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Metallopeptides as model systems for the study of Cu(ii)-dependent oxidation chemistry

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Title:
Metallopeptides as model systems for the study of Cu(ii)-dependent oxidation chemistry
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Book
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English
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Tay, William Maung
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Bacitracin
Histatin 5
Copper (II)
Oxygen
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Copper is one of the essential metal ions for aerobic organisms. Two well known functions of copper in the biological systems are electron transfer and molecular oxygen interaction. Thus, this metal can be found in haemocyanin, an oxygen carrier protein, and superoxide dismutase, an enzyme that involves in electron transfer. In addition, having a positive redox potential allows copper to be involved in redox chemistry. It is the redox properties of copper that are responsible for many important biochemical processes. Although the copper-containing oxidases have been well studied over the years, certain mechanistic details such as reaction intermediates remain to be elucidated. Several research groups have been trying to study this by trying to mimic the native systems, synthesizing bulky organic molecules with copper-binding and oxidative capabilities. However, these model systems are only applicable in organic solvents at low temperatures. In this study, three naturally occurring peptides, amyloid-β, bacitracin, and histatin 5, have been shown to display the oxidative chemistry when complexed with CusuperscriptII. A combination of spectroscopic (UV-Vis and NMR) and reactivity was used in studying their metal-binding properties as well as in elucidating their catalytic mechanism.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
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by William Maung Tay.
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Title from PDF of title page.
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Document formatted into pages; contains 209 pages.
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Includes vita.

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ABSTRACT: Copper is one of the essential metal ions for aerobic organisms. Two well known functions of copper in the biological systems are electron transfer and molecular oxygen interaction. Thus, this metal can be found in haemocyanin, an oxygen carrier protein, and superoxide dismutase, an enzyme that involves in electron transfer. In addition, having a positive redox potential allows copper to be involved in redox chemistry. It is the redox properties of copper that are responsible for many important biochemical processes. Although the copper-containing oxidases have been well studied over the years, certain mechanistic details such as reaction intermediates remain to be elucidated. Several research groups have been trying to study this by trying to mimic the native systems, synthesizing bulky organic molecules with copper-binding and oxidative capabilities. However, these model systems are only applicable in organic solvents at low temperatures. In this study, three naturally occurring peptides, amyloid-§, bacitracin, and histatin 5, have been shown to display the oxidative chemistry when complexed with Cu[superscript]II. A combination of spectroscopic (UV-Vis and NMR) and reactivity was used in studying their metal-binding properties as well as in elucidating their catalytic mechanism.
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Histatin 5
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Oxygen
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Metallopeptides As Model Systems For The Study Of Cu(II)-Dependent Oxidation Chemistry by William Maung Tay 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 Major Professor: Li-June Ming, Ph.D. Steven H. Grossman, Ph.D. Kirpal S. Bisht, Ph.D. Peter Zhang, Ph.D. Jun Tan, M.D., Ph.D. Date of Approval: April 1, 2008 Keywords: amyloid, bacitracin, histatin 5, copper (II), oxygen, catechol oxidation, antimicrobial peptides, Alzheimer's dis ease, hydrogen peroxide, kinetics, NMR spectroscopy Copyright 2008 William Maung Tay

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DEDICATION Overall, my graduate career had been a special journey a nd a very rewarding experience. In addition to the academic en richment, I had the opportunity to gain life experiences and make life-long friends. None of these would have been possible without the strong support of my family. First and foremost, I would like to dedicate this work to my parents Ma Khin Win and Kian Chwan Ta y, who sacrificed a lot to come to the United States in search of a better life for their children. Their strong will and dedication have been an inspiration all throughout my academic career. With this accomplishment, I hope to set an example in perseverance to my si blings as well as to future generations. I would also like to dedicate this accomplis hment to my girlfriend Jennifer Wellborn for always being there during difficult times and loving me for who I am. Next, I would like to make a special dedication to my grandfather, who is currently suffering from end-stage Alzheimers disease. He had always been a loving and caring family man of great character as well as a strong supporter of education. I would also like to extend my dedication to Jens late grandmot her Mary Wellborn, who also succumbed to this debilitating disease. Granny was a devoted grandmother and is greatly missed by us.

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ACKNOWLEDGEMENT First, I would like to ack nowledge Dr. Li-June Ming for being a great mentor and providing me with the opportunity to do rese arch in his lab. Next, I must acknowledge my big sister Dr. Vasiliki Lykourinou and big brother Dr. Giordano F. Z. da Silva for their emotional support as well as professiona l guidance. I must also acknowledge my other sisters Dr. Brianne OL eary, Dr. Brenda Held, and Erin for being supportive during difficult times. I would also like to acknowledge Kashmir Juneja, who is like a younger brother, for always providing a good laugh and being a great opponent in basketball. William Wagner, Alaa Hashim, and Justin Moses must be acknowledged for being great friends and colleagues, who have been very s upportive in times of n eed. Finally, I would like to acknowledge my committee members Dr. Steven Grossman, Dr. Kirpal Bisht, Dr. Peter Zhang, and Dr. Jun Tan for their patience and guidance.

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NOTE TO THE READER Note to Reader: The original of this document contains color that is necessary for understanding the data. The or iginal dissertations is on file with the USF library in Tampa, Florida.

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TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v LIST OF ABBREVIATIONS ix ABSTRACT xii CHAPTER 1: COPPER-OXYGEN CHEMISTRY: A BRIEF OVERVEW 1 1.1 Introduction 2 1.2 Cu Proteins 3 1.2.1 Dinuclear Copper Proteins 4 1.2.2 Mononuclear Copper Proteins 6 1.3 Biomimetic Synthetic Models Complexes 16 1.4 Metallopeptides 21 REFERENCES 24 CHAPTER 2: ANTIOXIDATIVE PR OPERTIES OF A NATURALLY OCCURRING FLAVONOID, QUERCETIN : IMPLICATIONS TOWARD TREATMENT STRATE GIES FOR ALZHEIMERS DISEASE 38 2.1 Amyloid 39 2.1.1 Flavonoids 49 i

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2.2 Materials and Methods 52 2.2.1 Kinetic Studies 53 2.2.2 Optical Studies 54 2.2.3 NMR Studies 55 2.3 Results and Discussion 55 2.4 Conclusion 95 REFERENCES 98 CHAPTER 3: ELUCIDATION OF THE IN VITRO OXIDATION CHEMISTRY OF COPPER(II)BACITRACIN COMPLEX 117 3.1 Bacitracin 118 3.1.1 Structure of Bacitracin 119 3.1.2 Metal Binding and Antibacterial Mechanism 120 3.2 Materials and Methods 124 3.2.1 Kinetic Studies 124 3.2.2 Optical Studies 126 3.2.3 Anaerobic Studies 127 3.2.4 DNA Cleavage Assay 127 3.3 Results and Discussion 128 3.4 Conclusion 152 REFERENCES 155 ii

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CHAPTER 4: ELUCIDATION OF THE IN VITRO OXIDATION CHEMISTRY OF COPPER(II)HISTATIN 5 COMPLEX BY MEANS OF REACTIVITY STUDIES 166 4.1 Traditional Antibiotics 167 4.1.1 Antimicrobial Peptides 168 4.1.2 Candida albicans 170 4.1.3 Histatin 5 171 4.2 Materials and Methods 177 4.2.1 Kinetic Studies 177 4.2.2 Optical Studies 178 4.2.3 NMR Studies 179 4.3 Results and Discussion 179 4.4 Conclusion 197 REFERENCES 199 ABOUT THE AUTHOR End Page iii

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LIST OF TABLES Table 2.1: Full assignment of the 1H NMR spectrum of the 1:1 Co2+Qr complex from 1D saturation transf er and 2D EXSY experiments and their corresponding T1 relaxation times. 78 Table 2.2: Full assignment of the 1H NMR spectrum of the 1:1 Yb3+Qr complex from 1D saturation transf er and 2D EXSY experiments and their corresponding T1 relaxation times. 89 iv

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LIST OF FIGURES Figure 1.1: Proposed Mechanism for tyro sinase. 7 Figure 1.2: Crystal structure of tw o oxygen-binding functional units of Octopus hemocyanin (PDB ID: 1JS8). 8 Figure 1.3: Crystal structure of Cu,Z n-superoxide dismutase with the two metal center shown in ball and stick confi guration (PDB ID: 2c9v). 10 Figure 1.4: Mechanism of Cu,Zn-superoxide dismutase. 12 Figure 1.5: Crystal structure of peptidylglycine -hydroxylating monooxygenase (PDB ID: 3PHM). 14 Figure 1.6: Proposed mechanism for peptidylglycine -hydroxylating monooxygenase and dopamine -monooxygenase. 15 Figure 1.7: End-on versus side-on binding mode (top) and a bidentate (LPy1) versus a tridentate (LPy2) ligand (bottom). 19 Figure 1.8: Ligands used for the pr eparation of Cu complexes: TPAR (Karlin); trenR,R (Tolman); HB(3t Bu-5i Prpz)3 (Kitajima). 20 Figure 2.1: Proposed mechanism for CuII-centered oxidation of catecholcontaining substrates in the presence (AD) and absence (FH and BD) of H2O2. 46 Figure 2.2: Proposed mechanism for the CuII-centered hydroxylation and oxidation of phenol-like substrate 47 Figure 2.3: Structure of quercetin (I), 5-hydroxyfla vone (II), and catechin (III). 51 Figure 2.4: Quercetin inhibition of catechol oxidation by CuIIA 1. 60 v

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Figure 2.5: Lineweaver-Burk analysis of quercetin inhibi tion of catechol oxidation by CuIIA 1 and replot of the slope versus inhibitor concentrations. 61 Figure 2.6: Optical titration of CuII to quercetin in DMSO. 64 Figure 2.7: Optical Job plot of CuII binding by quercetin in DMSO. 66 Figure 2.8: Optical titration of CoII to quercetin in DMSO. 67 Figure 2.9: Optical Job plot of CoII binding to quercetin in DMSO. 69 Figure 2.10: Optical CoII titration of catechin and 5-hydroxyflavone. 70 Figure 2.11: 1D NMR spectrum of CoII-quercetin-triethylamine in a 1:5:1.5 ratio in d6DMSO. 73 Figure 2.12: The 2D 1H EXSY spectrum of the 1:1 CoIIquercetin complex in d6-DMSO. 75 Figure 2.13: 1D saturation-transfer results for the CoII-quercetin complex. 76 Figure 2.14: Optical titration of CaII to quercetin in DMSO. 80 Figure 2.15: Calcium influence on quercetin inhibition of catechol oxidation by CuIIA 1. 83 Figure 2.16: 1D NMR spectrum of YbIII-quercetin-triethylamine in a 1:1:0.7 ratio in d6DMSO. 85 Figure 2.17: The 2D 1H EXSY spectrum of the 1:1 YbIIIquercetin complex in d6-DMSO. 87 Figure 2.18: The 2D 1H EXSY spectrum of the expanded region of ~12 to ppm for Fig. 2.17 of the 1:1 YbIIIquercetin complex in d6-DMSO. 88 Figure 2.19: Optical titration of YbIII to quercetin at 20.0 M in DMSO. 91 Figure 2.20: Optical Job plot of YbIII binding to quercetin in DMSO. 93 vi

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Figure 2.21: Ytterbium influence on querce tin inhibition of catechol oxidation by CuIIA 1. 94 Figure 3.1: Structures of bacitracin congeners (a dopted from ref. 2). 121 Figure 3.2: Oxidative cleavage of 225 ng of plasmid DNA by 25.0 M CuII-bacitrcin with 0.05% H2O2 in 100.0 mM HEPES buffer at pH 7.0 and 25 C. 129 Figure 3.3: Catechol oxidation by CuII-bacitrcin in 100.0 mM HEPES at pH 7.0 and 25 C. 132 Figure 3.4: 4,5dichlorocatechol (DCC) binding study of CuII-bacitracin in DMF. 133 Figure 3.5: Optical Job binding study of DCC toward the CuII-bacitrcin complex. 135 Figure 3.6: Interaction between the CuII-bacitracin and cate chol using the Job method. 136 Figure 3.7: The oxidation of 2.5 mM catechol by 100.0 M CuII-bacitracin under anaerobic condition. 138 Figure 3.8: Addition of air into anaerobic sample containing 100.0 M CuII-bacitracin and 2.5 mM catechol. 140 Figure 3.9: Hydrogen peroxide infl uence on catechol oxidation by 2.0 M CuII-bacitracin at pH 7.0 in 100.0 mM HEPES buffer at 25C. 143 Figure 3.10: Hanes analysis of oxidation of catechol by CuII-bacitracin at different concentrations of H2O2 (kinetic data from Figure 3.9 right). 145 Figure 3.11: ZnII (A), CoII (B), and NiII (C) dilution of CuII for the analysis of mononuclear versus dinuclear metal center in the catalysis of catechol oxidation by CuII-bacitracin. 149 Figure 3.12: Proposed mechanism for catechol oxidation by CuII-bacitracin vii

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through a mononuclear CuII-centered catalysis. 151 Figure 4.1: Optical CuII binding study of histatin 5 (200.0 M) in 100.0 mM HEPES buffer at pH 7.0 and 25 C. 181 Figure 4.2: The continuous-wav e (CW) EPR spectra of CuII-histatin 5 complexes in DMF. 183 Figure 4.3: NMR spectra of CoII-histatin 5 at 2:1 (bottom) and 6:1 (top) ratios with 8k scans. 184 Figure 4.4: Catechol oxidation by CuII-histatin 5 (4:1) in 100.0 mM HEPES at pH 7.0 and 25 C. 186 Figure 4.5: CuII binding of histatin 5 monito red with catechol oxidation activity. 188 Figure 4.6: CuII binding of Hn5 monitored with cat echol oxidation activity. 190 Figure 4.7: 4,5-dichlorocatech ol binding study of CuII-Hn5 in DMF. 192 Figure 4.8: Hydrogen peroxide infl uence on catechol oxidation by 0.2 M CuII-histatin 5 at pH 7.0 in 100.0 mM HEPES buffer at 25 C. 194 Figure 4.9: Hanes analysis of oxidation of catechol by CuII-Hn5 at different concentrations of H2O2 (kinetic data from Figure 4.7). 196 viii

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LIST OF ABBREVIATIONS CuII: Copper(II) CoII: Cobalt(II) NiII: Nickel(II) FeIII: Iron(III) ZnII: Zinc(II) ROS: Reactive Oxygen Species AD: Alzheimers Disease A : AmyloidCaII: Calcium YbIII: Ytterbium AMPs: Antimicrobial Peptides Qr: Quercetin Cat: Catechin Vmax: Maximum Velocity KM: Michaelis-Menten Constant KA: Affinity Constant ix

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KD: Dissociation Constant Ki: Inhibition Constant kcat: Catalytic Turnover ATP: Adenosine 5' Triphosphate EDTA: Ethylenediaminetetraacetic acid H2O2: Hydrogen Peroxide O2: molecular oxygen or dioxygen Bc: Bacitracin Hn: Histatins Hn5: Histatin 5 UV-Vis: Ultraviolet-Visible nm: Nanometer ppm: Parts-per-million Hz: Hertz 1H NMR: Proton Nuclear Magnetic Resonance EPR: Electron Paramagnetic Resonance ORD: Optical Rotatory Dispersion EXAFS: Extended X-ray Absorption Fine Structure DFT: Density Functional Theory d6-DMSO: deuterated Dimethyl Sulfoxide HEPES: N-(2-HydroxyEthyl)-Pipe razine-N'-2-EthaneSulfonic Acid x

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MES: 2-Morpholino-Ethanesulfonic Acid MBTH: 3-Methyl-2-BenzoThiazolinone Hydrazone Hydrochloride CA: Catechol DTBC: Di-Tert-Butyl Catechol DCC: Di-Chloro-Catechol MALDI-TOF: Matrix Assi sted Laser Desorption Mass Spectrometry Time Of Flight CD: Circular Dichroism DI: Deionized Water TEA: Triethylamine 5HF: 5-hydroxyflavone CN: Catechin xi

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Metallopeptides As Model Systems for th e Study of Cu(II)-Dependent Oxidation Chemistry William Maung Tay ABSTRACT Copper is one of the esse ntial metal ions for aerobic organisms. Two well known functions of copper in the bi ological systems are electron transfer and molecular oxygen interaction. Thus, this meta l can be found in haemocyanin, an oxygen carrier protein, and superoxide dismutase, an enzyme that involv es in electron transfer. In addition, having a positive redox potential allows copper to be involved in redox chemistry. It is the redox properties of copper that are responsible fo r many important biochemical processes. Although the copper-containing oxidases have b een well studied over the years, certain mechanistic details such as reaction intermed iates remain to be elucidated. Several research groups have been trying to study th is by trying to mimic the native systems, synthesizing bulky organic molecules with copper-binding and oxidati ve capabilities. However, these model systems are only a pplicable in organi c solvents at low temperatures. In this study, three naturally occurring peptides, amyloid, bacitracin, and xii

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xiii histatin 5, have been shown to display th e oxidative chemistry when complexed with CuII. A combination of spectro scopic (UV-Vis and NMR) and reactivity was used in studying their metal-binding properties as we ll as in elucidating their catalytic mechanism.

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CHAPTER 1 COPPER-OXYGEN CHEMISTRY: A BRIEF OVERVIEW 1

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1.1 Introduction Copper is essential for various biological processes, but can be very toxic when presence in excess. Some of the biologica l processes that involve Cu are antioxidant defenses, pigmentation, mitochondrial respira tion, neurotransmitter synthesis, connective tissue formation, peptide amidation, and iron metabolism.1 As a result, it is strictly regulated. Disturbances in this delicate balance can lead to detrimental disorders such as Menkes (i.e. Cu deficiency)2 and Wilson (i.e. Cu excess) disease.3 In the case of Cu deficiency, the inability to incorporate this metal into important enzymes like dopamine -monooxygenase and Cu,Zn-superoxide di smutase reduces the bodys ability to synthesize neurotransmitters and defend against oxidative stress, respectively. Thus, the clinical symptoms of Menkes disease are ne urological defects (such as severe metal retardation and neurodegeneration), gr owth retardation, hypothermia, and hypopigmentation.4 On the other extreme, the compli cations in Wilson disease are due to inability to remove Cu, leading to accumulation of Cu in the liver and brain. As a result, the victim typically suffers from liver failure and neurodegeneration.5 The toxicity of Cu is partially associat ed with its potential to bind and activate dioxygen (O2), leading to generation of reactive oxygen species (ROS).6 Ironically, the same harmful chemical property of Cu is th e one that has enabled us to survive and evolve into multicellular organims under th e oxidizing conditions of the Earths atmosphere.7 This is evident in the essential involvement of Cu in O2 transfer (i.e. hemocyanin), energy production (i.e. cytoch rome oxidase), and antioxidation (i.e. 2

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Cu,Zn-superoxide dismutase). The versatility and reactivity of Cu is largely owing to its flexibility in ligand binding by both po ssible oxidation states (i.e. CuI and CuII).7 Thus, it is essential to unde rstand the redox chemistry of Cu with respect to how the O2 is activated in order to better unders tand its pathological roles. In this respect, a general overview of Cu-O2 chemistry on selected dinuclear and mononuclear Cu centers in biological system s will be discussed herein. Then, a brief discussion of progresse s in the study of Cu-O2 chemistry using biomimetic approach through synthetic model complexes will follow. Finally, the chapter is concluded with an introduction to metallo-peptides with a relevance to Cu oxidation chemistry. 1.2 Cu Proteins Copper in biological systems can be classi fied into three groups named type I, type II, and type III.20 c Type I Cu consists of a group of small mononuclear Cucontaining proteins known as the blue Cu prot eins, owing to their visible intense blue color. This intense color is a result of a lig and-to-metal charge transfer between sulfur of Cys to CuII.8 A typical ligand environment of type I Cu consists of two His, one Cys, and an axial Met through a dist orted tetrahedral geometry. These blue Cu proteins are mainly involved in a single electron transfer processes. Type II Cu proteins contain a regular tetragonally distorted Cu ceter and can bind to a variety of amino acids to give different coordination geometry. These Cu proteins can participate in oxidation and oxygenation chemistry.19 c Type III Cu systems are composed of two CuII bridged by 3

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H2O or OH, and the two CuII ions are antiferromagnetically coupled. Type III Cu proteins can also be involved in oxidation and oxygenation chemistry.9 1.2.1 Dinuclear Cu Proteins Tyrosinase, Catechol Oxidase, and Hemocyanin. Tyrosinase is a type III di-CuII protein ubiquitously ex pressed in nature.10 The molecular weight of tyrosinase can vary from 14 to 43 kDa depending on the source.11 In plants, sponges, arthropods, and many invertebrates, they are involved in w ound healing and primary immune response.12 In mammals, they are expressed in melanocytes of the retina and skin and are responsible for melanin formation.13 Furthermore, this enzyme is the cause of fruit and vegetable browning. By activating molecular oxyge n, tyrosinase can exhibit two types of activities toward phenol-containing substrates: cresolase or monophenolase activity and catecholase or diphenolase activit y. The di-Cu center can be in four different states: CuICuI ( deoxy); CuICuII ( half met ); CuII-OH-CuII ( met ); and CuII-O2 2-CuII ( oxy).11 In the met state, the CuII centers are bridged by OH; whereas, the O2 2 serves as a bridge in the oxy state. The cresolase activity requires the oxy state, while the catecholase activity can occur through either the met in the presence of H2O2 or oxy state. In the cresolase activity, the phenol substrat e is hydroxylated at the ortho position to give a catechol, which can be further oxidized to o -quinone (catecholase activity).11 A crystal structure of tyrosinase from Streptomyces Castaneoglobisporus is recently proposed with a small 4

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protein bound to the active site.14 This protein named ORF378 is suggested to be important for crystallizat ion and insertion of CuII ions into the active site of tyrosinase. The overall structure of tyrosinase is -helical and both CuII ions are coordinated by 3 histidine residues. From the crystal structure, the distances between different states are 4.1, 3.3.9, and 3.4 for the deoxy met and oxy states, respectively.14 According to the proposed mechanism for tyrosinase (Figure 1.1),15 oxygen initially binds to the deoxy di-CuII center in a side-on bind ing mode, forming a peroxobridged di-CuII center. In the monophenolase or cres olase activity, the phenolic substrate binds to one of the CuII atoms in the axial position (Figure 1.1). The CuII center is then rearranged to place the ortho position of the substrate closer to one of the peroxo oxygen for hydroxylation.15 The phenolic substrate is proposed to be subsequently hydroxylated by mechanism consistent with electrophilic aromatic substitution, forming a diphenol or catechol. In the diphenolase or catecholase activity, the catec hol substrate binds to both CuII at the same time and oxidized and released as o-quinone (Figure 1.1). Finally, the deoxy di-CuI site is regenerated by the re lease of bridging OH as water.15 Catechol oxidase, also known as o-diphenol oxidase a nd 1,2-benzenediol oxygen oxidoreductase, catalyzes the conversion of catechol to o-quinone.16 This enzyme can be found in plant tissues, in sects, and crustaceans.17 Like tyrosinase, the molecular weight of catechol oxidase ranges between 30 and 60 kDa, depending on the source. Based on extended X-ray absorption fine structure (E XAFS) and X-ray absorption near edge 5

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structure (XANES) studies, th e distances between the CuII ions are 2.9 and 3.8 for met and oxy states, respectively.18 Hemocyanin, a structurally related protein to tyrosinase and catechol oxidase, is an oxygen carrier in arthropods and mollusks. Hemocyanin has also been proposed to form a large protein aggregate with up to 8 MDa.19 Although the di-CuII center is conserved among all three proteins, each pr otein has a different function. While hemocyanin is involved in O2 transfer, both catechol oxidase and tyrosinase can perform the oxidation of catechol to o-quinone, whereas tyrosina se also has a monophenolase activity. The presence of an amino acid residue (which varies among di fferent species) in the active site of hemocyanin and catechol oxi dase has been proposed to be correlated to the inability to perform cresolase activity of tyrosinase. 1.2.2 Mononuclear Copper Proteins Cu,Zn-Superoxide dismutase. Copper-zinc superoxide dismutase (Cu,Zn-SOD) is an enzyme containing a type-II mononuclear Cu center, essential for catalyzing the decomposition of the highly reactive superoxide (O2 ) to O2 and H2O2.20 It is expressed in both bacteria and eukaryotes.21 In bacteria, this enzyme is found in the periplasmic space.21 e, f In eukaryotes, Cu,Zn-SOD is largely found in the cytosol, nuclei, lysosomes, peroxisomes, and intermembrane space of mitochondria.21 ad However, most structural and mechanistic studies are done on the eukary otic forms (yeast, bovine, and human) of the enzyme.22 6

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Figure 1.1 Proposed Mechanism for tyrosinase.15 The monophenolase activity (or phenol ohydroxylation) only occurs through the oxy state of di-Cu center; whereas, the diphenolase (or catecholase activ ity) can occur through both the met and oxy states of the di-Cu center. This illustration is adopted from ref. 15. 7

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Figure 1.2 Crystal structure of two oxygen-binding functional units of Octopus hemocyanin (PDB ID: 1JS8).23 The giant protein is composed of 10 subunits, and each subunit is consisted of 7 oxygen-binding functional units. The blue spheres represent Cu atoms, and the black spheres are oxygen atoms bound in a side-on mode. 8

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The human form of Cu,Zn-SOD is compos ed of homodimers of 32 kDa, in which each has one CuII and one ZnII atom.24 Each domain is composed of eight stranded sheets forming a Greek key -barrel and three ex tended loop regions (Figure 1.3). In its oxidized form, the CuII is penta-coordinated by four hi stidine residues (i.e. His-46, His48, His-63, and His-120 in human form) in a di storted square planar coordination and a weakly axial-coordinated water at 2.5 away from the metal. The ZnII ion is located approximately 6.6 from the CuII, and the two metals are bridged by the imidazole nitrogens of His-63. The ZnII atom is coordinated by Hi s-63, His-71, His-80, and Asp-83 in a nearly tetrahedral geometry.20 Furthermore, the active site is stabilized by extensive network of hydrogen bonds. The dismutation of O2 to occur at the CuII center. However, the ZnII binding has been proposed to help position the amino acid residues Glu-132, Glu-133, and Lys-136 in the correct conformation for substrate recognition.25 In general, the dismutation process can be explained in two parts (Eq. 1 and 2). In the first part, CuII is reduced to CuI by one O2 molecule. According to a proposed mechanism, the superoxide is initially non-sp ecifically protonated to give a hydroperoxyl radical (HO2 ) (Figure 1.4).26 Then, the bond between CuII and the imidazole group is broken upon HO2 binding to the CuII center. The hydroperoxyl radical is deprotonated upon binding, followed by the protonation of the imidazole nitrogen. Next, an electron is transferred to the CuII center to afford CuI and O2 (Figure 1.4 c). In the second part, CuI is reoxidized to CuII by another HO2 molecule.26 Upon binding to the CuI, 9

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Figure 1.3 Crystal structure of Cu,ZnSOD with the two metal center shown in ball and stick configuration (PDB ID: 2c9v).27 ZnII and CuII are displayed in green and orange, respectively. 10

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HO2 is reduced to hydroperoxide while CuI is oxidized to CuII. Finally, the hydroperoxide is protonated to give hydrogen peroxide by th e proton from the imidazole group of His-63 (Figure 1.4 f). The deprotonated imidazole group then can bind to CuII and bridge the two metal centers. CuIIZnIISOD + O2 CuIZnIISOD + O2(1) CuIZnIISOD + O2 + 2H+ CuIIZnIISOD + H2O2 (2) Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder where 20% of familial ALS, has been asso ciated with mutations in the Cu,Zn-SOD gene, sod1. Over 100 mutations of sod1 have been identified, which ranges from amino acid substitutions to frameshifts.28 Furthermore, protein ag gregates containing mutant Cu,Zn-SOD have been found from ALS patient s, ALS transgenic mice, and cell culture model systems.29 Currently, there is no cure for this devastating disease. Peptidylglycyine -Hydroxylating Monooxygenase (PHM) and Dopamine Monooxygenase (D M). Peptidylglycine -hydroxylating monooxygenase (PHM) and dopamine -monooxygenase (D M) are another group of proteins with mononuclear Cu center.30 While PHM hydroxylates C-terminal glycine-extended peptides, D M converts dopamine to norepin ephrine by hydroxylating the carbon. Both enzymes are exclusively expressed in higher eukaryot es, and their primary sequences are 27% identical and 40% similar.31 PHM and D M can be found in the secretory vesicles of the pituitary gland32 and the chromaffin granules of the adrenal gland,33 respectively. 11

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Figure 1.4 Mechanism of Cu,Zn superoxide dismutase. This illustration is adapted from ref. 4. Steps ac constitute disproportionation on one O2 molecule, where the oxidation state of Cu is 1+ and the imidazo le nitrogen is protonated. Steps df account for catalysis of a second O2 molecule. 12

PAGE 30

Each enzyme contains two CuI sites, which are 11 apart (Figure 1.5).34 In PHM, the copper centers are named CuH and CuM (i.e. in D M, CuH = CuA and CuM = CuB), and CuM has been proposed to be the site of substrate and oxygen interaction. On the other hand, CuH is suggested to be involved in el ectron storage and transfer. As a result, PHM and D M are considered as mononuclear Cu proteins during their catalysis. Furthermore, the amino acids involved in Cu binding are highly conserved between them. The CuH atom is coordinated by three histidine residues, whereas CuM is bound by two histidine and a methionine residue.30, 34 According to the proposed hydroxylation mechanism (Figure 1.6), the substrate binds close to the CuM site and not at the metal center.30 Upon O2 binding to CuM, an electron is transferred from the Cu atom to O2 to afford CuIIO2 species. Then, a protontunneling occurs from the substrate to the CuIIO2 complex to give CuIIOOH (hydroperoxo) complex. At this stage, two pathways have been proposed. In the first proposal, the electron from CuH reductively cleaves the hydroperoxo complex to give CuM IIoxo radical, which reacts with the substr ate-derived radical to give alcohol product.35 The second route involves a direct hydroxyl radical abstraction by the substrate-derived radical to afford CuM IIoxo, which is then reduced by the electron transfer from the CuH atom to afford CuM IIOH.36 Finally, CuI is regenerated by ascorbic acid.30 13

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Figure 1.5 Crystal structure of peptidylglycine -hydroxylating monooxygenase (PDB ID: 3PHM).37 The two Cu atoms are displayed in orange, and they are ~11 from each other. In addition, they are fully exposed to solvent and yet the el ectron transfer from CuH (right orange sphere) occurs to CuM (left orange sphere). 14

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Figure 1.6 Proposed mechanism for peptidylglycine -hydroxylating monooxygenase and dopamine -monooxygenase. This illustrati on is adapted from ref. 30. 15

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1.3 Biomimetic Synthetic Model Complexes Even among the type III di-Cu proteins with a very well conserved active site, such as hemocyanin, catechol oxidase, and ty rosinase, nature has fine-tuned them to function differently. While the same di -Cu center is used for reversible O2 transfer in hemocyanin, this active site is also applicable in both o-hydroxylation of phenol to catechol and oxidation of catechol to o-quinone in tyrosinase a nd the latter in catechol oxidase. These three are only a few of many Cu -containing proteins th at are involved in oxidation and oxygenation chemistry. The interactions between O2 and different types of Cu centers vary, and the reaction mechanism can become complicated with respect to the active site environment. These structural and mechan istic complexities have long interested inorganic chemists such as the late Nobumasa Kita jima, William B. Tolman, Shinobu Itoh, and Kenneth D. Karlin just to name a few. Th eir approaches have been to elucidate the O2 intermediates during catalysis by Cu proteins through synthe tic organic complexes that mimic the protein active site. Since histidin e is the most common ligand in Cu proteins, most of these synthetic ligands contain nitr ogen, involved in Cu binding. Their findings have made significant contribu tions in understanding the O2 chemistry of Cu proteins. A classic example is the very firs t crystal structure of a di-CuII-peroxo complex supported by the [(HB(3,5-iPr2pz)3)]2 ligand with O2 bound in a side-on binding mode determined by Kitajima and co-workers in 1989.38 Prior to his discovery, the end-on binding between the two Cu centers had been well accepted for O2 binding to the active site of 16

PAGE 34

hemocyanin. He revealed that oxygen binds between the two Cu centers in a side-on mode.38 His finding was also demonstrated 5 years later on oxy -hemocyanin from arthropod with X-ray structural determination.39 Since then, many ( 2: 2-peroxo)Cu2 II complexes of different bidentate and tridenta te nitrogen ligands have been synthesized and studied.40 Although a catalytic mechanism of tyrosinase has been proposed,41 the O2 transfer from the dinuclear CuII-peroxo center to phenol (i.e. o-hydroxylation or monophenolase activity) still has not been well explained. Th is step of the mechanism has been long investigated with dinuclear CuII model systems. dication olate the 42 One common finding is the formation of C-C coupled dimer at the ortho position of phenol. Th is is the in that phenoxyl radicals have been gene rated. Since there are two possible O2 binding modes, end-on bis( -oxo) and side-on ( 2: 2), a bidentate (LPy1) and a tridentate (LPy2) ligand that give end-on and side-on complexes (Figure 1.7) respectively, are used to determine the hydroxylation step of the mechanism in a recent study.43 Using phen as a substrate in organic solv ent at 4 C, the dinuclear CuII-peroxo complex gives oxygenated product catechol, whereas, using neutral phenol only resulted in the dimer product. However, the -oxo complex does not give catec hol regardless of the type of substrate used. By correlating the kinetic data to the oxidation potential of the substrate, the authors to propose the hydroxylation to occur through an electrophilic aromatic substitution mechanism.43 17

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Unlike dinuclear Cu-O2 complexes, the studies of mononuclear Cu-O2 complexes have a big short coming. This is due to a higher tendency of CuI and O2 to form stable dinuclear Cu-O2 complexes.40 b However, a number of mononuclear Cu-O2 complexes have been prepared based on spectroscopic evidence and structure determined.44 Transient mononuclear end-on CuII-superoxo complexes with tripodal tetradentate ligands such as TPAR and trenR, R (Figure 1.8) have been observed to show a strong absorption at ~410 nm prior to assembling into ( trans -1,2-peroxo)CuII 2 complexes.45 The resonance Raman results from isotope -labeling experiments indicate these intermediates have stretchi ng vibrations at ~1120 cm ( = 16O16O) and ~60 cm ( = 16O18O), which have been suggested to be consistent with the formation of CuIIsuperoxo.45 Kitajima and co-workers have suc cessfully isolated and characterized a mononuclear side-on CuII-superoxo complex with the ligand HB(3t Bu-5i Prpz)3 (Figure 1.8), a monoanionic tridentate liga nd having a bulky alkyl substituents.46 Other mononuclear Cu-O2 complexes have been proposed, but spectroscopic results alone are not enough to unambiguously assign the oxygen intermediates in some cases.47 In general, both the size of the attached groups48 and the hydrogen bonding interaction49 between the oxygen and ligand help st abilize the complex. Nevertheless, most of these mononuclear Cu-O2 complexes are inactive toward external substrates.50 Although slow, progress has been made in characterization of mononuclear Cu-O2 complexes with the help of a lowtemperature stopped-flow technique.51 18

PAGE 36

CuO O O O Cu h1end-on h2side-on N D D N N D D N N LPy1LPy2 Figure 1.7 End-on versus side-on binding mode (top) and a bidentate (LPy1) versus a tridentate (LPy2) ligand (bottom).43 19

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N N N N R R R N N N R R' N R' R R R' TPARtrenR,R' Figure 1.8 Ligands used for the preparation of Cu complexes: TPAR (Karlin);45 b trenR,R (Tolman);40b HB(3t Bu-5i Prpz)3 (Kitajima).46 20

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1.4 Metallopeptides Recently, there is a new interest in th e study of peptides with metal binding capabilities, owing to their versatile nature, in which de novo sequences can be generated to exhibit various functions. Some of their well known functions are DNA/RNA recognition and cleavage,52 57, 58 heavy metal binding,53 antibacterial and antifungal activities,54 and fluorescent probes.55 These peptides contain one or more metal binding sites which increase the stability of the ove rall complex or provide the active site for various catalyses.56 Cowan and co-workers have shown one of many potential applications of designed metallopeptides in DNA and RNA recognition and cleavage.57 58 In a recent study, they have constructed polypeptides ranging from 21 amino acid residues containing N-terminal AT CUN (Amino Terminal CUII and NiII binding)59 metal binding motif and C-terminal HIV Rev response elem ent (RRE) RNA recogni tion peptide with a glycine linker with different lengths.58 Then, a green fluorescent protein is co-expressed with RRE RNA in the N-terminus of the DNA oligonucleotide sequence in pET-21 plasmid vector in E. coli. The CuII complexes of ATCUN-RRE RNA recognition peptides are included during the expression. The damage of RRE RNA is determined based on the decrease in fluorescence inte nsity, which reflects the population of translatable mRNA. Significant reduction in the fluorescence intensity is observed only in the presence of CuII-ATCUN-RRE RNA complexes and not ATCUN or RRE RNA sequences alone.58 In addition, Cowan and co-workers have shown the damage of 21

PAGE 39

polynucleotides by CuII complexes of short ATCUN peptides is through oxidative cleavage.57 In a study by Imperiali et al., the versatility and specificity of synthetic peptidyl templates are well exploited.55 A family of peptides is designed, modeled after the Zn finger domain,60 to be used as a fluorescent sensor specifically toward ZnII ions. The chemosensor is composed of a Zn finger domain, an L-amino alanine, and a fluorophore. The L-amino alanine is essential for the incorporation of the fluorophore. Before removing the newly synthesized polypeptide, the L-amino alanine can be selectively deprotonated with a PdII catalyst under mild conditions.61 Then, the fluorophore can be coupled to L-amino alanine. Thus, incorporation of L-amino alanine in any part of the sequence also en ables the attachment of the fluorophore at any desired part of the peptidyl sequence. Since Zn fingers have been known to undergo metal-induced reversible folding,62 the chelation can be sensitively detected by placing the fluorophore close to the hydrophobic resi dues, which potentially involve in the folding process. As a result, the binding of ZnII ions to the Zn finger motif is reported to show great sensitivity. Due to the high ZnII binding affinity of the Zn finger domain, the specificity can be achieved.55 The above two studies clearly illustrate the versatilit y, specificity, and applicability of peptide templa tes in studies involving metal i ons. In the same respect, I have proposed the use of three naturally o ccurring peptidyl systems in studying the CuIIO2 chemistry with relevance to their biological activities in the following chapters. The 22

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23 peptides of interests are short N-terminal amyloidfragment, A 10 (the full length peptide found in the plaques is olated from the brain of Alzh eimers patients.), bacitracin (a metal binding peptide antibiotic with specificity toward Gram positive bacteria.), and histatins 5 peptide (a saliva ry peptide with high potency toward the opportunistic yeast Candida albicans ). In chapter 2, the possible therapeu tic effect of a naturally occurring flavonoid, quercetin, toward metalcentered oxidation chemistry of CuIIA 1 is presented. Then, in chapters 3 and 4 the oxidation chemistry of CuII complexes of bacitracin and histatin 5 are discussed.

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24 References 1 Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Copper homeostasis and neurodegenerative disorders (Alzheimers, Prion, and Parkinsons diseases and amyotrophic lateral scleosis). Chem. Rev. 2006, 106, 1995. 2 (a) Menkes, J. H.; Alter, M.; Steigleder, G. K.; Weakley, D. R.; Sung, J. H. A sexlinked recessive disorder with retardation of growth, peculiar hair, and focal cerebral and cerebellar degeneraton. Pediatrics 1962, 29, 764. (b) Danks, D. M.; Campbell, P. E.; Walker-Smith, J.; Stevens, B. J.; Gillespie, J. M.; Blomfield, J.; Turner, B. Menkes kinky-hair syndrome. Lancet 1972, 1, 1100. 3 Ala, A.; Walker, A. P.; Ashkan, K.; Dooley, J. S.; Schilsky, M. L. Wilsons disease. Lancet 2007 369, 397. 4 (a) Kaler, S. G. Metabolic and molecular bases of Menkes diseas e and occipi tal horn syndrome. Pediatr. Dev. Pathol. 1998, 1, 85. (b) Mercer, J. F. Menkes syndrome and animal models. Am. J. Clin. Nutr. 1998, 67S, 1022S. (c) Tumer, Z.; Horn, N. Menkes disease: underlying genetic defect and new diagnostic possibilities. J. Inherit. Metab. Dis. 1998, 21, 604. 5 Gitlin, J. D. Wilson disease. Gastroenterology 2003, 125, 1868. 6 Stohs, S. J.; Bogchi, D. Oxidative mechanisms in the toxicity of metal ion. Free Rad. Biol. Med. 1995, 18, 321.

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25 7 Da Silva, J. J. R. F.; Williams, R. J. P. Copper: extracytoplasmic oxidases and matrix formation. in The Biological Chemistry of the El ements. The Inorganic Chemistry of Life 2nd Ed.; Oxford University Press: New York, 2001; Chapter 15. 418. 8 Nersissian, A. M.; Shipp, E. L. Blue copper-binding domains. Adv. Protein Chem. 2002, 60, 271. 9 (a) Solomon, E. I.; Chen, P.; Metz, M.; L ee, S. K.; Palmer, A. E. Oxygen binding, activation, and reduction to water by copper proteins. Angew. Chem. Int. Ed. Engl. 2001, 40, 4570. (b) Rosenzweig, A. C.; Sazinsky, M. H. Structural insights into dioxygen-activating copper enzymes. Curr. Opin. Struct. Biol. 2006, 16, 729. 10 (a) Mayer, A. M.; Harel, E. Polyphenol oxidases in plants. Phytochemistry 1978, 18, 193. (b) van Gelder, C. W.; Flurkey, W. H.; Wichers, H. J. Sequence and structural features of plant and fungal tyrosinases. Phytochemistry 1997, 45, 1309 1323. 11 Claus, H.; Decker, H. Bacterial tyrosinases. Sys. Appl. Microb. 2006, 29, 3. 12 (a) Cerenius, L.; Soderhall, K. Th e prophenoloxidase-activ ating system in invertebrates. Immunol. Rev. 2004, 198, 116. (b) Riley, P. A. Melanogenesis and melanoma. Pigment Cell Res. 2003, 16, 548. 13 Garc a-Borro n, J. C.; Solano, F. Molecular anatomy of tyrosinase and its related proteins: beyond the histidine bound metal catalytic center. Pigment Cell Res. 2002, 15, 162.

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26 14 Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. Crystallographic evidence that he dinuclea r copper center of tyrosinase is flexible during catalysis. J. Biol. Chem. 2006 281, 8981. 15 Wilcox, D. E.; Porras, A. G.; Hwang, Y. T.; Lerch, K.; Winkler, M. E.; Solomon, E. I. Substrate Analogue Binding to the Coupl ed Binuclear Copper Active Site in Tyrosinase. J. Am. Chem. Soc. 1985, 107, 4015. 16 Solomon, E. I.; Sundaram, U. M.; M achonkin, T. E. Multicopper oxidases and oxygenases. Chem. Rev. 1996, 96, 2563. 17 Hughes, A. L. Evolution of the arthropod prophenoloxidase/hexamerin protein family. Immunogenetic 1999, 49, 106. 18 Gerdemann, C.; Eicken, C.; Krebs, B. Th e crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Acc. Chem. Rev. 2002, 35, 183 191. 19 (a) Decker, H.; Sterner, R. Nested a llostery of arthropodan hemocyanin (Eurypelma californicum and Homarus americanus). The role of protons. J. Mol. Biol. 1990, 211, 281. (b) Loewe, R. Hemocyanin in spid ers: v. flourimetric recording of oxygen binding curves, and its applica tion to the analysis of allosteric interactions in Eurypelma californicum hemocyanin. J. Comp. Physiol. 1978 128B 161. 20 (a) Valentine, J. S.; Doucette, P. A.; Potte r, S. Z. Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis. Annu. Rev. Biochem. 2005, 74, 563. (b)

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27 Culotta, V. C.; Yang, M.; OHalloran, T. V. Activation of superoxide dismutases: putting the metal to the pedal. Biochim. Biophys. Acta 2006 1763, 747. (c) MacPherson, I. S.; Murphy, M. E. P. Type-2 copper-containing enzymes. Cell. Mol. Life Sci. 2007 64, 2887. 21 (a) Field, L. S.; Furukawa, Y.; OHalloran, T. V.; Culotta, V. C. Factors controlling the uptake of yeast copper /zinc superoxide dismutase into mitochondria. J. Biol. Chem. 2003, 278, 28052. (b) Lindenau, J.; Noack, H.; Possel, H.; Asayama, K.; Wolf, G. Cellular distribution of superoxi de dismutases in the rat CNS. Glia 2000, 29, 25 34. (c) Sturtz, L. A.; Diekert, K.; Jensen, L. T.; Lill, R.; Culotta, V. C. A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondriaa phys iological role for SOD1 in guarding against mitochondrial oxidative damage. J. Biol. Chem. 2001 276, 38084. (d) Chang, L. Y.; Slot, J. W.; Geuze, H. J.; Crapo, J. D. Molecular immunocytochemistry of the CuZnsuperoxide dismutase in rat hepatocytes. J. Cell Biol. 1988, 107, 2169 2179. (e) Benov, L.; Sage, H.; Fridovich, I. The copperand zinc-containing superoxide dismutase from Escherichia co li: molecular weight and stability. Arch. Biochem. Biophys. 1997, 340, 305. (f) Kroll, J. S.; Langford, P. R.; Wilks, K. E.; Keil, A. D. Bacterial [Cu,Zn]-superoxide di smutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all! Microbiology 1995, 141, 2271.

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28 22 (a) Richardson, D. C.; Bier, C. J.; Richardson, J. S. Two crystal forms of bovine superoxide dismutase. J. Biol. Chem. 1972, 247, 6368. (b) Thomas, K. A.; Rubin, B. H.; Bier, C. J.; Rich ardson, J. S.; Richardson, D. C. The crystal structure of bovine Cu2+, Zn2+ superoxide dismutase at 5.5resolution. J. Biol. Chem. 1974, 249, 5677. (c) Richardson, J. S.; Thomas, K. A.; Rubin, B. H.; Richardson, D. C. Crystal structure of bovine Cu,Zn superoxi de dismutase at 3 resolution: chain tracing and metal ligands. Proc. Natl. Acad. Sci. USA 1975, 72 1349. 23 Cuff, M. E.; Miller, K. I.; Holde, K. E. ; Hendrickson, W. A. Cr ystal structure of a functional unit from Octopus hemocyanin. J. Mol. Biol. 1998 278, 855. 24 Carrico, R. J.; Deutsch, H. F. The presen ce of zinc in human cytocuprein and some properties of the apoprotein. J. Biol. Chem. 1970, 245, 723. 25 Getzoff, E. D.; Cabelli, D. E.; Fisher, C. L.; Parge, H. E.; Viezzoli, M. S.; Banci, L.; Hallewell, R. A. Faster superoxide dismutase mutants designed by enhancing electrostatic guidance. Nature 1992, 358, 347. 26 Pelmenschikov, V.; Siegbahn, P. E. Copper-z inc superoxide dismutase: theoretical insights into the catalytic mechanism. Inorg. Chem. 2005, 44, 3311. 27 Strange, R. W.; Antonyuk, S. V.; Hough, M. A. ; Doucette, P. A.; Valentine, J. S.; Hasnain, S. Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn a nd as-isolated wild type enzymes. J. Mol. Biol. 2006, 356, 1152.

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29 28 Cleveland, D. W.; Rothstei n, J. D. From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat. Rev. Neurosci. 2001 2, 806. 29 Bruijn, L. I.; Miller, T. M.; Cleveland, D. W. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu. Rev. Neurosci. 2004, 27. 723. 30 Klinman, J. P. The copper-enzyme family of dopamine -monooxygenase and peptidylglycine -hydroxylating monooxygenase: resolving the chemical pathway for substrate hydroxylation. J. Biol. Chem. 2006, 281, 3013. 31 (a) Lamouroux, A.; Vigny, A.; Biguet, N. F.; Darmon, M. C.; Franck, R.; Henry, J.-P.; Mallet, J. The primary structure of human dopamine-hydroxylase: insights into the relationship between the soluble and th e membrane-bound forms of the enzyme. EMBO J. 1987, 6, 3931. (b) Southan, C.; Kruse, L. I. Sequence similarity between dopamine -hydroxylase and peptide -amidating enzyme: evidence for a conserved catalytic domain. FEBS Lett. 1989, 255 116. (c) Eipper, B. A.; Quon, A. S. W.; Mains, R. E.; Boswell, J. S. ; Blackburn, N. J. The catalytic core of peptidylglycine -hydroxylating monooxygenase: i nvestigation by site-directed mutagenesis, Cu X-ray absorption spectrosc opy, and electron paramagnetic resonance. Biochemistry 1995, 34, 2857. 32 Eipper, B. A.; Stoffers, D. A.; Mains, R. E. The biosynthesis of neuropeptides: peptide -amidation. Annu. Rev. Neurosci. 1992 15, 57.

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30 33 Stewart, L. C.; Klinman, J. P. Dopa mine beta-hydroxylase of adrenal chromaffin granules: structure and function. Ann. Rev. Biochem. 1988, 57, 551. 34 Klinman, J. P. Mechanisms whereby mononuclear copper proteins functionalize organic substrates. Chem. Rev. 1996, 96, 2541. 35 Evans, J. P.; Ahn, K.; Klinman, J. P. Evidence that dioxygen a nd substrate activation are tightly coupled in dopamine -monooxygenase. J. Biol. Chem. 2003, 278, 49691 49698. 36 Chen, P.; Solomon, E. I. Oxygen activation by the noncoupled binuclear copper site in peptidylglycine -hydroxylating monooxygenase. React ion mechanism and role of the noncoupled nature of the active site. J. Am. Chem. Soc. 2004, 126, 4991. 37 Prigge, S. T.; Kolhekar, A. S.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Substratemediated electron transfer in peptidylglycine -hydroxylating monooxygenase. Nat. Struct. Biol. 1999, 6, 976. 38 Kitajima, N.; Fujisawa, K.; Moro-oka, Y. 2: 2-peroxo binuclear copper complex, [Cu(HB(3,5-pPr2pz)3)]2(O2). J. Am. Chem. Soc. 1989, 111, 8975. 39 Magnus, K. A.; Hazes, B.; Ton-That, H.; Bonaventura, C.; Bonaventura, J.; Hol, W. G. J. Crystallographic analysis of ox ygenated and deoxygenated states of arthropod hemocyanin shows unusual differences. Proteins 1994, 19, 302. 40 (a) Mirica, L. M.; Ottenwaelder, X.; Stack, T. D. P. Structure and spectroscopy of copper-dioxygen complexes. Chem. Rev. 2004, 104, 1013. (b) Lewis, E. A.;

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31 Tolman, W. B. Reactivity of dioxygen-copper systems. Chem. Rev. 2004, 104, 1047 1076. (c) Hatcher, L. Q.; Karlin, K. D. Ligand influences in copper dioxygen complex-formation and substrate oxidations. Adv. Inorg. Chem. 2006, 58, 131. (d) Itoh, S.; Tachi, Y. Structure and O2-reactivity of copper (I) complexes supported by pyridylalkylamine ligands. Dalton Trans. 2006, 4531. 41 Solomon, E. I.; Sundaram, U. M.; M achonkin, T. E. Multicopper oxidases and oxygenases. Chem. Rev. 1996, 96, 2563. 42 (a) Kitajima, N.; Koda, T.; Iwata, Y. ; Moro-oka, Y. Reaction aspects of a -peroxo binuclear copper (II) complex. J. Am. Chem. Soc. 1990, 112, 8833. (b) Paul, P. P.; Tyeklr, Z.; Jacobson, R. R.; Karlin, K. D. Reactivity patterns and comparisons in three classes of synthe tic copper-dioxygen {Cu2-O2} complexes: implication for structure and biological relevance. J. Am. Chem. Soc. 1991, 113, 5322. (c) Obias, H. V.; Lin, Y.; Murthy, N. N.; Pidcock, E.; Solomon, E. I.; Ralle, M.; Blackburn, N. J.; Neuhold, Y.-M.; Zuber-bhler A. D.; Karlin, K. D. Peroxo-, oxo-, and hydroxo-bridged dicopper complexes: observation of exogenous hydrocarbon substrate oxidation. J. Am. Chem. Soc. 1998, 120, 12960. (d) Mahapatra, S.; Halfen, J. A.; Wilkinson, E. C. Jr.; Tolman, W. B. Modeling copper-dioxygen reactivity in proteins: aliphatic CH bond activation by a ne w dicopper(II)-peroxo complex. J. Am. Chem. Soc. 1994, 116, 9785. (e) Mahadevan, V.; Henson, M. J.; Solomon, E. I.; Stack, T. D. P. Differe ntial reactivity between interconvertible side-

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32 on peroxo and bis-oxodicopper isomers using pera lkylated diamine ligands. J. Am. Chem. Soc. 2000, 122, 10249. (f) Osako, T.; Ohkubo, K.; Taki, M.; Tachi, Y.; Fukuzumi, S.; Itoh, S. Oxidation mech anism of phenols by dicopper-oxygen (Cu2/O2) complexes. J. Am. Chem. Soc. 2003, 125, 11027. 43 Itoh, S.; Fukuzumi, S. Monooxygenase ac tivity of type 3 copper proteins. Acc. Chem. Res. 2007, 40, 592. 44 Itoh, S. Mononuclear copper active-oxygen complexes. Curr. Opin. Chem. Biol. 2006, 10, 115. 45 (a) Karlin, K. D.; Wei, N.; Jug, B.; Kader li, S. Niklaus, P.; Zuberbhler, A. D. Kinetics and thermodynamics of formati on of copper-dioxygen adducts: oxygenation of mononuclear copper(I) complexes cont aining tripodal tetrad entate ligands. J. Am. Chem. Soc. 1993, 115, 9506. (b) Zhang, C. X.; Kaderli, S.; Costas, M.; Kim, E.; Neuhold, Y.-M.; Karlin, K. D.; Zuberbhler A. D. Copper(I)-dioxygen reactivity of [(L]CuI] + (L = tris(2-pyridylmethyl)amine): kinetic/thermodynamic and spectroscopic studies concerningthe formation of Cu-O2 and Cu2-O2 adducts as a function of solvent medium and 4-pyridyl ligand substituent variations. Inorg. Chem. 2003, 42, 1807 1824. (c) Komiyama, K.; Furutachi, H.; Na gatomo, S.; Hashimoto, A.; Hayashi, H.; Fujinami, S.; Suzuki, M.; Kitagawa, T. Dioxygen reactivity of copper(I) complexes with tetradentate tripodal ligands having aliphatic nitr ogen donors: synthesis, structures, and properties of pe roxo and superoxo complexes. Bull. Chem. Soc. Jpn.

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33 2004, 77, 59. (d) Weitzer, M.; Schindler, S.; Brehm, G.; Schneider, S.; Hrmann, E.; Jung, B.; Kaderli, S.; Zuberbhler, A. D. Reversible binding of dioxygen by the copper(I) complex with tris(2-dimethylaminoethyl)amine (Me6tren) ligand. Inorg. Chem. 2003, 42, 1800. 46 Fujisawa, K.; Tanaka, M.; Moro-oka, Y. ; Kitajima, N. A monomeric side-on superoxocopper(II) complex: Cu(O2)(HB(3-tBu-5-iPrpz)3). J. Am. Chem. Soc. 1994, 116, 12079. 47 (a) Fry, H. C.; Scaltrito, D. V.; Karlin, K. D.; Meyer, G. J. The rate of O2 and CO binding to a copper complex, determined by a flash-and-trap t echnique, exceeds that for hemes. J. Am. Chem. Soc. 2003, 125, 11866. (b) Schatz, M.; Raab, V.; Foxon, S. P.; Brehm, G.; Schneider, S.; Re iher, M.; Holthausen, M. C.; Sundermeyer, J.; Schindler, S. Combined spectroscopic a nd theoretical evidence for a persistent endon copper superoxo complex. Angew. Chem. Int. Ed. Engl. 2004, 43, 4360. (c) Chaudhuri, P.; Hess, M.; Weyhermller, T. ; Wieghardt, K. Aerobic oxidation of primary alcohols by a new mononucl ear Cu(II)-radical catalyst. Angew. Chem. Int. Ed. Engl. 1999, 38, 1095. (d) Spencer, D. J. E.; Aboelella, N. W.; Reynolds, A. M.; Holland, P. L.; Tolman, W. B. -diketiminate ligand backbone structural effects on Cu(I)/O2 reactivity: unique copper-superoxo and bis( -oxo) complexes. J. Am. Chem. Soc. 2002, 124, 2108. (e) Aboelella, N. W.; Kryatov, S. V.; Gherman, B. F.; Brennessel, W. W.; Young, V. G. Jr.; Sara ngi, R.; Rybak-Akimova, E. V.; Hodgson, K.

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34 O.; Hedman, B.; Solomon, E. I. Dioxygen activa tion at a single copper site: structure, bonding, and mechanism of formation of 1:1 Cu-O2 adducts. J. Am. Chem. Soc. 2004, 126, 16896. 48 Chen, P.; Root, D. E.; Campochiaro, C.; Fujisawa, K.; Solomon, E. I. Spectroscopic and electronic structure studies of th e diamagnetic side-on CuII-superoxo complex Cu(O2)[HB(3-R-5-iPrpz)3]: antiferromagnetic coupling vers us covalent delocalization. J. Am. Chem. Soc. 2003, 125, 466. 49 (a) Yamaguchi, S.; Nagatomo, S.; Kitagawa, T.; Funahashi, Y.; Ozawa, T.; Jitsukawa, K.; Hideki, M. H. Copper hydroper oxo species activated by hydrogen-bonding interaction with its distal oxygen. Inorg. Chem. 2003, 42, 6968. (b) Yamaguchi, S.; Kumagai, A.; Nagatomo, S.; Teizo, K. T.; Funahashi, Y.; Ozawa, T.; Jitsukawa, K.; Masuda, H. Synthesis, characterization, and thermal stability of new mononuclear hydrogenperoxocopper(II) complexes with N3O-typetripodal liga nds bearing hydrogenbonding interaction sites. Bull. Chem. Soc. Jpn. 2005, 78, 116. 50 (a) Reynolds, A. M.; Lewis, E. A.; Aboelell a, N. W.; Tolman, W. B. Reactivity of a 1:1 opper-oxygen complex: isolation of a Cu(II)-o-iminosemiquinonato species. Chem. Commun. 2005, 2014. (b) Fujii, T.; Naito, A.; Yamaguchi, S.; Wada, A.; Funahashi, Y.; Jitsukawa, K.; Nagatomo, S.; K itagawa, T.; Masuda, H. Construction of a square-planar hydroperoxo-coppe r(II) complex inducing a hi gher catalytic reactivity. Chem. Commun. 2003, 2700.

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35 51 (a) Osako, T.; Nagatomo, S.; Tachi, Y.; Kitagawa, T.; Itoh, S. Low-temperature stopped-flow studies on the reactions of copper(II) complexes and H2O2: the first detection of a mononuclear copper(II)-peroxo intermediate. Angew. Chem. Int. Ed. Engl. 2002, 41, 4325. (b) Osako, T.; Nagatomo, S.; Kitagawa, T.; Cramer, C. J.; Itoh, S. Kinetics and DFT studies on the reaction of copper(II) complexes and H2O2. J. Biol. Inorg. Chem. 2005 10, 581. 52 Long, E. C. Ni(II)-Xaa-Xaa-His metallopeptideDNA/RNA interactions. Acc. Chem. Res. 1999, 32, 827. 53 Ghosh, D.; Pecoraro, V. L. Probing metalprotein interactions using a de novo design approach. Curr. Opin. Chem. Biol. 2005, 9, 97. 54 (a) Cabras, T.; Patamia, M.; Melino, S.; Inzitari, R.; Messana, I.; Castagnola, M.; Petruzzelli, R. Pro-oxidant activity of hi statin 5 related Cu(II)-model peptide probed by mass spectrometry. Biochem. Biophy. Res. Comm., 2007, 358, 277. (b) Melino, S.; Gallo, M.; Trotta, E.; Mondello, F.; Paci M.; Petruzzelli, R. Metal-binding and nuclease activity of an antimicrobial pep tide analogue of the sa livary histatin 5. Biochemistry 2006, 45, 15373. (c) Brewer, D; Lajoie, G. Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatins 3 and 5 by electrospray ionizati on mass spectrometry. Rqpid Comm. Mass Spec. 2000, 14, 1736 1745. (d) Melino, S.; Rufini, S.; Sette, M. ; Morero, R.; Grottesi, A.; Paci, M.; Petruzzelli, R. Zn2+ ions selectively induce antimicrobi al salivary peptide histatin-5 to

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36 fuse negatively charged vesicles. Identifica tion and characterizati on of a zinc-binding motif present in the functional domain. Biochemistry 1999, 38 9626. 55 Walkup, G. K.; Imperiali, B. Fluorescent chemosensors for divalent zinc based on zinc finger domains. Enhance oxidative stab ility, metal binding affinity, and structural and functional characterization. J. Am. Chem. Soc. 1997, 119, 3443. 56 Svg, I.; sz, K. Metal ion select ivity of oligopeptides. Dalton Transactions 2006 3841. 57 Cowan, J. A.; Jin, Y. DNA cleavage by copperATCUN complexes. Factors influence cleavage mechanism a nd linearization of dsDNA. J. Am. Chem. Soc. 2005, 127, 8408. 58 Cowan, J. A.; Jin, Y. Cellular activity of Rev response element RNA targeting metallopeptides. J. Biol. Inorg. Chem. 2007, 12, 637. 59 Harford, C.; Sarkar, B. Amino terminal Cu(II)and Ni(II)-binding (ATCUN) motif of proteins and peptides: metal bindin g, DNA cleavage, and other properties. Acc. Chem. Res. 1997, 30, 123. 60 Berg, J. M.; Merkle, D. L. On the Metal Ion Specificity of Zinc Finger Proteins. J. Am. Chem. Soc. 1989, 111, 3759. 61 Kates, S. A.; Daniels, S. B.; Albericio, F. Automated allyl cleavage for continuousflow synthesis of cyclic and branched peptides. Anal. Biochem. 1993, 212, 303.

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37 62 Eis, P. S.; Lakowicz, J. R. Time-resolv ed energy transfer measurements of donoracceptor distance distributions and intramol ecular flexibility of a CCHH zinc finger peptide. Biochemistry 1993, 32, 7981.

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CHAPTER 2 ANTIOXIDATIVE PROPERTIES OF A NATURALLY OCCURRING FLAVONOID, QUERCETIN : IMPLICATIONS TOWARD TREATMENT STRATEGIES FOR ALZHEIMERS DISEASE 38

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2.1 Amyloid Alzheimers disease (AD) is a debilitating neurodegenerative disorder that mainly affects the elderly population a nd currently without a cure. The average age-onset is 60, and the risk increases with aging. According to the National Institute of Health, as many as 4.5 million Americans suffer from this disorder.1 While approximately only 5% of the population between the ages 65 has this dis ease, up to 50% have been estimated for the age group 85 and older. AD is characte rized by a progressive deterioration of the brain; the average life-span of the diagnosed individuals is 5 years, although some may live up to 20 years. It is a memory disorder, initially characterized by inability to recall recent events. As the disease progres ses, memory loss and cognitive impairments become a part of the patients daily life. AD was init ially recognized by Dr. Alois Alzheimer in 1906. He discovered a noticeabl e amount of tissue masses accumulated in the brain of a female patient, suffering from a strange mental illness. These masses were later termed amyloidplaques.1 Amyloid(A ) is a proteolytic product of am yloid precursor protein (APP), consisting of 39 amino acids, and aggregatio n of this peptide in the brain is the hallmark of Alzheimers disease.2 APP is a membrane-bound synaptic protein, where the Nand C-terminal ends are extracellularl y and intracellularly situated, respectively.3 In addition, APP has two evolutionarily rela ted cousins, APLP-1 and APLP-2, in mammals.4 All three synaptic proteins are es sential for developmental and postnatal functions, as have been shown by the double and triple knock-out studies in mice. 39

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Cranial abnormalities and early postnatal lethality are two phenotypes common among all knock-out studies.5 Different proteolytic cleavage of APP by secretases decide the fate of A The sequential processing of APP by and -secretases results in th amyloidogenic and toxic form of A while the proteo lytic product of -secretase is considered non-toxic since this enzyme cleaves APP within the A peptide. e 6 The full length peptide can be further cleaved into small soluble fragments (e.g. A 1) from the N-terminal end by and -secreatases7 as well as insulin-degrading enzyme.8 Despite extensive studies, th e physiological function of A is still unclear. Nevertheless, a commonality in the brains of postmortem AD patients is the presence of plaques, resulted from the aggregated A peptide. The two most common forms of A peptide are 1 and 1. While the former is found in larger quantity, the latter is suggested to be more toxic. The primary sequence of A is as follows: DAEFRH DSGY10 EV HH QKLVFF20 AEDVGSNKGA30 IIGLMVGGVV40 IA.9 The A peptide is amphipathic in nature, and its conformational state is influenced by a few factors, such as pH, temperat ure, and solvent environment.10 At pH < 4 or > 7, the peptide is suggested to rema in as a monomeric form in -helical or random coiled conformation. Within the pH range 4, however, it becomes less stable and tends to form oligomers, which eventually lead to aggregation. The N-terminal 20 amino acids are more hydrophilic and can adopt -helical or random coil conformation in aqueous environment, depending on the conditions.10 c, g The C-terminus is quite hydrophobic and 40

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is suggested to form stable -sheet structures with a high tendency to aggregate, regardless of the conditions and environment.10g Furthermore, the plaques have been shown to contain a high concentration of metal ions such as CuII, FeIII, and ZnII, determined to be 390, 940, and 1055 M, respectively.11 Comparing to the age-matched contro ls, the concentration of these metal ions in the brain of AD patients ha ve increased 6, 3, and 3 times for CuII, FeIII, and ZnII, respectively. All three metal ions can cause A aggregations in vitro ; a process that is reversed by metal chelator such as EDTA.12 Thus, it is important to determine the interaction of A with metals. Contrary to exhaustive metal bindi ng studies using numerous powerful spectroscopic techniques,13 the coordination environment is still under debate. The matter is complicated by experimental varia tions such as solvent system, pH, and the length of peptide. Neverthele ss, it is generally accepted that only the N-terminal region (1 residues) of the A peptide is involved in metal bindi ng and that the affinity for the metal ion is in the micromolar range,13 e, h although an earlier study suggested an attomolar affinity, which mistakenly combined a coagulation constant into the affinity constant.13g The N-terminus contains three hi stidine (at positions 6, 13, and 14) and several acidic residues (Asp-1, Glu-3, Asp-7, and Glu-11) that can potentially involve in metal binding. The involvement of all three His residues in CuII and ZnII binding is universally accepted.13, 63 The lack of AD in rats clearl y demonstrates the involvement of these histidine residues in AD, since the rat variant of A peptide contains the following 41

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three mutations: Arg-5 to Gly, Tyr-10 to Phe, and His-13 to Arg.14 The ability to bind metal is greatly reduced by the H13R muta tion, and thus, rats do not suffer from AD.14 In addition to the histidine residues, several candidates ha ve been proposed to be the fourth ligand in metal binding, including the amino group of the N-terminal Asp-1,13 d, e, f the phenolate group of Tyr-10,13c, g and the carboxylate group of Glu-11.13 b According to the amyloid cascade hypothesis, the plaques are the culprits of neurodegeneration.15 However, this theory was quic kly invalidated by postmortem brain analysis of AD victims. Ther e is a discrepancy between the amount of aggregates present and the severity of deterioration.16 Closer analysis of the plaques led to evidence of a possible oxidative stress in the brain.17 Metal ions in biological systems are cl osely regulated, especially redox-active metals such as CuII and FeIII.22 The availability of Fe in cell involves two transporters, DMT1 (influx) and Ireg1 (efflux),18 and the extracellular transport and storage are responsible by high affinity binding tran sferrin and highly efficient ferritin, respectively.19 Similarly, the influx of CuII ions is performed by Ctr1, DCT1, and DMT1, while the excretion is controlled by Menkes (MNK) and Wilson (WND) ATPases.20 21 In addition, the intracellular CuII concentration is highly regulated by multiple chaperone proteins.22 However, these regulatory processes lose efficiency wit aging, leading to accumulation of the unbound metal ions. h ically 23 Redox-active metals such as CuII and FeIII can generate highly reactive hydroxyl (OH) radical through Fenton chemistry in the presen ce of molecular oxygen.24 This radical can attack and chem 42

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modify biological molecules such as lipid molecules in membranes, amino acids in proteins, and nucleic acids (DNA a nd RNA), rendering them dysfunctional.17 A recent study displaying the ability of A to generate hydrogen peroxide (H2O2), a reactive oxygen species (ROS), in the pr esence of biological reducing agents and CuII confirms the involvement of redox chemistry in the etiology of AD.25 In fact, there is direct evidence of oxidative damages in the A plaques.17 It is possible that oxidative stress leads to neurodegeneration, and the plaq ues are the end result of a protective role played by the A peptide.26 The same has been proposed for other aggregate-forming neurodegenerative disorders, such as Prion-related and Parkinsons diseases.27 In this proposed theory, the A peptide attempts to chelate a nd remove the accumulated free pool of metal ions. The situation is exacerbated instead when the CuII/FeIIIA complexes themselves can act as the redox centers to generate ROS. The free and complexed peptides may be chemically modified and subsequently aggregated in the process.28 Recent findings show the ability of APP to bind and reduce CuII, suggesting a role in CuII regulation of this protein.29 In addition, APP has been shown to be neuroprotective in vivo further supporting the protectiv e role of its C-terminal A fragment.30 If the aggregated form of A is the aftermath, the soluble or oligomeric form of the peptide is truly responsible for the redox chemistry. In fact, recent studies have shown the oligomeric A peptide to be more toxic.31 This explains the lack of consistency between the disease stage and the amount of A aggregate. 43

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In addition to oxidative stress, interference in the intracellular CaII homeostasis by A was also suggested to cont ribute to the pathology of AD.32 Even though the dysfunction in CaII homeostasis does not play a role in the overall neurodegeneration, it is responsible for the cognitive impairments in AD.33 In the hippocampus, A aggregates have been shown to span lipid membrane, creating channels with poor ion selectivity.34 Moreover, the soluble forms of A can cause membrane depolarization and impair Na+/K+-ATPase, which can lead to an increase in CaII influx.35 36 The rise in the CaII level defects neurons and synaps es, deters long-term potentiati on in certain brain regions, and leads to impairments in learning and memory.37 Thus, regulation of redox-active metals as well as CaII influx in the hippocampus is e ssential toward possible prevention and treatment of AD. We have recently reported that the CuII complexes of the shorter proteolytic fragments, A 1 and A 1, are able to perform two-el ectron oxidation of polyphenol, catechol, and phenol to their respective o-quinone products consistent with Type-3 copper oxidases.38 63, 64 In the proposed catechol oxida se-like mechanism (Figure 2.1),63 the reducing substrate catechol can bind directly to the di-CuII center bridged by a hydroxyl group and readily oxidi zed into the corresponding orthoquinone (Figure 2.1 steps F and G). This process involves 2e transfers, and the di-CuII is reduced to 2CuI. In the presence of dioxygen, the metals can resume its oxidized states by forming a dinuclear dioxygen-bound CuII complex, where the species present can be one of the three isoelectric states: 44

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3 2 2 2 2 11 2 2 22, :, : CuoxoorCuperoxo Cuperoxo (B in Fig. 2.1).This peroxo-bridged di-Cu2+ center can go through another cycle of oxidation, before returning to its hydroxyl-bridged di-CuII following the concomitant release of one of the bound-oxygen as water (Figure 2.1 steps C and D). In addition, in the presence of the peroxo-bridged di-CuII can form directly from the hydroxyl-bridged species (Figure 2.1 steps AD).63 In the phenol-oxidase-like mechanism (Fig. 2.2),64 the phenol substrate initially binds to a CuII center, followed by a transfer of an electron to give a CuI-(phenol radical) intermediate (step a). The radical is stabilized with the unpaired e situated on the ortho and para positions. Next, the binding of dioxygen to the CuI-phenol radical results in another electron transfer to afford a CuII superoxide structure (step b), which is potentially stabilized by binding of a second CuII center. Finally, the o -quinone product is formed by the transfer of an electron and an oxygen atom to the semi-quinone (Figure 2.2 steps c, d). The possi ble involvement of di-CuII center was shown by diluting redoxactive CuII with the redox-inactive ZnII.64 We also observed catecholamine neurotransmitters can be effectively oxidized by CuII-A with and without H2O2.38 Thus, in the presence of CuII the A peptide can potentially oxidize and deplete the catechola nd phenol-containing neurotransmitters (i.e. dopamine, serotonin, epinephrine, and norepinephrine) in the brain, causing neuronal death. The fact that oxidative stress is a meta l-dependent process raises a new interest in metal-chelation therapy as a treatment for AD. 45

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Figure 2.1. Proposed mechanism for CuII-centered oxidation of catechol-containing substrates in the presence (AD) and absence (FH and BD) of H2O2.63 46

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Figure 2.2. Proposed mechanism for the CuII-centered hydroxylation and oxidation of phenol-like substrate(s): (a) binding and elec tron transfer to the phenol substrate from the CuII center to give a CuI-phenol radical; (b) dioxygen and a second CuII center binding; (c) transfer of an electron and an oxygen atom to the phenol to result in an o quinone product (d).64 47

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One of the current treatment strategies toward AD rely on metal chelation as a key step in preventing the aggregated A plaques from forming, such as the use of CuII chelator clioquinol (5-chl oro-7-iodo-8-hydroxyquinoline).39 This drug was banned from its original usage in 1970 as an antibiotic due to a severe side effect, subacute myelooptic neuropathy, in the central nervous system.40 This toxicity has been explained to be the result of CoII chelation by clioquinol from Co balamine, leading to vitamin B12 deficiency. In a transgenic mice treatment study, clioquinol has been shown to alleviate up to 49% A deposition, accompanied by a small increase in the concentration of soluble A .41 A recent Phase II clinical trial invol ving 36 patients suggests this drug to be more effective toward those with greater disease severity. In this study, one of the clioquinol-treated subjects suffered from dysfunction in visual acuity and color vision with the symptoms go away upon discontinued treatment; two other patients suffered from leg numbness.42 In addition, an earlier study by Yagi et. al has shown clioqui nol can induce lipid peroxidation in cultured chick retin al cells in the presence of iron.43 A recent study supports the pro-oxidant activity of this drug by the killing of cultured neuronal cells in the presence of redox-active FeIII and CuII, and 90% cell viability was recovered in the presence of vitamin C and Trolox C.44 Thus, clioquinol, pos sessing these intrinsic toxicities, may not be suitable for use as a therapeutic treatment toward AD. Since oxidative stress in different regions of the brain is the underlying theme among the neurological pathologies, the possibility of using naturally occurring powerful 48

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antioxidants from plants has become an increasingly popular interest.45 59a One family of these antioxidative secondary plant metabolites are flavonoids 2.1.1 Flavonoids Flavonoids are polyphenolic compounds ubiqu itously distributed in plants. They display antimicrobial and antif ungal properties, which largel y contribute toward growth and development of plants.46 They show various beneficial biological activities such as antihepatotoxic, antiinflammatory, antiather ogenic, antiallergen ic, antiosteoporotic, anticancer, and neuroprotective properties.47 These biological activities can be credited to their involvements in signaling,48 interaction with enzymes,49 and ability to scavenge free radicals50 and chelate metal ions.51 Flavonoids have gained intense interest because of their consumptions through fruits, vegeta bles, and related food products, which can be correlated to a better health (e.g. re duce the risk of cardiovascular diseases).52 Many in vitro studies have shown antioxidant and anti tumor capabilities of flavonoids toward multiple cell lines.47 Moreover, they have a larg e variation in functional groups, rendering them useful for applications as temp lates toward future drug design. However, the molecular basis and mechanism of the antioxidation activity of the structurally diverse flavonoids and their capability of scavenging free radicals have not been conclusively revealed. In general, th e phenoxyl groups on the flavonoids have been associated with radical scavenging property.50 53 Thus, those with a higher number of hydroxyl substituents are considered better antioxidants. 49

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Quercetin (Qr), 3,3',4',5,7-pentahydroxyflavone, is one of the extensively studied flavonoids with potent antioxi dant activity (Figure 2.3 I).51 52 It is largely found in apple, onion, tea, red wine, blue berry, and grape.54 The structure of Qr contains a hydroxylated pyrone ring (C ring) conjugated to a meta -diphenolic ring (A ring) and an ortho diphenolic ring (B ring). Like most flavonoi ds, Qr has the ability to scavenge free radicals and chelate metal ions. Studies ha ve shown that Qr binds metal ion(s) in a bidentate manner.51 b,d There are three possible meta l-binding sites on quercetin: -ketophenolate (4-carbonyl and 5-phenolate), -keto-enolate (4-carbonyl and 3-enolate), and catecholate (3'and 4'-phenolate) moiety (Figur e 2.3 I). The metal-binding ability of Qr has been extensively studied with potentiome tric and different spec troscopic techniques, using alkaline-earth (i.e. CaII and MgII), transition (i.e. CuII and FeIII), and lanthanide (i.e. YbIII) metal ions. Nevertheless, the coordination environment and metal binding modes have not reached consensus.51 The redox-active metal dependent oxidative stress is proposed to be the culprit of neurodegeneration in the pathology of AD. We have confirmed this by showing the catecholand phenol-oxidase-like chemistry of small A fragments in the presence of redox-active CuII in vitro .38 63, 64 Thus Qr, with its radical-scavenging and metalchelating properties, is one of many candidates as an inhib itor toward the metal-induced oxidative stress. 55 Qr can potentially degrade the ROS generated by the oxygen-bound CuII center or can directly bind to the active CuII center to prevent further chemistry. The latter has been shown by the ability of Qr and related flavonoi ds in inhibiting the 50

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O O OH O OH OH OH OH OH I II O O OH OH OH OH OH 7 5 4 3 3' 4'A C B III2' 5' 6' 6 8 Figure 2.3. Structure of quercetin (I), 5-hydroxyf lavone (II), and catechin (III). There are three potential metalbinding sites on quercetin: -keto-phenolate (4-carbonyl and 5phenolate); -keto-enolate (4-carbonyl and 3-enol ate); and catecholate (3'and 4'phenolate), while 5-hydr oxyflavone only has the -keto-phenolate moiety and catechin only possesses the catecholate moiety for metal binding. 51

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chemistry of tyrosinase, a dinuclear CuII-containing oxidase capable of converting phenol derivatives to o -quinone.56 Both Qr and kaempferol (a Qr analogue with a missing OH group at 3' position) competitively inhi bit tyrosinase activity, with an estimated IC50 values were 0.13 and 0.23 mM, respectively.57 58 The mechanism of inhibition was suggested to be due to metal chelation at the active site. Qr along with several other flavonoids have been suggested to have neuroprotective properties.59 To better understand the neurop rotective activity of Qr, the redox chemistry and coordination properties mu st be clearly revealed to provide a relationship between their struct ures and activities. In this study, the antioxidative mechanism of Qr toward the observed CuII-centered oxidation chemistry of A was determined through optical, NMR, and reactivity studies. The A peptide and aggregates have been proposed to cause a rise in the intracellular CaII concentration, leading to memory loss and cognitive impairments in AD patients. Thus, the metal binding properties of Qr and the influence of CaII on the antioxidative prope rty of Qr was also determined. 2.2 Materials and Methods The N-terminal amyloid(A 10) was synthesized at the University of South Florida Peptide Synthesis and Mass Spectom etry Center and the accuracy and purity checked with a MALDI-TOF mass spectromete r. Quercetin (Qr), Pyrocatechol, ~99% (CA), deuterated dimethyl sulfoxide d6-DMSO (99.9%), and 3-methyl-benzyl 52

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thiazolinone hydrochloride (MBTH), the lant hanide(III) chloride salts (99.99%) and CaCl2 were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI); CuSO4 and 4-(2-hydroxyethyl)-1-piperazin eethanesulfonate buffer (HEPES) from Fisher Scientific Co. (Fair Lawn, NJ); and CoCl2 6H2O from Mallinckrodt (Paris, KY). All chemicals were used without further purification. All other solvents and reagents were the highest grade available from the commercial s ources. The deionized water (18.2 M ) was obtained from a Millipore Milli-Q system. Plasticware and glassware were demetallized with EDTA and extensively rinsed prior to use. Due to the low solubility of Qr in aqueous solution, some experiments were performed in DMSO or d6-DMSO. Both the metal and the ligand solutions were freshly prepar ed just prior to the experiments. Quartz cuvettes were used in all the kinetic and optical measurements. 2.2.1 Kinetic Studies The 1:1 CuIIA 1 complex was prepared in deio nized water. The oxidation of catechol by 1.0.0 M CuIIA 1 in the presence or absence of different concentrations of Qr, CaII, and YbIII was monitored in 100.0 mM HEPES at pH 7.0 and 25 C. Equal amounts of substrate and of the specific o -quinone indicator, MBTH, were used in the assay.60 All components were dissolved in DI wa ter, and the final volume in the cuvette was fixed at 1.0 mL. The fo rmation of the quinone product was followed photometrically ( = 32,500 M cm for o -quinone-MBTH complex)60 at 500 nm with a Varian CARY50 spectrophotometer equi pped with a water peltier PCB-150 thermostable cell 53

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(Varian, Palo Alto, CA), and the initial rate was determined from the slope of the change in the absorbance with respect to ti me (0 mins) in the linear region. Inhibition/activation of the reaction was performed w ith various amounts of an inhibitor/activator, and the rate determined and fitted to appropriate rate law to reveal the inhibition pattern. Sigma Plot 8.0 was used for plotting and fitting the data. 2.2.2 Optical Studies All optical studies were performed in DMSO; triethylamine (TEA) was added as needed to enhance metal binding to Qr. The electronic spectra were acquired on the cary50 spectrophotometer. For metal-binding studies, 20.0 M Qr was added with different concentrations of metal to a to tal volume of 1.0 mL, and the mixture was monitored from 200 nm. An optical Job plot61 can be utilized for revealing the metal-to-ligand stoichiometry, and has prev iously been demonstrated useful for the determination of complicated metal-drug binding stoichiometry.62 In the plot, a distinct absorption of the complex as a function of metal mole fraction (XM) or ligand mole fraction (XL) was obtained with a constant amount of the total concentration ([M] + [L]), wherein the ratio of XM:XL at which the absorption reaches the maximum in the plot reflects the stoichiometry of the M-L complex in solution. For all the Job plot studies, the metal and ligand concentra tions were varied from 0.0 M while keeping the overall concentration constant. 54

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2.2.3 NMR Studies 1D and 2D 1H NMR spectra were acquired on a Varian INOVA500 spectrometer (at 500 MHz 1H resonance) with a 5-mm bio-TR (triple resonance) probe. A 90 pulse (~5 s) was used for th e acquisition of 1D 1H NMR spectra with 8 K data points, whereas 1024 512 data points were used for 2D EX SY (EXchanged SpectroscopY) experiment which reveals chemical exchange and the exch ange pairs, with a presaturation pulse as well as superWEFT technique68 for solvent suppression. In 1D 1H saturation transfer experiments to reveal chemical exchange, a signal of the exchange partners is saturated with a presaturation pulse, and the different spectrum is obtained from the irradiated and the reference spectra (the presaturation pulse was applied at a signa l-free region of the spectrum). A line-broadening of 40 Hz was applied to improve the signal-to-noise ratio of the paramagnetically shifted signals The spin-lattice relaxation times (T1) for the paramagnetically shifted signals were determ ined using the inversion-recovery method ( 1-1802-90 -FID). The peak intensities versus the values were fitted with a threeparameters fitting program on the spectrometer to afford the T1 values. 2.3 Results and Discussion The presence of ZnII along with redox-active CuII and FeIII in amyloid plaques is evidence that AD is a metal-dependent path ology. Numerous studies have shown the possible involvement of CuII and FeIII as the cause of oxidative stress, leading to damages that can potentially cause neurodegeneration in the brain. The ability of CuIIA 1 and 55

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CuIIA 1 to perform catecholand phenol-oxidase-like chemistry has been recently proposed.63 64 The suggested metal-centered re dox catalysis involves two electron transfers from the CuII complex to the substrate for the oxidation of catechol to orthoquinone. In the presence of O2 or H2O2, the continuation of oxidative chemistry is enhanced.63 64 The ability of these CuIIA complexes to oxidize neurotransmitters, such as dopamine and serotonin, has been shown through in vitro studies,38 suggesting a possible mechanism for the oxidative stress. Owing to the fact that oxidative stress is in this case metal-dependent chelation therapy has gained much interest in AD search. Two well known attributes of Qr in term s of antioxidant activity are metal chelation and free radical scavenging. As a result, this natural metal chelator is a potential candidate for use as a therapeutic ag ent or a template for future drug design for the treatment of AD. In fact, several studies have indicated the ne uroprotective role of this ubiquitous natural product.45 The structure of Qr has three potential metal-binding sites, rendering it a very effective metal chel ator. In addition, the B ring of Qr has a catechol moiety, which may allow this flavonoid to potentially act as a suicide substrate. This, in turn, may prevent the deplet ion of catechola nd phenol-containing neurotransmitters, such as dopamine and serotonin, in the brain. The hypothesis was tested by checking the influence of Qr toward the metalcentered oxidation of catechol by CuIIA 1. The catechol oxidation was followed by monitoring the red adduct formed between the oxidized product, o -quinone, and MBTH, an o -quinone-specific indicator, at 500 nm. When different concentrations (0.6 M) 56

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of Qr was incubated with 3.0 M CuIIA 1, a sharp decrease in the oxidation activity toward 400.0 M catechol was observed in a concentr ation dependent manner, and then followed by a less drastic decrease (Figure 2. 4 inset). From this observation, Qr can effectively inhibit the oxidation chemistry of CuIIA 1 at submicromolar concentrations, and at roughly 10.0 M Qr, the loss of 50% activity is observed. A full inhibition study was performed in order to reveal th e inhibition pattern (Figure 2.4). Different concentrations of catechol were incubated with 3.0 M CuIIA 1 at several concentrations of Qr in 100.0 mM HEPES at pH 7.0 and 25 C. At all concentrations of Qr, the data show an in itial increase in catechol oxidation, followed by a saturation at higher substrate concentrations. This pa ttern suggests a possible preequilibrium kinetics, which is in agreement with previous studies.63 64 The preequilibrium kinetics (Eq. 1) can be described as follows: [Cu(II)-Amyloid-CA] [Cu(II)-Amyloid] + [CA] [Cu(II)-Amyloid] + o-quinone k1k-1kcat where CAK CAVM Max 0 (1) and VMax are the initial and maximum velocity, respectively, and 1 1k kk Kcat M is the virtual dissocia tion constant of the CuIIA 1-CA complex. At high concentration of the substrate, the activities of Qr are similar, suggesting similar maximum velocity values, Vmax. The dissociation constant, KM when substrate 57

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concentration is ~0.5* Vmax, for the CuIIA 1-CA complex increases with increasing Qr concentration. These behaviors are typi cal to that of competitive inhibition.65 In competitive inhibition, the substrate [CA] and the inhibitor [Qr] are considered mutually exclusive according to this relationship: [Cu(II)-Amyloid-CA] [Cu(II)-Amyloid] + [CA] [Cu(II)-Amyloid] + o-quinone KCAkcat+ [Qr] [Cu(II)-Amyloid-Qr] KI The equation 2 below represents a competitive inhibition: CA K Qr K CAVI CA 1max (2), where KI is the inhibition constant and KCA is the dissociation constant of the CuIIA 1-CA complex. The observed behaviors in the data can be described accordingly. The only difference between Eq. 1 and 2 is KM versus I CAK Qr K 1, and in the absence of inhibitor, KCa = KM. Thus, in competitive inhibition the Vmax values at all inhibitor concentrations ar e similar and changes are only seen in the apparent KM values.65 The data at each Qr concentration was fitted to Eq. 2 to obtain 58

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individual KI values. The fitted inhibition cons tant (the average of individual KI values) for the Qr inhibition of catechol oxidation by CuIIA 1 is 4.26 M. The data were also plotted and fitted linearly, as the Lineweaver-Burk plots (1/ vs. 1/[catechol]) (Eq. 3). The reciprocal form of the velocity equation for competitive inhibition is: max max11 1 1 VCAK Qr V KI M (3) Qr KV K V K slopeI CA M max max (4) where KI = [Qr] when slope = 0 and KI is the inhibition constant which can be easily obtained from a secondary plot (Eq. 4). Th e point of convergence from the fitted lines can also suggest the type of inhibition presen t. The lines from the fits converge at the yaxis, suggesting a competitive inhibition (Figure 2.5 left).65 This conclusion is in congruence with the above non-linear behavior s. From a secondary plot of the fitted slopes versus Qr (inhibitor) concen trations, an inhibition constant, KI, of 4.24 M, ( KI = x-intercept) is determined, which is in ag reement with the value from the non-linear fit (Figure 2.5 right). The observed competi tive inhibition is in agreement with the inhibition of the di-CuII center protein tyrosinase ( KI = 38.6 M) by O2. The inhibition of tyrosine by Qr was credited to the metal binding property of Qr.56 57 59

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[Catechol] (M) 0.000 0.001 0.002 0.003 500 (Ms -1 ) 0 1e-8 2e-8 3e-8 4e-8 5e-8 0 M Qr 2 M Qr 4 M Qr 8 M Qr [Quercetin] (M) 05e-51e-42e-42e-4 500 (Ms -1 ) 0 2e-9 4e-9 6e-9 8e-9 1e-8 Figure 2.4. Quercetin inhibition of catechol oxidation by CuIIA 1. Different concentrations of catechol (0. 2 mM) were incubated with 3.0 M CuIIA 1 with different concentrations of Qr (0.0 M) in 100.0 mM HEPES pH 7.0 and 25C. The inset is the Dixon plot or direct titration of Qr, m onitored with the activity. 60

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[Catechol] -1 (M -1 ) 02e+34e+36e+38e+31e+4 -1 (M -1 s) 0 1e+8 2e+8 3e+8 4e+8 5e+8 0 mM Qr 2 mM Qr 4 mM Qr 8 mM Qr [Quercetin] (M) -2e-602e-64e-66e-68e-6 Slope 2e+4 3e+4 4e+4 Figure 2.5. Lineweaver-Burk analysis of querce tin inhibition of catechol oxidation by CuIIA 1 (left) and replot of the slope versus inhibitor concentra tions (right). The Lineweaver-Burk fitted lines intersect at x = 0, which is an indication of a competitive inhibition pattern. The inhibition constant, KI, is equal to the negative of the x-intercept of the secondary plot of the slope from th e plot on the right or a function of [Qr]. 61

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The mechanism of inhibition by Qr can be due to metal chelation or free radical scavenging. In our recent publication, we have shown the inability of mannitol and DMSO, hydroxyl and superoxide free radical scavengers, respectively, to stop the oxidation chemistry.63 Thus, the inhibitory action may be from the metal chelation property of Qr. Based on the observed competitive inhibition, it is possible that Qr binds to the CuII center and prevents the substrate from binding. This may be proposed from the non-linear plots of the inhibition study, where the KM (approximately 0.5*Vmax), the binding or dissociation constant for the CuIIA 1-catechol complex, increases as the Qr concentration is increased (Figure 2.4). Qr may be competing against catechol in binding to the CuII center. As a result, the binding between the catalyst and substrate is weakened as indicated by the increasing KM at higher Qr concentrations. Metal-binding studies of Qr have been previously done, using multiple physical techniques.51 Qr has three potential metal-binding sites: -keto-phenolate (4-carbonyl and 5-hydroxyl); -keto-enolate (4-carbonyl and 3-hydroxyl ); and catecholate (3'and 4'hydroxyl) (Figure 2.3). However, no consensu s has been reached on the metal binding mode of Qr.51 Since the antioxidant activity of Qr may be due to metal-binding, it is important to determine which metal-binding si te and how well this flavonoid can chelate the metal ion in solution. Thus, the meta l binding by Qr was studied with UV-Vis and NMR spectroscopy in DMSO. Qr (20.0 M) was slowly titrated with copper sulfate (CuSO4) in DMSO and monitored from 200 nm. Triethylamine (TEA), was added as needed to ensure full deprotonatio n of all metal-binding sites. 62

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The electronic spectrum of Qr in the ab sence of metal shows two absorptions at ~280 and ~380 nm (Figure 2.6); they have been previously assigned to the benzoyl (A ring including the -keto-phenolate moiety) and cinna moyl (C ring including the -ketoenolate and catecholate moieties), respectively.51 b, c Upon addition of CuII, a new absorption appears at 450 nm, accompanied by a decrease in the absorption of free ligand at 380 nm. This new absorption along with two isosbestic points at 325 and 410 nm indicates the formation of CuII-Qr complex (Figure 2.6). The CuII-to-Qr stoichiometry has been proposed from 1:1 up to 2:3, depending on the phase (i.e. solid vs. liquid) as well as solvent (i.e. EtOH vs. aqueous).51 In this study, the plot of the molar absorptivity value at 450 nm versus the equivalents of CuII added shows a saturation approximately at 1.2 equivalents of CuII in DMSO (Figure 2.6 inset). Then, the plot of the molar absorptivity value at 450 nm against CuII concentration can be fitted to a 1:1 metal-toligand binding quadratic equation to afford an affinity constant, KCu, of 1.06 106 M (Figure 2.6 inset). The preferred stoich iometry between CuII and Qr was determined using the Job method. It is a continuous variation method that can suggest the optimum binding between a metal and a ligand or interac tion between a catalyst and a substrate.61 62 In the optical Job study, the concentrations of CuII and Qr were arrayed while keeping the overall concentration constant. The molar absorptivity of the CuII-Qr complex at 450 nm 63

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Wavelength (nm) 300400500600700800 (M-1cm-1) 0 5000 10000 15000 20000 25000 30000 [Cu(II)]/[Qr] 0.00.51.01.52.02.5 Abs450 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Figure 2.6. Optical titration of CuII (0.0 equivalents) to Qr (20.0 M) in DMSO. An isosbestic point at 410 nm along with an incr ease in the absorption at 450 nm indicates the formation of CuII-Qr complex. Fitting the absorbance values with respect to [CuII] affords an affinity constant, KCu, of 1.06 106 M (the inset). 64

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was plotted against th e mole fraction of CuII ( QrCu CuII II ) (Figure 2.7 inset). The result shows an increase in the absorption of the complex with Cu(II) and reaches a maximum at Cu(II) = 0.5, followed by the decrease (Fi gure 2.7 inset). The result can be fitted to a stoichiometry of 1:1 for the binding between CuII and Qr (Figure 2.7 inset). The slow electronic relaxation of CuII can broaden the NMR signals beyond detection, rendering it useless in the metal-bindi ng study by means of NMR spectroscopy. However, the fast relaxing CoII has been effectively utilized as a paramagnetic NMR probe in studying the metal-coordi nation site of various systems.66 To ensure that CoII binds Qr in a similar way to CuII, the optical studies were repeated with CoII. A gradual addition of CoII into 20.0 M Qr was monitored with a spectrophotometer from 200 nm. Similar to the CuII binding study, a new absorption appears at 450 nm, accompanied by a corresponding decrease in the absorption at 380 nm. The absorption at 450 nm and the two isosbestic points at 305 and 410 nm suggest the formation of the CoII-Qr complex in a single equilibrium (Figure 2.8). The affinity constant KCo of 5.58 105 M was determined by fitting the plot of molar absorptivity value of the complex versus CoII concentration (Figure 2.8 inset). The stoi chiometry of the bound species was further determined with the Job method. Similar to the data from CuII binding study, a gradual increase in the absorption of the CoII-Qr complex (450 nm) with Co(II) reaches a maximum at Co(II) = 0.5, followed by a decrease (Figur e 2.9 inset). This suggests that a 1:1 species is the preferred stoichiometry between CoII and Qr under the experimental 65

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Wavelength (nm) 300400500600700800900 (M-1cm-1) 0 5000 10000 15000 20000 25000 [Cu(II)]/([Cu(II)]+[Qr]) 0.00.20.40.60.81.0 Absorbance 450 0.00 0.02 0.04 0.06 0.08 0.10 Figure 2.7. Optical Job plot of CuII (0.0 M) binding by Qr (0.0 M) in DMSO. The molar absorptivity values at 450 nm of the complex was plotted against the mole fraction with respect to the CuII concentration (the inset). Th e data was fitted to a simple 1:1 metal-to-ligand binding with the r2 value of 0.93. 66

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Wavelength (nm) 300400500600700800 (M-1cm-1) 0 10000 20000 30000 40000 50000 [Co(II)]/[Qr] 02468101214 Abs450 0.0 0.1 0.2 0.3 0.4 0.5 Figure 2.8. Optical titration of CoII (0.8 equivalents) to Qr (20.0 M) in DMSO. An isosbestic point at 405 nm along with an incr ease in the absorption at 450 nm indicates the formation of a CoII-Qr complex. Fitting the absorbance values at 450 nm with respect to [CoII] affords an affinity constant KCo of 5.58 105 M (the inset). 67

PAGE 85

conditions herein. Overall, Qr binds CuII and CoII similarly, and the decrease in the absorption at 380 nm for both studies suggests the possible involvement of -keto-enolate or catecholate moiety (the cinnamoyl group) in chelating metal ion.51 b, c To further elucidate the preferred bindi ng site among the three possible moieties on Qr in solution, the optical CoII binding to 5-hydroxyflavone (5HF) and catechin (CN) were performed. While the structure of 5HF only has the -keto-phenolate moiety for metal binding, the only functional group available for chelating metal on CN is the catecholate moiety (Figure 2.3). The electron ic spectrum of free CN shows an absorption at approximately 285 nm. Upon CoII addition to CN, a new abso rption appears at 313 nm (an isosbestic point at 298 nm) with a corre sponding minor decrease in the absorption at 285 nm, indicating the complexation between CoII and CN (Figure 2.10 left). Similarly, the increase in a new absorpti on at 410 nm (with an isosbe stic point at 368 nm) coupled with the decrease in the absorption of fr ee 5HF at 335 nm upon gradual addition of CoII marks the formation of a CoII-5HF complex (Figure 2.10 right). The absorptions of the CoII-catecholate (313 nm) and -keto-phenolate (410 nm) co mplexes can be clearly distinguished from that of the CoII-Qr complex, which appears at 450 nm, indicating the CoII binding site cannot be the -keto-phenolate and the catechol ate site. By default, the CoII binding site in Qr is -keto-enolate. However, the possibility of CoII-ketophenolate binding cannot be completely ruled out since the absorptions of the complexes are close. The metal-binding site was fu rther confirmed by means of NMR spectroscopy. 68

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Wavelength (nm) 300400500600700800 (M-1cm-1) 0 5000 10000 15000 20000 25000 [Co(II)]/([Co(II)]+[Qr]) 0.00.20.40.60.81.0 Abs450 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 Figure 2.9. Optical Job plot of CoII (0.0 M) binding to Qr (0.0 M) in DMSO. The plot of the molar absorptivity values at 450 nm versus the mole fraction with respect to the mole fraction of CoII fits well to a 1:1 species with the r2 = 0.95 (the inset). 69

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Wavelength (nm) 350400450500 0.00 0.05 0.10 0.15 0.20 Wavelength (nm) 260280300320340360380400 Absorbance 0.00 0.05 0.10 0.15 0.20 Figure 2.10. Optical CoII titration of catechin (left) a nd 5-hydroxyflavone (right). The new absorption at 313 nm for catechin and 410 nm for 5-hydroxyflavone along with their corresponding isosbestic points at 298 and 368 nm, respectively, indicates the formation of CoII-ligand complexes. 70

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NMR is a powerful spectroscopic te chnique for studying the coordination environment of paramagnetic metal ions, such as CoII, FeII, and NiII as well as some lanthanideIII ions, in both simple complexes and metallo-proteins.66 Because of the overwhelming electron magnetic moment of th e unpaired electron(s) in the paramagnetic systems (658 times that of the proton), the co ordination environment is very sensitive to changes due to electron-nuclear hyperfine interaction. As a result, metal ions such as CoII and YbIII have been commonly utilized as paramagnetic NMR probes for studying the metal coordination environments in ZnIIand CaII-containing metalloproteins, respectively, upon replacing the native me tal ions in the proteins with CoII and YbIII ions.66 The same electron-nuclear hyperfine in teraction is responsib le for shifting the signals of the coordinated ligand and outside the typical diamagnetic spectral region (0 15 ppm). In addition, the fast electronic relaxation of these pa ramagnetic agents can enhance nuclear relaxation; thus the relaxation time of the signals close to the metal ion is considerably shortened. Th e slow electronic relaxation of CuII (10s) can reduce the nuclear relaxation time and can l ead to broadening of the signals beyond detection. The fast relaxing CoII (103s), however, can afford relatively sharp shifted signals.66 Thus, CoII was used in place of CuII for the determination of the metal binding site on Qr. A comp lete assignment of the 1H NMR spectrum of the CoII-Qr complex is accomplished by the use of 1D a nd 2D saturation transfer NMR techniques, 62 67 owing to the presence of exchange between the metal-bound and metal-free Qr according to the equilibrium, M + Qr MQr. 71

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The 1H NMR spectrum of CoII-Qr acquired in d6-DMSO displays 8 paramagnetically shifted signals (out of 10 pr otons) within the spect ral region of 60 to 80 ppm (Figure 2.11). TEA was added as needed to ensure full depr otonation of all three metal-binding sites on Qr. The proton spectrum in Fig. 2.11 was acquired with the superWEFT (modified Water Elim inated Fourier Transform) ( 1-180 2-90 -FID),68 a solvent suppression technique for paramagnetic systems that selectively suppresses the slowly relaxing diamagnetic signals. As a result, the diamagnetic signals appeared distorted. An exchanging system allows the use of 2D saturation transfer experiment, EXSY (EXchanged SpectroscopY), for the assignm ent of paramagnetic signals once the diamagnetic free ligand signals are fully assigned.67 From the EXSY spectrum, 5 out of 8 shifted paramagnetic signals can be conn ected to their corresponding diamagnetic counterparts. The shifted signals at 17.0 and 16.3 ppm of the CoII-Qr complex are in exchange with the signals of the 3'-OH and 4'-OH protons, respectiv ely, of the catechol moiety of free Qr (3' and 4' cross peaks Figur e 2.12). This assignment clearly shows that the catecholate moiety on Qr does not involve in metal binding, otherwise the 3 and 4 protons should not be detected since th ey deprotonate upon metal binding. This conclusion is in agreement with the finding from optical studies. The remaining three signals are correlated with 6-H, 8-H, and 5'-H protons (Fig. 2.12 and Table 2.1). 72

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Figure 2.11. 1D NMR spectrum of Co(II)-(25.0 mM) Qr-TEA in a 1:5:1.5 ratio in d6 DMSO. The spectrum was acquired with th e spectral width of 110k ( to 100 ppm) on the Varian INOVA500 using superWEFT (Water Eliminated Fourier Transform)68 technique to selectively obser ve the fast-relaxing paramagne tically shifted signals over the diamagnetic ones. The signal intensities are quite different owing to their very different relaxation times which result in si gnal saturation to different extents. The inverted signals are due to diamagnetic protons far away from the paramagnetic metal center, which do not relax to gain positive intensity after the 180 pulse and the short delay. 73

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The broadness of the three furthest shifte d signals (56.4, 49.8, and .1 ppm) did not allow the assignment by 2D EXSY experime nt. However, they can be assigned with 1D saturation transfer technique. The irradiation of the mo st upfield shifted signal at 75.1 ppm reveals saturation transfer to the si gnal at 12.5 ppm corres ponding to 5-H of the hydroxyl proton of the -keto-phenolate moiety on the structure of Qr. Furthermore, the protons of the two most downf ield shifted signals at 56.4 and 49.8 ppm are in exchange with 2'-H and 6'-H protons respectively (Figure 2.13). The solvent exchangeable hydroxyl OH signals can be easily identified with the addition of a few drops of D2O to the d6-DMSO solution. The OH signals are identified as the signals at 17.0, 16.3, and 75.1 ppm, correlating to 3'-H, 4' -H, and 5-H. They are in ag reement with the assignments by 2D EXSY and 1D saturation transfer experiments. After all the paramagnetic si gnals have been assigned, the two proton signals left unaccounted for are the hydroxyl protons at 7-H and 3-H. The former is a free-standing hydroxyl group and unlikely to be the preferred metal binding site. The latter, however, is the hydroxyl proton for the -keto-enolate moiety, and the absence of a paramagnetic counterpart may indicate the de protonation of the enol group and binding of the metal ion. On the other hand, the shif ted signal for 5-H suggests that the -keto-phenolate site is still protonated. The protons closest to the metal ion will be most affected by the electron-nuclear interaction, and they are expected to have larger paramagnetic shifts as well as shorter relaxation times. With the conclusion above that Qr binds CoII at the keto-enolate moiety, the protons in the closest proximity are the 5-H on the -keto74

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* * *Figure 2.12. The 2D 1H EXSY spectrum of the 1:1:0.1 CoII-(31.0 mM) Qr-TEA complex in d6-DMSO. The data was acquired w ith the mixing time of 90 ms. The paramagnetic signals (marked with asterisks) of the complex are correlated to their diamagnetic counterparts, and the numbers ne xt to the crosspeaks represent the protons according to Figure 2.3. 75

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Figure 2.13. 1D saturation-transfer results for the CoII-Qr complex. The difference spectra from the 1D 1H saturation transfer experiments for the three farthest shifted signals, .1, 49.8, and 56.4 ppm show connections with their corresponding diamagnetic protons (7.5, 7.6, and 12. 5 ppm, bottom to top) of the 1:1 CoII-Qr complex. 76

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phenolate site and both 6'-H and 2'-H, since the single bond allows the rotation of the B ring (Figure 2.3). Thus, the 5-H, 2'-H, and 6' -H protons are expected to have the largest chemical shifts, .1, 49.8, and 56.4 ppm, respectively, in comparison to the remaining shifted signals. In addition, T1 relaxation times for all shifted signals were determined with the standard inve rsion recovery method ( 12) and are reported in Table 1. As expected, 5-H, the closest proton to the CoII ion, has the lowest T1 value (4.0 ms) among all, followed by the 6'-H (5.4 ms) and 2' -H (6.2 ms) signals. From the combined results, the metal binding on Qr can be unambiguously assigned to the -keto-enolate moiety. Physiological studies have shown that soluble A peptides of the sequence 1, 1, and 25 can increase the intracellular CaII concentration in hippocampus, deteriorating learning ability and memory.33 Both the soluble and aggregated forms of the amyloidpeptides can span lipid membrane which creates channels with poor ion selectivity35 and can cause membrane depolarization and impair Na+/K+-ATPase which leads to an increase in CaII influx.36, 37 The rise in the CaII level can defect neurons and synapses, deter long-term potentia tion in certain brain regions, and lead to impairments in learning and memory.32 37 As shown earlier, Qr is a potent inhibitor ( KI = 4.24 M) against the catechol oxidation chemistry of CuIIA 1. A possible mechanism of inhibition is chelation of the CuII center by Qr; this prevents the substrate from binding to the CuII center. 77

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1H Position dia (ppm) para (ppm) T 1 (ms) 6'-H 7.5 56.4 5.4 2'-H 7.6 49.8 6.2 5'-H 6.9 19.1 89 3'-OH 9.3 17.0 213 4'-OH 9.6 16.3 408 8-H 6.4 13.0 158 6-H 6.2 .3 134 5-OH 12.5 .1 4.0 Table 2.1. Full assignment of the 1H NMR spectrum of the 1:1:0.1 CoII-(31.0 mM) QrTEA complex from 1D satu ration transfer and 2D EX SY experiments and their corresponding T1 relaxation times. 78

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Alternatively, Qr can act as a suicid e substrate and can be oxidized by the CuIIA 1 complex, competing with other physiologically important substrates. There are three possible sites on Qr for interacting with th e metal ion, and the mode of binding can suggest the possible mechanism of inhibition. For example, if the catecholate moiety is the binding site, Qr is potentially oxidized since CuIIA 1 has been shown to have both catecholand phenol-oxidase activity.63 64 The metal binding site of Qr has been determined to be at the -keto-enolate moiety by means of optical and NMR spectroscopy discussed above. Thus, the an tioxidative mechanism of Qr is possibly through chelation of the metal center. This finding is important sin ce Qr is a naturally occurring antioxidant which may be applicable as a therapeutic agent against the redox metal-dependent oxidative stre ss in the pathology of AD. Since Qr is a good metal chelator,51 it may indiscriminately bind CaII at the same site and blocks its binding to the metal in CuIIA 1. As a result, the increased intracellular CaII concentration may reduce the antioxidativ e effect of Qr in the brain. To gain further information about the metal-bind ing versatility of Qr the binding of the biologically significant CaII to Qr was investigated. A new absorption at 450 nm and an isosbestic point at 405 nm (Fig. 2.14) are an alogous to the transition metal complexes of Qr (Figs. 2.6 and 2.8), confirming the formation of CaII-Qr complex. Fitting of the molar absorptivity value (at 40 5 nm) as a function of CaII concentration gives a smaller affinity constant KCa of 530 M. 79

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Wavelength (nm) 300400500600700800 (M-1cm-1) 0 5000 10000 15000 20000 25000 30000 [Ca(II)]/[Qr] 02e+24e+26e+28e+21e+3 Abs450 0.00 0.05 0.10 0.15 0.20 F igure 2.14. Optical titration of CaII (0.2 equivalent s) to Qr (20.0 M) in DMSO. An isosbestic point at 405 nm along with an increase in the absorption at 450 nm indicates the formation of CaII-Qr complex. Fitting the absorbance values versus [CaII] affords an affinity constant, KCa, of 5.35 102 M (the inset). 80

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The influence of CaII on the antioxidative activity of Qr was probed with respect to catechol oxidation. Se veral concentrations of CaII (0 mM) were incubated with 3.0 M CuIIA 1 and a fixed concentration of ca techol in the presence of 4.0 M Qr in 100 mM HEPES at pH 7.0 and 25 C. The cat echol oxidation gradually increases with increasing CaII concentrations and eventually reaches a plateau at high millimolar (mM) concentrations of CaII (Figure 2.15 inset). The saturati on profile potentiall y indicates that the presence of CaII is some how promoting the substrat es ability to interact with the redox CuII center, since CaII is a redox-inert metal. It is possible that CaII is interfering with the CuII center chelated by Qr. Even though the affinity of Qr toward CaII is not as strong as that of CuII, the binding and removing of Qr from the CuII center may occur in the presence of high concentrations of CaII. This is evident in the requirement of mM concentration in order to clearly see the effect. The effect of CaII was further investigated with ki netics. Different concentrations of catechol (0.2 mM) were incubated with 4.0 M Qr ( KI concentration) and 3.0 M CuIIA 1 with different c oncentrations of CaII (0.0 mM) in 100.0 mM HEPES pH 7.0 and 25 C. At all concentrations of CaII, the data show an initial increase in catechol oxidation, followed by a saturation at higher subs trate concentrations (Figure 2.15). This pattern suggests a possible pre-equi librium kinetics, which indicates CaII binding to Qr. A rate law (Eq. 5) is derived according to the following relationship: 81

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[Cu(II)-Amyloid-CA] [Cu(II)-Amyloid] + [CA] [Cu(II)-Amyloid] + o-quinone KCatecholkcat+ [Qr] [Cu(II)-Amyloid-Qr] KI+ [Cal] [Cal-Qr] KCal CA K KCa IK K CAVI Cal II Cal Catechol 0 0 max1 (5) where [I]0 = experimental [Qr], [Ca]0 = experimental [CaII], KI = Qr inhibition constant, KCatechol = intrinsic (in the absence of Qr and Ca ) dissociation constant of catechol, and KCal = dissociation constant for Ca The results are fitted to Eq. 5 to afford KCal = 0.263 M. This large dissociation constant for Ca is consistent with the small affinity constant observed from the optical titration, wh ich also suggests th e influence of Ca on Qr inhibition of catechol oxidation chemistry by Cu A is the result of binding to Qr. Since the effect of CaII toward Qr inhibition of catechol oxidation by CuIIA 10 is potentially due to binding to Qr and removing of Qr from the CuII center for oxidation, it is essential to know how Qr binds Ca in order to make a correlation between the structure and observed activity. Thus, the Ca binding mode of Qr was elucidated by means of NMR spectroscopy. Since Ca is spectroscopically inert, the lanthanide Yb II II II II II II II II III 82

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[Catechol] (M) 0.000 0.001 0.002 0.003 500 (Ms-1) 0 1e-8 2e-8 3e-8 4e-8 5e-8 6e-8 0 mM Ca 2+ 16 mM Ca 2+ 48 mM Ca 2+ 80 mM Ca 2+ [Calcium] (M) 0.000.040.080.120.16 500 (Ms -1 ) 6.0e-9 8.0e-9 1.0e-8 1.2e-8 1.4e-8 1.6e-8 Figure 2.15. Calcium influence on quercetin inhibition of catechol oxidation by CuIIA 1. Different concentrations of catechol (0.2 mM) were incubated with 4.0 M Qr at KI concentration and 3 M CuIIA 1 with different concentrations of CaII (0.0 mM) in 100.0 mM HEPES pH 7.0 and 25 C. The inset is the dire ct titration of CaII in the presence of the inhibitor, Qr monitored with the activity. 83

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was used as a paramagnetic NMR probe. Th e lanthanides have similar radii and ligand binding preferences as CaII and thus are good CaII substitutes in biological systems.69 Similar to the CoII binding study, the 1HNMR spectrum of the YbIII-Qr complex shows the exchange between the free and metal-bound form of Qr. As a result, 1D and 2D saturation transfer experiments, such as 1D NOE difference and 2D EXSY, were used in the assignment of paramagnetically shifted signals. The 1H NMR spectrum displays 9 shifted signals within the range of 20 to ppm (marked, Figure 2.16). Unlike the CoIIQr spectrum, some signals of the YbIII-Qr complex appear within the diamagnetic spectral region (0 ppm). This is due to the difference between the transition and lanthanide metal ions in their electronnuclear interaction. While the through-bond interaction mainly occurs in the transiti on metal complexes, only the through-space or dipolar interaction can take place in the lanthanide complexes, since the f orbitals in the lanthanides are shielded and do not overlap with the ligand orbitals. Furthermore, the dipolar interaction only occurs in the presence of magnetic anisotropy, and the direction and magnitude of magnetic anisotropy dete rmine the direction and magnitude of the dipolar shift.66 From the EXSY spectra, 8 shifted signals of the YbIII-Qr complex can be assigned to their diamagnetic counterparts of metal-free Qr (Figures 2.17 and 2.18). Half of these signals are determined to be solven t-exchangeable OH protons based on the disappearance of signals upon D2O addition. Among them, the signals at 19.2, 4.1, and 84

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* * * * * Figure 2.16. 1D NMR spectrum of Yb(III)-Q r-TEA in a 1:1:0.7 ratio in d6DMSO. The spectrum was acquired with the spectral width of to 100 ppm on the Varian INOVA500 using superWEFT technique. 85

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2.1 ppm are exchanging with the diamagnetic signals at 12.5, 9.6, and 9.3 ppm, corresponding to 5-H, 4'-H, and 3'-H on the structure of Qr (Figure 2.3). The one remaining signal at 6 ppm was assigned to 6-H with the 1D saturation transfer experiment. Similar to the results from the CoII binding study, th e protons at the -ketophenolate (5-H) and cate cholate (4'-H and 3'-H) sites ar e present, which indicate their noninvolvement in metal binding. On the opposite note, the absence of 3-H signal along with short T1 relaxation times of the nearby protons (5-H, 6'-H, and 2'-H) clearly indicate the -keto-enolate site to be the YbIII binding site. The exchange correlations along with the corresponding T1 values are summarized in Table 2.2. Based on the YbIII binding study, Qr may potentially bind CaII at the same site as CuII and CoII, which is at the -keto-enolate moiety. This may explain the effect of CaII toward the Qr inhition of catechol oxidation by CuIIA 1. At sufficiently high concentration of CaII, it can compete with CuII in binding to Qr, si nce Qr binds both CaII and CuII at the same site. As a result, the CuII center of the CuIIA 1 complex is made available for binding and oxidati on of catechol. In order to confirm this hypothesis, the influence of YbIII on the Qr inhibition was determined with optical and kinetic studies. While the optical study ensures the similarity between CaII and YbIII binding to Qr, the kinetic study will help to determine if the CaII interference in the Qr inhibition is the result of its binding to Qr. Consistent with the transition metals and CaII binding studies, a new absorption appears at approximately 440 nm upon addition of YbIII to Qr (Figure 2.19). A 86

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Figure 2.17. The 2D 1H EXSY spectrum of the 1:1:0.7 YbIII-Qr-TEA complex in d6DMSO. The data was acquired with mixing time of 8.3 ms. The protons shifted the farthest with respect to the diamagnetic enve lope, indicating their cl ose proximity to the metal center. 87

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Figure 2.18. The 2D 1H EXSY spectrum of the expanded region of ~12 to ppm for Fig. 2.17 of the 1:1:0.7 YbIII-Qr-TEA complex in d6-DMSO. The data was acquired with a mixing time of 8.3 ms. The presence of a crosspeak for 3'-OH eliminates the catechol moiety from being a possible metal-binding site. 88

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1H Position dia (ppm) para (ppm) T 1 (ms) 5-OH 12.5 19.2 8.3 7-H 10.9 8.8 832 6-H 6.2 6.0 420 3'-OH 9.3 2.1 638 8-H 6.4 .1 554 5'-H 6.9 .5 339 2'-H 7.8 .6 32.1 6'-H 7.7 .2 24.3 Table 2.2. Full assignment of the 1H NMR spectrum of the 1:1:0.7 YbIII-Qr-TEA complex from 1D saturation transfer and 2D EXSY experiments and their corresponding T1 relaxation times. 89

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corresponding decrease in the absorption of the free Qr in 380 nm is observed along with the isosbestic point at roughly 400 nm, which indicates the formation of YbIII-Qr complex. Fitting the molar absorptivity at 440 nm versus YbIII concentration to a simple 1:1 binding pattern affords an affinity constant KYb = 5.60 105 M (Figure 2.19, inset). The stoichiometry of the binding between YbIII and Qr was further determined with the Job method as described above. The plot of the molar absorptivity values at 440 nm versus the mole fraction of YbIII shows a maximum absorption intensity at Yb(III) = 0.5, which indicates the preferred stoichiome try to be 1:1 (Figure 2.20, inset). The influence of YbIII on the antioxidative activity of Qr was further investigated with respect to catechol oxidation. Several concentrations of YbIII (0.0 M) were incubated with 3.0 M CuIIA 1 and a fixed concentration of catechol in the presence of 4.0 M Qr in 100.0 mM HEPES at pH 7.0 and 25 C. An increase in catechol oxidation was observed with increasing YbIII concentration and reaches a plateau at high micromolar ( M) concentration. The effect of YbIII was further investigated with kinetics. Different concentrations of catechol (0. 2 mM) were incubated with 4.0 M Qr ( KI concentration) and 3.0 M CuIIA 1 with different concentrations of YbIII (0.0 M) in 100.0 mM HEPES pH 7.0 and 25 C. At all concentrations of YbIII, the data show an initial increase in catechol oxidation, followed by a saturation at higher substrate c oncentrations (Figure 2.21). This pattern suggests a possible pr e-equilibrium kinetics, which indicates YbIII binding to Qr. A rate law (Eq. 6) is de rived according to the following relationship: 90

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Wavelength (nm) 300400500600700800 (M-1cm-1) 0 10000 20000 30000 40000 50000 [Yb(III)]/[Qr] 0246 Abs440 0.0 0.1 0.2 0.3 0.4 Figure 2.19. Optical titration of YbIII (0.4 equivalents) to Qr (20.0 M) in DMSO. An isosbestic point at ~400 nm along with an in crease in the absorption at 440 nm indicates the formation of YbIII-Qr complex. Fitting the molar abso rptivity values with respect to the added YbIII equivalent affords an affinity constant, KYb, of 5.60 105 M (the inset). 91

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[Cu(II)-Amyloid-CA] [Cu(II)-Amyloid] + [CA] [Cu(II)-Amyloid] + o-quinone KCatecholkcat+ [Qr] [Cu(II)-Amyloid-Qr] KI+ [Yb] [Yb-Qr] KY CA K KYb IK K CAVI Y III Y Catechol 0 0 max1 (6) where [I]0 = experimental [Qr], [YbIII]0 = experimental [YbIII], KI = Qr inhibition constant, KCatechol = intrinsic (in the absence of Qr and Yb ) dissociation constant of catechol, and KY = dissociation constant for Yb The results are fitted to Eq. 6 to afford KY = 4.85 10 M. Compared to Ca a smaller dissociation constant for Yb is expected since its affinity to ward Qr is approximately 1000 times higher than that of CaII based on the affinity constants obtained from the optical titration experiments. III III II III 92

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Wavelength (nm) 300400500600700800 (M -1 cm -1 ) 0 5000 10000 15000 20000 25000 30000 [Yb(III)]/([Yb(III)]+[Qr]) 0.00.20.40.60.81.0 Absorbance 440 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 Figure 2.20. Optical Job plot of YbIII (0.0 M) binding to Qr (0.0 M) in DMSO. The plot of the absorbance at 440 nm versus the mole fraction of YbIII shows a maximum absorption intensity at Yb(III) = 0.5, which indicates the pref erred stoichiometry to be 1:1 (the inset). 93

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[Catechol] (M) 0.0000.0010.0020.0030.0040.0050.006 (Ms-1) 0 2e-8 4e-8 6e-8 8e-8 1e-7 0 M Yb 80 M Yb 180 M Yb 600 M Yb [Ytterbium] (M) 0.04.0e-48.0e-41.2e-31.6e-3 500 (Ms -1 ) 1.2e-8 1.6e-8 2.0e-8 2.4e-8 2.8e-8 Figure 2.21. Ytterbium influence on quercetin inhibition of catechol oxidation by CuIIA 1. Different concentrations of catechol (0.0 mM) were incubated with 4.0 M Qr ( KI concentration) and 3.0 M CuIIA 1 with different concentrations of YbIII (0 600.0 M) in 100.0 mM HEPES pH 7.0 and 25C. The inset is the direct titration of YbIII in the presence of the inhibitor, Qr, monitored with the activity. 94

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2.4 Conclusions It is originally thought that the aggregation of A peptide is the cause of neurodegeneration in the pat hology of Alzheimers disease.15 However, there is inconsistency in the amount of plaque pres ent and the severity of neurodegeneration.16 Furthermore, the presence of high concentr ation of redox-active metal ions such as CuII and FeIII in the plaques11 along with oxidized biological molecules17 indicates that the metal dependent oxidative stress may be a culpri t of neuronal damages in the brain. As a result, the metal chelation th erapy has become a center of attention. A good metal chelator such as clioquinol has been proposed.39, 41 However, the testing of this drug has been stopped at the Phase II clinical trials due to its intrinsic toxicities.40 44 Recently, naturally occurring flavonoids have gained the interests of researchers due to their biological properties.47 One of the most studied among them is Qr. Qr is well known for its free radical scave nging and metal chel ating abilities.50 51 We have recently shown the ability of short N-terminal A fragments (A 16 and A 1, which contains the metal binding domain) to perform catecholand phenol-oxidase-like chemistry in the presence of CuII.63, 64 In this study, the antioxidant ability of Qr toward the observed oxidation chemistry is determined Qr can potentially serve two purposes. It can be a metal chelator (block the CuII center and prevent the substrate from binding) or a suicide substrate (the C ring contains a catechol moiety, which can be oxidized). Based on kinetic studies, Qr has been determined to competitively inhibit catechol oxidation by CuIIA with a KI of 4.24 M. Qr has three possible metal binding sites: 95

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keto-phenolate, -keto-enolate, and catecholate moiety The site of binding may suggest the mechanism of inhibition. For example, if the metal is bound through the catecholate moiety, Qr is potentially oxidized by CuIIA acting as a suicide substrate. Thus, the metal binding studies were performed by means of optical and NMR spectroscopy. According to NMR results, the metal binding site can be unambiguously assigned to the -keto-enolate moiety, suggesting that the m echanism of inhibition by Qr may be due to CuII center chelation. A recent study showing tyrosinase inhibition by Qr supports this conclusion.57 The A peptide has been shown to cause a disruption in CaII homeostasis in AD, leading to the rise in intracellular CaII concentration. Memory loss and cognitive impairments have been associated with an increase in CaII level.32 37 Since Qr can bind metal indiscriminately, the presence of CaII may interfere with its antioxidant ability, or on the other hand, the free CaII can be chelated by Qr whic h may decrease the damage caused by the extra free CaII. As expected, the recovery of catechol oxidation by CuIIA was observed in the presence of high concentration of CaII. The fact that the interference is due to CaII binding was shown by using YbIII as a spectroscopic probe (for spectroscopically inert CaII) in optical and NMR studies. Once again, the NMR results showed that Qr binds YbIII at the -keto-enolate site. This indicates that CaII binds Qr at the same site as CuII and thus releasing the CuII center for catechol oxidation. These findings suggest that Qr may have dual protective roles in AD, preventing both the metal96

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97 centered oxidative stress by binding to the metal in CuIIA as well as CaII-induced memory and cognitive impairments by chelating CaII. Since Qr-containing fruits and vegetables make up a large part of our daily diets, this compound is relatively safe.47 Thus, Qr may be potentially used as a therapeutic agent toward AD. However, one main concer n for the flavonoids is their bioavailability, especially for Qr. The hydrophobic nature of the polyphenolic structures greatly reduces its absorbability into the body. Furthermore, metabolic processes can potentially change or modify the chemical st ructure(s), leading to the reduction or loss of function.70 Another possibility that may c ontribute to this discrepancy is the inability of flavonoids to be at the places when they are needed, which includes being presence when they are not needed (e.g. when ROS homeostasis is well maintained) and possible interaction with biomolecules (e.g. hydrogen bonding through the hydroxyl group).71 As a result, the efficacies of naturally occurring antioxidants in vitro cannot be directly translated to the situations in vivo Thus, further in vivo studies in animal models are needed. Nonetheless, Qr may be a good starting point serving as a template for future drug design.

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98 References 1 National Institute on Aging, a division of U.S. National Institutes of Health. http://www.nia.nih.gov/Alzheim ers/Publications/adfact.htm (accessed March 3, 2008). 2 Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald, B. L.; Beyreuther, K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4245. 3 Chen, Y.; Tang, B. L. The amyloid precursor protein and postnatal neurogenesis/neuroregeneration. Biochem. Biophys. Res. Comm. 2006, 341, 1. 4 Coulson, E. J.; Paliga, K.; Beyreuther, K.; Masters, C. L. What the evolution of the amyloid protein precursor supergene fa mily tells us about its function. Neurochem. Int. 2000, 36, 175. 5 (a) Heber, S.; Herms, J.; Gajic, V.; Hainfellner, J.; Aguzzi, A.; Rulicke, T.; von Kretzschmar, H.; von Koch, V.; Sisodia, S. S. ; Tremml, P.; Lipp, H. P.; Wolfer, D. P.; Muller, U. Mice with Combined Gene K nock-Outs Reveal Essential and Partially Redundant Functions of Amyloid Precu rsor Protein Family Members. J. Neurosci. 2000, 20, 7951. (b) Wang, P.; Yang, G.; Mosier, D. R.; Chang, P.; Zaidi, T.; Gong, Y. D.; Zhao, N. M.; Dominguez, B.; Lee, K. F.; Gan, W. B.; Zheng, H. Defective Neuromuscular Synapses in Mice Lacking Amyloid Precursor Protein (APP) and APP-Like Protein 2. J. Neurosci. 2005, 25, 1219. (c) Herms, J.; Anliker, B.;

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105 Matushita, T. Metal [MLx; M ) Fe, Cu, Co, Mn]/Hydrope roxide-Induced Activation of Dioxygen for the Oxygenation of Hydro carbons: Oxygenated Fenton Chemistry Acc. Chem. Res. 1996, 29, 409. 25 Opazo, C.; Huang, X.; Chernyl, R. A.; Moir, R. D.; Roher, A. E.; White, A. R.; Cappai, R.; Masters, C. L.; Tanzi, R. E.; Inestrosa, N. C.; Bush, A. I. Metalloenzymelike Activity of Alzheimers Disease -Amyloid: Cu-Dependent Catalytic Converstion of Dopamine, Cholesterol, and Biologi cal Reducing Agents to Neurotoxic H2O2. J. Biol. Chem. 2002, 277, 40302. 26 Zou, K.; Gong, J. S.; Yanagisawa, K.; Michikawa, M. A Novel Function of Monomeric Amyloid -Protein Serving as an Antioxidant Molecule against MetalInduced Oxidative Damage. J. Neurosci. 2002, 22 4833. 27 (a) Lee, H.-G.; Zhu, X.; Takeda, A.; Perry, G.; Smith, M. A. Emerging evidence for the neuroprotect ive role of -synuclein. Exper. Neurol. 2006, 200, 1. (b) Quilty, M. C.; King, A. E.; Gai, W.-P.; Pountney, D. L.; West, A. K.; Vickers, J. C.; Dickson, T. C. Alpha-synuclein is upregulated in neur ones in response to chronic oxidative stress and is associated with neuroprotection. Exper. Neurol. 2006, 199, 249. (c) Nadal, R. C.; Abderlraheim, S. R.; Brazier, M. W.; Rigby, S. E. J.; Brown, D. R.; Viles, J. H. Prion protein does not redox-silence Cu2+, but is a sacrificia l quencher of hydroxyl radicals. Free Rad. Biol. Med. 2007, 42, 79.

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106 28 Bush, A. I. The metallobiology of Alzheimer's disease. Trends in Neurosciences 2003, 26, 207. 29 (a) Atwood, C. S.; Scarpa, R. C.; Huang, X.; Moir, R. D.; Jones, W. D.; Fairlie, D. P.; Tanzi, R. E.; Bush, A. I. Characteriza tion of Copper Interac tions with Alzheimer Amyloid Peptides. Identification of an Attomolar-Affin ity Copper Binding Site on Amyloid 1-42. J. Neurochem. 2000, 75, 1219. (b) Opazo, C., Barria, M. I., Ruiz, F. H., Inestrosa, N. C. Copper reduction by copper bindi ng proteins and its relation to neurodegenerative diseases. Biometals 2003, 16, 91. (c) Ruiz, F. H., Gonzalez, M., Bodini, M., Opazo, C., Inestrosa, N. C. Cysteine 144 Is a Key Residue in the Copper Reduction by the -Amyloid Precursor Protein. J. Neurochem 1999, 73, 1288. 30 Cerpa, W. F., Barria, M. I., Chacon, M. A., Suazo, M., Gonzalez, M., Opazo, C., Bush, A. I., Inestrosa, N. C. The N-te rminal copper-binding domain of the amyloid precursor protein protects against Cu2+ neurotoxicity in vivo. FASEB J. 2004, 18, 1701. 31 (a) McLean, C. A.; Cherny, R. A.; Fraser, F. W.; Fuller, S. J.; Smith, M. J.; Beyreuther, K.; Bush, A. I.; Mast er, C. L. Soluble pool of A amyloid as a determinant of severity of neurodegenerati on in Alzheimers disease. Ann. Neurology 1999, 46, 860. (b) Lue, L.-F.; Kuo, Y.-M.; roher, A. E.; Brachova, L.; Shen, Y.; Sue, L.;

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107 Beach, T.; Kurth, J. H.; Rydel, R. E.; R ogers, J. The Magnitude of Brain Lipid Peroxidation Correlates with the Extent of Degeneration but Not with Density of Neuritic Plaques or Neurofib rillary Tangles or with AP OE Genotype in Alzheimers Disease Patients. Amer. J. Pathol. 1999, 155, 853. (c) Dahlgren, K. N.; Manelli, A. M.; Stine, W. B. J.; Baker, L. K.; Krafft, G. A.; LaDu, M. J. Oligomeric and Fibrillar Species of AmyloidPeptides Differentially Affect Neuronal Viability. J. Biol. Chem. 2002, 277, 32046. (d) Walsh, D. M.; Klyubin, I.; Fadeeva, J. V.; Cullen, W. K.; Anwyl, R.; Wolfe, M. S.; Rowan, M. J.; Selkoe, D. J. Naturally secreted oligomers of amyloid protein potently inhibi t hippocampal long-term potentiation in vivo Nature 2002, 416, 535. (e) Wang, H.-W.; Pasternak, J. F.; Kuo, H.; Ristic, H.; Lamber, M. P.; Chromy, B. ; Viola, K. L.; Klein, W. L.; Stine, W. B.; Krafft, G. A.; Trommer, B. L. Solubl e oligomers of b amyloid (1-42) inhibit longterm potentiation but not long-term depression in rat dentate gyrus. Brain Research 2002, 924, 133. 32 Chen, Q.-S.; Kagan, B.L.; Hirakura, Y. ; Xie, C.-W. Impairment of Hippocampal Long-Term Potentiation by Alzheimer Amyloid -Peptides. J. Neurosci. Res. 2000, 60, 65. 33 Xie, C.-W. Calcium-regul ated signaling pathways. Neuromol. Med. 2004, 6 53.

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108 34 Arispe, N.; Rojas, E.; Pollard, H.B. Alzheimer disease amyloid 8 protein forms calcium channels in bilayer membranes: Blockade by tromethamine and aluminum. Proc. Natl. Acad. Sci. USA 1993, 90, 567. 35 Good, T.A.; Smith, D.O.; Murphy, R.M. -Amyloid Peptide Blocks the Fastinactivating K+ Current in Rat Hippocampal Neurons. Biophysical Journal 1996, 70, 296. 36 Mark, R.J.; Hensley, K.; Butterfield, D.A.; Mattson, M.P. Amyloid -peptide Impairs Ion-Motive ATPase Activities: Evid ence for a Role in Loss of Neuronal Ca2+ Homeostasis and Cell Death. J. Neuroscience 1995, 15, 6239. 37 Cullen, W.K.; Suh, Y.-H.; Anwyl, R.; Rowan M.J. Block of LTP in rat hippocampus in vivo by b-amyloid precursor protein fragments. NeuroReport 1997, 8 3213. 38 da Silva, G. F. Z.; Ming, L.-J. Metallo-ROS in Alzheimers disease: oxidation of neurotransmitters by CuII-amyloid and neuropathology of the disease. Angew. Chem. Int. Ed. 2007, 46, 3337. 39 Raman, B.; Ban, T.; Yamaguchi, K.-I.; Saka i, M.; Kawai, T.; Naiki, H.; Goto, Y. Metal Ion-dependent Effects of Clioquinol on the Fibril Growth of an Amyloid Peptide. J. Biol. Chem. 2005, 280, 16157. 40 Yassin, M. S.; Ekblom, J.; Xilinas, M.; Go ttfries, C. G.; Oreland, L. Changes in uptake of vitamin B12 and trace metals in brains of mice treated with clioquinol. J. Neuro. Sci. 2000, 173, 40.

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109 41 Cherny, R. A.; Atwood, C. S.; Xilinas, M. E.; Gray, D. N.; Jones, W. D.; McLean, C. A.; Barnham, K. J.; Volitakis, I.; Fraser, F. W.; Kim, Y.-S.; Huang, X.; Goldstein, L. E.; Moir, R. D.; Lim, J. T.; Beyreuther, K.; Zheng, H.; Tanzi, R. E.; Masters, C. L.; Bush, A. I. Treatment with a Copper-Zinc Chelator Markedly and Rapidly Inhibits _Amyloid Accumulation in Alzheimers Disease Transgenic Mice. Neuron 2001, 30, 665. 42 Regland, B.; Lehmann, W.; Abedini, I.; Blennow, K.; Jonsson, M.; Karlsson, I.; Sjogren, M.; Wallin, A.; Xilinas, M.; Gottfries, C. G. Dement. Geriatr. Cogn. Disord. 2001, 12, 408. 43 Yagi, K.; Ohtsuka, K.; Ohishi, N. Experientia 1985, 41, 1561. 44 Benvenisti-Zarom, L.; Chen, J.; Regan, R. F. The oxidative neurotoxicity of clioquinol. Neuropharmacology 2005, 49, 687. 45 (a) Hirohata, M.; Hasegawa, K.; Tsutsumi-Yasuhara, S.; Ohhashi, Y.; Ookoshi, T.; Ono, K.; Yamada, M.; Naiki, H. The anti -amyloidogenic effect is exerted against Alzheimers -amyloid fibrils in vitro by prefer ential and reversible binding of flavonoids to the amyloid fibril structure. Biochemistry 2007, 46, 1888. (b) Vafeiadou, K.; Vauzour, D.; Spencer, J. P. E. Neuroinflammation and its modulation by flavonoids. Endo. Metab. Immun. Dis. Drug Targets 2007, 7 211. (c) Sharma, V.; Mishra, M.; Ghosh, S.; Tewari, R.; Basu A.; Seth, P.; Sen, E. Modulation of

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110 interleukin-1 mediated inflammatory response in human astrocytes by flavonoids: implications in neuroprotection. Brain Res. Bull. 2007, 73, 55. 46 Havsteen, B. H. The biochemistry and medical significan ce of the flavonoids Pharmacol. Therapeu. 2002, 96, 67. 47 (a) Cao, G.; Sofic, E.; Prior R. L. Antioxidant and Prooxi dant Behavior of Flavonoids: Structure-Ac tivity Relationships. Free Rad. Biol. Med. 1997, 22, 749 760. (b) Carlo, G.D.; Mascolo, N.; Izzo, A.A.; Capasso, F. Flavonoids: Old and New Aspects of a Class of Natural Therapeutic Drugs. Life Sciences 1999, 65, 337. (c) Erlund, I. Review of the flavonoids querc etin, hesperetin, and naringenin. Dietary sources, bioactivities, bioava ilability, and epidemiology. Nutr. Res. 2004, 24, 851. (d) Mandel, S.; Amit, T.; Reznichenko, L.; Weinreb, O.; Youdim, M. B. H. Mol. Nutr. Food Res 2006, 50, 229. 48 Williams, R. J.; Spencer, J. P. E.; Ri ce-Evans, C. FLAV ONOIDS: ANTIOXIDANTS OR SIGNALLING MOLECULES? Free Rad. Biol. Med 2004, 36, 838. 49 (a) Da Silva, EL; Tsushida, T.; Terao J. Inhibition of Mammalian 15-LipoxygenaseDependent Lipid Peroxidation in Low-Density Lipoprotein by Quercetin and Quercetin Monoglucosides. Arch. Biochem. Biophys. 1998, 349, 313. (b) Nagao, A.; Seki M.; Kobayashi H. Inhibition of Xanthine Oxidase by Flavonoids. Biosci. Biotechnol Biochem. 1999, 63, 1787.

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111 50 Furusawa, M.; Tanaka, T.; Ito, T.; Nishikawa, A.; Yamazaki, N.; Nakaya, K.-I.; Matsuura, N.; Tsuchiya, H.; Nagayama, M.; Iinuma, M. Antioxidant Activity of Hydroxyflavonoids. J. Heal. Sci. 2005, 51, 376. 51 (a) Bodini, M. E.; Copia, G.; Tapia, R.; Leighton, F.; Herrera, L. Iron complexes of quercetin in aprotic medium. Redox chemistr y and interaction with superoxide anion radical. Polyhedron 1999, 18, 2233. (b) Bravo, A.; Anacona, J.R. Metal complexes of the flavonoid quercetin : antibacterial properties. Trans. Metal Chem. 2001, 26, 20. (c) Zhou, J.; Wang, L.-f.; Wang, J.-y.; Tang, N. Synthesis, characterization, antioxidative and antitumor ac tivities of solid quercetin rare earth(III) complexes J. Inorg. Biochem. 2001, 83, 41. (d) Zhou, J.; Wang, L.; Wang, J.; Tang, N. Antioxidative and anti-tumour activities of solid quercetin metal(II) complexes. Trans. Metal Chem. 2001, 26, 57. (e) Nest, G.L.; Caille, O., Woudstra, M.; Roche, S.; Guerlesquin, F.; Lexa, D. Inorg. Chim. Acta 2004, 357, 775. (f) Nest, G.L.; Caille, O.; Woudstra, M.; Roche, S.; Burlat, B.; Belle, V.; Guigliarelli, B.; Lexa, D. Inorg. Chim. Acta 2004, 357 2027. (g) Erdogan, G.; Karadag, R. Rev. Anal. Chem. 2004, 24, 9. (h) Esparza, I.; Salinas, I.; Santamaria, C.; Garca-Mina, J.M.; Fernndez, J.M. Electrochemical and theoretical complexation studies for Zn and Cu with individual polyphenols. Anal. Chim. Acta 2005, 543, 267. (i) Torreggiani, A.; Tamba, M. ; Trinchero, A.; Bonora, S. Copper(II)Quercetin complexes in aqueous solutions: spectroscopic and kinetic properties. J. Mol. Struc.

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112 2005, 744, 759. (j) Kosti D. A.; Mileti G. Z.; Miti S. S.; Rai, I. D.; ivanovi V. V. Chem. Pap. 2007, 61, 73. 52 (a) Kamada, C.; da Silva, E. L.; Ohnishi-Kameyama, M.; Moon, J.-H.; Terao, J. Attenuation of lipid peroxidation and hyperlipid emia by quercetin glucoside in the aorta of high cholesterol-fed rabbit. Free Rad. Res. 2005, 39, 185. (b) Grinberg, L. N.; Newmark, H.; Kitrossky, N.; Rahamim, E.; Chevion, M.; Rachmilewitz, E. A. Protective effects of tea polyphenols agains t oxidative damage of red blood cells. Biochem. Pharma. 1997, 54, 973. (c) Russo, G. L. Ins and outs of dietary phytochemicals in cancer chemoprevention. Biochem. Pharm. 2007, 533. 53 Pannola, A.; Rice-Evans, C.; Halliwell, B.; Singh, S. Inhibition of PeroxynitriteMediated Tyrosine Nitration by Catechin Polyphenols. Biochem. Biophys. Res. Commun. 1997, 232, 164. 54 (a) Hertog, M. G. L.; Hollman, P. C. H.; Katan, M. B. Content of Potentially Anticarcinogenic Flavonoids of 28 Vegetabl es and 9 Fruits Commonly Consumed in The Netherlands. J. Agric. Food Chem. 1992, 40, 2379. (b) Hertog, M. G. L.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D. Nutr. Cancer 1993, 20, 21. 55 de Boer, V. C. J.; Dihal, A. A.; van de r Woude, H.; Arts, I. C. W.; Wolffram, S.; Alink, G. M.; Rietjens, I.M.C.M.; Keijer, J.; Hollman, P. C. H. Tissue Distribution of Quercetin in Rats and Pigs. J. Nutr. 2005, 135, 1718.

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113 56 Parvez, S.; Kang, M.; Chung, H.-S.; Bae, H. Naturally occurring ty rosinase inhibitors: mechanism and applications in skin health cosmetics and agriculture industries. Phytother. Res. 2007, 21, 805. 57 (a) Chen, Q.-X.; Kubo, I. Kinetics of Mushroom Tyrosinase Inhibition by Quercetin. J. Agric. Food Chem. 2002, 50, 4108. (b) Kubo, I.; Kinst-Hori, I.; Yokokawa, Y. Tyrosinase Inhibitors From Anacardium Occidentale Fruits. J. Nat. Prod. 1994, 57, 545. 58 (a) Kubo, I.; Kinst-Hori, I. Flavonols fr om Saffron Flower: Ty rosinase Inhibitory Activity and Inhibition Mechanism. J. Agric. Food Chem. 1999, 47, 4121. (b) Kubo, I.; Yokokawa, Y. Two tyrosinase inhi biting flavonol glycosides from Buddleia coriacea. Phytochemistry 1992, 31, 1075. 59 (a) Zhu, J. T. T.; Choi, R. C. Y.; Chu, G. K. Y.; Cheung, A. W. H.; Gao, Q. T.; Li, J.; Jiang, Z. Y.; Dong, T. T. X.; Tsim, K. W. K. Flavonoids Possess Neuroprotective Effects on Cultured Pheochromocytoma PC12 Cells: A Comparison of Different Flavonoids in Activating Estrogeni c Effect and in Preventing -Amyloid-Induced Cell Death. J. Agric. Food Chem. 2007, 55, 2438. (b) Kim, H.; Park, B.-S.; Lee, K.G.; Choi, C. Y.; Jang, S. S.; Kim, Y.-H.; Lee, S.-E. Effects of Naturally Occurring Compounds on Fibril Formation and Oxidative Stress of -Amyloid. J. Agric. Food Chem. 2005, 53, 8537. (c) Marambaud, P.; Zhao, H.; Davies, P. Resveratrol Promotes Clearance of Al zheimers Disease AmyloidPeptides. J. Biol. Chem. 2005,

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114 280, 37377. (d) Heo, H. J.; Lee, C. Y. Protective Effects of Quercetin and Vitamin C against Oxidative Stre ss-Induced Neurodegeneration. J. Agric. Food Chem. 2004, 52, 7514. (e) Weinreb, O.; Mandel, S.; Amit, T.; Youdim, M. B. H. Neurological mechanisms of green tea polyphenols in Alzheimers and Parkinsons diseases. J. Nutr. Biochem. 2004, 15, 506. (f) Patil, C. S.; Singh, V. P.; Satyanarayan, P. S. V.; Jain, N. K.; Singh, A.; Kulkarni, S. K. Protective Effect of Flavonoids against Agingand Lipopolysacch aride-Induced Cognitive Impairment in Mice. Pharmacology 2003, 69, 59. (g) Ono, K.; Yoshiike, Y.; Takashima, A.; Hasegawa, K.; Naiki, H.; Yamada, M. Potent anti-amyloidogenic and fibrildestabilizing effects of polyphenols in vitro: impli cations for the prevention and therapeutics of Alzheimers disease. J. Neurochem. 2003, 87, 172. (h) Ishige, K.; Schubert, D.; Sagara, Y. Fl avonoids Protect Neuronal Cell s From Oxidative Stress by Three Distinct Mechanisms. Free Rad. Biol. Med. 2001, 30, 433. (i) Schroeter, H.; Williams, R. J.; Matin, R.; Iversen, L.; Rice-Evans, C. A. Phenolic Antioxidants Attenuate Neuronal Cell Death Following Uptake of Oxidized Low-Density Lipoprotein. Free Rad. Biol. Med. 2000, 29, 1222. 60 Srivatsan, S. G.; Nigam, P.; Rao, M. S.; Verma, S. Phenol oxidation by coppermetallated 9-allyladenine-DVB polymer: reac tion catalysis and polymer recycling. Applied Catalysis A 2001, 209, 327. 61 Polster, J.; Lachmann, H. Spectrometric Titrations; VCH: New York, 1989.

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115 62 Wei, X.; Ming, L.-J. Comprehensiv e 2D 1H NMR Studies of Paramagnetic Lanthanide(III) Complexes of Anthracycline Antitumor Antibiotics. Inorg. Chem. 1998, 37, 2255. 63 da Silva, G. F. Z.; Tay, W. M.; Ming, L.-J. Catechol oxidase-like oxidation chemistry of the 1 and 1 fragments of Alzheimers disease-related -amyloid peptide: their structure-activity correlation an d the fate of hydrogen peroxide. J. Biol. Chem. 2005, 280, 16601. 64 da Silva, G. F. Z.; Ming, L.-J. Alzheimers disease related copper(II)-amyloid peptide exhibits phenol monooxygenase a nd catechol oxidase activities. Angew. Chem. Int. Ed. 2005, 44, 5501. 65 Segel, I. H. Steady-State Kine tics of Multireactant Enzymes. Enzyme Kinetics: Behavior and Analysis of Rapid Equilib rium and Steady-State Enzyme Systems John Wiley & Sons, Incs.: New York, 1993. 66 (a) Bertini, I.; Luchinat, C.; Parigi, G. Solution NMR of Paramagnetic Molecules ; Elsevier Science B.V.: Amsterdam, The Netherlands, 2001. (b) Ming, L.-J. Nuclear Magnetic Resonance of Paramagnetic Meta l Centers in Proteins and Synthetic Complexes. In Physical Methods in Bioinorgani c Chemistry: Spectroscopy and Magnetism Que, Jr. L., Ed.; University Science Books: Sausalito, 2000. 67 Ming, L.-J.; Wei, X. An Ytterbium (II1) Complex of Duanomycin, a Model Metal Complex of Anthracycline Antibiotics. Inorg. Chem. 1994, 33, 4617.

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116 68 Inubushi, T.;Becker, E. D. Efficient Detection of Paramagnetically Shifted NMR Resonances by Optimizing WEFT Pulse Sequence. J. Magn. Reson. 1983, 51, 128 133. 69 (a) Bnzli, J.-C. G., Choppin, G. R., Eds. Lanthanide Probes in Life, Chemical and Earth Sciences Elservier: Amsterdam, 1989. (b) Ming, L.-J. Magn. Reson. Chem. 1993, 31, S104S109. (c) Ming, L.-J. Parama gnetic Lanthanide(III) Ions as NMR Probes for Biomolecular Structure and Function. In La Mar, G. N.; Ed. Nuclear Magnetic Resonance of Paramagnetic Molecules NATO-ASI, Kluwer: Dordrecht, Netherlands, 1995. 70 Halliwell, B.; Rafter, J.; Jenner, A. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenol s: direct or indirect eff ects? Antioxidant or not? Am. J. Clin. Nutr. 2005, 81, 268SS. 71 Shen, L.; Ji, H.-F.; Zhang, H.-Y. How to understand the dichotomy of antioxidants. Biochem. Biophy. Res. Comm. 2007, 362, 543.

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CHAPTER 3 ELUCIDATION OF THE IN VITRO OXIDATION CHEMISTRY OF COPPER(II)BACITRACIN COMPLEX 117

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3.1 Bacitracin Bacitracin (Bc) is an antibiot ic peptide, isolated from Bacillus subtilis and B. licheniformis .1 2 It was first isolated from an open wound of a young girl named Margaret Tracey.1 This antibiotic has come to be termed bacitracin It is active primarily against Gram-positive bacteria, including Staphylococcus, Streptococcus, and Clostridium difficile.3 In addition to its worldwide use in animal feed as a preventive drug for livestock,4 Bc makes up one of the ingredie nts in the commonly used topical triple antibiotic ointments, Neosporin and Polysporin, along with polymyxin B and neomycin since 1956.5 The three antibiotics have been suggested to work synergistically. This may be one of the r easons for the rare occurrences of bacterial resistance, despite more than 50 years of over-the-counter usage.6 In addition, bacitra has other well known functions, such as an inhibitor of disulfide isomerase cin s regation.8 7 as well a platelet and multiple agonist agg Although it is relatively safe for oral inta ke owing to the low absorption by the gastrointestinal tract, Bc can cause ne phrotoxicity when given systemically.3 5 The minute intestinal absorption has been previously shown in animal studies.9 It is generally safe for use in topical applications, but a growing concern in the topical usage of bacitracin is the increasing reports of allergic contact dermititis.10 A recent publication suggested this to be the result of improper or over usage of the drug (i.e. application on all surgical wounds in hospita ls and on fresh tattoo wounds).10 b The same study proposed white petrolatum to be used as an alternative for cleaning surgical wounds. 10 b 118

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Furthermore, bacitracin is one of the worl ds massively produced and used food additives for farm animals it is until recently banned in European countries, which is an act of precaution against bacterial resistance.11 Up to 60% resistance by bacteria has been proposed,12 but a full resistance to the drug has not been reported over 5 decades of application. Thus, relatively speaking bacitrac in resistance still is a rare occurrence, and this is possibly due to its unique structure.2 3.1.1 Structure of Bacitracin Bacitracin is synthesized non-ribosomally by bacitracin synthetase ABC, a multienzyme complex consisted of BacA (598 kD a), BacB (297 kDa), and BacC (723 kDa). Each synthetase is responsible for the incorp oration as well as chemical modification of different parts of the full-length peptide.13 14 This peptide is produced as a mixture of closely related peptides, possi bly as many as 50 variants.15 Among the congeners, bacitracin A1 (BcA) is the major component with the most potent antibiotic activity.16 17 Combined bacitracin A and B components ar e accountable for >90% of the biological activity although BcA2 has low activity.18 Bacitracin F is biologically inactive and has been proposed to be the cause of nephrotoxi city. It is the oxid ized product of BcA19 that can act as a vasoconstrictor toward kidneys.20 Bacitracin was initially is olated using counter-curre nt distribution technique,21 and the structure was determined by NMR.22 Several structures were proposed, but the structure was not fully characterized until th e early 1990s by means of modern 2D NMR 119

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techniques.23 The congeners can also be purif ied with reverse-phase HPLC and characterized with mass spect rometry and NMR spectroscopy. 24 The structure of BcA consists of a thiazoline ring formed by th e condensation reaction between the carboxylate of Ile-1 and the NH2 and the SH groups of Cys-2. It is a dodecapeptide, containing four D-amino acids, with a unique cyclic heptapeptide structure formed via an amide linkage between the side chain of Lys-6 and the C-terminus of Asn-12.23 In the characterized structure of Bc, the N-termin al end folds over the cyclic heptapeptide, placing the thiazoline ring, Gl u-4, and His-10 in proximity.23 It has been suggested that these unusual structural features may prev ent this peptide from being degraded by proteases.25 Structures of bacitracin conge ners are displayed in Figure 3.1. 3.1.2 Metal-Binding and Antibacterial Mechanism Bacitracin requires ZnII for its antibacterial activity26 and has been shown to bind several divalent meta l ions, including CoII, NiII, and CuII, in a 1:1 stoichiometry. 27 28 A previously proposed antibacterial mechanism of bacitracin involve s the inhibition of peptidoglycan cell wall synthesis. At the final step of the s ynthesis, the uridine diphosphate sugar molecule is transported to the cell wall by a lipid carrier known as C55isoprenyl pyrophosphate.29 After the incorporation of the disaccharide into the peptidoglycan layer, the lipid carrier is dephosphorylated to its monophosphate form in order to bind another sugar molecule. The metalII-Bc complex inhibits the cell wall 120

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Figure 3.1. Structures of bacitrac in congeners (adopted from ref. 2). Bacitracin is a dodecapeptide, containing four D-amino acids, with a uniqu e cyclic heptapeptide structure formed via an amide linkage be tween the side chain of Lys-6 and the Cterminus of Asn-12. In BcA, the two N-term inal amino acids form a thiazoline ring by the condensation reaction betw een the carboxylate of Ile-1 and the NH2 and the SH groups of Cys-2. 121

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synthesis by binding to the lipid carrier with high affinity (Kf = 1.05 106 M).30 Thus, metal coordination is an essential pa rt of its biological activity. Metal binding of Bc have been studied by means of multiple techniques, including proton release tit ration, ORD, EPR, EXAFS, 1H NMR, and UV-Vis spectroscopy.28 30, 31 One of the earlier studies proposed that the metal binding site is consisted of His-10 nitrogen, thiazoline ring (nitrogen or sulfur), and the amino group of Ile-1, based on a prot on release titration.31 b Nonetheless, later studies did not suggest the involvement of Ile-1 group. A nother metal binding study with CuII and MnII suggested the binding through the imidazole ring of His-10, and the carbonyl group of acidic amino acid residues such as Asp and Glu according to 13C NMR results.31 d EPR28 and EXAFS31f studies showed metal binding through thiazoline ring, Glu-4, and His-10 but disagreed on the fourth ligand, Asp-11 and Ile-1-NH2 for EPR and EXAFS study, respectively. Finally, the metal binding site of Bc was unambiguously assigned by the use of CoII as a paramagnetic NMR probe.24 The suggested metal-binding ligands are the thiazoline ring nitrogen, the N nitrogen of His-10, and the carboxylate side chain of DGlu-4.24 Furthermore, the study suggested a correlation between the structure and function of bacitracin. According to the authors, both BcA2 and BcF cannot effectively bind the metal ion to create a proper binding si te for the lipid carri er. Although Ile-1 is not involved in metal binding, it is important in creating a correct environment as seen in the inability of BcA2 (Dallo -Ile-1 versus L-Ile-1 of BacA1 at the N-terminus) to bind the 122

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metal properly. Likewise, BcF, having an oxidi zed thiazole ring instead of a thiazoline ring at the N-terminus, is only able to bi nd the metal ion weakly through the imidazole group of His-10. In addition, no metal binding by D-Glu-4 was observed in both congeners. The structure of a metal-bound bacitr acin was generated based on the T1 relaxation results with a molecular modeling program.24 According to the proposed structure, the four N-termin al amino acid residues fold back onto the His-10-bound metal ion to complete the tridentate coordination sphere. The structure also suggested the presence of a hydrophobic pocket (Ile-5, Phe-9, and Ile-10 are in close proximity). This hydrophobic binding site was proposed to play an important role in lipid pyrophosphate binding, aided by the stabilizat ion effect of Ile-1-NH3 + through H-bonding. As a result, inability of binding metal correlates to the biological activity of Bac congeners. 24, 2 Pyrophosphate and its several deriva tives were found to bind to CoII-bacitracin to form kinetically inert ternary complexes by means of NMR spectroscopy.2 b The study suggested the pyrophosphate moiety to be the minimum necessity for binding to the metallo-bacitracin complex, since sodium phosphate was shown only to bind very weakly.2 b The ability of the metalII-Bc complex (not free Bc) to bind pyrophosphate may suggest a potential in teraction with the phospho-group on DNA. Thus, in the presence of a redox-active metal such as CuII, bacitracin may bind and perform an oxidative damage of DNA. This hypothesis is supported by the CuII-dependent oxidation chemistry by metal-binding short peptides (e.g. the amyloidpeptides in the brain of the patients with 123

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AD, A 1 and A 1) has been previously proposed.32 33 In this study described herein, the catechol oxidation chemistry of CuII-Bc was explored using optical and kinetic methods. 3.2 Materials and Methods Bacitracin and pyrocatechol (~99%) were purchased from SigmaAldrich Inc. (St. Louis, MO), the plasmid pQE30Xa was pur chased from Qiagen (Valencia, CA), and 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate, 98%, was acquired from and Acros Organics (NJ) Copper Sulfate (anhydrous) and 4-(2hydroxyethyl)-1-piperazineethanesulfonate buffe r (HEPES) used for the oxidation study was purchased from Fisher Sc ientific Co. (Fair Lawn, NJ). All chemicals were used without further purification. All other solv ents and reagents we re the highest grade available from commercial sources. Dei onized (DI) water (18.2 M ) was obtained from a Millipore Milli-Q system. Plastic ware and glassware were demetallized with EDTA and extensively rinsed. All components were freshly prepared just prior to the experiments. Quartz cuvettes were used in all the kinetic and optical studies. 3.2.1 Kinetic Studies The catechol oxidation assay was carri ed out as previously described.32 33 Briefly, different concentrati ons of the substrate catechol ranging from 0.1 to 19.2 mM (along with each corresponding conc entrations of MBTH as an ortho -quinone trapper) 124

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were incubated with 20.0 M (or 2.0 M in H2O2 related experiments) CuII-bacitracin (1:1 metal-to-peptide stoichiometric ratio) in 100.0 mM HEPES buffer at pH 7.0 and 25 C in the presence and absence of different H2O2 concentrations (0 to 64.0 mM). All components were dissolved in DI water, and the final volume in the cuvette was fixed at 1.0 mL. The binding of oxidized oquinone to MBTH formed a red adduct, which was monitored at 500 nm ( = 32, 500 Mcm)34 on a Varian Cary50 Bio UV-Vis spectrophotometer, and the initial rate was dete rmined from the slope of the change in the absorbance with respect to time. The optimal ratio between the catalyst and substrate during catalysis was determined using the Job method with respect to catalytic activity. 35 The Job method is a continuous variation technique, where the concentrations of catalyst [CuII-Bc] and substrate [CA] were varied while keepi ng the total concentration constant ([CuIIBc]+[CA] = 50.0 M). The result, with respect to act ivity, is plotted versus the mole fraction of catalyst (Cu(II)-Bc) or substrate ( CA). The ratio of XCu(II)-Bc:XCA at which the activity reaches the maximum in the plot refl ects the preferred stoi chiometry of the (CuIIBc)-CA complex for the given experimental conditions. Since the oxidation reaction follows the enzyme-like kinetics (discussed in the result section), the kinetic constants, such as KB and VMax, were determined by fitting the data to the Michaelis-Menten equation with the Sigma Plot 8.0. 125

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3.2.2 Optical Studies The slow substrate 4,5-di-chloro-catechol (DCC) was used as a ligand in the binding study to show the interaction between the CuII-Bc complex and the catechol substrate. Due to low solubility in wate r, DCC was dissolved in DMF. The stock solution of the CuII-Bc complex was prepared in DI wa ter, since only the peptide and not the copper salt would dissolve in DMF. The experiment was run in DMF, and all components were completely miscible. Briefly, DCC ranging from ~60.0 to 600.0 M was gradually titrated into a 1.0 mL solution of CuII-Bc (300.0 M) with the sample cuvette left in the spectrometer to keep the baseline intact. Th e background was zeroed with only the solvent, and the mixture was scanned from 200 to 900 nm on the Varian Cary50 Bio UV-Vis spectrophotometer. The dilution factor from each addition was corrected in the final concentrations. The molar absorptivity value of the (CuII-Bc)-DCC complex was plotted with respect to the titrat ed DCC concentration, and the data fitted to simple 1:1 binding quadratic e quation in Sigma Plot 8.0 fo r the determination of the affinity constant, KDCC. The stoichiometry of (CuII-Bc)-to-DCC binding was determined with the Job method. As described in section 3.2.1, the metal complex [CuII-Bc] and ligand [DCC] were continuously varied while keepi ng a constant overall concentration ([CuIIBc]+[DCC] = 500.0 M). The maximum ratio ( Cu(II)-Bc: DCC) on the plot of the molar absorptivity of the (CuII-Bc)-DCC complex versus the mole fraction of DCC ( DCC) or 126

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CuII-Bc ( Cu(II)-Bc) indicates the preferred binding stoichiometry between the CuII-Bc complex and DCC. 3.2.3 Anaerobic Studies For anaerobic experiments, the disso lved oxygen from all components was removed through freeze-thawing on a vacuum-argon line. Briefly, the oxygenated solutions were first frozen in liquid nitrogen and then evacuated. Next, the frozen sample was thawed in warm water, followed by application of pure Ar gas. The process was repeated 58 times to ensure the rem oval of oxygen and replacement by Ar. All components, except the catalyst were transferred to the ev acuated quartz cuvette with a gas-tight syringe. The reac tion was immediately monitored following the addition of the catalyst (CuII-Bc). 3.2.4 DNA Cleavage Assay The 1:1 CuII-Bc stock solution was prepared by dissolving the peptide in ultrapurified water. The metal-complexes were freshly prepared in all experiments. The DNA cleavage assay contained 225 ng of plasmid DNA, 0.05% H2O2, and 25.0 M of CuII-Bc in 100.0 mM HEPES at pH 7.00 and 37 C in a volume of 25.0 L. A timecourse (0 mins) experiment was perfor med and analyzed in a 1.0 % agarose gel stained with ethydium bromide, and th en photographed on a transilluminator. 127

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3.3 Results and Discussion A divalent metal ion is an essential cofactor for the biological activity of bacitracin.26 The metal-bound complex can bind the sugar carrier, C55-isoprenyl pyrophosphate, with high affinity.30 Furthermore, the CoII-Bc complex has been shown to interact with various phosphate derivatives through NMR studies.2 b Thus, in the presence of redox-active metal ion such as CuII, Bc can potentially interact and cause oxidative damage of biomolecules, such as DNA. The ability of (1:1) CuII-Bc to perform oxidative DNA cleavage was studied in the presence of H2O2 following the previously proposed protocol with a slight modification.32 The plasmid DNA (225 ng) was incubated with 25.0 M CuII-Bc and 0.05% H2O2 in 100.0 mM HEPES at pH 7.0 and 37 C over a time course (0 mins). On the gel (Figure 3.2), the first lane is the 1 kb DNA molecular weight marker (M on gel), and the second lane is the reference (R on gel), which only contains the plasmid DNA (pQE30Xa) incubated over the same time course. Lanes 1 represent different incubation times (i.e. 5, 10, 20, 40, 60 mins) for the CuII-Bc complex. For the reference lane, both supercoiled (lower) and nicked circ ular (top) bands can be clearly seen. In lanes 1 corresponding to 5, 10, and 20 mins incubation, a middle band of approximately 3.5 kbp based on the marker corresponds to a linearized plasmid from the manufacturer. This indicates a double-stra nded DNA cleavage has occurred. In lanes 4 and 5, corresponding to 40 and 60 mins incubation, the plasmid DNA is completely cleaved. Short peptides with CuII binding ability have been shown to perform oxidative 128

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M R 1 2 3 4 5Figure 3.2 Oxidative cleavage of 225 ng of plasm i d DNA b y 25.0 M CuII-Bc with 0.05% H2O2 in 100.0 mM HEPES buffer at pH 7.0 and 25 C. The first lane is the 1 kb DNA molecular weight m a rker (M on gel), a nd the second lane is the ref e rence (R on gel). Lanes 1 represent different incuba tion tim e s (i.e. 5, 10, 20, 40, 60 m i ns) for the CuII-Bc complex. 129

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cleavage of plasmid DNA. Thus, the a bove results suggest the ability of CuII-Bc to perform oxidation chemistry. The oxidation chemistry catalyzed by the CuII-Bc complex was further studied with catechol (CA) as a substrate. Different concentrations of CA were incubated with 20.0 M CuII-Bc in 100.0 mM HEPES at pH 7.0 and 25C. The formation of the oxidized oquinone product was followed opticall y, by monitoring the red adduct formed between the product and MBTH (an o-quinone specific indicator). The catechol oxidation by the CuII-Bc complex increases with increasing amount of substrate and eventually reaches a plateau. The observed saturation at higher concentrations of substrate suggests a possibility of enzyme-like pre-equilibri um kinetics shown below. The rate law for this reaction can be desc ribed accordingly to Eq. (1), with the assumption that [CA]>>[Cu(II)-Bc-CA]: [Cu(II)-Bc-CA] [Cu(II)-Bc] + [CA] [Cu(II)-Bc] + o-quinone k1k-1kcat CAK CAVB Max 0 (1) where 0 and VMax are the measured and maximum velocity, respectively, and 1 1k kk Kcat B is the virtual dissociation constant of the (CuII-Bc)-CA complex. The data fits well to Eq. (1) to afford KB = 3.31 10 M, a first-order rate constant or the turnover number kcat ( VMax/[Cu(II)-Bc]) = 6.99 10 s, and a second-order rate 130

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constant or the catalytic efficiency kcat/KB = 2.11 M s (Figure 3.3). Compared to the auto-oxidation rate consta nt of catechol (4.74 10 s) in the absence of CuII-Bc,32 there is a 1.47 104-time increase with re spect to the first-order rate constant. The interaction between the CuII-Bc complex and the substrate catechol is confirmed by an optical binding study with a slow substrate, 4,5-dichlorocatechol (DCC). The activity of DCC is roughly 200 times sl ower than catechol with respect to kcat as a ligand.33 A 1.0-mL solution of 300M CuII-Bc was slowly titrated with DCC in DMF at 25 C. The formation of complex was monitored from 200 nm on the Varian Cary50 Bio UV-Vis spectrophotometer. Upon addition of DCC, a new absorption corresponding to a complex between CuII-Bc and DCC appears at approximately 304 nm (Figure 3.4). The catecholate moiety has been previously proposed to bind the metal ion in a bidentate manner.36 A recent CoII binding study of bacitracin by means of NMR spectroscopy and molecular modeling suggest ed the presence of a possible hydrophobic binding pocket and the metal coordination sphere to be a distorted 5 or 6 instead of 4 coordination.24 Thus, it may be possible for the ca techolate moiety on DCC (or catechol) to bind the metalII center in a 1:1 stoichiometry to gi ve a penta-coordinated sphere. The plot of molar absorptivity ( ) value of the CuII-Bc-DCC complex at 304 nm versus the added equivalence of DCC can be fitted well to a 1:1 binding quadratic formula to afford the affinity constant, KDCC, of 2.40 104 M (Figure 3.4 inset). Even though the affinity constant can be determined from the fitting, the stoichiometry of the binding cannot be clearly abstracted. 131

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[Catechol] (M) 0.0000.0020.0040.0060.0080.0100.012 500 (Ms -1 ) 0.0 2.0e-9 4.0e-9 6.0e-9 8.0e-9 1.0e-8 1.2e-8 Figure 3.3 Catechol oxidation by CuII-Bc in 100.0 mM HEPES at pH 7.0 and 25 C. The activity increases with increasing substr ate concentration and reaches saturation at higher concentration, which may suggest an enzy me-like kinetics. The data fits well to Eq. (1), affording KB, a first-order rate consta nt or the turnover number kcat ( VMax/[Cu(II)Bc]), and a second-order rate constant or the catalytic efficiency kcat/KB of 3.31 10 M, 6.99 10 s, and 2.11 M s, respectively. 132

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Wavelength (nm) 260280300320340360380400 (M-1cm-1) 0 500 1000 1500 2000 2500 3000 [DCC]/[Cu(II)-Bacitracin] 0.00.51.01.52.02.5 Absorbance304 0.0 0.2 0.4 0.6 0.8 Figure 3.4. 4,5dichlorocatechol (DCC) binding study of CuII-Bc in DMF. DCC is added in 60.0 M (0.2 equivalent) increment. The CuII-Bc-DCC complex gives a strong absorption at 304 nm. The changes in the abso rbance at 304 nm with respect to the added DCC equivalence afford an a ffinity constant of 2.40 104 M (the inset). The spectra are baselined with the CuII-Bc complex prior to the addition of DCC. 133

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Thus, the Job method was used to dete rmine the stoichiometry of the (CuII-Bc)DCC complex. The method calls for continuous ly varying the concentrations of the CuIIBc complex and DCC while keeping the overall concentration constant.35 For the optical study, the concentrations of th e two components were arrayed to a total concentration of 500.0 M in 100.0 mM HEPES buffer at pH 7.0 and 25 C (i.e. [CuII-Bc]:[DCC] = 500:0 M, 450:50 M,:500 M). The absorption of the complex (310 nm) in buffer slightly shifted toward the higher wavelength (Figure 3.5 ). As a result, the ratio that corresponds to the maximum intensity represen ts the preferred binding between CuII-Bc and DCC. The plot of the molar absorptivity value at 310 nm versus the mole fraction of DCC ( DCC) shows the maximum around 0.5 (0.5:0.5) which suggests that the stoichiometry of the preferred species is Cu(II)-Bc: DCC = 1:1 (Figure 3.5 inset). Similarly, since an equilibrium is present between the CuII-Bc complex and substrate the interaction betw een the catalytic center (CuII-Bc) and substrate (CA) was further investigated with the Job method, monitored with the activity toward catechol oxidation. The total co ncentration of [CuII-Bc]+[CA] was kept constant at 50 M, and the experiment was conducted in 100 mM HE PES buffer at pH 7.0 and 25 C. The result shows a gradual increase in activity with Cu(II)-Bc and reaches a maximum at Cu(II)-Bc = 0.5, followed by a decrease in the activity of subsequent mole fractions which can be fitted to a 1:1 stoichiometry (Figure 3.6). Fr om the result, the most active species has a ratio of (CuII-Bc):CA = 1:1 (i.e. 0.5:0.5), establis hing the equilibrium between reactants and the (CuII-Bc)-CA complex in the catalysis. 134

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Wavelength (nm) 240260280300320340360380400 (M-1cm-1) 0 1000 2000 3000 4000 [DCC]/([Cu(II)-Bc]+[DCC]) 0.00.20.40.60.81.0 Absorbance310 0.20 0.30 0.40 0.50 Figure 3.5. Optical Job plot of DCC binding tothe CuII-Bc complex. The absorbance at 310 nm versus the molar ratios between the CuII-Bc complex and DCC suggests that the stoichiometry of the pr eferred species is 1:1. 135

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[Catechol]/([Cu(II)-Bacitracin]+[Catechol]) 0.00.20.40.60.81 .0 500 (Ms -1 ) 0.0 2.0e-10 4.0e-10 6.0e-10 8.0e-10 1.0e-9 1.2e-9 1.4e-9 1.6e-9 Figure 3.6. Interaction between the CuII-Bc and catechol using the Job method. The total concentration of the molar ratio is 50.0 M. The assays were performed in 100.0 mM HEPES pH 7.0 buffer at 25 C, and the in teraction was monitore d with the oxidation activity. The plot of the initial rates vers us the molar ratios indicates the preferred stoichiometry to be 1:1. 136

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The results from the two binding studies (CuII-Bc:DCC = 1:1 and CuII-Bc:CA = 1:1) using the Job method clearly indicate th at the observed catechol oxidation chemistry occurs through a mononuclear CuII center. The oxidation chemistry of mononuclear CuII systems has been thoroughly studied with synthetic ligand complexes, several spectroscopic techniques, and density f unctional theory (DFT) calculations.37 These studies have proven the important role of dioxygen in mononuclear CuII-center catalysis. Since the oxidation of catechol to o-quinone is a 2e transfer process, the involvement of dioxygen in recycling of the metal center (CuI/CuII) is suspected. Thus, the ability of CuII-Bc to perform catechol oxidation was dete rmined in the absence of dioxygen (under anaerobic conditions). The dissolved oxygen in all components wa s removed by the method described in the experimental section. The reaction (100.0 M CuII-Bc and 2.5 mM CA) was performed in methanol, and the formation of o-quinone was monitored from 400 nm for 5 mins. (Figure 3.7). The o-quinone-MBTH adduct immediately appears upon CuIIBc addition approximately at 500 nm. A gradual and minor increase in the absorption from the first scan (Abs = 0.015) can be seen over 5-minute period and then stop increasing after 2 mins. The difference in the intensity is estimated to be 0.010 ( Abs = 0.025 0.015), and the overa ll concentration of o-quinone is roughly estimated to be 770 nM (based on MBTH = 32, 500 Mcm), which indicates a very inefficient catalysis. This is to be expected since a single CuII can only oxidize one of the two hydroxyl groups on catechol at a time. 137

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Wavelength (nm) 400 450 500 550 600 Absorbance 0.000 0.005 0.010 0.015 0.020 0.025 0.030 Figure 3.7 The oxidation of 2.5mM catechol by 100.0 M CuII-Bc under anaerobic condition. The reaction was immediately monitored upon CuII-Bc addition up to 5 minutes. A small, gradual increase was observed up to 2 mins. The change in absorbance is approximately 0.01. 138

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The involvement of oxygen was further shown by introducing some air into the same sample and scanning over the same region. An air-tight syringe was used to apply the air from the surrounding, and the listed air volume is based on the volume of the syringe. As a result, this is a qualitative experiment. From 5 to 40 mL of air was added into the mixture over 4 different occasions. The increase in the absorbance was observed after each addition (Figure 3.8). Quantification of the product based on the volume of air added is not feasible since factors such as the amount of oxygen in a given volume of syringe and the dissolvability of oxygen (which may be time-dependent) are not known. Qualitatively, however, this experiment clear ly shows the importance of dioxygen in the observed mononuclear CuII-centered catechol oxidation. The binding between copper and dioxygen depends on the oxidati on state of metal (CuI vs. CuII) and the binding mode of dioxygen (end-on vs. side-on). Two possible binding modes of dioxygen are end-on ( 1) and side-on (2) binding, where the dioxygen binds the metal ion in a monodentate and bidentate fashion, respectively.37 In the presence of a bidentate catechol, dioxygen may only be able bind the CuII-Bc center (a 3coordinated complex) through the end-on mode du e to steric hindrance. The steric effect is another important factor in the selecti on of dioxygen binding mode. A classic example is the very first synthetic CuII-ligand complex, showing the mononuclear CuII-superoxo complex with a side-on binding mode, by Kitajima et. al. in 1994.38 The side-on binding by dioxygen in this complex was induced by steric effect of the ligand. 139

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Wavelength (nm) 400 450 500 550 600 Absorbance 0.00 0.02 0.04 0.06 0.08 0.10 5 mL air 10 mL air 20 mL air 40 mL air Figure 3.8 Addition of air into an aerobic sample containing 100.0 M CuII-Bc and 2.5 mM CA. The same air-tight syringe was used to suck up and bubble in the air from surrounding, and the listed air volume is base d on the volume of the syringe. From 5 to 40 mL of air were added into the mixture over 4 different additions. 140

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Upon catechol binding, the CuII center may be reduced to CuI while the catechol is oxidized to a semiquinone. Under aerobic conditions, the CuI can interact with dioxygen to give the following three potential copper-oxygen intermediates: CuIIsuperoxo (-O2 ), CuII-hydroperoxo (-OOH) and CuIII-peroxo (-O2 2).37 The CuIII-O2 2 species may not be the intermediate since si de-on binding to the Cu center may not be possible (this binding mode will result in 7-c oordination of the Cu). On the other hand, both CuII-O2 and CuII-OOH species can have both end-on and side-on binding mode. In its protonated form, the CuII-OOH species forms an end-on intermediate. However, it can potentially form a side-on in termediate once deprotonated.37 The side-on binding mode may be too crowded in the metal coordination sphere as with the CuIII-O2 2. Furthermore, a recently solved crystal structure of a mononuclear CuII enzyme, peptidylglycine -hydroxylating monooxygenase (P HM), shows the end-on O2 binding of the Cu center.39 Thus, the end-on CuII-O2 or CuII-OOH species is very likely to be the intermediate. Unlike oxygen, hydrogen peroxide (H2O2) prefers to bind the CuII center through a side-on mode upon full deprot onation, possibly forming the CuII-Bc-O2 2 intermediate. Hence, H2O2 was included in the reaction in order to determine its influence on the observed chemistry. Different c oncentrations (0.0 mM) of H2O2 were incubated with 20.0 M CuII-Bc and 0.7 mM catechol in 100.0 mM HEPES buffer at pH 7.0 and 25 C. The increase in catechol oxidati on was observed with increasing H2O2 and reaches saturation at higher co ncentrations of H2O2 (Figure 3.9 left panel). As discussed above, 141

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the saturation profile indicates the possibility of an enzymelike kinetics, thus suggesting H2O2 as another substrate capable of binding to the metal center. The data can be fitted to Eq. 2 to afford a kcat of 9.37 10 s and an apparent binding or dissociation constant KH2O2 of 7.49 10 M for H2O2. This value compared to KB = 3.31 10 M, the binding constant for catec hol in the absence of H2O2, is approximately twice as higher, suggesting this substrate to bind ~2 lower than catechol toward the CuII-Bc complex. 22 22 022OHK OHVOH Max Background (2) Since both substrates (i.e. both catechol and H2O2 show saturation) can independently bind to the Cu2+ center, it is essential to de termine how the binding of one affects the other. Different concentrations (0.6 mM) of catechol were incubated with the CuII-Bc (2.0 M) complex in the presence of different H2O2 concentrations (0.0 mM). The enhancements in activity of catechol oxidation in the presence of H2O2 were clearly observed (Figure 3.9 right). While KB is increased by ~7 folds, the addition of 32.0 mM H2O2 in the assay increased the kcat and kcat/KB by 55 and 7 folds, respectively. Because the two substrates may bind to the CuII-Bc complex independently of each other, the Hanes plot can be used to determine th e influence or relationship between the two substrates. From the Hanes analysis, the apparent binding equilibrium constants for both substrates and an intrinsic binding consta nt for one substrate can be calculated.40 The 142

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[Catechol] (M) 0.0000.0020.0040.0060.0080.0100.012 (M s-1) 0 1e-7 2e-7 3e-7 0 mM h2o2 2 mM h2o2 4 mM h2o2 8 mM h2o2 16 mM h2o2 32 mM h2o2 64 mM h2o2 [H2O2] (M) 0.000.020.040.06 (Ms-1) 0.0 5.0e-8 1.0e-7 1.5e-7 2.0e-7 Figure 3.9 Hydrogen peroxide influen ce on the catechol oxidation by 2.0 M CuII-Bc at pH 7.0 in 100.0 mM HEPES buffer at 25C (right). The concentrations of H2O2 were varied from 0 to 64.0 mM (right). The titration of H2O2 at a constant catechol concentration of 0.7 mM displays saturati on, which indicates the direct interaction between the CuII-Bc center and H2O2 (left). 20.0 M CuII-Bc was used in the latter experiment (left). 143

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Hanes analysis requires an initial linear trea tment of the data in Figure 3.6 according to the following rate law (Eq. 3), 22 max max 22 022 221 1 OH K V K Catechol V OH K CatecholInt OH App Catechol App OH (3) where is the intrinsic binding constant for H2O2, and are the apparent binding constants for H2O2 and catechol, and Int OHK22App OHK220App CatecholK and are the experimental and maximum velocity, respectively (F igure 3.10 A). The ratio between the corresponding apparent and intrinsic equilibr ium constants for each substrate can suggest how the binding of one substrate affects the binding of another. Then, the secondary plots of the fitted y-intercept (Eq. 4) and slope (Eq. 5) values versus 1/[H2O2] afford the two apparent equilibrium constants = 1.91 10 M and = 8.38 10 M and the intrinsic binding constant = 9.03 10 M (Figure 3.10 B and C). maxVKApp OHK22Int OHK22App Catechol 22 max max1 int22OH V KK V K ercept yInt OH App Catechol App Catechol (4) 22 max max1 122OHV K V slopeApp OH (5) The ratio between the apparent and intrin sic binding constants is 2.53 for catechol ( /KB) and 2.11 for H2O2 (/ ), which suggests that both substrates are equally affected by the presen ce of the other. Since O2 2 has been shown to bind Cu in a App CatecholKApp OHK22Int OHK22 144

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[Catechol] (M) -0.0020.0000.0020.0040.0060.0080.0100.012 [Catechol]/ (s) -4.0e+0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 0 mM h2o2 2 mM h2o2 4 mM h2o2 8 mM h2o2 16 mM h2o2 32 mM h2o2 64 mM h2o2 A [H2O2]-1 (M1) 0100200300400500600 SlopeCatechol 0.0 5.0e+6 1.0e+7 1.5e+7 2.0e+7 2.5e+7 B C [H2O2]-1 (M1) 0100200300400500600 y-interceptCatechol 0.0 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 C Figure 3.10. Hanes analysis of the oxidation of catechol by CuII-Bc at different concentrations of H2O2 (kinetic data from Figure 3.9 right ). Plot B and C are the replots of the slope and y-inte rcept values from plot A with respect to [H2O2]. The apparent binding equilibrium constants for catechol ( ) and H2O2 ( ) as well as the intrinsic binding constant for H2O2 ( ) can be determined from the secondary plots. App CatecholKApp OHK22Int OHK22 145

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side-on (bidentate) mode,41 the reduction in the effectiven ess of binding by catechol (also a bidentate binding) is expected due to inability of the CuII center to support a 7coordination sphere. Thus, the observation described herein is more consistent with the end-on binding mode of HO2 when the catechol is bound to the metal center. In the attempt to differentiate be tween a mononuclear and dinuclear CuII-centered catalysis, redox-dilution experiments we re performed as previously proposed.33 Even though the studies above str ongly suggest a mononuclear CuII-based catalysis, these interesting results, which may be of mechanistic relevance, are included. Similar to the Job method, the concentrations of redox-inert ZnII to CuII were continuously arrayed with the total concentration kept constant ([ZnII]+[CuII] = 20.0 M). Two additional experiments with CoII and NiII in place of ZnII were performed. Th e interaction between the two metal ions and the peptide was mon itored with the catechol oxidation activity. The catechol (1.6 mM) oxidation by 20.0 M metal-Bc complex was performed in 100.0 mM HEPES buffer at pH 7.0 and 25 C. Since Bc can bind both metals, the presence of ZnII-Bc complex can essentially dilute the redox activity of the CuII-Bc complex. A mononuclear versus dinuclear mechanism may be suggested based on the resulted pattern. In a mononuclear mechanism, the ZnII complex has no influence on the redox activity of the CuII complex; thus, a linear trend is expected for the activity with respect to the CuII concentration. However, the redox-inactive ZnII species can essentially interfere with the redox chemistr y in the dinuclear mechanism by competing for the formation of the dinucle ar center. Thus, in the dinuc lear mechanism, only a slow 146

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and steady increase in the activ ity is expected up to 50% ZnII and 50% CuII ratio. Then, a sharper increase in the activity is exp ected due to the presence of more CuII species for the formation of dinuclear center when the CuII to ZnII ratio surpasses 1:1, affording a sigmoidal-like pattern. The data can be fitted to the Hill equati on (Eq. 6) which suggests the presence of cooperativity during the catalysis In the Hill equation, BacIICuK BacIICuVA Background )( )(max (6) and Vmax are the usual experimental and maximum velocity, respectively; KA is equivalent to the Michaelis constant accounting for all interactions; and is the fitted Hill coefficient that indicates the presence of cooperativity wh en greater than 1.33 The results for ZnII and CoII show an initial small and gradual increase in activity up to Cu(II) = 0.5, then followed by larger increase in activity (Figure 3.11 A and B). A similar trend is observed for NiII; however, the sigmoidal-lik e nature is not as apparent (Figure 3.11 C). The sigmoidal-like patterns in th e case of catechol oxidation by CuII-Bc in the presence of ZnII or CoII can be fitted to the Hill equation to afford = 1.93 (r2 = 0.99) and 2.37 (r2 = 0.99), respectively (Figure 3.11 A and B), suggesting the presence of cooperativity. For comparison, the results were also fitted with a simple 1:1 quadratic binding equation. As shown in Figur e 3.11, the 1:1 binding pattern for CuII binding to Bc may not be appropriate for fitting the data (the points deviate from the fitted dotted line), showing the r2 values of 0.92 and 0.89, respectively, for ZnII and CoII dilution. The 147

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difference in r2 values for the Hill (r2 = 0.991) and quadratic (r2 = 0.986) equation, unlike for ZnII and CoII, is less apparent for NiII. This is also reflected in the Hill coefficient of 1.38. At a first glance, the presence of cooperativity can suggest a di-CuII center catalysis. Nevertheless, the optical (DCC binding) and mech anistic (catechol oxidation) Job data strongly indicate a mononuclear CuII center oxidation chemistry. The results from the latter two experiments also s uggest the binding of catechol to the CuII center in a bidentate manner (i.e. Cu(II) = 0.5). A possible explanation for the presence of cooperativity is the electron tr ansfer between the nearby CuII-Bc complexes. Thus, when the metalII:CuII ratio is greater than 1:1 ( Cu(II)>0.5), the increase in CuII-Bc (higher probability to be in close proximity) speci es corresponds to increase in activity. Structural change upon metal binding was observed, which might account for the cooperativity.24 Although not common, the presence of cooperativity between two CuII centers in close proximity has been described in PHM and D M for a mononuclear CuII center oxidation chemistry.42 PHM is responsible fo r C-H bond hydroxylation of Cterminal glycine, and D M hydroxylates the benzylic C-H bond of dopamine to generate norepinephrine. Even though they are di-Cu enzymes, their Cu centers (CuM and CuH) are ~11 apart,37 and only CuM, the active site, directly inte racts with the substrate and oxygen.42 As a result, these pr oteins are still categor ized as mononuclear Cu monooxygenases. However, CuH has been proposed to perform a long-range transfer of an electron to the CuM site for regeneration of CuI from CuII.43 148

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nCu/[nCu+nZn] 0.00.20.40.60.81.01.2 500 (Ms -1 ) 0 1e-8 2e-8 3e-8 4e-8 nCu/[nCu+nCo] 0.00.20.40.60.81.01.2 500 (Ms -1 ) 0 1e-8 2e-8 3e-8 4e-8 B A nCu/[nCu+nNi] 0.00.20.40.60.81.01.2 500 (Ms -1 ) 0 1e-8 2e-8 3e-8 4e-8 C Figure 3.11 ZnII (A), CoII (B), and NiII (C) dilution of CuII for the analysis of mononuclear versus dinuclear metal center in the catalysis of catechol oxidation by CuIIBc. The experiments were followed by th e oxidation of catechol in pH 7.0 100.0 mM HEPES buffer at 25 C. The two metal ions were arrayed in differe nt ratios while the overall concentration of all the metal ions was fixed. The data can be better fitted to the Hill equation (solid trace) than the quadratic equation for a simple 1:1 binding (dashed trace). 149

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Based on the experimental results, the following mechanism is proposed for the oxidation of catechol by CuII-Bc through a mononuclear Cu center (Figure 3.12). First, catechol binds to the CuII center in a bidentate manner, followed by 1e transfer to CuII to afford a CuI-semiquinone complex (step A and B). The CuI-semiquinone complex binds O2 and transfers 1e to O2 give a CuII-superoxo-semiquinone complex (step C). The semiquinone is further oxidized to give o-quinone and a CuII-peroxo complex (step D). The CuII-peroxo complex can go through another cycle since O2 2 can take 2 more e before released as 2H2O, or H2O2 can be released with a regenerated CuII center (step E). 150

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Cu2+L L L + O O Cu2+L L L OH OH O O Cu+L L L +O2 O O Cu2+L L L O O Cu2+L L O O2L + O O +O O H H A B+2H+C D E Figure 3.12 Proposed mechanism fo r catechol oxidation by CuII-Bc through a mononuclear CuII center catalysis in the presence of O2. First, catechol binds to the CuII center in a bidentate manner, followed by 1e transfer to CuII to afford a CuI-semiquinone complex (step A and B). The CuI-semiquinone complex binds O2 and transfers 1e to O2 give a CuII-superoxo-semiquinone complex (step C). Then, the semiquinone is further oxidized to give o-quinone and a CuII-peroxo complex (step D). The CuII-peroxo complex can go through another cycle since O2 2 can take 2 more e before released as 2H2O, or H2O2 can be released with a regenerated CuII center (step E). In the absence of O2, the oxidation is not catalytic. 151

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3.4 Conclusions Bacitracin, a dodecapeptide with a cyclic stru cture, is a naturally occurring potent antibiotic specifically against Gram positive bacteria.2 It is an effective component of probably the most used topical antibiotic ointment in North America. While topical application of this antibiotic is considered safe, a syst emic administration has been proposed to cause nephrotoxicity.2 The proposed antibacterial mechanism involves the inhibition of bacterial cell wall synthesis by the tight binding of ZnII-Bc complex to the pyrophosphate group of lipid sugar carrier.26 29, 30 In this study, I have shown the abil ity of bacitracin to perform oxidation chemistry in the presence of redox-active CuII. A high affinity binding of metallo-Bc to C55-isoprenyl pyrophosphate29 as well as the NMR study of binding between CoII-Bc and several phosphate derivatives2 b led to the hypothesis that the CuII-Bc complex may bind DNA through the phosphate moiety and perform the oxidative cleavage of DNA. Indeed, oxidative cleavage of plasmid DNA by CuII-Bc was observed (Figure 3.2). The oxidation chemistry of CuII-Bc was studied with catechol as a substrate. The substrate was indeed observed to be oxidize d effectively by CuII-Bc in the absence a nd presence of 32.0 mM H2O2, showing first-order rate constant of 6.99 10 s and 9.37 10 s, respectively, accounting for a rate enhancement of 1.47 104 and 1.98 104 fold, respectively with respect to the au to-oxidation under the same condition. Studies using the Job method, an optical binding of a slow substrate, 4,5dichlorocatechol, (1:1 CuII-Bc-DCC) and a mechanistic binding of catechol (1:1 CuII-Bc152

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CA) with the CuII-Bc complex nicely suggest a mononuclear CuII center oxidation chemistry. Since catechol oxidation is a 2e transfer process, oxygen must be involved in the redox cycling of the Cu (CuII/CuI) center. The 1:1 binding indicates a bidentate binding of CA to the CuII center, which results in a 5-c oordination sphere. This further leads to the proposal of a monodentate endon oxygen binding. Furthermore, dilution experiments indicate the possible presence of cooperativity. A mechanism is postulated based on the experimental results (Figure 3.12). Upon catechol binding (in a bi dentate manner) to the CuII center, an electron is readily transferred to the Cu center to form a CuI-bound semiquinone (Figure 3.12 steps A and B). Next, dioxygen binds to the CuI center in an end-on ( 1) mode. This is immediately followed by transfer of an electron from the Cu center to the oxygen, affording a CuII-superoxo intermediate (Figure 3.12 st ep C). The semiquinone is then fully oxidized to o-quinone and released after another electron transfer to the Cu center (Figure 3.12 step D). The bound oxygen may be able to accept up to four electrons before released as two water molecules, or H2O2 and a regenerated CuII center may be released without further electr on transfer (Figure 3.12 step E).44 A possible presence of cooperativity between nearby CuII-Bc complexes may also be suggested. Furthermore, the cooperativity may be explained by th e bridging of dioxygen to two different CuII-Bc complexes, which may help stabilize the overall structure. The chemistry of di-CuII centers with oxygen has been extensively studied and well characterized with a combination of synthetic organic models, multiple 153

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M R 1 2 3 4 5 6 7 8 9 10spectro scopic techn i ques and DFT calculation s.45 Although our understanding of mononuclear CuI/II centers has in creased recently, it is still a work in pro g ress. The above proposed mechanism is deduced based on lim ited inform ation from the experim ental results and our understanding of m ononuclear Cu system s. Th us, further detailed kinetic experim ents com b ined with spectro scopic t echniques, such as EPR and resonance Ram a n spectroscopy, are essential to validate the oxidation m echanism of CuII-Bc. In general, b acitracin is a relatively safe antibio tic. Because of its low in tes tina l absorption, bacitracin has been globally utilized as anim al feeds to prevent bacterial inf ections in f a rm anim als.4 In addition, it has been larg ely used as wound prophylaxis in hospitals and on tattoo wounds.10 However, there is an increase in repo rted cases of allergic reactions from this drug recently. An earlier study has proposed these allergic reactions to be due to polym e rization of bacitracin into m acromolecules w ith high molecular w eight.46 Metal-induced conf or m ational changes an d aggregations are commonly observed am ong sm all peptides and proteins especially by redox-active CuII and FeIII.47 Thus, the oxidation chemistry of CuII-Bc reported in this chapter m a y potentially contribute toward the alle rgic reactio ns cause d by bacitracin. 154

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155 References 1 (a) Meleney, F. L.; Johnson, B. A. Bacitrain. Am. J. Med. 1949, 7, 794. (b) Johnson, B. A.; Anker, H. S.; Meleney, F. L. Bacitracin: a new antibiotic produced by a member of the B. subtilis group. Science 1945, 102, 376. (c) Haavik, H. Effects of amino acids upon bacitracin production by Bacillus licheniformis. FEMS Microbiol. Lett. 1981, 10, 111. 2 (a) Ming, L.-J.; Epperson, J. D. Metal bind ing and structureactivity relationship of the metalloantibiotic peptide bacitracin. J. Inorg. Biochem. 2002, 91, 46. (b) Epperson, J. D. Paramagnetic Cobalt(II) As a Nuclear Magnetic Resonance Probe for the Study of Metallo-macromolecules: from Peptides and Proteins to Dendrimers. Ph.D. Dissertation, University of South Florida, Tampa, FL, 1999. 3 Brewer, G. A.; Florey, K. (Editor) Analytical Profiles of Drug Substances 1980, 9, 1 69. 4 Hanson, D. J. Human health effect s of animal feed drugs unclear. Chem. Eng. News 1985, 63, 711. 5 Arky, R. Physicians Desk Reference for Nonprescription Drugs 18th Ed. Medical Economics Company: Montvale, NJ, 1997. 6 Bonomo, R. A.; Van Zile, P. S.; Li, Q.; Shermock, K. M.; McCormick, W. G.; Kohut, B. Topical tripleantibiotic ointment as a novel therapeu tic choice in wound

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156 management and infection prevention: a practical perspective. Expert Rev. Anti. Infect. Ther. 2007, 5, 773. 7 (a) Weston, B. S.; Wahab, N. A.; Roberts, T.; Mason, R. M. Bacitracin inhibits fibronectin matrix assembly by me sangial cells in high glucose. Kidney Inter. 2001, 60, 1756. (b) Lawrence, D. A.; Song, R.; Weber, P. Surface thiols of human lymphocytes and their changes after in vitro and in vivo activation. J. Leuk. Biol. 1996 60, 611. (c) Essex, D. W.; Chen, K.; Swiat kowska, M. Localization of Protein Disulfide Isomerase to the External Surf ace of the Platelet Plasma Membrane. Blood 1995, 86, 2168. (d) Ryser, H. J.; Levy, E. M.; Mandel, R.; DiSciullo, G. J. Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfi'de interchange upon virus-receptor interaction. Proc. Nat. Acad. Sci. USA 1994, 91, 4559. (e) Mandel, R.; Ryser, H. J.; Ghani, F.; Wu, M.; Peak, D. Inhibition of a reductive function of the plas ma membrane by bacitr acin and antibodies against protein disulfide-isomerase. Proc. Nat. Acad. Sci. USA 1993, 90, 4112. 8 (a) Essex, D. W.; Li, M.; Miller, A.; Feinma n, R. D. Protein Disulfide Isomerase and Sulfhydryl-Dependent Pathways in Platelet Activation. Biochemistry 2001, 40, 6070 6075. (b) Essex, D. W.; Li, M. Protein di sulphide isomerase mediates platelet aggregation and secretion. Brit. J. Haem. 1999, 104, 448. 9 Donoso, G.; Craig, G. O.; Baldwin, R. S. The distribution and excretion of zinc bacitracin-14C in rats and swine. Toxicol. Appl. Pharmacol. 1970, 17, 366.

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157 10 (a) Sowa, J.; Tsuruta, D.; Kobayashi, H.; Ish ii, M. Allergic contact dermatitis caused by colistin sulfate & bacitracin. Contact Dermatitis 2005, 53, 175. (b) Jacob, S. E.; James, W. D. Froam Road Rash to Top Allergen in a Flash: Bacitracin. Dermatol. Surg. 2004, 30, 521. (c) Belsito, D. V.; DeLeo, V. A.; Fowler, J. F. Jr. Dermatitis 2004, 15, 176. (d) Blas, M.; Briesacher, K. S.; L obato, E. B. Bacitracin Irrigation: A Cause Of Anaphylaxis in the Operating Room. Anesth. Analg. 2000, 91,1027. (e) Gall, R.; Blakley, B.; Warrington, R.; Bell, D. D. lntraoperative Anaphylactic Shock from Bacitracin Nasal P acking after Septorhinoplasty. Anesthesiology 1999, 91, 1545. (f) Saryan, J. A.; Dammin, T. C.; B ouras, A. E. Anaphylaxis to Topical Bacitracin Zinc Ointment. Am. J. Emerg. Med. 1998,16, 512. (g) Zaki, I.; Shall, L.; Dalziel, K. L. Bacitracin: a signif icant sensitizer in leg ulcer patients. Contact Dermatitis 1994, 31, 92. 11 Phillips, I. Withdrawal of growth-promoti ng antibiotics in Europe and its effects in relation to human health. Int. J. Antimicro. Agents 2007, 30, 101. 12 (a) Dutta, G. N.; Devriese, L. A. Observ ations on the in vitr o sensitivity of Grampositive intestinal bacteria of farm animals to growth promoting antibacterials. J. Appl. Bacteriol. 1984, 56, 117. (b) Dutta, G. N.; Devriese, L. A. Susceptibility of fecal streptococci of poultry origin to nine growth promoting agents. Appl. Envir. Microbiol. 1982, 44, 832.

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158 13 Frvshoy, L. The Production of Bacitracin Synthetase by Bacillus Licheniformis ATCC 10716. FEBS Lett. 1977, 81, 315. 14 Konz, D.; Klens, A.; Schrgendorfer, K.; Mara hiel, M. A. The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases. Chem. Biol. 1997, 4, 927. 15 Kang, J. W.; De Reymaeker, G.; Van Sche pdael, A.; Roets, E.; Hoogmartens, J. Analysis ofbacitracin by micellar electrokin etic capillary chromatography with mixed micelle in acidic solution. Electrophoresis 2001, 22, 1356. 16 Siegel, M. M.; Huang, J.; Lin, B.; Tsao, R. Structures of bac itracin A and isolated congeners: sequencing of cyclic peptides with blocked line ar side chains by electrospray ionizati on mass spectrometry. Biol. Mass Spect. 1994, 23, 196. 17 Morris, M. Primary structur al confirmation of components of the bacitracin complex. Biol. Mass Spect. 1994, 23, 61. 18 Tsuji, K.; Robertson, J. H. Improve d high-performance liquid chromatographic method for polypeptide antibiotics and its application to stud y the effects of treatments to reduce microbial levels in bacitracin powder. J. Chromatogr. 1975, 112, 663. 19 Konigsberg, W.; Craig, L. C. The oxidation and acid isomerization of bacitracin A. J. Org. Chem. 1962, 27, 934.

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159 20 Drapeau, G.; Petitclerc, E.; Toulouse, A.; Marceau, F. Dissociation of the Antimicrobial Activity of Bacitracin USP from its Renovascular Effects. Antimicrob. Agents Chemother. 1992, 36, 955. 21 (a) Barry, G. T.; Gregoly, J. D.; Craig, L. C. The nature of bacitracin. J. Biol. Chem. 1948, 175, 485. (b) Craig, L. C.; Weisiger, J. R.; Hausmann, W.; Harfenist, E. J. The separation and characterization of the bacitracin polypeptides. J. Biol. Chem. 1952, 199, 259. (c) Newton, G. G. F.; Abraha m, E. P. Some peptides of bacitracin polypeptides. Biochem. J. 1953, 53, 597. 22 (a) Coates, H. B.; McLaughlan, K. A.; Campbell, I. D.; McColl, C. E. Proton spin lattice relaxation time measurem ents at 90 MHz and 270 MHz. Biochim. Biophys. Acta 1973, 310, 1. (b) Reynolds, W. F.; Peat, I. R.; Freedman, M. H.; Lyerla, J. R. Determination of the tautomeric form of the imidazole ring of L-histidine in basic solution by C-13 magnetic resonance spectroscopy. J. Am. Chem. Soc. 1973, 95, 328 331. 23 (a) Kobayashi, N.; Takenouchi, T.; Endo, S.; Munekata, E. 1H NMR study of the conformation of bacitracin A in aqueous solution. FEBS Lett. 1992, 305, 105. (b) Pons, M.; Feliz, M.; Molins, M. A.; Giralt, E. Conformational analysis of bacitracin A a naturally occurring lariat. Biopolymers 1992, 31, 605.

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160 24 Epperson, J. D.; Ming, L.-J. Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structureactivity relationship. Biochemistry 2000, 39, 4037. 25 Pfeffer, S.; Hohne, W.; Branner, S.; Wils on, K.; Betzel, C. X-ray structure of the antibiotic bacitracin A. FEBS Lett. 1991, 285, 115. 26 Storm, D. R.; Strominger, J. L. Binding of Bacitracin to Cells and Protoplasts of Micrococcus lysodeikticus. J. Biol. Chem. 1974, 249, 1823. 27 Scogin, D. A.; Mosberg, H. I.; Storm, D. R. ; Gennis, R. B. Binding of nickel and zinc to bacitracin A. Biochemistry 1980, 19, 3348. 28 Seebauer, E. G.; Duliba, E. P.; Scogin, D. A.; Gennis, R. B.; Belford, R. L. EPR evidence of the copper(II)-bacitracin A complex. J. Am. Chem. Soc. 1983, 105, 4926 4929. 29 Siewert, G.; Strominger, J. L. Bacitraci n: an Inhibitor of the Dephosphorylation of Lipid Pyrophosphate, an Intermediate in Bios ynthesis of the Pepti doglycan of Bacterial Cell Walls. Proc. Nat. Acad. Sci. USA 1967, 57, 767. 30 (a) Storm, D. R.; Strominger, J. L. Co mplex Formation Between Bacitracin Peptides and Isoprenyl Pyrophosphates. J. Biol. Chem. 1973, 248, 3940. (b) Stone, K. J.; Strominger, J. L. Mechanism of action of bacitracin: complexation with metal ions and C55-isoprenyl pyrophosphate. Proc. Nat. Acad. Sci. USA 1971, 68, 3223.

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161 31 (a) Garbutt, J. T.; Morehouse, A. L.; Ha nson, A. M. Metal Binding Properties of Bacitracin. J. Agri. Food Chem. 1961 9, 285. (b) Craig, L. C.; Phillips, W. F.; Burachik, M. Bacitracin A. Isolatio n by counter double curr ent distribution and characterization. Biochemistry 1969, 8, 2348. (c) Cornell, N. W.; Guiney, D. G. Jr. Binding sites for Zinc(II) in bacitracin. Biochem. Biophys. Res. Comm. 1970, 40, 530. (d) Wasylishen, R. E.; Graham, M. R. A nuclear magnetic resonance study of the metal binding sites in bacitracin. Can. J. Biochem.1975, 53, 1250. (e) Mosberg, H. I.; Scogin, D. A.; Storm, D. R.; Gennis, R. B. Proton nuclear magnetic resonance studies on bacitracin A and its interaction with zinc ion. Biochemistry 1980, 19, 3353. (f) Drabls, F.; Nicholson, D. G.; Rnning, M. EXAFS study of zinc coordination in bacitracin A. Biochim. Biophys. Acta 1999, 1431, 433. 32 da Silva, G. F. Z.; Tay, W. M.; Ming, L.-J. Catechol oxidaselike oxidation chemistry of the 1 and 1 fragments of Alzheimers disease-related -amyloid peptide: their structure-activity correlation an d the fate of hydrogen peroxide. J. Biol. Chem. 2005, 280, 16601. 33 da Silva, G. F. Z.; Ming, L.-J. Al zheimers disease related copper(II)-amyloid peptide exhibits phenol monooxygenase a nd catechol oxidase activities. Angew. Chem. Int. Ed. 2005 44, 5501.

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162 34 Srivatsan, S. G.; Nigam, P.; Rao, M. S.;Verma, S. Phenol oxidation by coppermetallated 9-allyladenine-DVB polymer: r eaction catalysis and polymer recycling. Applied Catal. A 2001, 209, 327. 35 (a) Job, P. Ann. Chim. 1936, 6, 97. (b) Ming, L.-J.; Wei, X. An Ytterbium(III) complex of duanomycin, a model metal co mplex of anthracycline antibiotics. Inorg. Chem. 1994, 33, 4617-4618. (c) Wei, X.; Ming, L.-J. Comprehensive 2D 1H NMR studies of paramagnetic lanthanide(III) complexes of anthracycline antitumour antibiotics. Inorg. Chem. 1998, 37, 2255. 36 (a) Torreggiani, A.; Tamba, M.; Trinch ero, A.; Bonora, S. Copper(II)Quercetin complexes in aqueous solutions: spectroscopic and kinetic properties. J. Mol. Struct. 2005, 744, 759. (b) Bravo, A.; Anacona, J. R. Metal complexes of the flavonoid quercetin: antib acterial properties. Trans. Metal Chem. 2001, 26, 20. 37 Itoh, S. Mononuclear copper active-oxygen complexes. Curr. Opin. Chem. Biol. 2006, 10, 115. 38 Fujisawa, K.; Tanaka, M.; Moro-oka, Y. ; Kitajima, N. A monomeric side-on superoxocopper(II) complex: Cu (O2)(HB(3-tBu-5-iPrpz)3). J. Am. Chem. Soc. 1994, 116, 12079. 39 Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Dioxygen Binds End-On to Mononuclear Copper in a Preca talytic Enzyme Complex. Science 2004, 304, 864.

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163 40 (a) Segel, I. H. Steady-State Kinetics of Multireactant Enzymes. Enzyme Kinetics: Behavior and Analysis of Rapid Equilib rium and Steady-State Enzyme Systems. John Wiley & Sons, Incs.: New York, 1993. (b) Leskovac, V. Comprehensive Enzyme Kinetics, Kluwer/Plenum, Boston, 2002, p. 119. 41 (a) Spencer, D. J. E.; Aboelella, N. W. ; Reynolds, A. M.; Holland, P. L.; Tolman, W. B. -diketiminate ligand backbone structural effects on Cu(I)/O2 reactivity: unique copper-superoxo and bis( -oxo) complexes. J. Am. Chem. Soc. 2002, 124, 2108. (b) Aboelella, N. W.; Kryatov, S. V.; Gh erman, B. F.; Brennessel, W. W.; Young, V. G. Jr.; Sarangi, R.; Rybak-Akimova, E. V.; Hodgson, K. O.; Hedman, B.; Solomon, E. I. Dioxygen activation at a single copper si te: structure, bonding, and mechanism of formation of 1:1 Cu-O2 adducts. J. Am. Chem. Soc. 2004, 126, 16896. 42 Klinman, J. P. The copper-enzyme family of dopamine (-monooxygenase and peptidylglycine -hydroxylating monooxygenase: resolving the chemical pathway for substrate hydroxylation. J. Biol. Chem. 2006, 281, 3013. 43 Jaron, S.; Blackburn, N. J. Does Superoxide Channel between th e Copper Centers in Peptidylglycine Monooxygenase? A New Mechanism Based on Carbon Monoxide Reactivity. Biochemistry 1999, 38, 15086. 44 (a) Tabner, B. J.; Turnbull, S.; Fullwood, N. J.; German, M.; Allsop, D. The production of hydrogen peroxide during early-s tage protein aggregation: a common pathological mechanism in differe nt neurodegenerative disease? Biochem. Soc. Trans.

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164 2005, 33, 548. (b) Tabner, B. J.; El-Agnaf, O. M. A.; Turnbull, S.; German, M. J.; Paleologou, K. E.; Hayashi, Y. Cooper, L. J.; Fullwood, N. J.; Allsop, D. Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer di sease and familial British dementia. J. Biol. Chem. 2005, 280, 35789. 45 (a) Selmeczi, K.; Rglier, M.; Giorgi, M.; Speier, G. Catechol oxidase activity of dicopper complexes with N-donor ligands. Coor. Chem. Rev. 2003, 245, 191. (b) Karlin, K. D.; Hatcher, L. Q. Oxidant t ypes in copper-dioxygen ch emistry: the ligand coordination defines the Cun-O2 structure and subsequent reactivity. J. Biol. Inorg. Chem. 2004, 9, 669. (c) Tolman, W. B. Using synthetic chemistry to understand copper protein active sites: a personal perspective. J. Biol. Inorg. Chem. 2006, 11, 261. 46 Stewart, G. T. Proteinace ous and polymeric residues in -lactam antibiotics and bacitracin. Antimicro. Agents Chemother. 1970, 1969, 128. 47 (a) Kowalik-Jankowska, T.; Rajewska, A.; Jankowska, E.; Grzonka, Z. Products of Cu(II)-catalyzed oxidation of -synuclein fragments containing M1-D2 and H50 residues in the presence of hydrogen peroxide. Dalton Transactions 2008, 832. (b) Bharathi; Indi, S. S.; Rao, K. S. J. Copperand iron-induced differential fibril formation in -synuclein: TEM study. Neurosci. Letts. 2007, 424, 78. (c) Guilloreau, L.; Combalbert, S.; Sournia-Saque t, A.; Mazarguil, H.; Faller, P. Redox

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165 chemistry of copper-amyloid: the generation of hydroxyl ra dical in the presence of ascorbate is linked to redox-poten tials and aggregation state. ChemBioChem. 2007, 8, 1317. (d) Ricchelli, F.; Fusi, P.; Tortora, P.; Valtorta, M.; Riva, M.; Tognon, G.; Chieregato, K.; Bolognin, S.; Zatta, P. Destab ilization of non-path ological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis. Int. J. Biochem. Cell Biol. 2007, 39, 966. (e) Ali, F. E. A.; Barnham, K. J.; Barrow, C. J.; Separovic, F. Metal-catalyzed oxidative damage and oligomerizations of the amyloidpeptide of Alzheimers disease. Aust. J. Chem. 2004, 57, 511. (f) Yoshiike, Y.; Tanemura, K.; Murayama, O.; Akagi, T.; Murayama M.; Sato, S.; Sun, X.; Tanaka, N.; Takashima, A. New insights on how metals disrupt amyloid -aggregation and their effects on amyloidcytotoxicity. J. Biol. Chem. 2001, 276, 32293.

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CHAPTER 4 ELUCIDATION OF THE IN VITRO OXIDATION CHEMISTRY OF COPPER(II)HISTATIN 5 COMPLEX BY MEANS OF REACTIVITY STUDIES 166

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4.1 Traditional Antibiotics The serendipitous discovery of penicillin (isolated from Penicillium notatum ) by the bacteriologist and immunol ogist Sir Alexander Fleming of Ayrshire, Scotland in 1928 marked the beginning of the use of naturally occurring antibiotics.1 2 It is inarguably one of the most important discoveries in the field of medicine in the 20th century. Although Fleming published his findings in 1929, the clinical application of penicillin was not fully established until 1940 by chemist Ernst Boris Chain (Berlin, Germany) and pathologist Howard Walter Florey (Adelaide, South Austra lia). The three shared the Nobel Prize for their findings in 1945.1 2 Since then, antibiotics have b een irreplaceable weapons against disease-causing microbes. By definition, antibiotics are chemical substances produced by bacteria and fungi to selectively inhibit th e growth or survival of other organi sms. In general, they deter the growth of microorganisms by interacting with DNA/RNA (e.g. FeII-bleomycin can bind and cleave DNA and RNA molecules through O2 activation.3) and the cell membrane (e.g. gramicidin can penetrate and span the li pid bilayer, leading to eventual bacterial cell death.4), preventing cell wall synthesis (e.g. pe nicillin prevents bacterial cell wall synthesis by binding irrevers ibly to the DD-transpeptidas e or the penicillin binding protein, which is responsible for cross-linking polysaccharide chains.5), or inhibiting protein synthesis (e.g. tetracycline inhi bits protein synthesis by binding to the ribosome.6). They are effective against a broad spectrum of infectious microbes, ranging from Gram positive and negative bacteria to fungi. In addition, some have antitumor and 167

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anticancer properties.3 Thus, naturally-occu rring antibiotics such as aminoglycosides, cephalosporins, macrolides, and vancomycin were considered wonder drugs and were extensively used. As a result of such exte nsive as well as impr oper usages (such as incorrect dosing and treatment duration) over th e years, bacterial resistance has become an alarming issue. For example, streptococci became resistant to penicillin only after one year of use,7 while tetracycline is obs olete against many bacteria6 8 and vancomycin is gradually losing its potency.9 Furthermore, there are different levels of toxicity associated with many antibiotics, ranging fr om minor (i.e. headache, diarrhea, and nausea) to serious (i.e. car diotoxicity, ototoxicity, and ne phrotoxicity) side effects, reducing effectiveness in their applications. 4.1.1 Antimicrobial Peptides (AMPs) In the early 1970s, a new way of combati ng infectious microorganisms came with the discovery of peptides having antimicrobial activities. They are commonly called antimicrobial peptides (AMPs) and are found in both plants and animals.10 In human, AMPs are an essential part of the innate im munity, where they form the first line of defense against the invading pathogens in va rious locations of the body. These natural antibiotics are typically sma ll cationic peptides of 3 kDa. 11 Although their amino acid compositions vary, these peptides have been su ggested to have simila r structural motifs, a random coil in an aqueous environment and an -helical or a -sheet conformation in a hydrophobic environment.12 168

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AMPs display a wide range of antimicrobi al activities against Gram-positive and negative bacteria, fungi, parasites, protozoa and some viruses. A generally accepted antibacterial mechanism of th ese peptides is their ability to penetrate the bacterial membrane and create pores through charge charge (positively charged peptide and negatively charged cell membrane) interactions which lead to cell death as a result of cytoplasmic leakage.10 Nevertheless, AMPs have also been suggested to interact with DNA, RNA, and specific proteins or enzymes as well as inhibit DNA, protein, or cell wall synthesis. Buforin II, a 21-amino acid an timicrobial peptide, is isolated from the stomach tissue of the Asian toad, Bufo bufo garagriozans, and causes cell death by binding to DNA and RNA.13 In addition to their antimicrobial functi ons, these antibiotic peptides are also associated with the processes in immunomodul ation and detoxification. They can act as a flag in directing immune or inflammatory cells to the infectious sites, can enhance angiogenesis and wound healing, and can bind a nd neutralize lipopolysa ccharides (LPSs). LPSs are released from the lysed bacterial cell wall as a result of antibiotic treatment. These molecules can over-activate the hosts immune system, which can ultimately lead to lethal endotoxicity.14 In addition, a salivary peptide, histatin 5, prevents the toxic tannin from intestinal absorption by bindi ng and precipitating out the plant toxin.15 Moreover, some of these peptide antibiotics have antiviral or anti cancer properties other than their normal antimicrobial activities (a ntibacterial and antifungal). The three wellstudied AMPs in humans are defens ins, cathelicidins, and histatins.10c Other well known 169

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AMPs are the amphibian peptides, magainins and dermaseptins, and the insect peptides, cecropins and melittins.10b 4.1.2 Candida albicans Human oral mucosa, a highly permeable tissu e that lines the oral cavity, houses over 200 bacterial a nd fungal species.16 The oral cavity in whic h these microbes reside is consisted of the saliva, the tongue, and the tooth-associat ed supra-gingival and subgingival plaques. While the Streptococcus species make up the majority of the bacterial flora, the Candida species are the most predominant among the fungal families, including Candida albicans C. dubliniensis C. glabrata C. krusei and C. tropicalis .16 Up to 80% of healthy individuals have been suggested to retain these fungal microbes at various regions of the body (i.e. the oral cavity, lungs, the gast rointestinal tract, the vaginal tract, blood, and skin) but mostly in the oral cavit y. Furthermore, these fungi are prominent (65%) among healthy children, the elderly, and HIV patients.16 Under normal circumstances, these fungal microorganisms are kept under control by the hosts innate defense, the whole saliva Nevertheless, this delicate equilibrium is disrupted in immunosuppressed or immunocompr omised individuals, and conditions such as diabetes mellitus, immunosuppressive ther apy, Sjogrens syndrome, radiation therapy for head and neck tumour, and HIV infection can lead to candidiasis .16 Moreover, C. albicans makes up the largest per centile among the above-lis ted fungi and mainly implicated in HIV-related or al candidiasis (up to 90%).16 Thus, the severity of its 170

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infections can be used as an indicator for the disease progression in AIDS, owing to the prominent nature of oral candidiasis among immunocompromised patients.17 Although oral fungal infections are tran sient for the healthy individuals, they can be progressively infectious and may even be lethal for th e immunodeficient HIV-infected adult and pediatric patients. In fact, the rise in the antifungal dr ug resistance has increased the mortality rate among AIDS patients over the years.18 Whole saliva contains arrays of antimicr obial peptides and pr oteins that help control infections caused by these fungal mi crobes; some of the well-studied ones are mucins, histatins, defensins, calprotec tin, myeloperoxidase, lysozyme, lactoferrin, secretory leukoprotease inhibitor, an d proteolytic peptide fragments.16 In the case of fungal infections among the immunosuppresse d individuals such as HIV patients, a number of drugs have been used: 5-fluoroc ytosine, the polyenes (i.e. amphotericin B and nystatin), and the azole-base d antifungal drugs (i.e. triazo le fluconazole). However, the fungal drug resistance has become an al arming issue over the years similar to the antibiotic resistance in bacteria.19 As a result, scientists are intensely studying the naturally-occurring antimicrobial peptides in hopes of finding a new way to combat those opportunistic microorganisms. 4.1.3 Histatin 5 (Hn5) The search for a salivary component th at enhances the glycolysis of oral microorganisms led to the discove ry of a small basic peptide,20 which was later 171

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determined to have multiple congeners and termed histatins (Hn) owing to their histidinerich primary structures.21 22 The ability of these peptid es to bind hydroxyapatite with high affinity23 and the difficulty in the separation of the congeners initially interested researchers.24 Histatins are a family of histidine-ri ch cationic peptides secreted from the parotid and submandibular glands.22 Along with other salivary proteins and peptides, such as proline-rich proteins and defensin s, histatins possess antimicrobial (against Streptococcus mutans )25 and antifungal (against Candida albicans ) properties and are active especially toward one of the most prevalent and opportunistic pathogenic yeasts, Candida albicans .26 In addition, Hn5 show antifungal activity against Cryptococcus neoformans and Saccharomyces cerevisiae27 as well as azole-resistant strains of Candida.28 4.1.3.1 Structure of Histatins Histatins are present only in higher pr imates. Two genes, HTN1 and HTN2 localized on chromosome 4q13, are responsib le for the synthesis of histatin 1 (DpSHEKRHHGY10 RRKFHEKHHS20 HREFPFYGDY30 GSNYLYDN) and histatin 3 (DSHAKRHHGY10 KRKFHEKHHS20 HRGYRSNYLY30 DN), respectively.29 30 Histatin 1 is composed of 38 amino acids, w ith Ser-2 phosphorylated; whereas, histatin 3 is made up of 32 amino acids, having similar sequence as histatin 1.22 Shorter variants of these two peptides have been isolated and id entified, which have also been shown to be 172

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active toward C. albicans Histatin 5 (Hn5), consists of the first 24 amino acids of histatin 3, is the most active fragment at physiological concentrations (15 M). Hn5 is expressed at the highest c oncentration among the variants in human saliva. Hn1, 3, and 5 make up 85% of all histatin peptides in the whole saliva,22 and the variants are the proteolytic products with differe nt antifungal activities toward C. albicans .31 Based on a circular dichroism study, the full length along with several shorter length peptides (the shortest fragment is made up of 10 C-terminal amino acid residues) of Hn5 have different tendencies to form -helices in hydrophobic environments; whereas, the random coil conformation is favored in aqueous environments. The same study suggests that the C-terminal (a minimu m of 14 amino acids) but not the N-terminal residues of Hn5 are essential for appreciable candidacidal activity. The -helical conformation is not required for its function, since less active fragme nts can also assume such secondary structure.32 4.1.3.2 Antimicrobial Mechanisms of Histatin 5 Even though the antibacterial and antif ungal activities of hi statins have been confirmed,25, 26 the antibiotic mechanism is still un clear. Two mechanisms have been proposed in the explanation of antimicrobial activity of Hn5. Initially, Hn5 was proposed to damage cell membranes, which result the release of K+ ions that are associated with the loss of cell viability.25 26 However, unlike other antimicrobial peptides, Hn5 has a weak amphipathic nature and lack the abil ity to form pores in the bacterial cell 173

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membranes.33 The calcein dye permeability study by Edgerton et. al. supports this hypothesis by proposing that the releas e of intracellular calcein from C. albicans is the aftermath of cell death and is not associated with cytotoxicity.34 In this study, it was proposed that the peptide destabilizes the cell membrane, foll owed by the interaction with specific cellular components leading to cell death and cytoplasmic leakage (which takes place after cell death).34 The same group later proposed that Hn5 induces cell death through non-lytic release of ATP.35 In this mechanism, Hn5 intially interacts and binds to Ssa1/2, the heat shock protein on the cell wall of C. albicans and internalized.36 After entering the cell wall, Hn5 interacts with TRK 1, the potassium transpor ter, leading to the loss of cell integrity.37 Extracellular ATP, in turn, bi nds and activates purinergic-like receptors, leading to apoptosis.35 Oppenheim and co-workers proposed a di fferent mechanism that involves the disruption of the yeast s respiratory machinery.38 According to this group, the Hn5 peptide is first internalized, possibly by translocation down th e membrane-potential gradient. This is followed by further intern alization of the peptid e into the mitochondrion through a similar mechanism. The intern alization of Hn5 ha s been shown by the aggregated intensity of the fluorescent agents, tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate coupled to Hn5 in mitochondria.38 b Once inside, Hn5 interferes with the electron transfer processes, which leads to the generation of reactive oxygen species (ROS). The presence of ROS was confirmed by the accumulation of the oxygen radical sensitive fluorescent probe, dihydroethidium in mitochondria.38 a 174

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Furthermore, they suggested that cellular respiration is necessary for the candidacidal activity since respiratory inhi bitors such as cyanide and azide as well as anaerobic conditions reduce or prevent the antifungal ac tivity of Hn5. The di sruption of cellular respiration and the oxidative damages to bi ological molecules caused by ROS lead to eventual cell death.38 4.1.3.3 Metal-Binding of Histatin 5 Having multiple histidine residues allows Hn5 to have great flexibility in binding metal ions. Hn5 has a high-affinity CuII and NiII binding site (Asp-1-Ser-His known as ATCUN A mino T erminal Cu IIand N iII-binding)39 on the N-terminus and a preferential ZnII binding site (HEXXH motif)40 on the C-terminus.41 The affinity constants determined from an isothermal calorimetry study are 2.6 107 M and 1.2 105 M for the CuIIand ZnII-binding site, respectively.42 In addition to CuII and ZnII, Hn5 can bind NiII, CaII, and FeII, although the binding affinities for the latter two are very weak. The order of metal binding to Hn5 is CuII > NiII > ZnII in aqueous environments. Both Hn3 and Hn5 have been shown to be able to bind more than one (and up to 5) equivalent of metal ions.41 A different study shows the binding of Hn5 to divalent transition metals, such as CuII, NiII, and ZnII, and displays DNA nuclease activity.43 It was shown that the CuII complexes of synthetic variants of Hn5, containing both th e ATCUN and HEXXH regions, can damage plasmid DNA through redox chemistry. Based on NMR results, it 175

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was suggested that the peptide structure is stabilized by metal binding. In a recent study by the same group, another short ATCUN c ontaining synthetic variant of Hn5 was proposed to have a pro-oxidant activity.44 They proposed a possible generation of ROS by Hn5 in the presence of CuII, supporting the ROS-induced antimicrobial mechanism. ROS generation was proposed to follow oxygen activation by the CuII-Hn5 complex. It is apparent that there is a trend in the ability of CuII-binding peptides to perform oxidation chemistry. This has been demonstr ated in recent studies, where the short soluble and metal-binding frag ments of amyloid peptides have been shown to perform catecholand phenol-oxidase-like chemistry in the presence of CuII.45 46 The oxidation mechanism has been proposed to involve oxygen binding and activation through a transient di-CuII center. Another example is the antib iotic peptide, bacitracin, discussed in the previous chapter. Bacitracin, howe ver, appears to perform a mononuclear Cucentered catalysis, based on experiment al results. In both cases, only one CuII ion can bind to the peptide. With histatin 5, at least two CuII ions can bind to each peptide, owing to the presence of 7 histidine residues. Thus, it will be interesting to study the potential oxidation chemistry with a pep tide containing more than one binding site in the sequence which is the theme of this chapter. 176

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4.2 Materials and Methods Histatin 5 (Hn5) was synthesized and the purity determined using MALDI-TOF mass spectrometer at the University of South Florida Peptide Synthesis and Mass Spectometry Center. Copper Sulfat e (anhydrous) and 4-(2-hydroxyethyl)-1piperazineethanesulfonate buffer (HEPES) us ed for the reactivity experiments was purchased from Fisher Scientific Co. (Fai r Lawn, NJ). Pyrocatechol (~99%) and 3Methyl-2-benzothiazolinone hydrazone hydroc hloride monohydrate (98%), MBTH, were acquired from SigmaAldrich Inc. (St. Louis, MO) and Acros Organics (NJ), respectively. All chemicals we re used without further purification. All other solvents and reagents were of the hi ghest grade available from co mmercial sources. Deionized water (18.2 M ) was obtained from a Millipore Milli-Q system. Plastic ware and glassware were demetallized with EDTA and extensively rinsed. All components were freshly prepared prior to the experiments. 4.2.1 Kinetic Studies Catechol oxidation assays were carried out as previously described.46 Different concentrations (0.4 mM) of catechol (with equal concentrations of o-quinone specific indicator, MBTH) were incubated with 5.0 M CuIIHn5 (4:1 metal-to-peptide) in the presence and absence of H2O2 in 100.0 mM HEPES buffer at pH 7.0 and 25 C. The final volume in the cuvette is 1.0 mL. The oxidized o quinone can bind to MBTH to form a red adduct, which was monitored at 500 nm ( = 32, 500 Mcm)47 on a Varian Cary50 177

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Bio UV-Vis spectrophotometer equipped with a water peltier PCB-150 thermostable cell (Varian, Palo Alto, CA). The initial rate was determined from the slope of the change in the absorbance with respect to time (0 mins) in the li near region. The kinetic constants, KM and kcat, were determined by fitting the data to the Michaelis-Menten equation in the Sigma Plot 8.0. 4.2.2 Optical Studies The slow substrate, 4,5-di-chloro-catechol (DCC), was used as a ligand in binding studies to show the interaction between the CuII-Hn5 complex and the catechol substrate. Due to low solubility in water, DCC was di ssolved in DMF. The stock solution of the CuII-Hn5 complex was prepared in DI water, since only the peptide and not the copper salt would dissolve in DMF. The experiment was performed in DMF, and all components were completely miscible. Briefly, DCC ranging from 0 to 1.0 mM was gradually titrated into a 1.0mL solution of CuII-Hn5 (200.0 M) with the sample cuvette left in the spectrometer to keep the baseli ne intact. The background was zeroed with only the solvent, and the mixture was scanned fr om 200 to 900 nm on the Varian Cary50 Bio UV-Vis spectrophotometer. The dilution factor from each addition was corrected in the final concentrations. The molar absorptivity value of the CuII-Hn5-DCC complex was plotted with respect to the ti trated DCC concentration, and th e data was fitted to simple 1:1 binding quadratic equation in Sigma Plot 8.0 for the dete rmination of the affinity constant, KDCC. 178

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4.2.3 NMR Studies 1H NMR spectra were acquired on Vari an INOVA500 spectrometer (at 500 MHz 1H resonance) with a 5-mm bio-TR (triple resonance) probe. A 90 pulse (~9 s) was used for the acquisition of 1D 1H NMR spectra with 8 K data points. The superWEFT technique48 was used for the suppression of slowly relaxing signals. Both peptide and cobalt stock solutions were prepared in d6-DMSO. Hn5 peptide sample (0.5 mM) was titrated with CoII, and the complex formation was monitored over 300 ppm spectral width ( to 150 ppm). Triethylamine was added as needed. A line-broadening of 40 Hz was applied to improve the signal-to-noise ra tio of the paramagnetically shifted signals. 4.3 Results and Discussion There are 7 histidine residues on Hn5, whic h afford more than one metal-binding site. Metal-binding studies have been prev iously done for Hn5, and two possible sites were proposed: a high-affinity CuII and NiII binding site on the N-terminus (known as ATCUN A mino T erminal Cu IIand N iII-binding)39 and a preferential ZnII binding site on the C-terminus (HEXXH motif)40. However, up to 4 equivalents of CuII have been suggested to bind 1 equivalent of Hn5 peptide.41 Thus, a 4:1 CuII-to-Hn5 complex was used in the reactivity studies. Only two metal binding motifs have been proposed for Hn5 although this peptide of 24 amino acids has 11 possible metal bindi ng residues (i.e. 1 Asp, 1 Glu and 2 Tyr in 179

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addition to 7 His residues). A recent study on ZnII and CuII binding by means of isothermal calorimetry suggest s that Hn5 has three possible metal binding sites for both metals.42 Since the presence of 2 versus 3 CuII ions may react with catechol differently, it is essential to know how many CuII ions are bound per Hn5 peptide. Thus, CuII binding by Hn5 was studied by means of optical, EPR, and NMR spectroscopy to determine the stoichiometry. The optical CuII titration was performed in 100.0 mM HEPES buffer at pH 7.0 and 25 C on Cary50 spectrophotomer. Hn5 (200.0 M) was gradually titrated with CuII and the electronic spectrum collected from 200 nm. Upon CuII addition, two strong absorptions around 250 and 300 nm and a dd transition absorption at ~520 nm were observed (Figure 4.1). As more CuII is added, a gradual red sh ift by all three signals was observed. The sample was titrated with up to ~6.0 equivalents of metal, resulting in ~100 nm shift in the dd transition. The resulted electroni c spectra suggest the presence of more than one CuII-bound Hn5 species. Since the number of species present cannot be clearly distinguished from th e electronic spectra, the CuII binding was further investigated with EPR spectroscopy. The continuous-wave (CW) EPR spectra of CuII-Hn5 complexes were acquired on a Bruker Elexsys E580 cw X-band spectromete r assisted by Dr. Angerhofer at the University of Florida. The spectra were acquired on the following complexes of CuII 180

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Wavelength (nm) 220240260280300320340360380400 (M-1cm-1) 0 2000 4000 6000 8000 10000 Wavelength (nm) 400500600700800 (M-1cm-1) 0 50 100 150 200 250 300 Figure 4.1 Optical CuII binding study of Hn5 (200.0 M) in 100.0 mM HEPES buffer at pH 7.0 and 25 C. Approximately 100.0 M CuII (0.5 equivalent) is added for each spectrum. The inset is the expansion of the wavelength from 400 nm. A gradual shift in all three observed signals was observed upon addition of metal. The dd transition at ~520 nm shifted approximately 100 nm after up to ~6.0 equivalents of CuII has been added. 181

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-(250.0 M) Hn5 in DMF: 1:1, 2:1, and 4:1 (F igure 4.2 and 4.3). The spectra display characteristics typical of CuII complexes, having 4 hyperfin e splittings in an axial orientation (i.e. gx = gy < gz), with overshoot signals. The magnetic field is converted into g values, and the spectra are plotted with respect to g values (on x-axis). The gparallel values for 1:1 and 2:1 species are approximately 2.198 and 2.269, respectively, while the g-perpendicular value is estimated to be 2.062 for both species (Figure 4.2). In the spectrum for 1:1 CuII-Hn5, only one set of hyperfine sp littings can be clearly seen; whereas, in the 2:1 spectrum, a nother set of hyperfine signals in addition to the former is apparent. The difference in the g values al so indicates the presence of two different CuII species in 2:1 spectrum, and these values fall within the normal range for CuII species bound to nitrogen-donor ligand such as histidine.49 At 4:1 species, the hyperfine splittings of 1:1 and 2:1 species are replaced by a single set of hyperfine signals with gparallel and g-perpendicular values of 2.313 and 2.082, respectively (Figure 4.3). Based on the clear difference between 1:1 and 2:1 vers us 4:1 species indicates there may be two or more types of CuII centers present at 4 equivalents of CuII. Next, the metal coordination by Hn5 wa s further defined by means of NMR spectroscopy. The slow el ectronic relaxation of CuII can broaden the NMR signals beyond detection, rendering it useless in metal-binding studies by means of NMR spectroscopy. However, fast relaxing CoII has been effectively ut ilized as a paramagnetic NMR probe in studying the metal-coor dination site of various systems.50 A 0.5-mM apoHn5 peptide sample was gradually titrated with CoII in d6-DMSO, and the 182

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g-values 2.0 2.2 2.4 2.6 Arbitrary EPR Intensity -20000 -15000 -10000 -5000 0 5000 10000 15000 1:1 Cu II -to-Hn5 2:1 Cu II -to-Hn5 g = 2.269 g = 2.062 g = 2.198 Figure 4.2 EPR spectra of 2:1 (dotted line) and 1:1 (solid line) CuII-(250.0 M) Hn5 in DMF. The g-parallel values for 1:1 a nd 2:1 species are 2.198 and 2.269, respectively, while the g-perpendicular value is estimat ed to be 2.062 for both species. The 2:1 spectrum clearly shows the presence of two different species of CuII, which can be distinguished based on th eir g-parallel values. 183

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g-values 2.0 2.2 2.4 2.6 arbitrary EPR unit -50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 4:1 CuII-to-Hn5 g = 2.082 g = 2.313 Figure 4.3 EPR spectrum of 4:1 CuII-(250.0 M) Hn5 in DMF. The estimated g values are 2.313 and 2.082 for g-parallel and g-perpendicular, respectively. 184

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data was acquired on the INOVA 500 Varian spectrometer with a bio-triple resonance 5 mm probe at 25 C. Up to 6.0 equivalents of CoII were added to the peptide in small increments. No signal was observed upon addition of 0.5 equivalent of CoII within the monitoring spectral region ( to 150 ppm). At 1.5 equivalents, three very weak signals can be observed at ~50, ~60, and ~ 70 ppm, which can be clearly seen at 2.0 equivalents of CoII (Figure 4.4). The two signals at 58 and 67 ppm are broader compared to a relatively sharp signal at 50 ppm. A new signal appears approximately at 48 ppm after 2.5 equivalents of CoII, and continued addition of CoII up to 6.0 equivalents only enhances the intensity of the shifted signals. The typi cal chemical shift range for CoII-bound imidazole group of histidine resi due is approximate ly from 40 ppm.51 Addition of 30.0 uL D2O to the sample significantly reduces the signal intensity, which clearly shows these signals to be the solven t-exchangeable imidazole NH protons. Thus, the signals observed may be assigned to the CoII-bound histidine residues on Hn5. An important observation from this experi ment is the indication that there are only two types of metal binding modes. From 0.5 to 2.5 equivalents of CoII, one binding mode of CoII-Hn5 can be seen from a simultaneous increase of signal intensity at 50, 58, and 67 ppm. A second mode of metal bi nding becomes observable starting at 2.5 equivalents of CoII, showing a new signal at 47 pp m and no additional signals are observed up to 6.0 equivalents of CoII. The three signals from the first binding mode may be assigned to His-15, His-18, and His-19 in the HEXXH motif, since there is only one histidine in the ATCUN motif. In the ATCUN motif, the metal is bound through the 185

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Figure 4.4 1H NMR spectra of CoII-Hn5 at 2:1 (bottom) and 6:1 (top) ratios with 8k scans. Up to 2.5 equivalents of CoII, only three signals at 50, 58, and 67 ppm are observed, and from 2.5 to 6.0 equivalents of CoII, a new signal at 47 ppm appears. All signals are solvent-exchangeable (i.e. determined by signal reductions after D2O addition). The three signals may repres ent one metal binding mode by 3 histidine residues in HEXXH motif, and the latter signal at 47 ppm may be assigned to a single histidine residue in ATCUN motif. 186

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amide backbones of the first three am ino acids and the imidazole group of 3His. Thus, a single signal from the second binding mode may be assigned to the ATCUN site. Different concentrations of cat echol were incubated with 5.0 M CuII-Hn5 (4:1) in 100.0 mM HEPES buffer at pH 7.0 and 25 C. An increase in the rate of catechol oxidation was observed with increasing substr ate, which reaches saturation at higher concentrations of catechol (F igure 4.5). The saturation pr ofile suggests th e presence of enzyme-like kinetics, and the rate law can be described according to the following equations, with the assumption th at [CA] >> [Cu(II)-Hn5-CA]: [Cu(II)-Hn5-CA] [Cu(II)-Hn5] + [CA] [Cu(II)-Hn5] + o-quinone k1k-1kcat CAK CAVHn Max 5 0 (1) where 0 and VMax are the measured and maximum velocity, respectively, and 1 1 5k kk Kcat Hn is the virtual dissociation constant of the CuII-Hn5-CA complex. The data can be well fitted to Eq. 1 to afford KHn5, the first-order rate constant or the turnover number kcat ( kcat = VMax/[CuII-Hn5]), and the second-order rate constant or the catalytic efficiency kcat/KHn5 of 3.06 10 M, 4.53 10 s, and 14.83 M s, respectively (Figure 4.5). Compared to the auto-oxidation rate of catechol (4.74 10 s) in the absence of CuII-Hn5,45 there is a 9.56 104-times rate acceleration with respect to the first-order rate constant. 187

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[Catechol] (M) 0 2e-3 4e-3 6e-3 (Ms-1) 0 1e-9 2e-9 3e-9 4e-9 5e-9 6e-9 7e-9 Figure 4.5 Catechol oxidation by CuII-Hn5 (4:1) in 100.0 mM HEPES at pH 7.0 and 25 C. The saturation profile suggests a possibl e pre-equilibrium kinetics. The data are fitted to Eq. 1, an analogue of Mi chaelis-Menten equation, to afford KHn5, kcat, and kcat/KHn5. 188

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Since CuII-Hn5 can oxidize catechol, the CuII binding to Hn5 was monitored with catechol oxidation. Diffe rent amounts of CuII (0.0 equivalents) were incubated with 5.0 M Hn5 and 5.0 mM catechol/MBTH in 100 mM HEPES buffer at pH 7.0 and 25 C. From 0.0 equivalent of CuII, no significant activation was observed (Figure 4.6). However, a sudden increase in the activity wa s observed after the first equivalent of CuII and does not reach saturation until 16.0 equiva lents of metal. Since Hn5 has multiple metal binding residues and two proposed meta l binding sites on the sequence, the peptide may bind the CuII ion in such a way that no bindi ng site is left open for substrate interaction. In the presence of more CuII, however, more substrate binding sites can become available. As a result, the 1:1 CuII-Hn5 species appears inac tive, and an abrupt increase in the activity is observed at higher CuII concentrations. The interaction between the CuII-Hn5 complex and the catechol was confirmed by an optical binding study with a slow substrate, 4,5-dichloro catechol (DCC). The activity of DCC is roughly 200 times slower than catechol with respect to kcat as a ligand.45 A 1.0-mL solution of 200M CuII-Hn5 was slowly titrated with DCC in DMF at 25 C. The formation of complex was monitored from 200 nm on the Varian Cary50 Bio UV-Vis spectrophotometer. Upon addition of DCC, a new absorption corresponding to a complex between CuII-Hn5 and DCC appears at 298 nm (Figure 4.7). The plot of the (CuII-Hn5)-DCC complex against equivalents of DCC added shows the binding to reach a saturation at approximately 3.0 equivalents of DCC. The results suggest a possible presence of three metal bindi ng sites for substrate binding. 189

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[Cu(II)]/[Hn5] 024681012141618 (Ms-1) 0 5e-8 1e-7 2e-7 2e-7 3e-7 3e-7 Figure 4.6 CuII binding of Hn5 monitored with catechol oxidation activity. No significant activity was observed approxi mately up to 1.0 equivalent of CuII, which is followed by a sudden increase in the activity for subsequent assays. 190

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In the presence of H2O2, a significant rate enhancem ent in catechol oxidation was observed for the CuII-Bc system in the previous chapter. Similar experiments were performed to determine the effect of H2O2 on catechol oxidation by CuII-Hn5. Different concentrations (0.0 mM) of H2O2 were incubated with 0.5 M CuII-Hn5 and 3.2 mM catechol in 100.0 mM HEPES buffer at pH 7.0 and 25 C. The increase in catechol oxidation was observed with increasing H2O2 and reaches saturation at higher concentrations of H2O2 (Figure 4.8 inset). The saturation profile indicates a possible presence of a pre-equilibrium kinetics, suggesting H2O2 as another substrate capable of interaction with the metal center(s). The data can be fitted to Eq. 2 to afford an apparent binding or dissociation constant KH2O2 of 8.63 10 M for H2O2. 22 22 022OHK OHVOH Max Background (2) This compared to KHn5 = 3.06 10 M, the binding constant for catechol in the absence of H2O2, is approximately 2.8 times higher, sugges ting that this substrate binds ~3 lower than catechol toward the CuII-Hn5 complex. The fact that both substrates can independently bind to the CuII center (i.e. both catechol and H2O2 show saturation), it is essential to determine how the binding of one affects the other. Different concentrations (0.4 mM) of catechol were incubated with the CuII-Hn5 (0.2 M) in the presence of different H2O2 concentrations (0.0 mM). The enhancements in activity of cat echol oxidation in the presence of H2O2 were clearly observed (Figure 4.8). While KHn5 is increased by ~2 folds, the addition of 32.0 mM 191

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Wavelength (nm) 260280300320340360380400 (M-1cm-1) 0 2000 4000 6000 8000 10000 [DCC]/[Cu(II)-Hn5] 012345 Absorbance298 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Figure 4.7 4,5-dichlorocatechol binding of CuII-Hn5 in DMF. All the spectra have been zeroed with that of CuII-Hn5 complex alone. The (CuII-Hn5)-DCC complex plotted versus DCC added shows satura tion at 3.0 equivalents of DCC. 192

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H2O2 in the assay increased both the kcat and kcat/KHn5 by 7 and 4 folds, respectively. Because the two substrates may bind to CuII-Hn5 independently of each other, the Hanes plot analysis can be used to determine the interaction between the two substrates. From the Hanes analysis, the apparent binding equilibrium constants for both substrates and an intrinsic binding consta nt for one substrate can be calculated.52 The Hanes analysis requires an initial linear trea tment of the data in Figure 4.8 according to the following rate law (Eq. 3), 22 max max 22 022 221 1 OH K V K Catechol V OH K CatecholInt OH App Catechol App OH (3) where is the intrinsic binding constant for H2O2, and are the apparent binding constants for H2O2 and catechol, and Int OHK22App OHK220App CatecholK and are the experimental and maximum velocity, respectively (Figure 4.9 A). The ratio betw een the corresponding apparent and intrinsic equilibrium constants for each substrate can suggest how the binding of one substrate affects the binding of an other. The secondary plots of the fitted y-intercept (Eq. 4) and slope (Eq. 5) values versus 1/[H2O2] afford the two apparent equilibrium constants = 5.23 10 M and = 3.63 10 M and the intrinsic binding constant = 2.26 10 M (Figure 4.9 B and C). maxVApp OHK22HKAppKCatecholInt O22 193

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[Catechol] (M) 0 2e-3 4e-3 6e-3 (Ms-1) 0 2e-8 4e-8 6e-8 8e-8 1e-7 0 mM H 2 O 2 2 mM H 2 O 2 4 mM H 2 O 2 8 mM H 2 O 2 16 mM H 2 O 2 32 mM H 2 O 2 64 mM H 2 O 2 [H2O2] (M) 0.000.020.040.06 500 (Ms-1) 0 2e-8 4e-8 6e-8 8e-8 1e-7 Figure 4.8 Hydrogen peroxide influe nce on catechol oxidation by 0.2 M CuII-Hn5 at pH 7.0 in 100.0 mM HEPES buffer at 25 C. The concentrations of H2O2 were varied from 0 to 64.0 mM. The titration of H2O2 at a constant catechol concentration of 3.2 mM displays saturation, which indicates the direct interaction between the CuII-Hn5 center and H2O2 (the inset). 194

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22 max max1 int22OH V KK V K ercept yInt OH App Catechol App Catechol (4) 22 max max1 122OHV K V slopeApp OH (5) The ratio between the apparent and intrin sic binding constants is 1.19 for catechol ( /KHn5) and 2.31 for H2O2 (/ ), suggesting that the binding of H2O2 to the CuII-Hn5 complex does not affect the cat echol binding; whereas, the binding of H2O2 is reduced by half in the presence of catechol. App CatecholKApp OHK22Int OHK22 195

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[Catechol] (M) 0 2e-3 4e-3 6e-3 8e-3 [Catechol]/ (s) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1 2 e +6 0 mM H 2 O 2 2 mM H 2 O 2 4 mM H 2 O 2 8 mM H 2 O 2 16 mM H 2 O 2 32 mM H 2 O 2 64 mM H 2 O 2 [H2O2]-1 (M-1) -2000200400600 y-interceptCatechol 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 1.4e+5 1.6e+5 [H2O2]-1 (M-1) -2000200400600 SlopeCatechol 0 2e+7 4e+7 6e+7 8e+7 A B C Figure 4.9 Hanes analysis of the oxidation of catechol by CuII-Hn5 at different concentrations of H2O2 (kinetic data from Figure 4.8). Pl ot B and C are the replots of the slope and y-intercept values from plot A with respect to [H2O2]. The apparent binding equilibrium constants for catechol ( ) and H2O2 ( ) as well as the intrinsic binding constant for H2O2 ( ) can be determined from the secondary plots.App CatecholKApp OHK22Int OHK22 196

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4.4 Conclusions In this study, the oxidative activity of Hn5 peptide in the presence of CuII has been studied. Recent results have shown th e ability of short N-terminal metal binding fragments A (i.e. A peptide of 1 or 1 amino acids isolated from the plaques of Alzheimers disease patients.), A 116 and A 10, can perform catecholand phenoloxidase-like chemistry in the presence of CuII.45, 46 Furthermore, in the previous chapter I have shown catechol oxidation chemistry of the CuII complex of bacitracin, a dodecapeptide antibiotic. While the CuII-A complexes oxidize catechol to o-quinone through a di-CuII center catalysis, the CuII-Bc complex only requires a mononuclear CuII center. Both systems bind to CuII in a 1:1 ratio. Nevertheless, two CuII-A complexes have been proposed to assemble into a di-CuII center either by the substrate or oxygen during catalysis.45, 46 Thus, it will be interesting to determine how will the Hn5 peptide, having two or more potential metal binding si tes within per peptid e sequence, perform catechol oxidation in the pr esence of redox-active CuII. The CuII complex of Hn5 has been shown to effectively oxidize catechol with a rate enhancement of 9.56 104 times with respect to the first-order rate constant, compared to the auto-oxidati on rate of catechol (4.74 10 s).45 For CuIIA 45 and CuII-Bc, the presence of H2O2 significantly enhances the catechol oxidation. H2O2 also significantly enhances th e catechol oxidation by CuII-Hn5. The preliminary results from the above studies only suggest the ability of CuIIHn5 to perform catechol oxidation. Accordi ng to EPR and NMR st udies, there are two 197

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198 potential metal binding sites on Hn5. Because the metal binding sites are on the same peptide, they may be in close e nough proximity to perform a di-CuII oxygen chemistry. However, more studies are necessary to differentiate between the mononuclear and dinuclear CuII center. Thus, experiments such as optical DCC binding and catechol binding (i.e. with respect to activity) using the Job method ca n provide further evidence. In addition, similar studies with EPR and NMR spectroscopic tec hniques may provide more details on substrate binding; whereas, all the a bove studies under anaerobic conditions can provide a clear picture for the role played by dioxygen. In conclusion, redox-active metals such as CuII and FeIII are essential for biological processes but are heavily regulated due to oxidative damages they can impose on the biological molecules.53 Dysregulation of metal ions have been associated with many human pathologies, such as amyotrophic lateral sclerosis, Wilsons, Alzheimers, and Parkinsons disease, just to name a few.54 There seems to be a trend among some biologically available peptides, where they can effectively bind a wide range of metal ions.55 It is possible that these metal binding peptides play an important role in the regulation of these redox metals. Thus, understanding of the oxidation chemistry of these metal-peptide complexes may be essential to better understanding of rela ted pathologies. Even though the findings in this disserta tion may not be directly extended to in vivo situations, they are intended for providing fundamental understandings in hopes of serving as a building block for future innovations.

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ABOUT THE AUTHOR William Maung Tay was born in 1979 in Rangoon, Burma (currently known as Yangon, Myanmar). He immigrated to the Unit ed States of America at the age of 12. After overcoming the language barrier, he graduated with high honor with distinction from Lakeland Senior High School. He attende d Florida Southern College from 1998 to 2002 and graduated with a B.Sc. in both chemistry and mathematics. Then, he pursued his graduate studies in chemis try at the University of Sout h Florida under the supervision of Dr. Li-June Ming. From 2002 to 2004, he served as a teaching assistant for General and Organic Chemistry Labs. Then, he took on the NMR teaching assistant position from 2004 to 2007.