bruker MALD I-TOF mass spectrometer. Dopamine, ascorbic acid, and glutathione (GSH) were obtai ned from Sigma-Aldrich (St. Louis, MO), 3-methyl-2benzothiazolinone hydrazone hydrochlorid e monohydrate (MBTH) from Acros (Fairlawn, NJ), and H2O2, EDTA, L-methionine, and Cu(NO3)2 from Fisher Scientific (Swanee, GA). All plastic ware and glassware we re demetallized with EDTA and extensively rinsed with 18.0-M deionized water. The catechol oxidase assay toward dopa mine was performed as previously reported.20,24 Same equivalent of a substrate dopamine and the o -quinone indicator MBTH were mixed in 100 mM HEPES at pH 7.00 in a final volume of 1.0 mL. The MBTH-adduct of o -quinone was monitored at 505 nm ( = 27,200 M–1 cm–1) and 25 C for 5–10 minutes with a Varian CARY50 Bi o-UV-Vis spectrophotometer equipped with

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4 a CARY PCB-150 Water Peltier temperature regulation system, and the rates determined by the change in absorbance over time. Rates were fitted to appropriate rate laws and rate constants determined with non-linear regression (SigmaPlot 8.0), such as the enzymatic Michaelis-Menten-like kinetics.25 The effects by H2O2, ascorbate, GSH, and L-Met were determined similarly in th e presence of different amounts of the corresponding reagent and the inhibition/disso ciation constants determined accordingly. Binding of L-Met to CuA 1–20 was performed by direct titration of Met into CuA 1-20 in 100 mM HEPES at pH 7.0 and monitored w ith the CARY50 spect rophotometer. Electron paramagnetic resonance (EPR) e xperiments were performed on a Bruker Elexsys E580 cw/pulsed X-band spectrometer at the University of Florida with professor Alexander Angerhofer. For a typical cw EPR spectrum, the field was set wide enough to reveal a possible low-field tr ansition for magnetically coupled systems with a microwave frequency of 9.4 GHz, field modulation typica lly around 2 G, and time constant of 40-80 ms at ~5–6 K. The g and A tensors were obtained with numerical fittings using the “easyspin” toolbox for Matlab.26 Electron relaxation times were measured with the standard two-pulse Hahn’s sp in-echo method or the inversio n-recovery methods, wherein the signal intensity as a function of time is fitted to simple or double exponential decay to obtain the relaxation time. ESEEM (electr on spin echo envelope modulation) spectra were recorded with the usual /2/2-T/2 pulse sequence with a /2 pulse of 20 ns in order to study 14N and 2H nuclei in the ligand sphere. A t ypical ESEEM trace consisted of 1024 points taken at time intervals of 8 ns. Th e transient was first base-line corrected by subtracting the ordinary T1 exponential decay function and any remaining constant

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5 baseline offset, then zer o-appended to 2048 points. A Hamming window function was then applied to the time-domain spin-echo envelope, followed by Fourier transformation to afford the frequency-domain spectrum. An ESEEM spectrum can reveal those nucle i having super-hyperfine coupling with the Cu2+ center, including c oordinated His side chains and wa ter, which can thus serve as a very useful tool for th e investigation of the Cu2+ center in CuA The theoretical background27 of ESEEM is summarized herein. In the case of coordinated His side chains, the super-hyperfine coupling arises from the electron-nuclear interactions and the nuclear quadrupole interactions (NQI) of the remote non-coordinated nitrogen (14N, I = 1) on the imidazole ring of coordinated His side chains. At X-band, three zero-field nuclear quadrupole resonance lines and o are observed which can be determined from the NQI lines in the ESEEM spectrum (Eq. 1 and 2), wherein e2qQ is the quadrupole coupling constant and the asymmetry parameter ( = 0 for a complete axial symmetry and 1 for a pure rhombic symmetry). = 1/4(e2qQ)(3 ) (1) o = 1/2(e2qQ) (2) The e2qQ and values can thus be obtained from the NQI lines in the ESEEM spectra since ( + – o/2)/3 = 1/4(e2qQ). In the case of coordina ted water, the super-hyperfine coupling arises from the electron-nuclear in teractions and the NQI of the coordinated water molecules upon deuteration. The deuteriu m Zeeman interaction at X-band is much

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6 larger than the isotropic component of the electron-nuclear coupling. Thus, the deuterium peak in the ESEEM spectrum is f ound at the deuterium Zeeman frequency and split slightly by the el ectron-nuclear coupling. III. RESULTS Dopamine is oxidized by CuA 1–20 in the absence of H2O2 with rate constant of kcat = 0.0104 s–1 and Km = 0.89 mM (trace Fig. 5.1), consistent with the observations discussed in Chapter IV.20 Addition of L-Met to the reaction solution significantly increases the activity (Fig. 5.2). The reaction in the presence of saturating amount of Met seems to induce slight cooperativity which can be fitted to the Hill equation with a Hill’s coefficient = 2.3 (Solid trace Figure 5.1). The ac tivity reaches a plateau at high [Met] (Fig. 5.1), indicating dire ct Met binding to the active center of CuA Fitting of the kcat values as a function of [Met] to a simple equilibrium of [CuA + Met Met-CuA ] gives an affinity constant of 1,900 M ( Kd = 0.53 mM). Met fo llows a non-essential activation pattern toward dopamine oxidation by CuA 1–20 as shown in the LineweaverBurk plot (Fig. 5.1), i.e., both CuA -substrate and Met-CuA -substrate complexes are active. From the data, the dissoci ation constant for the activation KA can be obtained to be 0.087 mM, and the dissociation constant for the ternary [CuA 1-20-Met-dopamine] complex KTS is 0.125 mM. In the presence of H2O2, the activity is further

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7 [L-Dopamine] mM rate mM/s 0.0 1.0e-4 2.0e-4 Figure 5.1. Saturation kinetics curves of dopamine oxidation by CuA fitted with Michaelis-Menten kinetics (dotted traces) and with Hill equation (solid traces).

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8 [L-methionine] mM 024681012 kcat s-1 2e-2 4e-2 6e-2 8e-2 1e-1 Figure 5.2. Effect of L-Met on the the first order rate constant toward dopamine oxidation.

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9 1/[Dopamine] mM -10123451/rate 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 Figure 5.3. Lineweaver-Burk plot of showing the non-essential activation pattern for L-Met toward dopamine oxidation by CuA

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10 enhanced. Herein, both Met and H2O2 can activate dopamine oxidation which are not exclusive of each other and show a combined effect, i.e., the kcat value of 0.180 s–1 in the presence of saturating amount of Met and H2O2 (Fig. 5.4) is a combination of those in the saturating amount of Met (0.088 s–1 for Met activation) and H2O2 (0.099 s–1 for H2O2 activation), indicating their i ndependent activation mechanisms As opposed to Met, the reducing agents ascorbic acid and GSH act as inhibitors toward dopamine oxidation by CuA (Fig. 5.5), with the former being a competitive inhibitor ( Ki = 66 M) while GSH a non-competitive inhibitor ( Ki = 53 M). The electronic spectrum of CuA exhibits an intense charge -transfer transition at 316 nm upon addition of Met (Fig. 5.6), indicat ive of direct Met binding to the Cu2+ center. Such charge-transfer trans ition is not observed in CuA 1–40, suggesting that Met35 does not interact with the Cu2+ center. The d-d transition of CuA 1-20 at 600 nm is not significantly affected in the pr esence of saturating amounts of Met (Fig. 5.6; inset). The change in the intensity of the charge transfer transition as a function of [Met] can be fitted to the 1:1 binding pattern of [CuA + Met Met-CuA ] to yield a dissociation constant Kd = 0.25 mM. This value is comparable to that obtained from the activity profile discussed above, indicating that direct Met binding to the Cu2+ center affords the enhancement of CuA activity. The coordination of CuA and its binding with Met have been investigated with EPR spectroscopy. The EPR spectrum of CuA 1–20 (Figure 5.7) can be at tributed to a typical tetragonally distorted Cu2+ center, i.e., an elongation along the z axis due to the Jahn

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11 [dopamine] mM 0246810 rate mM/s 2.5e-4 5.0e-4 7.5e-4 1.0e-3 Figure 5.4. Rate of dopamine oxidation by CuA 1-20 in the presence of saturating amounts of L-Met and H2O2.

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12 1/[dopamine] mM1 0241/rate (mM/s)1 2e+5 4e+5 6e+5 1/[dopamine] mM1 024 1e+6 2e+6 A B Figure 5.5. (A) Glutathione (10.0 M, 20.0 M, and 40.0 M from bottom) inhibition and (B) ascorb ic acid inhibition (0.0, 0.12, 0.55, and 0.95 mM from bottom) toward dopamine oxidation by Cu-A 1–20 in 100.0 mM HEPES pH 7.0.

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13 wavelength nm 300400500600700800 M1cm1 0 500 1000 1500 2000 wavelength nm 400600800 0 30 60 90 [Met] equiv 012 0 500 1000 1500 Figure 5.6. Optical titration of L-me thionine to 0.2 mM CuA The insets show the low energy d-d transition and the saturation curve fitted to 1:1 ligand:metal stoichiometry.

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14 Teller effect, which can be slightly better fitted to a rhombic than an axial magnetic gz = 2.266, gx = 2.057, and gy = 2.080 and Az = 547, Ax = 30.6, and Ay = 51.1 MHz (Fig. 5.7) or g// = 2.268, g = 2.064, A// = 547, and A = 51.1 MHz. These values are consistent with those reported for several Cu2+ complexes of A and fragments,28 i.e., g// = 2.265– 2.269, g = 2.059–2.062, and A// = 465–577 MHz (from 166-206 G). Upon addition of Met, the EPR spectrum changes only slightly to gz = 2.257, gx = 2.055, and gy = 2.072 and Az = 564.9, Ax = 35.8, and Ay = 46.0 MHz (Fig. 4A) or g// = 2.257, g = 2.059, A// = 565, and A = 35.8 MHz, as opposed to the dramatic change in the el ectronic spectrum. The Cu2+ center in CuA 1–20 and its interaction with Met are further investigated with pulsed EPR. The X-band ESEEM spectrum of CuA 1–20 (Fig. 5.8) reveal three 14N NQI lines at o = 0.33, – = 1.11 (shoulder), and + = 1.45 MHz (Eqs. 1 and 2), the doublequantum transitions at ~4 Mz, and the combination lines at 2.33, 2.95, and 3.45 MHz. From which, e2qQ is obtained to be 1.71 MHz, a va lue typical of a coordinated His,27 and calculated to be 0.39 (Eqs. 1 and 2). The – line (a shoulder) can be better determined once the values of e2qQ and are determined from o and +. At least one coordinated water is also revealed which attributes to the deuterium modulation at 2.29 MHz when the spectrum was acquired from a sample in D2O buffer excited at 3391 G (dashed trace, Fig. 5.8), consistent with 2H resonance of 2.21 MHz at this field. This small discrepancy may be attributed to the presence of a small super-hyperfine coupling. An ESEEM spectrum for Met-bound CuA in D2O buffer was acquired under the same conditions (dotted trace, Fig. 5.8) to investigate the status of the coordinated water upon

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15 Magnetic Field (G) 280032003600 Figure 5.7. X-band EPR spectra of CuA 1-20 in the presence and absence of L-Met and simu lated spectra with rhombic g tensors (dashed traces).

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16 Frequency (MHz) 02468 Figure 5.8. ESEEM spectra of 1.0 mM CuA 1–20 in 100.0 mM HEPES buffer in H2O (solid trace) and in D2O (dashed trace) at pH(D) 7.0 and after addition of 8.0 equivalents Met (dash-dot trace).

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17 Met binding. The 14N NQI lines change only sligh tly upon Met binding, found at o = 0.15, – = 1.22 (shoulder), and + = 1.37 MHz which afford e2qQ = 1.73 MHz and a small = 0.17. Upon Met binding, the deuteriu m line is observed at 2.19 MHz when excited at 3351 G, right at the 2H resonance frequency at th is field which reflects a negligible superhyperfine coupling. IV. DISCUSSION The oxidation of catechol and phe nol and their derivatives by CuA in the presence and absence of H2O2 was demonstrated to be consistent with the mechanism of type-3 copper oxidases such as di-C u catechol oxidase (Fig. 5.9).19 Therein, the catecholcontaining substrate like dopamine binds to a di-Cu2+ active center (step i ) under aerobic conditions and is oxidized via 2-elec tron transfer to afford a di-Cu+ active center ( C ) and o -quinone product (step ii ). The reduced di-Cu+ can bind O2 (step iii ) to form a peroxo-Cu2+ 2 center ( D ) as demonstrated in both enzy me and chemical model systems, which may bind a substrate and followed by oxidation of the substrate (steps iv and v ). The presence of a reducing agent such as ascorbic acid (or Met35 as previously suggested9) can thus facilitate the aerobic pathway to yield the di-Cu+ active center ( C ) ready for O2 binding. In the meantime, a reducing agent can also result in H2O2 production through the reduction of the -peroxo-Cu2+ 2 center (step vi ). An alternative “short-cut” route, also known as the “p eroxide shunt” pathway in heme-containing peroxidase,29 for the oxidation to take pl ace is to form the -peroxo-Cu2+ 2 intermediate

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18 Cu2+Cu2+ X O H Cu2+Cu2+ O O R X O H O O R Cu+Cu+ X Cu2+Cu2+ O O R X O O O H H2O H2O2 H2O2 2H+2H+S 2H+S O2, H+3H+_ Cu2+Cu2+ X O O 2e ,2H+_ R O O 2+HOiii iii iv v vi vii viiiA B C D E Figure 5.9. Proposed mechanism for the catec hol oxidase-like activity of CuA toward the oxidation of dopamine. X i ndicates an endogenous ligand or a bound Met.

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19 by direct binding of H2O2 to the di-Cu2+ center (step vii ), followed by substrate binding (step iv ) to form the -peroxo-Cu2+ 2-substrate ternary complex ( E ) which then undergoes oxidation. The ternary complex E can also be formed upon H2O2 binding to the substrate-bound intermediate ( B ; step viii ), representing a random bi-substrate mechanism. Met35 in A has been suggested to be a reducing agent9 responsible for the initiation of the redox cycling of the Cu2+ center in CuA and leads to H2O2 production (i.e., step vi ).5 The oxidation of the thioether moiety of Met to its sulfoxide form in A has been implicated in aggregation, lipid peroxidation, and redox chemistry in association with the metal center.9 The activity profile of [Met] (Fig. 5.1) and the optical spectrum (Fig. 5.6) indicate direct Met binding to CuA rather than outer-sphere interaction, which potentially can reduce the Cu2+ center as proposed previously Herein, the amount of diCu+ ( C ) increases which in turn fo rms a larger amount of the -peroxo-Cu2+ 2 center upon O2 binding ( D ; step iii ), but fewer amount of di-Cu2+ ( A ) would be present for substrate binding as it is reduced by Met. However, once CuA ( A) is reduced, it cannot follow the peroxide shunt pathway anymore. The observation that H2O2 can still significantly enhance the activity in the presence of a satu rating amount of Met (F ig. 5.2) suggests the Cu2+ center is not significantly reduced by Met, which is de monstrated by the detection of the S = 1/2 EPR features (Fig. 5.7). Mo reover, the difference in electrode potentials between Cu2+-A and Met is 0.71 V,30 which gives a dramatic 68.5 kJ Gibbs free energy that is equivalent to a negligibly small equilibrium constant of 9.8 10–13 for oneelectron reduction of Cu2+-A by Met at 298 K.

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20 The non-exclusive nature of Met and H2O2 binding and their additive activations indicate that there ar e two pathways for the oxidation of dopamine in the presence of Met and H2O2, presumably the pathways i–v and vii–iv–v with a bound Met (the “X” ligand in Fig. 5.9), wherein the enhancemen t of the activity with Met al one is supposed to be due to a non-redox mechanism that fine-tune s the reduction potential of the Cu2+ to favor the oxidative catalysis. The sigmoidal activit y profiles for dopamine oxidation in the presence of Met (Fig. 5.1) and fr om the concerted action of both H2O2 and Met (Fig. 5.4) reflect possible presence of c ooperativity, such as the form ation of the dinuclear active center D Despite the lack of a Met and a ny redox-active amino acid, the fragments CuA 1–16 and CuA 1-20 exhibit significant metalcentered oxidative activity18–20 which indicates the redox role of Met35 mi ght have been overstretched. The oxidation state of the Cu center in CuA 1–20 upon Met binding can be concluded from the electronic and EPR spectra, wher ein the remaining d-d transition at 600-nm (Fig. 5.6) and EPR features (Fig. 5.7) indicate that Cu2+ is not reduced by Met as opposed to previous suggestions for the reductive role of Met35 in CuA 1–40.9 Moreover, the charge transfer transition is consistent with a thio-to-Cu2+ charge-transfer transition observed in Cu2+-methionine complexes.31 The well resolved EPR spectral features at g~2 (Fig. 5.7) upon Met binding confirm the d9 electron configuration of the Cu2+ center with S = 1/2. The lack of MS = 2 transition (i.e., between the MS = –1 and +1 levels in an antiferromagnetically coupled di-Cu2+ center) in the EPR spectrum at low field indicates magnetic coupling between the Cu2+ centers may not exist. Thus, the previously proposed His-bridged dinuclear Cu,Zn-superoxide dismutase center for

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21 CuA 1–28 32 is not present in the case herein. The small change in the EPR spectrum upon Met binding indicates the charge transfer tr ansition must be due to the binding of the thioether of Met through the non-magnetic dz2 orbital at an axial position of CuA 1–20. Otherwise, more significant changes in th e g and A tensors would be observed as reported for the binding of thio-g roups to Type-2 Cu proteins.33 The ESEEM spectrum of CuA 1–20 (Fig. 5.8) confirms the binding of Cu2+ to A through His side chains via the magnetic dx2–y2 orbital in equatorial positions which gives rise to the quadrupole coupling with the remote non-coordinated 14N on the coordinated His imidazole ring. There are at least two coordinated His side as reflected by the combination lines since a single coordinate d His does not give rise to these lines.33g,34 The small values indicate that the Cu2+ center is only slight rhombic, which has already been observed in the CW EPR spectra (Fig. 5.7). The detection of a deuterium line at 2.29 MHz suggests the presence of coordinated water (as D2O), presumably in an equatorial position via the magnetic dx2–y2 orbital.27 This signal is not much affected upon Met binding, once again suggesting Met binding to an axial position. It is interesting to note that the double-frequency peak at 4.6 MHz vanishes upon Met binding. This indicates that at least one of the weakly coupled deuterium atoms in the vicinity of the Cu2+ is replaced by the Met ligand. Since the binding of Met to CuA does not reduce the Cu2+ center, the enhancement in activity must be attributed to a ch ange in the reduction potential of the Cu2+ center. The axially coordinated Met liga nd in blue copper proteins ha s been suggested to play a role in controlling the reduction potential of th e protein since mutation of this Met results

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22 in a significant change in the potential.35 Based on the results herein, I propose the binding of methionine to Cu2+ in CuA may modulate the Cu2+/Cu+ potential to favor the redox catalysis, yet is not necessarily invol ved directly in the redox chemistry as a reducing agent as previously proposed.9 The oxidation of Met35 previously observed may follow the metal-centered mechanism as a catechol or phenol substrate via the reactive di-Cu2+-peroxo intermediate to resu lt in oxygenation reaction, ie., the coordinated Met “X” serves as a substrate via step iv and v in the mechanism without the catechol substrate (Fig. 5.9). The catalytic pathway of CuA is altered under reduc tion conditions (Fig. 5.9), wherein the reaction is locked into a H2O2-producing cycle ( iii and vi ). The competitive inhibition of ascorbate toward the oxidation of dopamine by CuA (Fig. 5.5A) may be because of possible chela tion and reduction of the Cu2+ center by ascorbate. GSH may bind to and reduce the Cu2+ center as a monodentate li gand which does not prevent substrate from binding to the Cu2+ center to form the inactive inhibitor-CuA -substrate complex, thus exhibiting non-comp etitive pattern (Fig. 5.5B). Th e inhibitory effect of the reducing agents toward metal-centered catal ysis is consistent with the proposed mechanism wherein H2O2 is generated (step vi ), also report ed previously.5 Thus, the ability of CuA to oxidize biologically relevant mo lecules such as dopamine is highly dependent on the redox state of its envi ronment. Although reducing agents can potentially inhibit the oxidation of neurotransmitters by CuA the production of H2O2 may still exacerbate the situation of oxidative stress.

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23 Metabolic malfunctions of catecholamines ne urotransmitters have been suggested to be related to the neuropathology of AD.36 Of these neurotransmitters, dopamine has been directly linked to the neurode generative Parkin sonÂ’s disease37 and has been proposed to be associated with adult neurogene sis in the subventricular zone.38 I discuss in chapter IV that CuA can catalyze oxidation of catecholamine neurotransmitters such as dopamine20 to generate neurotox ic quinone products.39 Dopamine quinone can result in polymerization of tau protein into fibrils40 and covalent modification of dopamine transporter which directly affects dopamine uptake.41 A possible mechanism for reduction in the delay-period activity and s hort-term memory, lack of attention, and change in mood and motivation experience d by AD patients may probably be partially due to long-term oxidation of dopamine by CuA Hence, the acceleration of dopamine oxidation via metal-centered mechanism that can be modulated by small molecules such as reducing agents, H2O2, and Met may well hint at th e significant role of CuA in oxidative stress in the brain of AD patients. V. CONCLUDING REMARKS Taken together, the results herein presen t more structural information about the metal center and further support for metaloxygen/peroxo-centered redox chemistry of CuA and provide additional mechanism for th e oxidative stress in the brain of AD patients. The role of Met35 has been rede fined and the dual mech anistic character of reducing agents (i.e., inhibition and H2O2 generation) in the redox cycle of CuA is

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24 clearly defined. The overall picture of AD neurop athology is likely to be composed of the pieces of information uncovered thus far, including generation of ROS, metaldependent aggregation of A and the largely overlooked me tal-centered degradation of biomolecules. Treatment and prevention stra tegies hence must address all of these pathways, including inhibitions toward H2O2 production and oxidative damage of neurotransmitters and other biomolecu les by the di-copper-peroxo active center.

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25 VI. LIST OF REFERENCES 1) (a) A.J. Nez-Sells, Antioxi dant therapy: Myth or reality? J. Braz. Chem. Soc. (2005), 16, 699-710. (b) M. Valko, M. Izakovic, M. Mazur, C.J. Rhodes, J. Telser, Role of oxygen radicals in DNA damage and cancer incidence Mol. Cell. Biochem. (2004), 266, 37-56. (c) A.C. Maritim, R. A. Sanders, J.B. Watkins, Diabetes, oxidative stress, and antioxidants: A review J. Biochem. Mol. Toxicol. (2003), 17, 2438. (d) L. Kruidenier, H.W. Verspaget, Review article: oxi dative stress as a pathogenic factor in inflammatory bow el disease—radicals or ridiculous? Alim. Pharmacol. Therap. (2002), 16, 1997-2015. (e) A. Spect or, Review: Oxidative stress and disease J. Ocul. Pharmacol. Ther. (2000), 16, 193-201. 2) (a) A.I. Bush, The Metallobiology of Alzheimer's Disease. Trends Neurosci. (2003), 26, 207-214. (b) D.A. Butterfield, Amyloid beta-peptide (1-42 )-induced oxidative stress and neurotoxicity: Im plications for neurodegeneration in Alzheimer's disease brain: A review. Free Rad. Res. (2002), 36, 1307-1313. 3) T.E. Golde, D. Dickson, M. Hu tton, Filling the gaps in the A cascade hypothesis of Alzheimer’s disease. Curr. Alzhei. Dise. (2006), 3, 421-430. 4) M.J. Del Rio, C. Velez-Pardo, The hydrogen peroxide and its importance in Alzheimer's and Parkinson's disease Curr. Med. Chem. (2004), 4, 279-285. 5) C. Opazo, X. Huang, R.A. Cherny, R.D. Moir, A.E. Roher, A.R. White, R. Cappai, C.L. Masters, R.E. Tanzi, N.C. Inestrosa, A.I. Bush, Metalloenzyme-like activity of Alzheimer's disease -amyloid. J. Biol. Chem. (2002), 277, 40302-40308.

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26 6) R. Sultana, S. Newman, H. Mohmmad-Abdul J.N Keller, D.A. Butterfield, Protective effect of the xanthate, D609, on Alzheimer's amyloid -peptide (1-42)-induced oxidative stress in primary neuronal cells. Free Rad. Res. (2004), 38, 449-458 7) H. Engelberg, Pathogenic Factors in Vasc ular Dementia and Alzheimer's Disease. Demen. Geriat. Cogn. Disord. (2004), 18, 278-298. 8) D.A. Butterfield, Amyloid -peptide [1-42]-associated fr ee radical-indu ced oxidative stress and neurodegeneration in Alzhei mer's disease brain: Mechanisms and consequences. Cur. Med. Chem. (2003), 10, 2651-2659. 9) (a) G.D. Ciccotosto, K.J. Barnham, R.A. Cherny, C.L. Masters, A.I. Bush, C.C. Curtain, R. Cappai, D. Tew, Methionine oxidation: Implications for the mechanism of toxicity of the -amyloid peptide from Alzheimer's disease. Lett. Pept. Sci. (2003), 10, 413-417. (b) M.E. Clementi, G.E. Martorana, M. Pezzotti, B. Giardina, F. Misiti, Methionine 35 oxidation reduces toxic effects of the amyloid -protein fragment (3135) on human red blood cell. Int. J. Biochem. Cell Biol. (2004), 36, 2066-2076. (c) C. Sch neich, D. Pogocki, G.L. Hug, K. Bobr owski, Free Radical Reactions of Methionine in Peptides: Mechanisms Relevant to -Amyloid Oxidation and Alzheimer's Disease. J. Am. Chem. Soc. (2003), 125, 13700-13713. (d) F.E. Ali F. Separovic, C.J. Barrow, R.A. Cherny, F. Fraser, A.I. Bush, C.L. Masters, K.J. Barnham, Methionine regulates copper/hydr ogen peroxide oxid ation products of A J. Pep. Sci. (2005), 11, 353-360. (e) F.E. Ali, K.J. Barnham, C.J. Barrow, F. Separovic, Copper catalysed oxidation of amino acids and Alzheimer's disease. Lett.

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27 Pep. Sci. (2003), 10, 405-412. (f) D.A. Butterfield, A.I. Bush, Alzheimer's amyloid beta-peptide (1-42): involvement of methi onine residue 35 in th e oxidative stress and neurotoxicity properties of this peptide Neurobiol. Aging. (2004), 25, 563-568. 10) K. Schuessel, S. Leutner, N.J. Cairns, W. E. Mueller, A. Eckert, Impact of gender on upregulation of antioxidant defense mech anisms in Alzheimer's disease brain. J. Neural Trans. (2004), 111, 1167-1182. 11) J. Apelt, M. Bigl, P. Wunderlich, R. Sc hliebs, Aging-related increase in oxidative stress correlates with developmental pa ttern of beta-secretase activity and betaamyloid plaque formation in transgenic Tg2576 mice with Alzh eimer-like pathology. Inter. J. Develop.l Neurosci. (2004), 22, 475-484. 12) J.T. Rogers, D.K. Lahiri, Metal and infl ammatory targets for Alzheimer's disease. Curr. Drug Targets (2004), 5, 535-551. 13) D.R. Richardson, Novel chelators for centr al nervous system di sorders that involve alterations in the metabolism of iron and other metal ions. Ann. N.Y. Acad. Sci. (2004), 1012, 326-341. 14) N.G.N. Milton, Role of hydrogen peroxide in the etiology of Alzheimer's disease: implications for treatment Drugs & Aging (2004), 21, 81-100. 15) I.H. Cheng, J.J. Palop, L.A. Esposito, N. Bien-Ly, F. Yan, L. Mucke, Aggressive amyloidosis in mice expressing human amyl oid peptides with the Arctic mutation. Nature Med. (2004), 10, 1190-1192.

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28 16) K. Jin, V. Galvan, L. Xie, X.O. Mao, O.F. Gorostiza, D.E. Bredesen, D.A. Greenberg, Enhanced neurogenesis in Al zheimer's disease transgenic (PDGFAPPSw,Ind) mice. Proc. Natl. Acad. Sci. USA (2004), 101, 13363-13367. 17) D. Boyd-Kimball, R. Sultana, H. Mohmmad-Abdul, D.A. Butterfield, Rodent A beta(1-42) exhibits ox idative stress properties similar to those of human A beta(1-42): Implications for proposed mechanisms of toxicity. J. Alzheimer’s Dis. (2004), 6, 515525. 18) G.F.Z. da Silva, L.-J. Ming, Alzheimer’s Disease-Related Copper(II)-Amyloid Peptide Exhibits Phenol Monooxygenase and Catechol Oxidase Activities. Angew. Chem. Int. Ed. (2005) 44, 5501-5504. 19) G.F.Z. da Silva, W.T. Tay, L.-J. Ming, Catechol Oxidase-Like Oxidation Chemistry of the 1-20 and 1-16 Fragments of Alzheimer’s Disease-Related -Amyloid Peptide: Their Structure-Activity Correlation and the Fate of Hydrogen Peroxide. J. Biol. Chem. (2005), 280, 16601-16609. 20) G.F.Z. da Silva, Ming L.-J, “Metallo-ROS” in Alzheimer’s Disease : Metal-Centered Oxidation of Neurotransmitters by CuII-Amyloid Provides an Alternative Perspective for the Neuropathol ogy of Alzheimer’s Disease. Angew. Chem. Int. Ed. (2007), 46, in press. 21) C.D. Syme, R.C. Nadal, S.E.J. Rigby, J.H. Viles, Copper Binding to the Amyloid(A ) Peptide Associated with Alzheimer’s Di sease. Folding, Coordination Geometry, pH Dependence, Stoichiome try, and Affinity of A (1–28): Insights from a Range of

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29 Complementary Spectroscopic Techniques. J. Biol. Chem (2004) 279, 18169– 18177. 22) (a) E.A. Lewis, W.B. Tolman, R eactivity of dioxygen-copper systems. Chem. Rev. (2004), 104, 1047-1076. (b) W.B. Tolman, Making and breaking the dioxygen O–O bond: New insights from studies of synthetic copper complexes. Acc. Chem. Res. (1997), 30, 227–237. 23) C. Gerdemann, C. Eicken, B. Krebs, The crystal structure of catechol oxidase: New insight into the function of type-3 copper proteins. Acc. Chem. Res. (2002), 35, 183191. 24) S.G. Srivatsan, P. Nigam, M.S. Rao, S. Verma, Phenol oxidation by coppermetallated 9-allyladenine-DVB polymer: r eaction catalysis and polymer recycling. Applied Catal. A: General (2001), 209, 327-334. 25) Leskovac V. Comprehensive Enzyme Ki netics. Kluwer/Plenum, Boston, MA (2002). 26) S. Stoll, A. Schweiger, EasySpin, a co mprehensive software package for spectral simulation and analysis in EPR J. Mag. Res. (2006), 178, 42. 27) N.D. Chasteen, P.A. Snetsinger, ES EEM and ENDOR Spectroscopy In Physical Methods in Bioinorganic Chemistry, Spectro scopy and Magnetism, L. Que Jr. Ed.; University Science Books, (2000). 28) J.W. Karr, H. Akintoye, L.J. Kaupp, V.A. Szalai, N-Terminal Deletions Modify the Cu2+ Binding Site in Amyloid. Biochemistry (2005), 44, 5478 – 5487. 29) J.S. Valentine J.S. in Bioinorganic Chemistr y (I. Bertini, H.B. Gray, S.J. Lippard, J.S. Valentine Eds. ) pp. 253-313, University Science Books, Mill Valley, CA (1994).

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30 30) C. Schneich, Methionine oxidation by r eactive oxygen species: reaction mechanisms and relevance to Alzheimer's disease. Biochim. Biophys. Acta (2005), 1703, 111-119. 31) K. Vrnagy, B. Bka, I. Svg, D. Sanna, P. Marras, G. Micera, Potentiomentric and spectroscopic studies on the copper(II) and nickel(II) comp lexes of tripeptides of methionine. Inorg. Chim. Acta. (1998), 275-276, 440-446. 32) C.C. Curtain, F. Ali, I. Volitakis, R.A. Cherny, R.S. Norton, K. Beyreuther, C.J. Barrow, C.L. Masters, A.I. Bush, K.J. Barnham, Alzheimer's disease amyloidbinds copper and zinc to generate an a llosterically ordered membrane-penetrating structure containing superoxide dismutase -like subunits. J. Biol. Chem. (2001), 276, 20466-20473. 33) (a) D.M. Dooley, C.E. Cot, Inactivation of beef plasma amine oxidase by sulfide. J. Biol. Chem. (1984), 259, 2923-2926. (b) L. Morpurgo, A. Desideri, A. Rigo, R. Viglino, G. Rotilio, Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovi ne carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes. Biochim.Biophys. Acta (1983), 746 168-175. (c) S. Suzuki, T. Sakurai, A. Nakahara, O. Oda, T. Manabe, T. Okuyama, Copper binding site in serum amine oxidase treated with sodium diethyldithiocarbamate. Chem. Lett. (1982), 487-490. (d) L. Morpurgo, G. Rotilio, A. Finazzi Agr, B. Mondovi Anion complexes of copper(II) bovine carbonic anhydrase. Arch. Biochem. Biophys. (1975), 170 360-367. (e) L. Morpurgo, A. Finazzi Agr, G. Rotilio, B. Mo ndovi, Anion complexes of copper(II) and cobalt(II) bovine carbonic a nhydrase as models for the c opper site of blue copper

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31 proteins. Eur. J. Biochem. (1976), 64, 453-457. (f) J. Peisach, W.E. Blumberg, Structural implications derived from the an alysis of electron paramagnetic resonance spectra of natural and arti ficial copper proteins. Arch. Biochem. Biophys (1974), 165, 691–708. (g) F. Jian, J. Peisach, L.-J. Ming, L. Que Jr., V.J. Chen, Electron Spin Echo Envelope Modulation Studies of the Cu(II)-Substituted Derivative of Isopenicillin N Synthase, a Stru ctural and Spectroscopic Model. Biochemistry (1991), 30, 11437–11445. 34) S.S. Eto, J. Dubach, G.R. Eaton, G. Thurman, D.R. Ambruso, Electron Spin Echo Envelope Modulation Evidence for Ca rbonate Binding to Fe(III) and Cu(II) Transferrin and Lectoferrin. J. Biol. Chem. (1990), 265, 7138–7141. 35) (a) H. Li, S.P. Webb, J. Ivanic, J.H. Je nsen, Determinants of the relative reduction potentials of type-1 copper sites in proteins. J. Am Chem. Soc. (2004), 126, 80108019. (b) J.F. Hall, L.D. Kanbi, R.W. Strange S.S. Hasnain, Role of the axial ligand in type 1 Cu centers studied by point mutations of Met148 in rusticyanin. Biochemistry (1999), 38, 12675-12680. 36) (a) W. Fu, H. Luo, S. Parthasarathy, M.P. Mattson, Catecholamines potentiate amyloid -peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis. Neurobiol. Disease (1998), 5, 229243. (b) J.I. Friedman, D.N. Adler, K.L. Davis, The role of norepinephrine in the pathophysiology of cognitive disorders: potenti al applications to the treatment of cognitive dysfunction in Schizophr enia and Alzheimer’s disease. Biol. Psychiatry (1999), 46, 1243-1252. (c) R.J. Gruen, J. Ehrlic h, R. Silva, J.W. Schweitzer, A.J.

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32 Friedhoff Cognitive factors and stress-induced changes in catecholamine biochemistry. Psychiatry Res. (2000), 93, 55-61. (d) H. Umegaki, N. Tamaya, T. Shinkai, A. Iguchi, The metabolism of plasma glucose and catcholamines in Alzheimer’s disease. Experiment. Gerontol. (2000), 35, 1373-1382. 37) D.B. Calne, J.W. Langston, Aetiology of Parkinson’s disease. Lancet (1983), 322, 1457-1459. 38) (a) N. Ohtani, T. Goto, C. Waeber, P.G. Bhide, Dopamine modulates cell cycle in the lateral ganglionic eminence. J. Neurosci (2003), 23, 2840–2850. (b) A. Borta, G.U. Hglinger, Dopamine and adult neurogenesis. J. Neurochem. (2007), 100, 587–595. 39) (a) Graham, D.G. (1978) Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol. Pharmacol 14, 633-643. (b) Graham D.G., Tiffany S.M., Bell W.R.Jr., Gutkn echt W.F. (1978) Autooxidation versus covalent binding of quinones as the m echamism of toxicity of dopamine, 6hydroxydopamine and related compounds toward C1300 neuroblastoma cells in vitro. Mol. Pharmacol. 644-653. (c) Hastings T.G. (1995) Enzymatic oxidation of dopamine: the role of prostaglandin H synthase. J. Neurochem.b 919-924. 40) (a) M.J. LaVoie, T.G. Hastings, Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine: Evidence against a role for extracellular dopamine. J. Neurosci. (1999)19, 1484-1491. (b) I. Santa-Maria, F. Hernandez, M.A. Smith, G. Perry, J. Avila, F.J. Moreno, Neurotoxic dopamine quinone facilitates the assembly of tau into fibrillar polymers. Mol. Cell. Biochem. (2005), 278, 203-212.

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33 41) S.B. Berman, M.J. Zigmond, T.G. Hasti ngs, Modification of dopamine transporter function: effect of reactive oxygen species and dopamine. J. Neurochem (1996), 67, 593-600.

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220 CHAPTER VI. THE ASTACIN FAMILY OF ENDOPEPTIDASES AND EMBRYOGENESIS I. INRODUCTION The astacin family of zinc-dependent endopeptidases is a class of enzymes ubiquitously distributed acr oss all phyla and part of the superfamily of metzincins.1 Approximately thirty members of the astacin family have been characterized at the protein level, in cluding meprins, bone morphogenetic protein1 (BMP-1), and tolloid while several ot hers have been id entified through gene sequencing, including a large number in Caenorhabditis. elegans .2 The signature active site sequence for this family of enzymes is H EXXH GXXH EXXRXDR (Figure 6.1), where one Zn2+ atom coordinates to three histidines, a tyrosine, and a water molecule (Figure 6.2).3 Most members share common expression features such as the preand pro-enzyme sequences immediately located NH2

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221 Figure 6.1. Active site motif common to all astacins.2

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222 Figure 6.2. Relaxed eye stereo view of the active-site structure of astacin with transition stat e analogue (PDB ID 1QJI).

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223 terminal to the protease domain. Severa l members contain one or two copies of epidermal growth factor EGF-like domains, a nd complement-like domains (Clr, Cls) near the COOH-terminus. The shuffling of differe nt domains in relation to the catalytic (protease or astacin) domain creates a variety of proteins with several different structures and functions. Embryo development is an area which remains unresolved in a number of more complex animals, including human embryogene sis and other representatives of subphylum vertebrata due to the inherent comple xities of deuterostome embryogenesis. The challenge of resolving proteoly tic signaling, that is likely re sponsible for modification of the extra cellular matrix (ECM), important in embryogenesis events, has partially been due to the lack of convenient model systems; a particular challenge is the isolation of ECM components in pure form in mice and human models. Sea-urchins hence are a good model system for the study of morphogenesis due to the facil ity of isolation of pure and large amounts of protein components of the ECM.4 Another advantage of the sea-urchin model is that its genome has recently been sequenced which will f acilitate the connection of upstream genetic events w ith its downstream message and proteolytic mechanisms. The presence of several astacin family enzy mes in the development of the sea-urchin embryo (i.e. suBMP, SpAN, envelysin, and BP10) makes it a target to study similar proteolytic processes in higher organisms, especially those containing enzymatic processes analogous to tolloid and BMP-1, two enzymes proposed to be similar to BP10 in structure .5,6 BP10 is an interesting enzymatic mode l system because it contains similar

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224 Figure 6.3. Phylogeny of the astacin family catalytic domains.6

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225 domains as other complex astacins such as tolloid and BMP-1, but is unique in domain arrangement which will be discussed further.5 Originally characterized by the use of immunoblotting techniques and sequence analysis of the gene and m-RNA transcript,5, 6 BP10 has remained an unstudied member of this class of enzymes. The focus of research has shif ted to other members of the astacin class of enzymes (astacin itself bei ng a crayfish digestive enzyme and hence only a novel prototype in catalytic mechanism) such as BMP-1 and tolloid due to their potential role in unraveling developmenta l information about human embryos (BMP-1) and the facility of handling a well esta blished model system (tolloid from Drosophila ). However, from an evolution standpoin t, echinoderms (Echinodermata, the deuterostomes) are more closely related to humans than a fruitfly (Arthropoda, the ecdysozoans).7 BP10 hence is a good candidate as a functional model system of BMP-1 and tolloid. The transcription of the BP10 ge ne is transiently activ ated around the 16to 32-cell stage and the accumulation of BP10 mRNA is limited to a short period at the blastula stage. Temporarily, the highest BP10 activity is de tected approximately 1.5 hours after expression of the sea urchin hatchi ng enzyme (envelysin) reaches a maximum.5 The BP10 transcripts are spatially expressed and onl y detected in a limited area of the blastula in the animal half of the embryo. The protein is first detected in early blastula stages. Its level reaches a peak in late cleavage, and d eclines abruptly before ingression of primary mesenchyme cells and remains c onstant in late development.5 The likely role of zymogen activator has been assigned to BP10, since th e presence of an EGF domain is a highly conserved motif in proteolytic cas cades or activation of precursors.8 However, it was

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226 accurately described5 that blocking BP10 activity prior to hatching with the use of an antibody resulted in deformed embryos, wher eas after hatching the embryos developed normally when treated with the same antibody, though basal levels of the enzyme can be detected after hatching. The morphogenetic studies conducted by R unnstrm and Horstadius determined that developmental processes occur across a gr adient in where the ectodermal structures are controlled by the animal axis and the mesodermal (skeleton) is controlled by the vegetal pole. This established mode l has been challenged by recent studies.9,10 Hence it becomes clear that sole analysis of gene stru cture and transcriptiona l levels is not enough to provide irrefutable evidence about animal development. A multi-disciplinary approach is needed where the skills of a developmenta l biologist and the deta iled information about structure/function of enzymes th at a biochemist can provide are needed in order to fully understand such questions. The BP10 protease is constructed of iden tical domains as BMP-1 and tolloid, and is described as a factor invol ved in cell differentiation in mi d to late blastula. Human BMP-1 has been characterized as a splice varian t of the tolloid gene which is required for correct dorsal-ventra l patterning of the Drosophila embryo. In preliminary studies, BP10 has not been shown to affect axis formati on; hence the structure/function paradox does not hold true for BP10, BMP-1, and tolloid. How do similar global mechanisms of development differ at the molecular level? What are the mechanisms of evolution

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227 Figure 6.4. Domain organization of proteins from the astacin/CUB/EGF subfamily.6

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228 involved in animal development across the de uterostomes? And fi nally, what are the regulatory networks during embryogenes is in humans, sea-urchins, and Drosophila ? BMP-1 has been assigned the role of a procollagen C-protease and its minimal domain structures necessary for activ ity and secretion have been identified.11 However BMP-1 is also able to cleav e non-collagenous substrates su ch as dentin and laminin, hinting at a wide distribution of biological regulation factors that may be attributed to this enzyme.12-15 BP10 may serve a similar purpose in the sea urchin embryo. The developmental regulation of BP 10 expression shows that it is an important and necessary event in embryogenesis.5 Recent studies have also show n that another astacin family enzyme found in sea urchins, SpAN, is able to regulate BMP signaling.16 BP10 may be part of the signaling events which involve the enzymes suBMP, SpAN and BP10 itself, or perhaps play the role as a less discriminate activator of othe r important molecular events. Since BP10 is homologous to tolloid and BM P-1 (Figure 6.4), it is possible that the sea urchin enzyme is part of a regulatory set of interactions with polypeptides of the TGFfamily. The involvement of BP10 in a process similar to that of BMP-1 is unlikely and most likely resembles tolloid in function.5 However, the high similarity in sequence and structural motifs makes BP10 an important comparis on model in a lower species. The mammalian BMP-1-related proteas es are all capable of activating the TGF protein growth differentiation factor (myosta tin), by freeing it from a noncovalent latent complex with its cleaved prodomain.17 Similarly, BMP-1 and the splice variant mTLL-1 but not tolloid or TLL-2 is able to free the TGFmorphogens BMP-2 and BMP-4 from latent complexes with the extracellular antagonist chordin.18 Thus, BMP-1/tolloid-like

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229 proteases may orchestrate formation of the ECM with signaling by various TGF-like proteins in morphogenetic events. BP10 is also a unique system that may c ontribute important information about the relationship of structural features such as EGF-Ca2+ binding domain and its CUB domains and the catalytic domain of these pr oteases (Figure 6.2). It is unique to BP10 that the EGF-Ca2+ domain is located between the cat alytic domain and the proposed regulatory sequences of CUB1 and CUB2. Often, these EGF-Ca2+ domains are located between CUB sequences. CUB domains have been implicated both in activity and regulation in BMP-1.11 The kinetic properties of BP10 should then compare and contrast nicely with those of other BMP1 and tolloid-lik e proteins. Ca2+ may have a synergistic effect on the reaction catalyzed by BP10 due to its close proximity to the catalytic domain. From a protein evolution viewpoint, BP 10 contains modules proposed to be involved in the domain shuffli ng (i.e evolution through exon s huffling). Characterization of BP10 with simultaneous analysis of its gene structure will add furt her insight into this mechanism. Moreover, once ot her sea urchin enzymes invol ved in embryogenesis, such as SPAN and suBMP are overexpressed and char acterized, and their ge ne structures fully characterized, a better model of module domain shuffling protein evolution can be presented, since BP10, SPAN and suBMP are part of the same family of enzymes, acting in the same organism, and involved in so me functional role during embryogenesis. In the case of astacin, BMP-1, tolloid, and BP10, the presence of a metalcoordinated tyrosine is a rather unusual mech anistic motif due to the reduction of Lewis

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230 acidity induced by the coordi nation of a negatively char ged phenolate to the metal center.19, 20 This is not the case in astacin wh ere the coordination of the Tyr-phenolate under physiological conditions does not affect its catalysis under neutral conditions.21,22 A metallotriad mechanism has been proposed based on kinetic, optic al, and EPR studies on the Cu2+ derivatives of as tacin and serralysin.21, 22 Further analysis of the physical characteristics of astacin-type enzymes using spectroscopically-active metal substitutes in concert with classical enzyme kinetics (i.e. pH profiles, inhibition st udies, etc.) analysis of splice variants of reco mbinant BP10 (whole enzyme and astacin domain truncation) will add further insight into the catalytic mech anism of these enzymes. Concomitant with physical studies on the native system, active si te mutants will allow for comparison both in kinetic parameters and the physical prope rties of BP10. Previ ous site directed mutagenesis studies have been conducted; how ever, the choice of mutations have shed no actual mechanistic insight toward the catal ytic mechanism of astacins, other than establishing that the conserved glutamate and tyrosine residues are catalytically important.23 The status of the coordinated tyrosine has been proposed to be an inhibitory process in the metal centered hydrolysis of peptide bonds.22 In this proposed metallotriad mechanism (Figure 6.5), the active-site Zn2+ coordinated by three His, tyrosine, and a water molecule can be activated via detach ment of the phenolate with a concomitant

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231 Figure 6.5. Proposed metallotriad mechanism in astacin and serralysin.22

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232 general base deprotonation via a gl utamate residue. The metal bound OH– is able to attack and hydrolyze th e scissile bond after Zn2+ creating electrostatic strain in the peptide bond by interaction w ith the carbonyl of the scissi le bond. The confirmation of this mechanism can shed valuable insight into numerous enzymatic processes across all members of the astacin family, from the numerous astacin-like enzymes in C.elegans to the astacin/EGF/CUB members in higher organisms.

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233 IV. LIST OF REFERENCES 1) J.S. Bond, R.J. Beynon, The astaci n family of metalloendopeptidases. Prot. Sci. (1995), 4, 1247-1261. 2) F. Moehrlen, H. Hutter, R. Zwilling, The astacin protein family in Caenorhabditis elegans Euro. J. Biochem. (2003), 270, 4909-4920. 3) F.X. Gomis-Rth, W. Stcker, H. Hube r, R. Zwilling, W. Bode, Refined 1.8 X-ray crystal structure of astacin, a zinc -endopeptidase from the crayfish Astacus astacus L.: Structure determination, refinement, molecular structure amd comparison with thermolysin. J. Mol. Biol. (1993), 229, 945-968. 4) V.D. Vacquier, Isolation and preliminary analysis of the hyaline layer of sea urchin eggs. Exp. Cell Res. (1969), 54, 140-2. 5) T. Lepage, C. Ghiglione, C. Gache, Spa tial and temporal expre ssion pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Dr osophila dorsal-ventral patterning gene tolloid. Development (1992), 114, 147-163. 6) T. Lepage, C. Ghiglione, C. Gache, Stru cture of the gene en coding the sea urchin blastula protease 10 (BP10), a memb er of the astacin family of Zn2+-metalloproteases. Eur. J. Biochem. (1996), 238, 744-751. 7) S.F. Gilbert, Developmental Biolog y, 6th Ed., Sinauer, Sunderland, MA, (2000). 8) E. Appella, I.T. Weber, F. Blasi, Struct ure and function of epid ermal growth factorlike regions in proteins. FEBS letters (1988), 231, 1-4.

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234 9) E.H. Davidson, Lineage Specific gene expres sion and the regulative capacities of the sea urchin embryo: aproposed mechanism. Development (1989), 105, 421-445. 10) F.H. Wilt, Determination and morphogenesis in the sea urchin embryo. Development (1987), 100, 559-576. 11) N. Hartigan, L. Garrigue-Antar, K.E. Ka dler, Bone morphogentic protein-1 (BMP-1): Identification of the minimal domain struct ure for procollagen Cproteinase acivity. J. Biol. Chem. (2003), 278, 18045-18049. 12) E. Kessler, K. Takahara, L. Biniam inv, M. Brussel, D.S. Greenspan, Bone morphogentic protein-1: the type I proco llagen C-proteinase. Science (1996), 271, 360-362. 13) A. Borel, D. Eichenberger, J. Farjanel E. Kessler, C. Gleyzal, D.J. Hulmes, P. Sommer, B. Font, Lysyl-oxidase-like prot ein from bovine aorta. Isolation and maturation to to an active form by bone morphogenetic protein-1. J. Biol. Chem (2001), 276, 48944-48949. 14) S. Amano, I.C. Scott, K. Takahara, M. Koch, M.F. Champliaud, D.R. Gerecke, D.L. Hudson, T. Nishiyama, S. Lee, D.S. Grees npan, Bone morphogenetic protein 1 is an extracellular processing enzyme of the 5 2 chain. J. Biol. Chem. (2000), 275, 2272822735. 15) N. Suzuki, P.A. Labosky, Y. Furuta, L. Hargett, R. Dunn, A.B. Fogo, K. Takahara, D.M. Peters, D.S. Greenspan, B.L.Hogan, Failure of ventral body wall closure in mouse embryos lacking a procollagen Cproteinase encoded by Bmp1, a mammalian gene related to Drosophila tolloid. Development (1996), 122, 3587-3595.

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235 16) F.C Wardle, L.M. Angerer, R.C. Angere r, L. Dale, Regulation of BMP signaling by the BMP1/TLD-related metalloprotease, SpAN. Develop. Biol. (1999), 206, 63-72. 17) N. Wolfman, A.C. McPherron, W.N. Papppano, M.V. Davies, K. Song, K.N. Tomkinson, J.F. Wright, L. Zhao, S.M. Seba ld, D.S. Greenspan, S.J. Lee, Activation of latent myostatin by the BMP-1/toll oid family of metalloproteinases. Proc. of the Natl. Acad. Sci. USA (2003), 100, 15842-15846. 18) I.C. Scott, I.L. Blitz, W.N. Pappano, Y. Imamura, T.G. Clark, B.M. Steiglitz, C.L. Thomas, S.A. Maas, K. Takahara, K.W. Y. Cho, D.S. Greenspan, Mammalian BMP1/Tolloid-Related Metalloproteinases, In cluding Novel Family Member Mammalian Tolloid-Like 2, Have Differential Enzymatic Activities and Distributions of Expression Relevant to Patte rning and Skeletogenesis. Develop. Biol (1999), 213, 283-300. 19) E. Kimura, Macrocyclic Polyamine Zinc (II) Complexes as Advances models for Zinc(II) enzymes. Prog. Inorg. Chem (1994), 41, 443. 20) E. Kimura, T. Koike, Intrinsic Properties of zinc(II) ion pertin ent to zinc enzymes. Adv. Inorg. Chem. (1997), 44, 229. 21) H.I. Park, L.J. Ming, The mechanistic ro le of coordinated ty rosine in astacin. J. Inorg. Biochem. (1998), 72, 57-62. 22) H.I. Park, L.J. Ming, Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism. J. Bio. Inorg. Chem (2002), 7, 600-610.

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236 23) I. Yiallouros, B.E. Grosse, W. Stcker, The roles of Glu93 and Tyr149 in astacin-like zinc peptidases. FEBS Letters (2000), 484, 224-228.

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237 CHAPTER VII. OVEREXPRESSION AND CHARACTERIZATION OF BLASTULA PROTEASE 10 (BP10) FROM PARACENTROTUS LIVIDUS1 I. INTRODUCTION Blastula Protease 10 (BP10) is a metalloen zyme assigned to the astacin family of Zn-dependent endopeptidases involved in sea urchin embryogenesis. It contains conserved structural motifs consistent w ith astacin, tolloid, and bone morphogenetic protein 1 (BMP-1). Astacin, a gut enzyme, a nd serralysin, a bacterial enzyme, have been proposed to carry out hydrolysis via a “met allotriad” mechanism that involves a metalcoordinated tyrosine. It has not been determined if the more structurally complex members of this family involved in eukaryot ic development share this mechanism. The recombinant BP10 has been overexpressed in E.coli its metalloenzyme nature confirmed, and its catalytic properties characterized thr ough kinetic studies. BP10 shows significant 1 This work has been published: G.F.Z. da Silva, R.L. Reuille, L.-J. Ming, B.T.Livingston J. Biol. Chem. 2006 281, 10737-10744.

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238 hydrolysis toward gelatin both in its native Zn-containing form a nd copper derivative. The copper derivative of BP10 shows a remark able 960 % rate-acceleration toward the hydrolysis of the synthetic s ubstrate N-benzoyl-arginine-p-ni troanilide when compared to the Zn form. The enzyme also shows calcium -dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-t ype enzymes in deuterostomes which can add supporting data to corroborate the metall otriad mechanism proposed for astacin. The astacin family of zinc-dependent endopeptidases is a class of enzymes ubiquitously distributed across all phyla and part of the superfamily of metzincins.1 Approximately thirty members of the astaci n family have been characterized at the protein level;2 including meprins, bone morphogenetic protein-1 (BMP-1 ), and tolloid, while several others have been identified through gene sequencing, including a large number in Caenorhabditis elegans.3 The signature of the primary sequence active site motif for this family of enzymes is H EXX H GFX H EXXRXDR, where one Zn2+ ion coordinates with three histidines, a tyrosine, and a water molecule.4, 5 Most members of this family share common domain structures such as the preand pro-enzyme sequences immediately located N-terminal to the protea se domain. Several members contain one or two copies of epidermal growth factor EG F-like domains, and complement-like domains (Clr, Cls) near the C-terminus.2 The shuffling of different domains in relation to the catalytic protease domain creates a variety of proteins with different structures and functions.

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239 Originally isolated and characterized as a developmentally regul ated gene in sea urchin embryos,6,7 BP10 protein has remained unchara cterized. It shares sequence similarity with other members of the astacin fa mily of enzymes. The simplest member of the family, astacin from crayfish digestive fl uid is a digestive enzyme and hence a novel prototype in catalytic mechanism important in development such as BMP-1 in vertebrates and tolloid in Drosophila Whereas BP10 is a good candi date as a functional model system of BMP-1 and tolloid. The BP10 proteas e is constructed of identical domains as BMP-1 and tolloid, but has different arrangemen t of these domains. The transcription of the BP10 gene is transiently activated around the 16to 32-cell stage and the accumulation of BP10 mRNA is limited to a short period at the blastula stage. Temporarily, the highest BP10 activity is detected approximately 1.5 hours after expression of the sea urchin hatching enzyme (envelysin) reaches a maximum.6 The BP10 transcripts are detected in a limited area of the blastula. The protein is first detected in early blastula stages, its level peaks in late cleavage, declin es abruptly before ingression of primary mesenchyme cells, and remains invariable in late development.6 A likely role of zymogen activator has been suggested for BP10, since the presence of an EGF domain is a highly conserved motif in proteolytic cascades or activation of precursors.8 Blocking BP10 activity prior to ha tching with the use of an antibody resulted in abnormal embryos. BP10 has a unique arrangement of structural features6, 7 such as EGF-Ca2+ binding domain, two adjacent CUB domains, an d a catalytic domain that is highly homologous to astacin. In particular, the EGF-Ca2+ domain is located between the

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240 catalytic domain and the proposed regulator y CUB sequences. More often, these EGFCa2+ domains are located between CUB sequences CUB domains have been implicated in activity and regulation in BMP-1.9 Astacin family enzymes and serine proteases have been implicated in remodeling the pericellu lar space in sea urchin embryos, which is composed of the extracellular ma trix and transmembrane proteins.10, 11 Moreover, several studies have reported gelatinase and collagena se activities from enzymes located in the sea urchin egg and embryo which were characterized as metalloenzymes due to inactivation with EDTA and 1, 10-phenanthroline.12-15 Beside the interesting distribution of astacin-like enzymes across phyla and the numerous functional roles of these enzymes, mechanistic questions about these highly conserved hydrolases domains still remain to be answered. Within the metzincin superfamily of enzymes, minor differences of active site function have been observed, which are likely to account for different substrate specificities.16 In the case of astacin as well as BMP-1, tolloid and BP10, a metalcoordinated tyrosine is a rather unusual metal-binding motif due to the reduction of Lewis acidity induced by the coordination of the negatively charged phenolate to the metal center.17, 18 However, the coordination of the Tyr-phenolate does not seem to affect astacin catalysis under neutral conditions. The status of the coordinated tyrosine has b een proposed to be an inhibitory process in the metal centered hydro lysis of peptide bonds.5 A metallotriad mechanism has been proposed for astacin and serralysin based on kinetic and spectrosc opic studies of the native enzymes and their Cu2+ derivatives.5,19 In the proposed metallotriad mechanism, the active-site Zn2+ coordinated by three His, a tyrosi ne, and a water molecule can be

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241 activated via detachment of the Tyr-phenolat e with a concomitant deprotonation of the coordinated water assisted by a gl utamate residue. The metal-bound OH– is able to hydrolyze the scissile bond with Zn2+ creating electrostatic strain in the peptide bond by interaction with the carbonyl group of the scissile bond. I di scuss in this chapter the overexpression and thorough mechanistic st udy of recombinant BP10, a model system for astacin-type developmentally regulated metalloenzymes. Further analysis of BP10 will add insight into the catalytic mechanis m of members of the astacin family of enzymes, and the degree to which the mechanism is conserved among the enzymes found in deuterostomes. II. EXPERIMENTAL The expression vector pQE30Xa, Ni-NTA agarose, mouse anti-His primary antibody were from Qiagen (Valencia, CA), XL1-Blue chemically competent E. coli from Invitrogen (Carlsbad, CA), Rosetta Blue chemically competent E. coli and Factor Xa removal kit were from Novagen (San Die go, CA), all primers were from Integrated DNA Technologies (Coralville, IA), all modi fying and restriction enzymes were from Promega (Madison, WI), Eppendorf Perfect plas mid preparation kit was from Eppendorf (Westbury, NY), BM purple phosphatase substr ate was from Roche (Indianapolis, IN), EDTA, ZnCl2, Cu(NO3), Ca(NO3)2, glycerol, ninhydrin, gua nidine hydrocholoride, bovine serum albumin, sodium dodecyl sulf ate, Triton X-100, Tween 20, imidazole, NaH2PO4, Na2HPO4, Tris-HCl, acrylamide, bis-acrylamide, TEMED, ammonium

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242 persulfate, NaN3, dimethyl sulfoxide (Me2SO), sodium citrate, acetic acid, guanidine hydrochloride, and propanol were from Fisher (Swanee, GA), Type A porcine gelatin 300 bloom, N-benzoyl-argininep -nitroanilide (BAPNA ), urea, isopropylthiogalactopyronoside (IPTG), phenylmethyl sulfonyl fluoride (PMSF), benzamidine, urea, lysozyme, bicinchoninic acid, arginine-hydroxamate and HEPES, CAPS, TAPS, and MES buffers were from Sigma-Aldrich (St. Louis, MO), 1,10-phenanthroline was from Acros (Fairlawn, NJ). All reagents were of enzyme or molecular biology grade when available, all others were reagent gr ade. All glassware and plasticware were extensively rinsed with EDTA to remove metal contamin ants and thoroughly washed with 18 M water to remove the chelator. All bu ffers contained Chelex resin to remove metal contaminants. All spectrophotometric measurements were performed on a Varian CARY 50 Bio-Spectrophotometer equipped wi th a PCB-150 water Peltier thermostable cell holder. Overexpression, purification, and re folding of recombinant BP10 : The cDNA coding for Paracentrotus lividus BP10 cloned into the pBlues cript plasmid (pBP10) was a generous contribution from Christian Gache and Thierry Lepage (Unit de Biologie Cellulaire, Center National de la Recherche Scie ntifique et Universit de Paris VI, Station Marine, 06230 Villefranche-sur-Mer, France). PCR primers coding for both 5Â’ and 3Â’ regions were designed according to the pr oposed full length BP10 to subclone the cDNA into the pQE30Xa overexpression ve ctor. The 5Â’ primer: 5Â’-PO4AAACTAATACTTTCCCTTTCGGGATTG-3Â’ codes for the first 9 codons in the proposed nucleotide sequence in BP10 and is 5Â’ phosphorylated for blunt-end cloning

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243 using the Stu I restriction site in pQ E30Xa; 3Â’ primer was de signed for cloning into the Xma I restriction site 5Â’AATT CCCGGG TTA GTTCAGACGAGGATCTC GGGT-3Â’ (bold = extra base pairs for melting te mperature optimization, underlined = Xma I restriction site, bold underlined = stop codon). The PCR product coding for BP10 was digested with Xma I and cloned into the pQE30Xa vector. The BP10 construct was transformed into Rosetta Blue competent ce lls. The colonies overexpressing recombinant BP10 were screened using a colony lift pr otocol according to Qiagen without modifications, where the production of BP10 wa s monitored using a mouse anti-His tag primary antibody. The active colonies were picked, propagated in liquid media to OD600 = 0.4. IPTG was added to a final concentra tion of 1.0 mM and the culture grown at 30 C and 300 rpm for 4.5 hours. The bacteria co ntaining recombinant BP10 were pelleted at 4,000 g at 4 C and resuspended in cell wall lysis buffer (50.0 mM NaH2PO4, 100.0 mM NaCl, 10.0 mM imidazole, 2.0 mM benzam idine, 2.0 mM PMSF, pH 8.0) containing 1.7 mg/mL lysozyme and incubated on ice ligh tly shaking for 60 minutes, then sonicated 6 10 seconds with 10 second intervals. The cultures were pelleted at 10,000 g at 4 C for 20 minutes and the inclusion bodies were resuspended in an urea buffer (8.0 M urea, 10.0 mM Tris, 100.0 mM NaH2PO4, 1.0 % Triton X-100, 2.0 mM benzamidine, 2.0 mM PMSF, pH 8.0) and incubated at 37 C at 200 rpm for 60 minutes. The solubilized inclusion bodies we re pelleted at 10,000 g at 4 C for 20 minutes, and 1.0 mL of NiNTA agarose was added to the supernatant. The Ni-NTA slurry was gently shaken at room temperature for 45 minutes then added to a gravity fed column and the recombinant BP10 eluted using a pH gradient, with bu ffers containing 6.0 M urea, 10.0 mM Tris,

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244 100.0 mM NaH2PO4, at pHÂ’s 6.3, 5.9, and 4.5. The recombinant BP10 was completely eluted at pH 4.5 and was immediatel y titrated to pH 7.4 using 0.5 M NaH2PO4. Overexpression and purification were mon itored on a time-dependent basis using 12.5 % SDS-PAGE according to Laemmli and Wester n blot techniques using the anti-His-tag antibody. The concentration of recombinant BP 10 was determined using standard BCA assay with a BSA standard curve. The recombinant protein was diluted 40 times by volume using 50.0 mM Tris with 50 mM NaCl then dialyzed extens ively with several changes against phosphate buffered sa line (PBS) containing 50 M ZnCl2 for 48 hours at 4 C. Recombinant BP10 was con centrated either under 18 psi N2 using an YM3 Amicon membrane or an Amicon Centricon YM3. Final BP10 concentrations were checked using BCA. The His-tag fusion was re moved using a Factor Xa His-tag removal kit from Novagen according to instruction. Circular Dichroism (CD) studies : CD spectra of urea-dena tured, and folded ZnBP10 and Cu-BP10 were collected in PBS us ing a 0.1 cm cell with a resolution of 0.5 nm. All absorbance readings were converted to molar ellep ticity and the pe rcent helicity and sheet content calculated. The -helical content was calcula ted according to published methods.20 Preparation of the copper derivative of BP10 : During the refolding of ureadenatured BP10, 1.0 mM 1,10-phenanthroline was added to the 50.0 mM Tris 50.0 mM NaCl pH 7.5 buffer, then extensively dial yzed against PBS cont aining 0.5 M guanidine. The guanidine-containing buffer was exchange d through dialysis with PBS buffer, and

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245 then with PBS containing 50 M Cu(NO3)2. Protein concentrations were determined with BCA assay. Gelatin Zymogram : Gelatin was incorporated into a polyacrylamide gel matrix according to standard protocols with modifications to fit current studies as listed below.21 A volumeof 1.25 mL of 1.4 M Tris at pH 8.8, 0.50 mL of 5.0 mg/mL gelatin solution in water, 25.0 L 10 % (w/v) APS, 200 L 10% (w/v) SDS, 25.0 L TEMED, 2.0 mL water, and 1.25 mL 30:1 acrylamide:bis-ac rylamide were mixed and allowed to polymerize in a mini-gel minus a stacking ge l. Several concentr ations of BP10 were mixed with non reducing gel-loading dye and incubated for 15 minutes at room temperature (standard Laemmli protocol minus mercaptoethanol or dithiothreitol22). These BP10 samples were loaded into each lane of the gel and run at 200 V and 4 C until the dye front reached the bottom of the plate. The running buffer did not contain SDS. The gel was washed two times in 0.25 % Triton X-100 for 15 minutes with gentle shaking, then incubated for 10 hours in 50.0 mM Tris pH 7.50, 1.0 M ZnCl2 0.5% Triton X-100, 0.02 % NaN3, and 2.0 mM CaCl2. After ten hours the gels were stained with 0.1% Coomassie brilliant blue in 40% propanol for 1.0 hour. The gel was destained in a 7% acetic acid soluti on to reveal the digestion, a nd then photographed on a light table with a digital camera. The enzyme with gelatinase activity is shown as unstained bands. Gelatinase Assay : The gelatinase activity of recombinant BP10 was monitored using a detection method for amino groups with ninhydrin as an indicator of peptide hydrolysis according to a standa rd protocol with modifications to fit current studies as

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246 described below.23 A 5.0 mg/mL gelatin solution was prepared in H2O and heated to 55 C for 15 minutes until completely dissolv ed. A ninhydrin detection solution was prepared by mixing 9.0 mL glycerol, 3.0 mL of 0.5 M sodium citrate at pH 5.50, and 3.75 mL of 1.0% (w/v) ninhydrin so lution in 0.5 M sodium citrate buffer. Gelatin and 1.0 M BP10 were mixed in PBS and incubated at room temperature. A 50.0 L sample was taken from the reaction at several time points and mixed with 950 L of ninhydrin detection solution then boiled for 12.0 minutes. The absorbance at 570 nm was determined using a sample containing undigested stock gelatin (the same gelatin used for the experiment, incubated under the same c onditions minus BP10) mixed with the same detection assay as the blank. The first order rate constant kobs was determined from an exponential curve fit. The molar absorptivity of Ruhemann’s purple ( 570 = 22,000 M–1cm–1)24 also allowed for monitoring the subs trate dependent hydrol ysis of gelatin. Several dilutions of a 10.0 mg/mL stock soluti on of gelatin were used according to the aforementioned protocol and rates were fitted as a function of substrate concentration according to the Michaelis-Menten equation, yielding kcat and Km parameters. Hydrolysis of BAPNA by Zn-BP10 and Cu-BP10 : BAPNA stock solutions were made in Me2SO and then diluted with 50.0 mM HEPES 50.0 mM NaCl pH 7.50. Less than 2% Me2SO by volume was present in each a ssay and found not to interfere with kinetic measurements. Several concentra tion of BAPNA were incubated with 2.17 M BP10 and rates determined colorimetrically from the release of the pnitroaniline product ( 405 = 10,150 M–1cm–1). Kinetic parameters were dete rmined by non-linear fitting to the Michaelis-Menten equation.

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247 Calcium activation assays : Gelatin was extensively di alyzed against an EDTA solution and then extensively dialyzed to re move the chelator. Calcium was carefully titrated under substrate saturation conditions to determine its effect on the hydrolysis of gelatin and BAPNA. Once saturating concen trations of calcium were determined, new kinetic parameters were obtained using su fficient (1.0 mM) calcium in all buffers. Inhibition studies : the effect of two inhibitors, 1,10-phena nthroline (OP) and arginine-hydroxamate (Arg-NHOH) toward th e hydrolysis of BAPNA were determined by running Michaelis-Menten kinetic s under several con centrations of e ach inhibitor. Inhibition constants were determined according to the inhibition patt erns for OP and ArgNHOH respectively. pH profiles : The pH profiles for Znand Cu-B P10 catalyses were constructed by monitoring gelatin and BAPNA hydrolysis under several di fferent pHÂ’s using 50.0 mM buffers containing 50.0 mM NaCl and 1.0 mM CaCl2 The following buffers were used: acetate (pH 5.0), MES (pH 5.5-6.5), HEPES (p H 7.0-8.0), TAPS (pH 8.5-9.0), CAPS (pH 9.5-11.0). The pH-dependent kinetic parameters were determined by non-linear fitting and pKa values were obtained from fitting th e data to a two-ionization process. Electronic spectrum of Cu-BP10 : The electronic spectrum of a 20.0 M Cu-BP10 was monitored from 350 to 800 nm and the tyro sine to copper charge transfer transition was observed at 454 nm. The quenching of this ligand to metal charge transfer transition (LMCT) was monitored spectrophotometr ically upon addition of Arg-NHOH. Homology modeling and substrate docking : The primary sequence of BP10 was overlaid over the crystal structure of serra lysin (PDB ID 1SAT) using BioCAChe 6.1.10

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248 (Fujitsu, Beaverton, OR). The struct ure was energy-minimized using MM3 and molecular dynamics calculations in a simu lated water box. The substrate BAPNA was docked into the active site using the st andard procedure (PF5) in BioCAChe. III. RESULTS AND DISCUSSION Overexpression and refoldi ng of recombinant BP10 : The recombinant enzyme was efficiently overexpressed, though it was in soluble and containe d within inclusion bodies (Figure 7.1, Lane 3). Urea solubilizat ion proved to be an efficient method for extracting the enzyme from inclusion bodies coupled with a fusion His-tag at the Nterminus to BP10 that allows for efficien t purification using Ni-NTA agarose. The overexpression and purification yields an av erage of 0.7 mg/mL of total recombinant BP10 after a pH gradient elution from the Ni-NTA agarose column (Figure 7.1). The protein overexpression was monitored on a tim e course using SDS-PAGE and Western blotting (Figure 7.2 B ). The detection of the Histag using an anti-His antibody upon induction with IPTG proved a sensitive a nd consistent method for monitoring the overexpression of the recombinant protein. Upon removal of the chaotropic reagent urea through extens ive dialysis, the protein was refolded and activity could be mon itored after removal of the His-tag. It was determined empirically that a 40 fold dilution was important since at higher concentrations the protein coagulates and precipitates out of solution. Recombinant BP10 showed no signs of degradation after refolding (Figure 7.2 A ). Upon removal of the

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249 Figure 7.1. SDS-PAGE gel (12.5 %) during purification of recombinant BP10. Lane 1, MW marker; lane 2 total soluble protein after 4 hour induction after lysozyme and sonication; lane 3, total protein solubilized with 8.0 M urea from inclusion bodies; lane 4, flow-thru unbound proteins from Ni-NTA column; lanes 5-6, buffer C wash; lanes 7-9, buffer D at pHÂ’s 6.3, 5.9, and 4.5 gradient elutio n, respectively. Lane 9 shows a homogenous band at 66 kDa, assigned to recombinant BP10. 1 2 3 4 5 6 7 8 9

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250 Figure 7.2 ( A ) SDS-PAGE (12.5 % ) showing intact BP10 after refolding. Lane 1, MW marker; lane 2, is tota l urea solubilized protein; lane3, recombinant BP10 after refolding. ( B ) Western blot showing the time course of overexpression. Lane 1, uninduced sa mple; lane 2, 1.0 hr; lane 3, 2.0 hr; lane 3, 2.0 hr; lane 4, 3.0 hr; lane 5, 4hr. ( C ) Gelatin zymogram showing concentration-dependent substrate hydr olysis by BP10; (Lanes 1-5) 0.25M, 0.50 M, 1.0 M, 1.50 M 1.75 M BP10. A B C 1 2 3 1 2 3 4 5 1 2 3 4 5

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251 His-tag using a Factor Xa removal k it from Novagen, the protein undergoes autohydrolysis and remained stable only for a few days at 4 C. However, the protein fused with the His-tag is stable indefinitely at 4 C. Hence, the His-tag was used as an efficient method for long-term storage of recombinant BP10 and removed only prior to running experiments. CD Spectra of ZnBP10 and CuBP10: The CD spectrum of urea-denatured BP10 (Figure 3, dotted trace) does not show the random -coil expected in the presence of a high concentration of chaotropic reagents. The CD spectrum of urea-denatured BP10 shows an overall -sheet-shape with blue shifts observed in the minimum and a red shift in the maximum of the spectrum. The characteristic minimum at 200 nm for random-coil peptides is not present, with only bathochromic shifts resulting in the denatu ration of BP10. This resistance to complete denaturation to a random-coil conformation may account for the efficient refolding of BP10 in the absence of reducing agents, due to partially formed secondary structures. The helical content of BP10 is 5.8 % which is consistent with the large content of sheet-like structures (i.e. -barrel) in CUB domain-containing proteins.25 According to sequence homology, only the helices present in the astacin-domain shoukd be present accounting for the low helical content of BP10. The CD spectra of Zn-BP10 and Cu-BP10 are virtuall y identical, suggesting no major conformational change in the overall structure of the protein due to metal substitution. Kinetics of gelatin hydrolysis : Recombinant BP10 was not able to hydrolyze casein, a commonly used substrate for endopep tidases. Porcine gelatin however proved to be a good substrate to monitor proteoly tic activity of BP10 w ith gelatin zymograms (Figure 7.2C) and a ninhydrin detection prot ocol (Figures 7.4 and 7.5), establishing the proposed role of BP10 as a protease. The formation of a colored ninhydrin-amino acid

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252 conjugate with a known molar absorptivity wa s a convenient method to monitor gelatin hydrolysis. The saturation kinetics can be fit to the Michaelis-Menten equation, to yield kcat = 0.013 s–1, Km = 51.3 M, and the second order rate constant kcat/ Km = 253.4 M–1s–1. Interestingly, BP10 shows a Ca2+-dependent activation (F igure 7.5, inset) yielding kcat = 0.77 s–1, Km = 46.5M, and kcat/ Km = 16,740 M–1s–1 at saturating [Ca2+]. Other gelatinases found in the sea ur chin embryo have shown a Ca2+-dependent activation.26, 27 However, whether or not the Ca2+ bound to the EGF domain is a cofactor in proteinprotein interaction or signa ling is not known for BP10 and cannot be determined in these studies. Further analys is of the rate consta nts can shed insight into the role of Ca2+ in catalysis. The Km values are similar in the presence and absence of Ca2+, while the kcat value for the catalysis in the presence of Ca2+ is much larger than in the absence of Ca2+. Since Km is defined as ( k–1+ kcat)/ k1, with k1 and k–1 the rate constants for substrate binding and dissociating from the enzymesubstrate (ES) complex, a significant increase in kcat with a constant Km value in the presence of Ca2+ reflects a smaller dissociation k–1/ k1 of the ES complex. This ob servation reveals that Ca2+ affects BP10 catalysis by enhancing substrate binding to the enzyme and by lo wering the activation energy (i.e., a larger kcat value). The activation of BP10 by Ca2+ is the first report of the effect of Ca2+ on the activity of astacin family metalloproteases, although Ca2+ dependent gelatinases have been identified26 in sea urchins; Mg2 + showed no effect in activation of BP10. BP10 contains an EGF-Ca2+ domain that could mediate the eff ect of calcium on catalysis.

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253 Elucidation of the role of the EGF domain and Ca2+ on BP10 activity may shed interesting insight once similarly functioning and structurally conserved enzymes have been characterized across all phyla. The kinetic parameters for Zn-BP10 towa rd gelatin hydrolysis were determined k k H Ka Ka H lim111 2 Equation (1) between pH’s 4.5-11.0. Plots of kcat and kcat/ Km against pH exhibit bell shaped curves (Figure 7.6 A,D ) indicating the presence of two i onizable groups in the catalytic mechanism of ZnBP10 and can be fitted to Equation (1). The two ionization constants from the kcat vs. pH profile yield pKa1 = 5.94 and pKa2 = 10.2 which can be assigned to the deprotonation of a Zn-bound water and to the coordinated tyrosine respectively upon substrate binding which is consistent with previous repor ts for serralysin.5 The role of the coordinated Tyr is furt her addressed below. Kinetics of BAPNA hydrolysis : The synthetic substrate BAPNA is a good substrate for BP10. This activity was previous ly observed for serralysin and is the only synthetic tripeptide of a series of diand tr ipeptide mimics, including glycine-, alanine-, valine-, leucine-, glutamate-, ly sine-, arginine-, trialanine -, and succinyl-trialanine-pnitroanilide that was hydrolyzed by BP10. The kinetics of BAPN A hydrolysis (Figure 7.7) fit Michaelis-Mente n kinetics to yield kcat = 0.079 s–1 Km = 0.66 mM and kcat/ Km = 120 M–1s–1 in the presence of Ca2+, and kcat = 1.83 10–3 s–1, Km = 1.55 mM and kcat/ Km = 1.18 M–1s–1 in the absence of Ca2+ (Figure 7.7 B). Like in gelatin hydrolysis by BP10, C 1 2 3 4 5 C

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254 nm 190200210220230240 (deg x cm 2 x dmol -1 ) -3000 -2000 -1000 0 1000 2000 Figure 7.3. CD spectra of Zn-BP10 in PB S with 8.0 M urea pH 7.4 (dotted trace), and renatured Zn(solid trace ) and Cu-BP10 in PBS pH 7.4 (dashed trace).

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255 time min. 050100150200 Abs570 2e-1 4e-1 6e-1 8e-1 Figure 7.4. First order kinetics of gelatin hydrolysis by Zn2+ ( ) and Cu2+ ( ) derivatives of BP10 in the presence of 1.0 mM Ca2+ at pH 7.5. The solid traces are the best fit to a pseudo-first-order rate law, which affords the rate constant kobs for each derivative.

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256 [gelatin] mM rate mM/s 0.0 4.0e-6 8.0e-6 1.2e-5 [Ca2+] mM 0.0010.010.1 V max (mM/s) 0 2e-4 4e-4 6e-4 8e-4 Figure 7.5. Gelatin hydrolysis by 1.0 M ZnBP10 in 50.0 mM HEPES pH 7.5, 50 mM NaCl, and 1.0 mM Ca(NO3)2. The solid line is the be st fit to the MichaelisMenten equation. The inset shows a Ca2+-dependent activat ion upon titration of Ca2+ to 60 M ZnBP10. The solid line in the inset is the best f it to a loose-binding equilibrium.

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257 Ca2+ activates BP10 by lowering th e activation energy and enhanc ing substrate binding as reflected in the rate constants a nd discussed above. The higher kcat and lower Km for gelatin hydrolysis than BAPNA also indicate a higher affini ty of gelatin than BAPNA binding to BP10, reflecting the endopeptidases nature of BP10. The pH profile for BAPNA hydrolysis by Zn-BP10 (Figure 7.6 B,E) compares well with that of gelatin, showing a bell shaped curve that can be fitted to Equation (1). The ionization constants also fall w ithin experimental range, yielding pKa1 = 5.39 and pKa2 = 9.18 for the first order rate constant kcat, and pKa1 = 5.83 and pKa2 = 8.98 for kcat/ Km. The inhibition of Zn-BP10 by the metal ch elator 1,10-OP toward the hydrolysis of BAPNA at pH 7.5 shows a noncompetitive patte rn which is consis tent with metal removal from metalloenzymes and gives Ki = 7.85 M (Figure 7.8). The mixed-type inhibition by Arg-NHOH (Fig. 7.8 B) is a good indication of a combination of specific interaction along with meta l chelation afforded by th e hydroxamate moiety. The inhibition pattern for Arg-NHOH can be fitte d to Equations (2) and (3) to yield Kic = 0.20 mM and Kiu = 0.90 mM, representing th e specific inhibition cons tant for the dissociation of the enzyme-inhibitor complex (EI) and the catalytic inhibition constant for the dissociation of the inhibitor from the en zyme-substrate-inhibitor complex (ESI), respectively.28 V K V K I Kapp m app m icmax max[] 1 Equation (2) V V I Kapp iumax max[] 1Equation (3)

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258 Mechanistic studies of the coppe r derivative of BP10 (Cu-BP10) : The spectroscopically inert Zn2+ ion found in astacins and several other metallohydrolases offers a poor probe for the metal coordination environment in the active site. Thus spectroscopically active metal derivatives of metalloenzymes can offer detailed insight into the catalytic mechanisms and structure within the active site.29 The formation of the Cu-BP10 derivative is evident by the intense Tyr-to-Cu2+ LMCT at 454 nm, analogous to that in Cu2+-astacin and Cu2+-serralysin. The activity of the Cu2+-substituted BP10 (CuBP10) is considerably higher than Zn-BP10 in terms of kcat (0.76 s–1) and kcat/ Km (5430 M–1 s–1) toward the hydrolysis of BAPNA, refl ecting a ~960 % increase in activity in terms kcat and ~485 % in terms of kcat/ Km. This is a rather unusual characteristic of metal derivatives of Zn enzymes,29 since most Cu2+ derivatives of Zn2+-enzymes are inactive. Increased activity of a Cu2+ derivative has been observed in serralysin, another astacin family member able to hydrolyze gelatin and BAPNA.5 Moreover, the overall proteolytic activity of Cu-BP10 toward gelatin hydrolysis is ~20% of that of the Zn derivative, which is also greater than many metal-substituted metallo-hydrolases previously reported.29 Metal-centered hydrolysis re lies on the Lewis acidity of metal ions, which can lower the pKa of metal-bound water molecu les by greater than 107 fold, generating a metal-hydroxide at neutral pH that can perf orm nucleophillic attack on the scissile peptide bond. The versatility of metal-centered hydrolysis has been widely demonstrated in synthetic Cu2+ model systems which show proficient peptidase activities and phosphodiesterase activities.30-32 Conversely, Cu2+ derivatives of metallohydrolases are generally inactive or exhibit considerably lower activities29, 33 despite the comparatively

PAGE 279

259 high Lewis acidity of Cu2+. Few examples of metal-substituted metallohydrolases in literature show considerable activation, with serralysin5 and astacin34 being the most significant representatives of metal-substitute d metallohydrolases that are activated by Cu2+. The poor activation of meta llohydrolases observed in Cu2+ derivatives may be attributed to Jahn-Teller distortion which can reduce the nucleophilicty of the metal bound water if positioned in the axial coordination of the metal center. The ability to use Cu2+ as a viable probe for mechanistic studies is characteristic for the astacin family of metalloenzymes. It is notewor thy that the metal ligands found in the astacin family are a unique example of metal-phenolate coordi nation in metallohydrol ases wherein the coordinated Tyr plays a “sw itch-off” role in catalysis5, 19 and may be involved in the unique Cu2+-activation observed in astacin family enzymes thus far characterized. The analysis of BP10 is consistent with this mechanism of hydrolysis discussed below. From analysis of kinetic parameters kcat and Km, there is an obvious requirement of the metal center for catalysis. Km value for the hydrolysis of BAPNA by Cu-BP10 in the presence of Ca2+ ( Km = 1.32 mM) is not significantly different (200 %)from the Zn form (0.66mM) when compared to the 960 % increase in kcat of Cu-BP10 compared to the native Zn-BP10. Once again, as previ ously discussed, a small change in Km concomitant with a large increase in kcat suggests a lowering of the activ ation energy and an increase in the affinity for the substrate in the ES complex. The hyperactive Cu-BP10 suggests that the metal center must be involved in cat alysis, most importantly in the turnover of the ES complexes to the product with a high kcat value.

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260 0.000 0.005 0.010 0.015 kcat s1 0.00 0.04 0.08 0 100 200 kcat/ Km M1s1 0 50 100 A B D E Figure 7.6. pH dependence of kcat and kcat/ Km for the hydrolysis of gelatin by Zn-BP10 ( A D ), hydrolysis of BAPNA by Zn-BP10 ( B E ). The solid traces are the best fit to the equation ] H [ 1 ] [H 12 1 lim a aK K k k to afford the two pKa values reported in the text.

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261 [L-BAPNA] mM rate mM/s 0 1e-6 2e-6 rate mM/s 0.0 4.0e-5 8.0e-5 1.2e-4 A B Figure 7.7. (A) Hydrolysis of L-BAPNA by Zn-BP10 in 50.0 mM HEPES, pH 7.5 in the presence of 50mM NaCl, and 1.0 mM Ca(NO3)2. (B) Same as in (A) in the absence of Ca2+.

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262 Because of the low concentrations ava ilable for BP10 (above ~ 70 M the protein precipitates), electronic spectra of the Cu2+ derivative was used to study the metal coordination environment in the activ e site. Upon substitution of Cu2+ during the refolding protocols, a 20 M sample can show significant ligand to metal charge transfer transition (LMCT) in the visible range. Unfortunately, the low energy d-d transition bands for tetragonally di storted octahedral Cu2+ have very low mola r absorptivity values (in the order of 100 M–1cm–1) and are too noisy to disti nguish in the spectrum (Figure 7.9). The large LMCT ( = 1220 M–1cm–1) observed at 454 nm are due to the tyrosinateto-Cu2+ charge transfer transition as dete rmined previously using Cu-astacin.34 This observation serves as support of EP R studies of Cu-astacin where g > g spectral features suggests that the metal center is te tragonaly distorted with a weak axial ligand.19 This is further proof of a similar as tacin-like active site structure for BP10. The inhibition of Zn2+ and Cu2+ derivatives of BP10 by Arg-NHOH at pH 7.50 (Figure 7.8, 7.10) displays a mixed type pattern as observed in serralysin,19 wherein the inhibitor is able to bind both the enzyme a nd the ES complex. The mixed type inhibition yields two different inhibition constants for the dissociation of the inhibitor form the EI and EIS complexes, yielding Kic = 1.58 M and Kiu = 3.93 M. The significantly different inhibition constants for Zn-BP 10 and Cu-BP10 are good evidence that the inhibitor binds directly to the metal center in the active site. Mixe d-type inhibition is often a good indicator of an alternative site fo r inhibitor and substrat e binding in the ES complex to afford an ES-I and ES-S ternary complexes.28

PAGE 283

263 -202461/rate (mM/s)1 5.0e+5 1.0e+6 1.5e+6 1/[L-BAPNA] mM1 0246 2e+4 4e+4 6e+4 8e+4 A B Figure 7.8. Inhibition of Zn-BP10 toward L-BAPNA hydrolysis in 50.0 mM HEPES pH 7.5, 50.0 mM, NaCl, 1.0 mM Ca(NO3)2 by 1,10 phenanthroline ( A ) and by Arg-NHOH ( B ). Inhibitor concentrations are as follows from bottom to top: ( A ) 0, 0.5, 1.0, and 2.0 mM; ( B ) 0, 0.25, 0.5, and 1.0 mM.

PAGE 284

264 The influence on the LMCT centered at 454 nm can serve as an indi cator for inhibitor binding directly to the meta l center (Figure 7.9). The quenching of the LMCT band upon inhibitor binding indicates that the metalcoordinated Tyr is detached upon inhibitor binding, a phenomenon observed in the stud ies of Cu-astacin and Cu-serralysin.5 The gradual decrease of the LMCT intensity upon inhibitor binding can be described according to Scheme (1), assuming that the binding of one equivalent of inhibitor per active site metal results in concomitant detachment of the coordinated Tyr. BP10(bound Tyr) +Arg-NHOH Arg-NHOH-BP10 + detached Ty r When fitting the quenching of the Tyr-to-Cu2+ charge transfer with respect to inhibitor concentration according to Scheme (1) without including [H+] gives an apparent association constant of 2.9 103 M–1 for Arg-NHOH binding to Cu-BP10 at pH 8.5 (Figure 7.9, inset). The sp ecific inhibition constant Kic for the inhibition of Cu-BP10 by Arg-NHOH at pH 7.5 (Figure 7.10) can be conve rted into an appa rent association constant of 6.33 105 M–1, greater than that at pH 8.5, i ndicating that the protonation of the metal-coordinated Tyr at lower pH assists the bi nding of Arg-NHOH. The kinetic parameters Km, kcat, and kcat/ Km for the hydrolysis of BAPNA by CuBP10 were determined between pH’s 5.0-9.5 and exhibit bell shaped curves that can be fitted to Equation (1) to give pKa1 = 5.48 and pKa2 = 7.98 for the pH dependence of kcat and pKa1 = 5.83 and pKa2 = 7.99 for the pH dependence of kcat/ Km (Figure 7.11). This describes a two ionization mech anism for the catalytic turnov er of the substrate by BP10. The similar crystal structures of di fferent metal derivatives of astacin34 suggest that the

PAGE 285

265 wavelength nm 400500600700800 M -1 cm -1 0 400 800 1200 [Arg-NHOH] mM 0246810 M -1 cm -1 3e+2 6e+2 9e+2 Figure 7.9. Optical titration of Arg-NHOH to Cu-BP10. The inset shows the decrease in the change of the molar absorptivity ( ) as a function [Arg-NHOH].

PAGE 286

266 1/[BAPNA] mM-1 02461/rate (mM/s)-1 5e+3 1e+4 Figure 7.10. Inhibition of Cu-BP10 by Arg-NHOH. Inhibitor concentrations are as follows from bottom to top: 0, 1.5, and 3.0 M.

PAGE 287

267 pH 4681012 k cat s -1 0.0 0.3 0.6 pH 4681012 k cat /K m M -1 s -1 0 200 400 A B Figure 7.11. pH profile of BAPNA hydr olysis by Cu-BP10.

PAGE 288

268 coordination sphere of Zn2+ and Cu2+ derivatives of BP10 would be similar. This would possibly attribute the differ ent ionization constants pKa1 and pKa2 for each metal derivative to the Lewis acidity of each metal, but not due to different metal environments within the active site of BP10. The low pKa2 approaching pKa1 for Cu-BP10 causes a considerable decrease in th e catalytic efficiency, from the intrinsic value of 851 M–1s–1 to the maximum fitted value of 580 M–1s–1 which means that only 64% of Cu-BP10 is active at pH 7.0. Conversely the intr insic and fitted values for kcat/ Km of Zn-BP10 differ only slightly with the in trinsic value of 119.7 M–1s–1 and the fitted value of 125.1 M–1s–1. This is consistent with the coordination sphere of the two metal derivatives of BP10 being similar, and suggest that the differences in io nization constants is due to the effects on the Lewis acidity of each metal. Ionizable groups must be coordinated or in very close proximity of the metal in the active site of an enzyme to be influenced by the metal ion, as re flected by a change of pKa values. In astacin the crystal structure s uggests that Tyr and Glu as well as a water molecule are bound to the metal center. This “metallotriad” framework of M––OH…–OOC is similar to the “catalytic tria d” of serine prot eases Ser–OH…His …–OOC, where the water nucleophile is sandwiche d by a Lewis acid (the active site metal) and a Lewis base (the carboxylat e in Glu) to serve in a gene ral-acid/general -base catalytic mechanism. This metal-centered tria d has also been confirmed in other metallohydrolases including serralysin,5 thermolysin,35 matrilysin,36 and carboxypeptidase A.37 We utilized the Cu2+ derivative of BP10 to determine if a similar mechanism is utilized by this enzyme.

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269 The 454 nm LMCT band does not show fu ll intensity at neutral and lower pH values (~150 M–1cm–1 at pH 6.0 versus 1255 M–1cm–1 at pH 8.5), indicating that they are pH dependent. The Tyr(phenolate)-to-Cu2+ CT in Cu-BP10 changes with pH in a sigmoidal manner (Figure 7.12). Thus, th e change can be described by a single ionization process5 for the ionization of the coordinated Tyr249 and simultaneous binding to the active site Cu2+ to give u = 109 and b = 1182 M–1cm–1 and a pKa value of 6.87 for the deprotonation of Tyr249, where u and b are the molar absorptivities due to the background and the Cu-bound Tyr249. However, the data do not fit well to a singleionization model (Fig. 7. 10, dotted trace) and the pKa value of 6.86 is not a close match to the kcat/ Km pKa2 value of 7.99. Since proposed models of astacin-type en zymes show that the Tyr side chain is Hbonded to the metal-coordinated water, the deprotonation of the phenolate moiety and subsequent binding to the active-site metal should be affected by the ionization of the coordinated water and reflected in the CT intensities. The data is much better fitted to a two-ionization process described previously5 with a fixed pKa1 = 5.83 (from the kcat/ Km pH profile of Cu-BP10, (Figure 7.11B) to afford pKa2 = 7.42 0.15, u = 26 15, W = 591 33, and b = 1425 M–1cm–1 (Figure 7.10 solid trace). Although the data can be reasonably fitted to a two-i onization process, the similar max throughout the titration suggests that it is likely to have only one species that affords the LMCT instead of two. Taken together our data is most consisten tly described as a si ngle species ionization involved in the LMCT, assigned to a Tyr249 to Cu2+ charge transfer. Herein, the increase in the LMCT due to Tyr binding corresponds to the decrease in act ivity, reflecting the

PAGE 290

270 pH 5678910 k cat /K m M -1 s -1 0 200 400 600 cm 0 500 1000 1500 Figure 7.12. The change in intensity of the LM CT transition of Cu-BP10 at 454 nm as a function of pH. The data is much bett er fitted to a two ioni zation processes (solid trace) than to a single ionization process (d ashed trace). The dashed bell-shaped curve is the best fit for the kcat/Km vs. pH profile of Cu BP10 from Figure 7.11B.

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271 inhibiting role of Tyr249 and its role as a “catalytic switch”. Homology Modeling : The catalytic domain of the astacin family of metallohydrolases is highly conserved across all sequenced enzymes. Because BP10 and serralysin are both able to cleave BAPNA effectively, homology modeling by the use of serralysin as the template for the catalytic domain and substrate binding was performed (Figure 7.11). Molecular m echanics calculations (MM3) a nd molecular dynamics were used to arrive at the final structure which shows Tyr249 within H-bonding distance of the metal-bound water and to the guanidine group of arginine in BAPNA. A predominant hydrophobic interaction is also observed between Trp165 and the benzoyl group of the substrate. The p-nitroanilide moiety of BAPNA is exposed to solvent which likely facilitates product rel ease after the cleavage of the substrate. IV. CONCLUDING REMARKS BP10 is the first member of the tolloidlike enzymes to be characterized with extensive kinetics and spectroscopic methods The studies show that BP10 is a metallohydrolase with a hydrolytic mechanis m consistent with other astacin-like proteases. The influence of Ca2+ toward the catalysis of gelatin and BAPNA by BP10 offers the important insight that Ca2+-signaling can serve an important function in regulation of the proteolytic ev ents in embryogenesis. The studies of the Cu-derivative support the metallotriad mechanism previously proposed for astacin and serralysin, with the involvement of the metal bound Tyr re sidue in catalysis Through homology

PAGE 292

272 Figure 7.13 Stereo view of the BP10 active site based on the crystal structure of serralysin (PDB ID 1SAT) as the homol ogy template with the use of molecular mechanics (MM3) and molecular dynamics calculations. The substrate BAPNA (ball-and-stick structure) is docked into the active site, with its carbonyl of the scissile bond pointing toward the active si te metal (larger sphere), the benzoyl moiety of BAPNA interacting with Trp165, and the guanidinium group of the Arg moiety interacting with Tyr249.

PAGE 293

273 modeling we observed the conserved astacin ca talytic motif and that the active-site Tyr may not only play a role in substrate bindi ng via detachment from the metal during a “resting” state, but also ma y assist in stabilization of the ES complex by directly interacting with the guanidine group of BAPNA via a charge interaction or H-bonding. The understanding of the detailed mechan ism of peptide hydrolysis by BP10 and revealing of the substrate specificity in vivo in future studies are important first steps in unraveling the proteolytic events during sea urchin embryogenesis. 2 3 4 5

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274 IV. LIST OF REFERENCES 1) J.S. Bond, R.J. Beynon, The astaci n family of metalloendopeptidases. Prot. Sci (1995) 4, 1247-1261. 2) N.M Hooper, (ed) Zinc Metalloproteases is Health and Disease Chap. 2, Taylor and Francis, Bristol, PA (1996). 3) F. Moehrlen, H. Hutter, R. Zwilling, X -ray absorption spectroscopy study of zinc coordination in tetanus neurotoxin, asta cin, alkaline protease and thermolysin. Euro. J. Biochem (2003), 270, 4909-4920. 4) F.X. Gomis-Rth, W. Stcker, H. Hube r, R. Zwilling, W. Bode, Refined 1.8 .ANG. xray crystal structure of as tacin, a zinc-endopeptidase fr om the crayfish Astacus astacus L. Structure determination, refine ment, molecular structure and comparison with hermolysin. J. Mol. Biol (1993), 229, 945-968. 5) H.I. Park, L.-J. Ming, Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism. J. Biol. Inorg. Chem (2002), 7, 600-610. 6) T. Lepage, C. Ghiglione, C. Gache, Spa tial and temporal expre ssion pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Dr osophila dorsal-ventral patterning gene tolloid. Development (1992), 114, 147-163.

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275 7) T. Lepage, C. Ghiglione, C. Gache, Stru cture of the gene en coding the sea urchin blastula protease 10 (BP10), a memb er of the astacin family of Zn2+-metalloproteases. Eur. J. Biochem (1996), 238, 744-751. 8) E. Appella, I.T. Weber, F. Blasi, F. Stru cture and function of epidermal growth factorlike regions in proteins. FEBS letters (1988), 231, 1-4. 9) N. Hartigan, L. Garrigue-Antar, K.E. Ka dler, Bone Morphogenetic Protein-1 (BMP-1). J. Biol. Chem (2003), 278, 18045-18049. 10) M.D. Sternlicht, Z. Werb, How matrix metalloproteinases regul ate cell behavior. Cell. Dev. Biol. (2001), 17, 463-516. 11) K. Imai, M. Kusakabe, T. Sakakura, I. Nakanishiki, Y. Okada, Susceptibility of tenascin to degradation by matrix meta lloproteinases and serine proteinases. FEBS Lett. (1994), 352, 216-218. 12) O. Vafa, D. Nishioka, Developmentally regulated pr otease expression during sea urchin embryogenesis. Mol. Reprod. Dev. (1995), 40, 36-47. 13) G. Karakiulakis, E. Papakonstantin ou, M.E. Maragoudakis, G.N. Miservic, Expression of type IV collagen-degr ading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis. J. Cell. Biochem. (1993), 52, 92-106. 14) J.P. Quigley, R.S. Braithwaite, P.B. Armstrong, Matrix metalloproteases of the developing sea urchin embryo. Differentiation (1993), 54, 19-23.

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276 15) C. Sharpe, J.J. Robinson, Characterizat ion of matrix metalloprotease activities induced in the sea urchin extraembryonic matrix, the hyaline layer. Biochem. Cell. Biol. (2001), 79, 461-468. 16) W. Stcker, F. Grams, U. Baumann, P. Reinemer, F.X. Gomis-Rth, D.B. McKay, W. Bode, The metzincins topological and sequential rela tions between the astacins, adamalysins, serralysins, and matrixins (col lagenases) define a superfamily of zincpeptidases. Prot. Sci. (1995), 4, 823-840. 17) E. Kimura, Macrocyclic polyamine zinc (II) complexes as advanced models for zinc(II) enzymes. Prog. Inorg. Chem (1994), 41, 443. 18) E. Kimura, T. Koike, Intrinsic properties of zinc(II) ion pertinent to zinc enzymes. Adv Inorg. Chem (1997), 44, 229. 19) H.I. Park, L.-J. Ming, The mechanistic role of the coordinated tyrosine in astacin. J. Inorg. Biochem. (1998), 72, 57-62. 20) Y.-H. Chen, J.T. Yang, H.M. Martinez, Dete rmination of the secondary structures of proteins by circular dichroism a nd optical rotatory dispersion. Biochemistry (1972), 11, 4120-4131. 21) G. Murphy, T. Crabbe, Gelatinases A and B. Methods in Enzymology (1995) 248, 470-475. 22) U.K. Laemmli, Cleavage of structural pr oteins during the assembly of the head of bacteriophage T4. Nature (1970), 227, 680-685.

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277 23) J.D. Grubb, Purification and assa ys of bacterial gelatinases. Methods in Ezymology (1994), 235, 602-606. 24) M. Friedman, Applications of the ninhydr in reaction for analysis of amino acids, peptides, and proteins to agricu ltural and biomedical sciences. J. Agric. Food Chem. (2004), 52, 385-406. 25) A.L. Sieron, A.S. Tretiakova, S. LundKatz, M.T. Khan, S.-W. Li, W. Stcker. Structure and Function of Procollagen C-Pr oteinase (mTolloid) Domains Determined by Protease Digestion, Circular Dichroism, Binding to Procollagen Type I, and Computer Modeling. Biochemistry (2000), 39, 3231-3239. 26) J. Mayne, J.J. Robisnon, Calcium-pr otein interactions in the extracellular environment: calcium binding, activa tion, and immunolocalization of a collagenase/gelatinase activity expr essed in the s ea urchin embryo. J. Cell. Biochem. (1998), 71, 546-558. 27) C. Calloway, C. Sharpe, J.J. Robinson, Id entification and partia l characterization of two inducible gelatin-cleavag e activities localized to th e sea urchin extraembryonic matrix, the hyaline layer. Biochim. Biophys. Acta (2003), 1621, 67-75. 28) A. Cornish-Bowden, Fundamentals of Enzyme Kinetics Portland Press, London (1995). 29) I. Bertini, H.B. Gray, S.J. Lippard, J.S. Valentine, (eds) Bioinorganic Chemistry Chap. 2., University Science Books, Sausalito, CA (1994).

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278 30) E.L. Hegg, J.N. Burstyn, Toward the deve lopment of metal-based synthetic nucleases and peptidases: a rationale and progress report in applying the principles of coordination chemistry. Coord. Chem. Rev. (1998), 173, 133-165. 31) S.T. Frey, N.N. Murthy, S.T. Weintraub, L.K. Thompson, K.D. Karlin, Hydrolysis of Unactivated Esters and Acetonitrile Hydr ation by a Hydroxo-Dicopper(II) Complex. Inorg. Chem. (1997), 36, 956-957. 32) V. Lykourinou-Tibbs, K. Bisht, L.-J. Mi ng, Effective heterogene ous hydrolysis of phosphodiester by pyridine-cont aining metallopolymers. Inorg. Chim. Acta. (2005), 358, 1247-1252. 33) E. Kimura, T. Koike, Intrinsic properties of zinc(II) ion pertinent to zinc enzymes Adv. Inorg. Chem. (1997), 44, 229-261. 34) F.X. Gomis-Rth, F. Grams, I. Yiallour os, H. Nar, U. Ksthardt, R. Zwilling, W. Bode, W. Stcker, Crystal structures, spect roscopic features, a nd catalytic properties of cobalt(II), copper(II), ni ckel(II), and mercury(II) de rivatives of the zinc endopeptidase astacin. A co rrelation of structure a nd proteolytic activity. J. Biol.Chem. (1994), 269, 17111-17117. 35) B.W. Matthews, Metalloaminopeptidases : Common functional themes in disparate structural surroundings. Acc. Chem. Res. (1998), 21, 333-340. 36) J. Cha, D.S. Auld, Site-directed mutage nesis of the active site glutamate in human matrilysin: Investigation of its role in catalysis. Biochemistry (1997), 36, 1601916024.

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279 37) D.W. Christianson, W.N. Lipscomb, Carboxypeptidase A. Acc. Chem. Res. (1989), 22, 62-69.

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ABOUT THE AUTHOR Giordano F.Z. da Silva was born in 1977 in the city of Rio de Janeiro, Brazil. Giordano graduated Magna Cum Laude from St Petersburg High School and attended St. Petersburg Junior College, graduating Magna Cum Laude in 1998 with an Associate of Arts degree with emphasis in the natural scie nces. In 1999 he enrolled at the University of South Florida and graduated in 2002 with a Bachelor of Arts. Giordano joined the graduate program in the Chemistry Departme nt at USF in 2002 working in Dr. Li-June MingÂ’s M etallo B iomolecule I nterest G roup (MBIG) and Dr. Bria n T. Livingston in the Department of Biology. In the five years in the graduate program Giordano has presented his work at the local, state, national, a nd international levels; taught a bioinorganic workshop at the National Chiayi University in Taiwan; published p eer-reviewed top-tier journal articles, and received several awards, including the Theodore Ashford and George Bursa departmental awards and the USF Successful Latino Award.


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