The Bioinorganic Chemistry Of Copper-Containing Systems: From Type-3 Systems Pertinent To Alzheimer’s Disease To Mononuclear Hydrolysis Involved In Biological Development by Giordano Faustini Zimmerer Da Silva A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Co-Major Professor: Li-June Ming, Ph.D. Co-Major Professor: Brian T. Livingston, Ph.D. Steven H. Grossman, Ph.D. Randy L. Larsen, Ph.D. Date of Approval: May 9, 2007 Keywords: kinetics, reactive oxygen species, metallopeptide, amyloid, enzymology, sea urchin, metalloprotease © Copyright 2007 , Giordano F.Z. Da Silva NOTE TO THE READER Note to Reader: The original of this document contains color that is necessary for understanding the data. The original dissertation is on file at the USF library in Tampa, FL. DEDICATION Although the graduate school experience is highly a personal journey, it would be foolish to believe that I was solely responsible for my accomplishments. I then would like to dedicate this work to all of those who have played a major role in this difficult yet rewarding part of my life. First my grandmother Ermelinda who is the sole reason why I am in the US, my parents Mauricio and Marta for sacrificing the presence of their son, and my sister Marcella for being sibling, friend and parent through part of my formative years. Second I must dedicate this work to the Kelley family for being my parents, brother, and sisters; Moms, Pops, John, Tere, and Abbie, I would not be here today without your love and support. Last and not least I must also dedicate this work to Rachel for being a friend, always in my heart, through difficult and joyous times, for her love and support without which I would have never remained in the US. I truly hope that the rest of my career will make the efforts of all of those herein mentioned worthwhile. ACKNOWLDGEMENTS I must acknowledge my friends Bri and Brenda, who were my sisters through the laughter, tears, and often difficult times, and hope to remain life-long friends. I must also acknowledge William and Kash for being brothers in arm, and Vaso for inspiration. I would also like to acknowledge Altan for being a patient mentor during my undergraduate years and believing in my potential. I would like to acknowledge my collaborator Dr. Alexander Angerhofer from UF for his availability and willingness to share his time and effort. Rae Reuille must be acknowledged for her patience and guidance while teaching molecular biology to a chemist. Dr. Randy Larsen and Dr. Steven Grossman must also be acknowledged for their efforts as my committee members and for always being present to discuss matters of science, life, history, and politics. Last and not least I must acknowledge Dr. Li-June Ming and Dr. Brian T. Livingston. In an ever-changing academic world it is often difficult to find mentors who still have a pure scholar’s mind. Through their efforts, patience, guidance, and discussions about science and life, I feel prepared to move on to the next stage of the scientific experience. They were teachers, mentors, and friends and I am eternally grateful for their time. I truly hope that the rest of my career will make the efforts of all of those herein mentioned worthwhile. TABLE OF CONTENTS List of Tables vi List of Figures vii List of Abbreviations xi Abstract xv Chapter I: Introduction 1 Background 1 Metabolic Generation of Reactive Oxygen Species 7 Superoxide and Superoxide Dismutase 10 Metal-Binding to Aβ and Structure of the Complex 19 Generation of ROS by Metallo-Aβ 22 Type-3 Copper Oxidase Models and Metallo-ROS 29 Concluding Remarks 52 List of References 53 Chapter II: Cu2+Aβ Complexes as Redox-Active Catalysts Toward the Oxidation of 1,2,3 Tri-Hydroxy Benzene 87 Introduction 87 Experimental 90 DNA cleavage 90 i THB Oxidation Assay 90 Metal Titration to Aβ 91 1H NMR Co2+ Titration to Aβ 92 Molecular Mechanics (MM3) 92 Results and Discussion 92 Oxidative Double-Stranded DNA Cleavage 92 Kinetics and Mechanism of Oxidative Catalysis by CuAβ 100 Metal Binding and Structure 112 Concluding Remarks 122 List of References 124 Chapter III: Catechol Oxidase and Phenol Monooxygenase Activities of CuAβ1-20 133 Introduction 133 Experimental 135 Phenol Monooxygenase MBTH Assay 135 Results and Discussion 137 Catechol Oxidase Activity 137 Zn2+ Dilution 139 Effect of H2O2 140 Phenol Hydroxylation 144 DCC Optical Titration 147 Concluding Remarks 149 ii List of References 150 Chapter IV: Metallo-ROS in Alzheimer ’s Disease: Metal-Centered Oxidation of Neurotransmitters by Cu2+-β- Amyloid Provides an Alternative Perspective for the Neuropathology of Alzheimer’s Disease. 154 Introduction 154 Experimental 155 Neurotransmitter Oxidation Assay 156 Results and Discussion 156 Catecholamine oxidation 156 Effect of H2O2 162 Effect of SDS 163 Effect of NADH and NADPH 165 DCC Titration to Aβ1-40 167 Serotonin Oxidation 174 Concluding Remarks 177 List of References 179 Chapter V: Methionine 35 is not a Reducing Agent for the Metal-Centered Oxidation Chemistry of Cu2+-β-Amyloid: Kinetic and EPR Studies 187 Introduction 187 Experimental 189 Catechol Oxidase MBTH Assay 189 L-Met Optical Titration 190 Effect of Reducing Agents 190 iii EPR and ESEEM 190 Results 192 Effect of L-Met 192 Effect of Reducing Agents 196 Electronic Spectrum of L-Met CuAβ1-20 196 CW-EPR of CuAβ1-20 and L-Met + CuAβ1-20 196 ESEEM Spectra CuAβ1-20 and L-Met + CuAβ1-20 200 Discussion 203 Concluding Remarks 209 List of References 211 Chapter VI: The Astacin Family of Endopeptidases and Embryogenesis 220 Introduction 220 List of References 236 Chapter VII: Overexpression and Characterization of Recombinant Blastula Protease 10 from Paracentrotus lividus 237 Introduction 237 Experimental 241 Overexpression, Purification, and Refolding of Recombinant BP10 242 Circular Dichroism (CD) Studies 244 Preparation of the Copper Derivative of BP10 244 Gelatin Zymogram 245 Gelatinase Assay 245 iv Hydrolysis of BAPNA by Zn-BP10 and Cu-BP10 246 Calcium Activation Assays 247 Inhibition Studies 247 pH Profiles 247 Electronic Spectrum of Cu-BP10 247 Homology Modeling and Substrate Docking 247 Results and Discussion 248 Overexpression and Refolding of Recombinant BP10 248 CD Spectra of ZnBP10 and Cu BP10 251 Kinetics of Gelatin Hydrolysis 251 Kinetics of BAPNA Hydrolysis 253 Mechanistic Studies of the Copper Derivative of BP10 (Cu-BP10) 258 Homology Modeling 271 Concluding Remarks 271 List of References 274 About the Author End Page v LIST OF TABLES Table 1: Standard Redox Potentials for Dioxygen in H2O. 15 Table 2: Kinetic Parameters for Dopamine Oxidation by CuAβ. 158 Table 3: Kinetic Parameters for the Oxidation of Catecholamines to o-quinone by CuAβ1–20. 164 vi LIST OF FIGURES Figure 1.1: Electronic states and standard reduction potentials for O2. 16 Figure 1.2: Types of enzymes that activate O2. 31 Figure 1.3: Structure of CuO intermediates identified in biomimetic complexes. 35 Figure 1.4: Early models of Type-3 oxidases by Karlin and Tolman . 41 Figure 1.5: Type-3 model systems by Itoh and Stack. 47 Figure 2.1: Concentration and metal-dependent assay of dsDNA cleavage. 94 Figure 2.2: Time course reactivity assay of dsDNA cleavage. 97 Figure 2.3: Time course reactivity assay toward the cleavage of 150 ng dsDNA. 98 Figure 2.4: Effect of peroxide concentrations on the rate of THB oxidation in the presence of 7.5 µM of CuAβ1-20. 102 Figure 2.5: H2O2 saturation profile of CuAβ1-16 and CuAβ1-20. 104 Figure 2.6: Hanes analysis of the kinetic data. 109 Figure 2.7: Proposed mechanism for the oxidation of THB by CuAβ. 111 2+ Figure 2.8: Cu titration to Aβ1-20 and Aβ1-16 monitored with their 113 oxidation activities. Figure 2.9: Electronic spectra of CuAβ1-20. 116 1 Figure 2.10: H NMR spectra of Aβ1-20 and Aβ1-16 in DMSO. 119 Figure 2.11: Proposed metal coordination and solution structure of CuAβ1-16. 121 vii Figure 3.1: Electronic spectrum of the MBTH-o-quinone adduct. 136 Figure 3.2: Saturation kinetics with catechol, phenol and deuterated phenol. 138 2+ Figure 3.3: Effect of Zn on the oxidative activity of CuAβ1-20 toward DTC 141 and phenol. Figure 3.4: The effect of H2O2 on the first order rate constant kcat toward the oxidation of phenol and catechol. 142 Figure 3.5: Hanes plot analysis of kinetic data from Figure 3.4. 143 Figure 3.6: Proposed mechanism for aerobic hydroxylation and oxidation of phenol. 146 Figure 3.7: Optical titration of DCC to 0.2 mM CuAβ. 148 Figure 4.1: Oxidation of catechol and dopamine by 1.47 µM CuAβ1-40 in 100 mM HEPES at pH 7.0 and 25° C. 157 Figure 4.2: Catecholamine oxidation by 2.5 µM CuAβ1-20. 160 Figure 4.3: H2O2 effect on oxidation of catecholamines. 162 Figure 4.4: Effect of SDS on the oxidative activity of CuAβ1-16,CuAβ1-20, 166 and CuAβ1-40 Figure 4.5: Influence of NAD(P)+ and NAD+ on the oxidative activity of CuAβ1–20 toward dopamine oxidation. 168 Figure 4.6: Phosphate inhibition toward catechol oxidation by CuAβ1–20. 169 Figure 4.7: Optical titration of DCC into 0.05 mM CuAβ1–40. 171 2+ Figure 4.8: Activity titration of Cu into 2.5 µM Aβ1–40 under 171 saturating conditions of catechol. Figure 4.9: Mechanism for the oxidation of catecholamine neurotransmitters and the cause of neurodegeneration by CuAβ. 173 Figure 4.10: Aerobic oxidation of serotonin by 1.47 µM CuAβ1-40.