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Interfacial Electrochemistry of Cytochrome C and Myoglobin

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Interfacial Electrochemistry of Cytochrome C and Myoglobin Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 1982 Interfacial Electrochemistry of Cytochrome c and Myoglobin Edmond F. Bowden Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Chemistry Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/4371 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. COLLEGE OF HUMANITIES AND SCIENCES VIRGINIA COMMONWEALTH UNIVERSITY This is to certify that the dissertation prepared by Edmond F. Bowden entitled "Interfacial Electrochemistry of Cytochrome £.and Myoglobin" has been approved by his committee as satisfactory completion of the dissertation requirement for the degree of Doctor of Philosophy. School Dean Date INTERFACIAL ELECTROCHEMISTRY OF CYTOCHROME C AND MYOGLOBIN A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by Edmond F. Bowden Director: Dr. Fred M. Hawkridge Professor of Chemistry Virginia Commonwealth University Richmond, Virginia May, 1982 Virginia Commonwealth Um�Ubrary ii ACKNOWLEDGEMENTS I offer my sincere thanks to the following people: Fred M. Hawkridge, without whom the whole show would not have been possible. Fred's guidance, trust, and intense interest as well as his leadership by example have provided inspiration for this work and for my future aspirations. Ronald and Nettie Bowden, my parents, who have continued to pro­ vide guidance and emotional and material support for my endeavors. Their lasting impact on my education and my father's influence on the development of my scientific outlook are invaluable. Joyce Stargardt, Charlene Crawley, Jim Castner, Eddie Thomas, and Dave Cohen, who, as fellow graduate students and good friends, made possible the positive intellectual and personal group environment that I feel very fortunate and proud to have been a part of. Their know­ ledge, generosity, and interest were invaluable. Henry Blount, for putting his ideas, laboratory and home at my dis­ posal, and for his inspiration and interest. Eric Bancroft, for his invaluable assistance with digital simula­ tion and various spectroelectrochemical experiments. Jan Chlebowski, for focusing attention on sample integrity and for providing the facilities and guidance in the purification of cyto­ chrome� samples. Diane Holmes, for her excellent and proficient typing of this the­ sis and for her hard work in meeting my completion deadlines. Carlos Armengol, for encouraging me to try graduate level re­ search. ii i My family and friends for their persistent encouragement and sin­ cere interest. I would also like to acknowledge financial assistance from the Na­ tional Science Foundation, National Institutes of Health, and the E. I. duPont Co. iv TABLE OF CONTENTS page LIST OF TABLES . vii LIST OF FIGURES viii LIST OF SYMBOLS X ABSTRACT . XV I. INTRODUCTION .......................................... A. Overview . .. .. .. .. .. .. .. .. .. .. .. 1 8 1 . References . .. .. .. .. .. .. .. .. .. .. B. Mitochondrial Oxidative Phosphorylation ............ 9 14 1 . References . .. .. .. .. .. .. .. .. .. .. .. 15 C. Cytochrome c . .. .. .. .. .. .. .. .. .. .. .. 1 . References . .. .. .. .. .. .. .. .. .. .. 29 D. Myoglobin . .. .. .. .. .. .. .. .. .. .. 33 1 . References . .. .. .. .. .. .. .. .. .. .. .. 35 E. Electrodes, Membranes, and Aqueous Inter- faces . .. .. .. .. .. .. .. .. .. .. .. .. .. 36 1. Metal Electrodes . 36 2. Metal Oxide Semiconductor Electrodes . .......... 45 3. Biological Membranes .. ... .. .. .. .. .. .. 57 4. References . .. .. .. .. .. .. .. .. .. 64 F. Direct Electrochemical Reduction and Oxi- dation of Heme Proteins . ... .. .. ... .. ..... ... .. 67 1. Cytochrome c/Mercury Electrodes ................ 70 2. Cytochrome C/Solid Electrodes .................. 91 3. Other Cytochromes . .. .. .. .. .. .. .. .. 105 4. 115 Myoglobin and Hemoglobin ... .. .. .. .. .. .. .. 5. Peroxidase and Catalase ........................ 121 6. Other Protein/Electrode Studies ................ 123 7 127 . Genera 1 i zat ion . .. .. .. .. .. .. .. .. 8. References . .. .. .. .. .. .. .. .. .. .. 128 II. THEORY 135 A. Electrode Kinetics 135 B. Cyclic Voltammetry 142 v TABLE OF CONTENTS (continued) page C. Chronoabsorptometry .. .. .. .. .. .. .. .. .. 143 D. References . .. .. .. .. .. .. .. .. 145 III. EXPERIMENTAL DETAILS . .. .. .. .. .. .. .. 147 A. Reagents and Materials . .. .. .. .. .. .. .. 147 B. Instrumentation .. .. .. .. .. .. .. 152 C. Electrochemical and Spectroelectro- chemical Cells .... .. .... .... .... .... .. .... .. .... 156 1. Standard OTE Cell . .. .. .. .. .. .. .. 156 2. Double-Sided OTE Cell ................. ....... .. 156 3. Light pipe OTE Cell ............................ 161 4. Blanketed Volta0111etry Cell .... .. .. .. .. .. .. .. ... 164 D. Techniques and Procedures . .... .. .. ... .. .. ... .. 164 1. Single Potential Step Chronoab- sorptometry .. .. .. .. .. .. .. .. .. 164 2. Asymmetric Double Potential Step Chronoabsorptometry .... .. .. .. .. .. .. .. 172 3. Linear Sweep and Cyclic Voltam- metry .. .. .. .. .. .. .. .. .. .. .. .. 172 4. Derivative Cyclic Voltabsorpto- metry .. .. .. .. .. .. .. .. .. .. .. .. 173 5. Gold Electrode Modification .................... 173 6. Ag/AgCl Reference Electrode Prepa- ration and Calibration ....................... 174 7. Cytochrome c Chromatographic Puri­ fication-:-.................................... 174 E. References . .. .. .. .. .. .. .. .. .. 181 IV. RESULTS .. .. .. .. .. .. .. .. .. .. .. .. 185 A. Cytochrome c (Purified) .. .. .. .. .. .. .. .. .. 185 - 1. Effects of Sample Purification ................. 185 2. Cyclic Voltan111etry and DCVA of Puri­ fied Cytochrome c at Indium Oxide OTEs ........... -:-. .. .. .. .. .. .. .. .. .. 188 3. Film Transfer/Cyclic Voltammetry of Purified Cytochrome c at Indium Oxide OTEs ......... -:-......................... 195 4. Cyclic Voltammetry of Purified Cyto- chrome cat Tin Oxide OTEs ................... 198 vi TABLE OF CONTENTS (continued) page 5. Cyclic Voltammetry of Purified Cy­ tochrome� at Au and Pt Electrodes 198 B. Cytochrome c (Commercial) ............................ 206 1. Reductive SPS/CA Kinetics of Commer- cial Cytochrome cat Tin Oxide OTEs ............ 206 2. Oxidative SPS/CA and ADPS/CA Kinetics of Commercial Cytochrome c at Tin Oxide OTEs ...... ........-:- ...................... 212 3. Cyclic Voltammetry of Commercial Cy- tochrome c at Indium Oxide and Tin Oxide Electrodes .. .. .. .. .. .. .. 220 C. Myoglobin . .. .. .. .. .. .. .. .. .. 225 1. Reductive SPS/CA Kinetics at Tin Ox- ide OTEs . .. .. .. .. .. .. .. .. .. 225 2. Reductive SPS/CA Kinetics at Methyl Viologen Modified Gold Electrodes ..... ......... 226 D. References .. .. .. .. .. .. .. .. .. .. 233 V. DISCUSSION . .. .. .. .. .. .. .. .. .. .. .. .. 234 A. Importance of Sample Purification .. .................. 234 B. Cytochrome� (Purified) . .. .. .. .. .. .. .. 235 C. Cytochrome � (Corrmerc i a 1 ) .. .. .. .. .. .. .. 245 D. Myoglobin . .. .. .. .. .. .. .. .. .. .. 248 E. Concluding Remarks .. .. .. .. .. .. .. .. .. 250 F. References 255 APPENDICES . .. .. .. .. .. .. .. .. .. .. .. .. .. .. 257 I. Methyl Viologen/Surface Hydrogen Reactions ..... ..... 257 II. Phosphate Buffers . .. .. .. .. .. .. .. .. 258 III. Tris/Cacodylate Buffers ...... .... .................... 259 IV. Gold Electrode Surface Area Determinations ..... ...... 260 VITA .. .. .. .. .. .. .. .. .. .. .. .. 262 vii LIST OF TABLES Table page 1. Literature Summary of Hemeprotein Elec- trode Reactions .. .. .. .. .. .. .. .. .. .. 71 2. Chromatographic Bands of Cytochrome£ . ............... 183 3. Heterogeneous Electron Transfer Kinetic Parameters for Purified Cytochrome c at ............ 191 Tin Doped Indium Oxide OTE's ......� ..... 4. Heterogeneous Electron Transfer Kinetic Parameters for Purified Cytochrome c at Fluorine Doped Tin Oxide OTE's ....--:- .................. 199 5. Heterogeneous Electron Transfer Kinetic Parameters for Purified Cytochrome c at Gold and Platinum Electrodes ......� ........... .... 203 6. Individual Heterogeneous Electron Trans­ fer Rate Constants for the Reduction of Commercial Cytochrome c at a Fluorine Doped Tin Oxide OTE ..--:- ............................... 209 7. Reductive Heterogeneous Electron Trans­ fer Kinetic Parameters for Commercial 213 Cytochrome£ at Tin Oxide OTE's ......... .. ......... 8. Oxidative Heterogeneous Electron Trans­ fer Kinetic Parameters for Commercial ...... 21G .. ...... .. Cytochrome£ at Tin Oxide OTE's . 9. Reductive Heterogeneous Electron Trans­ fer Kinetic Parameters for �1yoglobin at Methyl Viologen Modified Gold Electrodes ............. 227 viii LIST OF FIGURES Figure page 1. The mitochondrion .. .. .. .. .. .. .. .. .. .. .. .. .. 11 2. Hemes b and c . .. .. .. .. .. .. .. .. .. .. .. .. .. .. 17 - - 3. Cytochrome£ molecular structure . .... .......... 21 4. Electrode/solution double layer struc- ture .. .. .. .. .. .. .. .. .. .. .. .. .. .. 38 5. Electrode/solution potential-distance profi 1es . .. .. .. .. .. .. .. .. .. .. .. 41 6. Semiconductor band model . .. .. .. .. .. .. .. 48 7. Silica hydration .. ... .. .. .. .. .. .. .. .. .. 56 8. Membrane mode 1s . .. .. .. .. .. .. .. .. .. .. .. .. 59 9. Electron transfer
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