Studies of the Thermodynamics of Some Native and Ruthenated Metalloproteins by Spectroelectrochemistry Citation Chiang, Huey-jenn (1987) Studies of the Thermodynamics of Some Native and Ruthenated Metalloproteins by Spectroelectrochemistry. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8fcf-kt72. https://resolver.caltech.edu/CaltechTHESIS:10312019-170145504 Abstract The thermodynamic parameters of redox centers in some metalloproteins have been investigated using spectroelectrochemical techniques, with the employment of an OTTLE (Optically Transparent Thin-Layer Electrode) cell. With the aid of various carefully selected mediators, the temperature dependence of the formal redox potentials of type I copper (blue copper) ions in Rhus vernicifera (tree) laccase, as well as in native and in pentaammineruthenium-modified azurins from Pseudomonas aeruginosa , have been measured. Similar experiments have been carried out for the heme site of cis -[Ru(en) 2 (OH)(His)]-horse heart cytochrome c (en: ethylenediamine; His: histidine). Cyclic voltammetry has been used to study the pH-dependence of the formal redox potentials of the appended ruthenium ion in cis -[Ru(en) 2 (OH)(His)]-horse heart cytochrome c . Finally, the synthesis and spectroelectrochemistry of sperm whale myoglobin reconstituted with [Ru(MpIX)(DMSO) dicarboxylic acid]- moiety (MpIX: mesoporphyrin IX; DMSO: dimethyl sulfoxide) have also been carried out. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: Chemistry Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Gray, Harry B. Thesis Committee: Gray, Harry B. (chair) Collins, Terrence J. Hopfield, John J. Marcus, Rudolph A. Defense Date: 4 May 1987 Record Number: CaltechTHESIS:10312019-170145504 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:10312019-170145504 DOI: 10.7907/8fcf-kt72 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 11878 Collection: CaltechTHESIS Deposited By: Mel Ray Deposited On: 01 Nov 2019 00:25 Last Modified: 16 Apr 2021 23:05 Thesis Files Preview PDF - Final Version See Usage Policy. 49MB Repository Staff Only: item control page

STUDIES OF THE THERMODYNAMICS OF SOME NATIVE AND RUTHENATED MET ALLOPROTEINS BY SPECTROELECTROCHEMISTRY Thesis by Huey-jenn Chiang In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 1987 (Submitted May 4, 1987) 11 DEDICATED TO MY HERO, COMMANDER, AND FATHER MR. TAO CHIANG A GIANT WHO KEEPS HIMSELF GOING ON FOR SUCCESS WITH ONLY LIMITED LUCK AND RESOURCES 111 ACKNOWLEDGEMENTS My years at Caltech as a graduate student are the most cherished of . my student career. I am glad to have had my prime time of learn- ing this wonderful appreciation to those would have in concert express my spiration, it with not during my years here. experience. people, been I would like to whose help and in- me make progress without possible for to Forgive me for being unable to pay my grati- tude to each one of them by name. First of deserves all, my deepest and the financial His great Professor B. my appreciate I gratitude. Gray, support he has provided for personality matter what and Harry will where I continue will to research the advisor. scientific He guidance me during these years. affect my future be, he will always be the life. No hero image that I shall try to follow. Many Hopfield at for Caltech. Ph.D. thanks to Professor Terry J. their I guidance appreciate committee. and their help Collins and Professor John J. throughout excellent service my graduate studies as members of my Marcus for I also want to thank Professor Rudy A. being a committee member for my final oral examination on my hasty invitation. I want to thank Dr. Vernon T. Taniguchi for leading me into the field of throughout spectroelectrochemistry. the whole thesis. la bora tor, I would To Dr. His contribution Chi-Ming like to say thank you for Che, my can be felt longtime col- everything he has done IV for me. I would Robert J. also like to Crutchley. I have thank Professors Nenad M enjoyed having Kostic and the opportunity to coop- erate with them on projects involving azurin and with ruthenated myoglobin, respectively. for assistance his I in would the also field like of to thank Dr. electrochemistry. Hsue-Yang Liu My also thanks go to Ms. Ting-Lin Kao for her help in the laboratory, I wish her the best of luck in her future career. I would like to thank two of my chemistry teachers at Tamkang University. Professor Kee-Chuan Pan was the key person who educated me to be a still cian I have never to scientist. He ever Professor taken any the United met. classes States to is from the best Bor-Her physicist Chen, him officially, study. To these and mathemati- even though encouraged me people, I shall I to have travel always be grateful. In those Worley for years at Auburn being my research done for me. University, I thank advisor. Professor S. Davis I appreciate everything he has I learned a lot, and owe a lot to Professors Curtis H. Ward and Philip B. Shevlin. They treated me as one of their own stu- dents, and without their recommendations I do not think I would have been able to go one step further. The enthusiasm and cordiality of Dr. Arthur Er-terg Chiou set a standard for tolerance during the thanks me for to follow. I appreciate his friendship, guidance and also deserve Drs. Yu-Min these years. Mr. and Mrs. Tony Chang their warm friendship and hospitality. Tsou and Y. Henry Sun are the best friends I could possibly have. I v have also enjoyed many conversations with Professors Chao-Ying Meng and Yuk Ling Yung and Dr. Mary Zi-ping Luo; I appreciate the intellectual characteristics of our friendship. Mr. guy has warmed me up for twenty five years. Chih-herng Yin, the The only feeling I have is to keep it going forever and enjoy. Professor Ambrose Yaw-Shong Jong and Mr. Chien-Tsung Mickey Hu are my tutors on educating me to Jong, even designed he molecular be a a biology. I appreciate biological scientist. laboratory course their enthusiasm in Especially solely for Professor me without getting any credit and, of course, without pay. I appreciate the companionship of all the present and bers of Professor R. Winkler, Gray's Walther R. group. I want to thank Ellis, Jr., Michael D. Lieber, Michael Albin, and Mr. past mem- especially Hopkins, Drs. Jay Charlie M Tad L. S. Fox for their understanding and help. Dr. Jimmy A. Cowan deserves my special thanks for proofreader of my thesis. I can certainly think of how being painful the job is. I appreciate his enthusiasm and patience. Professor Aron Kuppermann also deserves my special thanks. I am sorry that I was not able to keep up my interest of being a theoretician while I was a member in his group. I will always appreciate his understanding and support. I would like to dedicate my limited success to my families, father Tao Chiang, my mother Chu-ying Cheng, my father-in-law kuo Ming, my mother-in-law feng, and Huey-ying, my my Chien- Yueh-er Ting, my sisters Huey-fang, Hueyelder cousin Shih-hsiung Cheng, for every- vi thing they have ever done for me. thesis to the memory of my it still hurts me so deeply. been with me stage director, since I supporter, I would also like to dedicate this younger brother Huey-lin. Sixteen years, I thank my wife Ming Feng-ying who has arrived and at Caltech. cheerleader ies; she deserves half of the credit for this thesis. For being throughout my my producer, Ph.D. stud- Vll ABSTRACT The thermodynamic talloproteins have been techniques, with Thin-Layer Electrode) ted tials the mediators, the of laccase, type as azurins from periments of redox investigated using employment of cell. the With temperature I well parameters an copper) as native and in Pseudomonas aeruginosa, various in Rhus have been [R u(en) 2 (0H)(His)]-horse heart cytochrome c (en: His: voltammetry ion in redox been potentials of cis-[Ru(en) 2 (0H)(His)]-horse heart synthesis and of stituted with [Ru(MpiX)(DMSO) spectroelectrochemistry poten- vernicifera (tree) of heme formal redox Similar ex- the dependence of the selec- measured. for has Transparent pentaammineruthenium-modified out Cyclic me- carefully carried histidine). been ions some (Optically dependence of the formal (blue have of in spectroelectrochemical OTTLE aid copper in centers used study the appended whale dicarboxylic acid]- cis- ethylenediamine; to cytochrome sperm site c. the ruthenium Finally, myoglobin moiety pH- (MpiX: oporphyrin IX; DMSO: dimethyl sulfoxide) have also been carried out. the reconmes- viii TABLE OF CONTENTS A cknow led gemen ts Ill Abstract Vll Table of Contents viii List of Figures X List of Tables xviii List of Schemes xxiii Chapter 1: Introduction Overview 2 Scope of the Thesis 26 References 28 Chapter II: Spectroelectrochemistry and the OTTLE Cell 32 Introduction 33 Spectroelectrochemistry 34 OTTLE Cells 37 Fabrication of OTTLE Cells 39 Spectroelectrochemistry of Biological Molecules 44 Nonisothermal Configuration of the Variable Temperature OTTLE Cells References 50 57 Chapter III: Thermodynamic Studies of Type-1 Copper in Tree Laccase 61 Introduction 62 Materials and Methods 79 ix Results and Discussion 83 References 114 Chapter IV: Redox Chemistry of Native, Singly, and Doubly Pentaammineruthenium-Modified Bacterial Azurin 118 Introduction 119 Materials and Methods 138 Results and Discussion 149 References 206 Chapter V: Ruthenium-Modified Proteins. Reaction of cis-[Ru(NH 3) 4 (0H 2)2]2+ and cis-[Ru(en) 2 (0H 2)2]2+ with Azurin, Myoglobin, and Cytochrome c 209 Introduction 210 Materials and Methods 211 Results and Discussion 215 References 241 Chapter VI: Synthesis and Spectroelectrochemistry of [R u(MpiX)(DMSO) dicar box ylic acid]- Reconstituted Myoglobin 244 Introduction 245 Materials and Methods 255 Results and Discussion 274 References 299 X LIST OF FIGURES Chapter I Figure I. The electron transport chain in mitochondrial oxidative phosphorylation 3 Figure 2. The electron transport chain in photosynthetic bacteria 5 Figure 3. Bimolecular electron transfer reactions between metalloproteins and small inorganic complexes 7 Figure 4. Semi-synthetic metalloproteins containing two redox centers with fixed and known distances 10 Figure 5. X-ray crystallographic structure of horse heart cytochrome c 13 Figure 6. Structural model for selected parts of [Ru(NH 3 ) 5 (His-33)]-tuna ferricytochrome c 15 Figure 7. Mechanisms of through-bond and through-space intramolecular electron transfer reactions 17 Figure 8. Intramolecular electron transfer pathway of pen taammineru theni um(His-33 )-f erricytochrome c 20 Figure 9. Optical density changes in flash photolysis of PFeiii /[Ru(bpy) 3]2+/EDTA and PFe 111 -Ru III/ [Ru(bpy) 3]2 +/EDT A solutions 22 Figure 10. Temperature dependence of the rate constant for the intramolecular electron transfer reaction in ruthenated cytochrome c 24 XI Chapter II. Figure I. Block diagram representation of spectroelectrochemical techniques 35 Figure 2. Dimension specifications for the OTTLE cell body 40 Figure 3. OTTLE cell assembly 45 Figure 4. Mediator-titrant mediates electron exchange between electrode and protein via an outer-sphere electron transfer mechanism 48 Figure 5. Isothermal and nonisothermal configurations for electrochemical cells 53 Chapter III Figure I. Structure of oxidized poplar plastocyanin 71 Figure 2. The copper-binding site of oxidized poplar plastocyanin 73 Figrue 3. Schemes of reduction and reoxidation mechanisms of laccase Figure 4. UV-VIS absorption spectrum of tree laccase 77 81 Figure 5. UV-VIS overlay spectra of [Fe(CN) 6] 3- and of [Fe(CN) 6 ]4 - 85 Figure 6. Thin-layer spectroelectrochemistry of native tree laccase 87 Figure 7. N ernst plot of spectroelectrochemical titration of type-1 copper center in tree laccase Figure 8. Temperature dependence of the formal reduction 89 xii potential for the type-1 copper center in tree laccase 95 Figure 9. Temperature dependence of the Gibbs free energy change of tree laccase 98 Figure 10. Temperature dependence of the formal reduction potentials for the type-1 copper center in fungal laccase 108 Figure 11. Temperature dependence of the formal reduction potentials for the type-1 copper centers in tree and in fungal laccases 110 Chapter IV Figure I. The folding of the polypeptide backbone in azurin 121 Figure 2. Absorption spectra of the hexapeptide containing His-83 and aqueous solution of [Ru(NH 3) 5 (His)]Cl 3 128 Figure 3. View of selected parts of the molecular skeleton of [a 5 Ru(His-83)]-azurin 130 Figure 4. (A) First-order kinetic plot for the reduction of Cu 11 in flash-generated [a 5 Ru 11 (His-83)]-Az(Cu II) at 23 °C, and (B) temperature dependence of the rate constant for long-range Run~ Cull electron transfer Figure 5. The pH-dependence of the rate constant for 139 Xlll long-range intramolecular electron transfer reaction in [a 5 Ru 11 (His-83)]-Az(Cu 11 ) at 23 °C 141 Figure 6. The pH-dependence of the rate constant for long-range intramolecular electron transfer reaction in (a 5 Ru 11 )2 -Az(Cu II) at 23 °C 143 Figure 7. Temperature dependence of the formal reduction potential for the type-1 copper center in Pseudomonas aeruginosa azurin at pH 7.0 155 Figure 8. Temperature dependence of the formal reduction potential for the type-1 copper center in Pseudomonas aeruginosa azurin at pH 6.0 159 Figure 9. Temperature dependence of the formal reduction potential, £ 0 ' , for the type- I copper center in Pseudomonas aeruginosa azurin at pH 5.0 163 Figure 10. Temperature dependence of the formal reduction potential for the type- I copper center in Pseudomonas aeruginosa azurin at pH 7.0, at pH 6.0, and at pH 5.0 165 Figure 11. Temperature dependence of the formal reduction potential for the type-1 copper center in [Ru(NH 3) 5 (His-83)]-azurin from Pseudomonas aeruginosa at pH 7.0 Figure 12. Temperature dependence of the formal reduction potential for the type-1 copper center in [Ru(NH 3) 5 (His-83)]-azurin from Pseudomonas 171 xiv aeruginosa at pH 5.0 175 Figure 13. Temperature dependence of the formal reduction potential for the type-1 copper center in [Ru(NH 3 ) 5(His-83)]-azurin from Pseudomonas aeruginosa at pH 7.0 and at pH 5.0 177 Figure 14. Temperature dependence of the formal reduction potential for the type-1 copper center in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurin at pH 7.0 183 Figure 15. Temperature dependence of the formal reduction potential for the type-1 copper center in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurin at pH 5.0 187 Figure 16. Temperature dependence of the formal reduction potential for the type-1 copper center in doubly pen taammineru therni urn modified Pseudomonas aeruginosa azurin at pH 7.0 and at pH 5.0 189 Figure 17. Temperature dependence of the formal reduction potential for the type-1 copper center in native, in [Ru(NH 3) 5(His-83)]-modified, and in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurins at pH 7.0 Figure 18. Temperature dependence of the formal reduction potential for the type-1 copper center in native, in [Ru(NH 3 )5(His-83)]-modified, and 194 XV in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurins at pH 5.0 198 Figure 19. The pH-dependence of the formal reduction potential for the blue-copper center in native, in [Ru(NH 3) 5(His-83)]-modified, and in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurins at pH. 203 Chapter V Figure I. Proton NMR spectra of horse heart cytochrome c and cis-[Ru(en) 2 (0H)(His-33)]-horse heart cytochrome c in the region of imidazole 1H resonances 218 Figure 2. UV-VIS absortion spectra of cis-[Ru(en) 2 (0H) (His-83)]-azurin from Pseudomonas aeruginosa and cis-[Ru(NH 3 )4 (0H)(His-83)]-azurin from Pseudomonas aeruginosa 221 Figure 3. Thin-layer spectroelectrochemistry of cis-[Ru (en) 2 (0H)(His -33)]-horse heart cytochrome c 224 Figure 4. Nernst plot of spectroelectrochemical titration of the iron center in cis-[Ru(en) 2 (0H)(His-33)] -horse heart cytochrome c 226 Figure 5. Temperature dependence of the formal reduction potentials for the heme center in cis-[Ru(en) 2 (OH)(His-33)]-horse heart cytochrome c Figure 6. Temperature dependence of the formal reduction 230 xvi potential for the heme center in native horse heart cytochrome c 234 Figure 7. Temperature dependence of the formal reduction potential for the heme centers in native and in cis-[Ru(en) 2(0H)(His-33)]-modified horse heart cytochrome c 236 Chapter VI Figure I. Structure of iron protoporphyrin IX 246 Figure 2. Structure of the reduced tuna cytochrome c 248 Figure 3. Structure of oxidized sperm whale myoglobin 251 Figure 4. The amino acid sequence of sperm whale myoglobin 253 Figure 5. Structure of ruthenium mesoporphyrin IX 260 Figure 6. Absorption spectra of ruthenium(II)(CO) mesoporphyrin IX dicarboxylic acid 263 Figure 7. The overlay spectra of Ru 11 (MpiX)(CO) dicarboxylic acid as a function of photolysis time 265 Figure 8. The absorption spectrum of [Ru 11 (MpiX)(DMSO) dicarboxylic acid]-reconstituted sperm whale myoglobin Figure 9. The absorption spectrum of K 4 [W(CN) 8]•2H 2 o 270 272 Figure 10. Thin-layer spectroelcctrochemistry of [Ru(MpiX)(DMSO) dicarboxylic acid]-reconstituted sperm whale myoglobin Figure 11. Nernst plot of spectroelectrochemical titration 275 XVll of ruthenium center in [Ru(MpiX)(DMSO) dicarboxylic acid]-reconstituted sperm whale myoglobin 278 Figure 12. Temperature dependence of the formal reduction potentials for [Ru(MpiX)(DMSO) dicarboxylic acid]-reconstituted sperm whale myoglobin 282 Figure 13. Reaction entropies of redox couples for selected transition metal complexes as well as native and modified metalloproteins 285 Figure 14. Temperature dependence of the formal reduction potentials for native sperm whale myoglobin 290 Figure 15. Temperature dependence of the formal reduction potentials for the sperm whale myoglobin and for the [Ru(MplX)(DMSO) dicarboxylic acid]reconstituted sperm whale myoglobin 292 XVlll LIST OF TABLES CHAPTER III Table I. Some blue and non-blue copper-containing proteins together with the types of copper ions present and their functions 63 Table 2. AI tern a ti ve classification for copper proteins 69 Table 3. Formal reduction potentials for the blue copper center in tree laccase 91 Table 4. Temperature dependence of the formal reduction potentials for the blue copper ion in tree laccase Table 5. Thermodynamic parameters for metalloproteins 93 101 Table 6. Comparison of spectral, formal reduction potential, and other thermodynamic properties of some blue 104 copper proteins Table 7. Temperature dependence of the formal reduction potentials for the blue copper ion in fungal 106 laccase Chapter IV Table I. Thermodynamic parameters for the reduction of [a 5 Ru(His)] 3+ and the blue-copper center in native and pentaammincruthenium modified azurins Table 2. Spectroscopic properties of the blue-copper sites in native and pentaammineruthenium modified 132 xix azurins 134 Table 3. Temperature dependence of the formal reduction potentials for the blue-copper ion in Pseudomonas aeruginosa azurin at pH 7.0 153 Table 4. Temperature dependence of the formal reduction potentials for the blue-copper ion in Pseudomonas aeruginosa azurin at pH 6.0 157 Table 5. Temperature dependence of the formal reduction potentials for the blue-copper ion in Pseudomonas aeruginosa azurin at pH 5.0 161 Table 6. Thermodynamic parameters of the type-1 blue-copper center in native Pseudomonas aeruginosa azurin at pH 7.0, at pH 6.0, and at pH 5.0 167 Table 7. Temperature dependence of the formal reduction potentials for the blue-copper ion in [Ru(NH 3) 5 (His-83)]-azurin from Pseudomonas aeruginosa at pH 7.0 169 Table 8. Temperature dependence of the formal reduction potentials for the blue-copper ion in [Ru(NH 3) 5 (His-83)]-azurin from Pseudomonas aeruginosa at pH 5.0 173 Table 9. Thermodynamic parameters of the type-1 blue-copper center in [Ru(NH 3 )5 (His-83)]-azurin from Pseudomonas aeruginosa at pH 7.0 and at pH 5.0 Table 10. Temperature dependence of the formal reduction 179 XX potentials for the blue-copper ion in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurin at pH 7.0 181 Table 11. Temperature dependence of the formal reduction potentials for the blue-copper ion in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurin at pH 5.0 185 Table 12. Thermodynamic parameters of the type-1 blue-copper center in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurin at pH 7.0 and at pH 5.0 191 Table 13. Thermodynamic parameters of the type-1 blue-copper centers in native, in [R u(NH 3 ) 5 (His-83 )]-modified, and in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurins at pH 7.0 196 Table 14. Thermodynamic parameters of the type-1 blue-copper centers in native, in [Ru(NH 3 ) 5 (His-83)]-modified, and in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurins at pH 5.0 200 Chapter V Table 1. Preparation and characterization of ruthenated proteins Table 2. Temperature dependence of the formal reduction potentials for the heme center in cis-[Ru(en) 2 216 XXI (OH)(His-33)]-horse heart cytochrome c 228 Table 3. Temperature dependence of the formal reduction potentials for the heme center in native horse heart cytochrome c 232 Table 4. Thermodynamic parameters of the heme centers in native and in cis-[Ru(en) 2 (0H)(His-33)]-modified horse heart cytochrome c 238 Chapter VI Table 1. Thermodynamic parameters of the heme centers in native and in (NH 3 )5 Ru 3 +/ 2+ modified horse heart cytochrome c as well as in native and (NH 3 ) 5Ru 3+/ 2 + modified sperm whale myoglobins 256 Table 2. Intramolecular long-range electron transfer kinetics in (NH 3 ) 5 Ru 3+/ 2+ modified horse heart cytochrome c and sperm whale myoglobin 258 Table 3. Temperature dependence of the formal reduction potentials for [Ru(MpiX)(DMSO) dicarboxylic acid] -reconstituted sperm whale myoglobin 280 Table 4. Temperatur dependence of the formal reduction potentials for the heme center in native sperm whale mypglobin Table 5. Thermodynamic parameters of iron centers in native and in [Ru(NH 3) 5(His-48)]-modified sperm whale myoglobins, as well as of ruthenium center 288 xxii in [Ru(MpiX)(DMSO) dicarboxylic acid]-reconstituted sperm whale myoglobin Table 6. Heme geometry in myoglobin derivatives 294 296 XXlll LIST OF SCHEMES CHAPTER IV Scheme 1. Preparation and purification scheme of pentaammineruthenium modified azurins 124 Scheme 2. Peptide mapping of [Ruii 1(NH 3) 5(His-83)]Az(Cuii) 126 Scheme 3. The scheme of flash kinetic spectrometry used in the study of the intramolecular long-range electron transfer reaction in [a 5Ru 111 (His-83)]Az(Cull) 136 CHAPTER I INTRODUCTION 2 Overview electron transfer studies l-l 2 of considerable attention in years. Long-range have drawn basis for an understanding photosynthesis 15 •16 , which are of oxidative the most involved in biological energetics. 17 tion as a series consecutive pairs of proteins. ly, so in I These the phosphorylation 13 • 14 and Electron metabolic transport transfer processes chains func- reactions between couple efficient- evolved energy in the electron and 2 18 depict a simplified view of the flow respectively. The most out- though they often they conserve and in photosynthesis, standing characteristic in these involve relatively redox forms have mitochondria the This important electron molecules proteins that Figures of recent biological large centers, the processes A or say 10 reaction are surprisingly rates of redox are many that transfer distances 19 •20 ' cause it is so unique in terms chemistry, is to scientists what is known interested m of electrons more, about events. between fast. 21 Be- conventional understanding the factors that control the rates of long-range electron transfers. Biological ent ways: siding in between been electron either transfer reactions can intermolecular electron transfer two protein distinct sites numerous actions between es. 22 - 33 A molecules, within · a investigations profile single-site of these of or single occur in two between intramolecular electron differ- sites re- transfer protein molecule. There have intermolecular electron transfer re- metalloproteins and experiments is shown inorganic complex- in 3, Figure how- 3 Figure 1. The electron transport phosphorylation. The chain in mitochondrial diagram shows the oxidative last stages in the conversion of sugars to C0 2 and H 2 0; the transfer of electrons (and hydrogen ions) to oxygen to form water. course of this across the membrane, ATPase process and complex, generating several these protons can molecules are then of transferred return ATP, as an energy source for other biochemical processes. In the via which the serve 4 Redox potential NADH' / NAD"'" -320 mV ADP Dehydrogenase (Flavoprotein complex) ~------~r-------~ -20 mV / ' ~ ATP Succinate'/ Fumarate Succinic dehydrogenase (Flavoprotein complex) +250 mV +600 mV 5 Figure 2. The electron transport chain in photosynthetic The diagram shows first stage in sunlight to pheophytin the chemical molecules, energy. together with the bacterial reaction center found of these bacteria. One molecule formed when protons, during the light-induced conversion of The chlorophyll and some others, up make in the plasma membrane of transferred return via the A TPase complex. the bacteria. electron A TP per photon across the membrane transfer reactions, IS 6 Redox potential -1000 mV -500 mV -tOO mV +500 mV Bacterio-chlorophyll complex 7 Figure 3. Bimolecular electron transfer reactions between talloproteins and inorganic complexes. The small es with hydrophobic ligands can ior of the protein structure, native possible distance However, both types for of penetrate intermolecular complexes into seeking complexthe the electron exchange me- intershortest transfer. electrons proteins via outer-sphere electron transfer mechanism. with e'fo+ ~+ hydrophobic ML-+ or (0 I+ M.. ~ 2. k12 (hydrophilic· ML+) decreases rapidly as R increases I. k12 (hydrophobic ML+) >k 12 Ctrtck~ilic M..+) hydrophilic ML+ + ML+ kl2 Metolloprotein Electron Trmsfer Rates 00 9 ever, the rate cases contained electrostatic constants of intermolecular electron bimolecular contributions from effects in addition transfer diffusion to the unimolecular been recognized as the in these rates and contributions of intramolecular electron transfer. The following factors have key parameters governing the electron transfer reactions: ( 1) Donor-to-acceptor distance. 3 (2) Thermodynamic driving force the between the difference for reduction i.e., reaction, potentials of the acceptor and donor. 34 •35 changes Geometric (3) redox of i.e., centers, inner sphere reorganization of donor and acceptor. Relative (4) orientation of the involved orbitals between the acceptor and donor sites. 36-38 In (5) Nature of the intervening medium. 39 - 41 ( 6) Solvation. 42 - 44 order to explore the transfer unimolecular electron designed in which the between two fused redox processes, in experiments long-range have centers, been reaction in a modified metalloprotein, illustrates the idea a Due to current transfer metalloprotein. tations, most of the tance, driving studies have concentrated and factors transfer electron transfer kinetics. these electron Figure force, of intramolecular observed. 45 - 55 4 effects geometric on of is semi-synthetic experimental limi- the dis- reorganization effects on the of electron 10 Figure 4. Semi-synthetic centers with molecular cally modifying and reduced form. two redox known distances. The intra- transfer proteins are by chemi- the with complexes containing and fixed electron talloproteins metal metalloproteins are surface residues transition metal substition inert made of single complexes. m both the site meThese oxidized II z Cl) .:.:. ~ z ~ ~ 6 0 w -LXL ·(/) 0: 12 Distance data between donors and acceptors are extracted from Xray crystallographic structural states and differences remains solvated although the debate between molecules m the With unresolved. state are edge-to-edge distances between electron transfer experiment performed distance estimation, demonstrate the results native horse heart nium-(histidine 33) electrochemical studies, tein's conformation native modification. to the of resolution, the However, is this on argument solid can centers for the Figures 5 and 6 the crystallographic for pentaammineruthe- Based on spectroscopic may change and the pro- perturbed 56 by other due that in modifying complexes used. structural the we significantly not solvated identical, overwhelming is the c, cytochrome evidence hydrophobicity of the the based and both redox solution. c. 49 •54 cytochrome that structurally in concerning solid assumption proteins the of the the estimate from results, cases At this level changes due to different provide details of the oxidation states are inevi ta bl y ignored. Crystallography can also relative tation and intervening medium between donors and acceptors. of these tremely factors m interesting. biological There through-bond/through-space coupling source information such as formal trochemistry. are potentials transfer processes is ex- several important issues, such as or Thermodynamic (Figure 7) and related to them. forces of closely pertaining to free The role electron mechanisms which electronic of are orien- driving energy parameters, changes, such as donor-acceptor The redox comes changes main centers, from of elec- enthalpy 13 Figure 5. X-ray crystallographic c, in which residues are acid the of non-heme-bound highlighted. side chain, to rentheses. structure The horse heart cytochrome histidine and methionine distance from each amino the closest heme edge, is given in pa- 14 . 26 (II , . -Hts a) 15 Figure 6. Structural model for selected parts of c, based on (histidine- 33 )]-fer ricytochrome for the tuna that the imidazole of membered ring of Trp-33 ture. protein. In this His-33 view is in the it has coincident tuna coordinates been with assumed the five- cytochrome c struc- 17 Figure 7. This figure depicts line connecting the His-18 idea and of His-33) shortest distance between His-18 intramolecular electron pentaammineruthenium c. through-bond and and (histidine-33)-horse curved through-space His-33) transfer (the (the mechanisms of reactions heart in cytochrome 18 > 00 Q 0 '<n I ~ ot, )( <.P <D oc::x: C\J •. -.§: (\Jf(') -a:: ::s I I </) ~ ~ )( C\J 19 and vs. potential the can entropy, entropy, be calculated temperature. are very based These important on the relationship thermodynamic in terms of of formal data, in particular obtaining a mechanistic understanding of electron transfer processes. Kinetic studies can be carried out in one of two different ways, each using method, a different flash photolysis, which is generated strong oxidizing tron distribution relaxation time of radiolysis, 57 by or agent. Again, the electron density between reasonable stants The the rate for 10) monitored 9). The second short pulse (- of the The electron comparison solvated 30 electrons of the redox this created obtained by valuable species species as a state of elec- (Figure 8). The in method, called pulse of electron beam ps) is first spectroscopically then centers The excited an acting as nonequilibrium temperature give system. nonequilibrium then constants can a is relaxation agreement. (Figure parameters the photons, agent, Using centers is (Figure of time. light. of the electronically redox state instead of pulse a a perturbing an among the employs for involves reducing this domain method reducing distribution followed these a two as a a of function methods are in dependence of the rate information on the activation the vital transfer, and can often experiments and theories. be Structural con- link in reorganiza- tion can also be understood on the basis of thermodynamic and kinetic studies. Hopefully, reactions will a clear picture be revealed by of protein-mediated systematic studies of electron transfer experiments using 20 Figure 8. The illustration shows fer pathway of pen taammineru theni urn ferricytochrome c. Here, Ru 3+/ 2 + for [Ru(NH 3)5(His-33)] 3+/ 2+, for stands [Ru(bpy) 3]2+*, free the intramolecular the surface Ru 2+ stands for free Ru 3 + stands for [Ru(bpy) 3 ]3+, respectively. electron trans- (histidine-33)histidine Ru 2+* bound stands [Ru(bpy) 3 ]2+ and 21 .1 22 Figure 9. Traces of the change in 550 from flash photolysis of (A) PFeiii /(Ru(bpy) 3]2+ /EDT A and (B) PFe 1Il-Ru III /(Ru(bpy) 3 ]2+/EDT A The vertical detector, and the been flash pulse. normalized rate constants. axis = refers 0 on The to nm to optical solutions reflect the of resulting at 22 °C. intensity at the axis coincides with light the horizontal intensities density the two differences traces in have quenching 23 0 ~ C/') o E co .: 0 v - cwu oss) ·a ·o 24 Figure 10. Temperature dependence of the rate constant for molecular electron transfer PFeii-Ru III at pH 7, 1.1 = inset is a duplication of Figure 9. PFe 111 -Ru II ~ phosphate buffer. The reaction 100 mM, the intra- 25 u 0 26 various combinations of proteins and complexes. strategies New also needed, in order to settle questions that are beyond are current ex- perimental capabilities. Scope of the Thesis The material contained in this thesis is confined to the thermodynamic ically aspects of electron transfer modified by ruthenium complexes, which techniques electrochemical such metalloproteins, or been studied have cyclic as native chemby voltammetry and electrochemical tech- Some details spectroelectrochemistry. Chapter niques of for II summarizes determining of an fundamentals redox formal have also spectroelectrochemistry procedures the OTTLE (Optically of potentials. been of the The fabrication Thin-Layer Electrode) presented. Transparent cell are also mentioned. Chapter copper III ion in presents Rhus the vernicijera spectroelectrochemistry of (tree) formal laccase. The type I blue- redox po- tentials of these proteins have been studied in the range 4 to 40 °C. Apparently, there are two ions in laccase exhibit. formal potentials different mechanisms that the four copper The comparison in temperature dependence of between tree laccase and fungal laccase has been made. Chapter azurin, as IV 1,48 well as describes the singly doubly and thermodynamic data from native pentaammineruthenium-modified 27 azurins, from tively. A few Pseudomonas aeruginosa at comparisons have made in modification, and the effect been of pH on 5.0, pH the 6.0, terms redox 7.0, and of respec- the effect of potentials of azurin have also been shown. v 58 Chapter cations using pentaammine. demonstrates ru theni urn the ammine cis-[Ru(en) 2 (0H)]- and possibilities complexes for other protein than cis-[Ru(NH 3 ) 4 (0H)]- modifiru theni urn are the two examples in this new direction. Chapter VI sperm whale myoglobin dicarboxylic acid]- moiety. opportunities for lines of illustrates new tron transfer processes. the synthesis and reconstituted of thermodynamic with This experiment study on biological studies [R u(MpiX)(DMSO) provides possible long-range elec- 28 References 1. Gray, H. B., Chem. Soc. Rev .. 15. 17 (1986). 2. Mayo, S. L., Ellis, W. R., B., Science . 233, 948 (1986). 3. Hush, N. S., Coord. Chern. Rev., 64, 135 (1985). 4. Marcus, (1985). 5. Scott, R. A., 932 (1985). 6. DeVault, D., Quantum Mechanical Tunnelling in Systems. Cambridge University Press, Cambridge, ed. 2, 1984. 7. Dreyer, J. L., Experientia, 40, 653 (I 984). 8. Isied, S. S., in Progress in Inorganic Chemistry, Lippard, S. J., ed ., Wiley, New York, 1984, p. 443. 9. Chance, B., DeVault, D. C., Frauenfelder, H., Marcus, R. A., Schrieffer, J. R., and Sutin, N., eds., Tunneling in Biological Systems. Academic Press, New York, 1979. 10. Hopfield, Membrance 471. 11. Wherland, S., and Gray, H. B., in Biological Aspects Inorganic Chemistry, Addison, A. W., Cullen, W. R., Dolphin, and James, B. R., eds., Wiley, New York, 1977, P. 289. 12. Hopfield, J . J ., Proc. Nat/. Acad. Sci. USA, 71, 3640 (1974). 13. Hatefi, Y., in Annual Review of Biochemistry, ed., Annual Reviews Inc., Palo Alto, 1985, P. 1015. 14. Dixit, B. P. Bioenergetics, 1984, P. 159. R. A., and Mauk, Jr., Crutchley, R. J., Sutin, N., Biochem. Biophys. A. and Gray, H. B., G., J. J., in Electrical Level, Roux, E., ed., J. and Gray, H. Acta, 811, 265 Chem. Educ., 62, Biological Volume Phenomena at the Elsevier, Amsterdam, S. N., and Vanderkooi, J. Volume 13, Lee, C. P., M., ed., 32, Biological 1978, P. Richardson, C. of D., C., in Current Topics in Academic, Orlando, 15. Michel-Beyerle, M. E., ed., Antennas and Reaction Photosynthetic Bacteria, Springer-Verlag, Berlin, 1985. Centers of 16. Govindjee, ed., Photosynthesis: Energy Conversion Bacteria, Volume 1, Academic Press, New York, 1982. Plants and by 29 17. Ernster, L., ed., Bioenergetics, Elsevier, Amsterdam, 1984. 18. Chance, B, Mueller, Today, 33, 32 (1980). 19. 20. DeVault, D., Deisenhofer, J., Michel, Sci., 10, 243 (1985). H., and Huber, R., Trends Deisenhofer, Miki, K., Huber, R., and J., Epp, P., 0., and Powers, L., Physics Biochem. Michel, H., J. Mol. Bioi., 180, 385 (1984). 21. DeVault, D., and Chance, B., Biophys. J., 6, 825 (1966). 22. Brunschwig, B. S., DeLaive, P. J., English, A. M., Goldberg, M., Gray, H. B., Mayo, S. L., and Sutin, N., Inorg. Chern., 24, 3742 (I 985). 23. English, A. M., Lum, V. R., DeLaive, P. Am. Chern. Soc., 104, 870 (I 982). 24. Mauk, A. G., Bordignon, E., and Gray, H. B., 104, 7654 ( 1982). 25. Gray, H. B., Coyle, C. L., Dooley, D. M., Grunthaner, P. J., Hare, J. W., Holwerda, R. A., McArdle, J. · V., McMillin, D. R., Rawlings, J., Rosenberg, R. C., Sailasuta, N., Solomon, E. 1., Stephens, P. J ., Wherland, S., and Wurzbach, J. A., in Advances in Chemistry Series, No. 162, Bioinorganic Chemistry-ll, Raymond, K. N., ed., American Chemical Society, Washington, D. C., 1977, P. 145. 26. McArdle, J. V., Coyle, C. L., Gray, H. B., Yoneda, Holwerda, R . A., J. Am. Chern. Soc. , 99, 2483 (I 977). 27. Holwerda, R. A., Wherland, S., and Gray, H. of Biophysics and Bioengineering, Volume ed ., Annual Reviews Inc., Palo Alto, 1976, P. 363. 28. Cummins, D., and Gray, H. B., Inorg. Chem., 20, 3712 (1981). 29. Holwerda, R. A., Knaff, D. B., Gray, H. B., Clemmer, J. D., Crowley, R., Smith, J. M., and Mauk, A. G., J. Am. Chern. Soc., 102, 1142 (I 980). 30. McArdle, J. V., 99, 4141 (1977). Yocum, K ., and 31. Coyle, C., and 1122 (I 976). Gray, H. B., J., and Gray, H. B., J. Gray, H. Biochem. J. Am. Chern. G. S., Soc., and B., in Annual Review 5, Mullins, L. J., B., J. Am. Biophys. Res. Chern. Soc .. Comm., 73, 30 32. Wherland, S., 2950 (1976). 33. McArdle, J. V., Gray, H. Chem. Soc., 96, 5737 (1974). 34. Ellis, W. R., Jr., Ph.D. Thesis, Technology, Pasadena, United States, 1986. 35. Miller, J. R., Beitz, J. Soc., 106, 5057 (1984). 36. Cave, R. J., Klippenstein, Phys., 84, 3089 (1986). 37. Siders, P., Cave, 5613 (1985). J., and Marcus, R. A., J. 38. Makinen, M., Schichman, Science, 222, 929 (1983). S. A., S. C., and 39. Higuchi, Y., Kusunoki, M., Matsuura, Kakudo, M., J. Mol. Bioi., 17 2, I 09 (1984). Y., Yasuoka, 40. Pierot, M., and Gray, R. Haser, R., H. Proc. B., B., Creutz, V., and S. C., Acad. and California and Payan, F., USA, N., 73, J. Am. Institute of K., J. R. A., Marcus, Hill, M., Sci. Sutin, Huddleston, R. J., Frey, Nat/. Am. Chem. Chem. Chem. J. Phys., 81, Gray, H. B., N., and Astier, J.-P., H., J. Chem. J. J., J. Phys. J. Chem. Phys. , and J. Bioi. Chem., 257, 14341 (1982). 41. Kimura, K., Nakahara, Phys., 70, 3317 (1979). 42. Onuchic, J. N., Beratan, Chem., 90, 3707 (1986). 43. Garg, A., Onuchic, 83, 4491 (1985). 44. Kosower, E. M., J. Am. Chem. Soc., 107, 1114 (1985). 45. Crutchley, R. J., Ellis, W. R., Jr., and Gray, H. B., in Frontiers in Bioinorganic Chemistry, Xavier, A. V., ed., VCH erlagsgesellschaft, Weinheim, F. R. G ., 1986, P. 679. 46. Crutchley, R. J., Ellis, W. Chem. Soc., 107, 5002 (1985). 47. Isied, S. S., Kuehn, 106, 1722 (1984). 48. Margalit, R., H.-J., Pecht, J. Y., Tagi, D. N., C., N., and R., and Kostic, N. M., I., Shelton, J. T., and and Inokuchi, Hopfield, Ambegaokar, Jr., and Worosila, V., Gray, G., Che, C.-M., B., Shelton, J. H. B., J. Am. Am. Chem. Soc., Blair, D. F., Chiang, J. R ., Schroeder, W. 31 A., and (1984). Gray, H. Proc. B., Nat/. Acad. Sci. USA, 81, 6554 49. Nocera, D. G., Winkler, J. R ., Yocom, K. M., Gray, H. B., J. Am. Chern. Soc. , 106, 5145 (1984). 50. Kostic, N. M., Margalit, R., Che, C.-M., Chern. Soc. , 105, 7765 (1983). and Gray, H. 51. Margalit, R., Pecht, 105, 301 (1983). B., 52. Yocom, K. M., Winkler, J. R., Nocera, D. Gray, H. B., Chern. Scr., 21, 29 (1983). G., Bordignon, E., and 53. Isied, S. S., Worosila, Soc., 104, 7659 (1982). S. J., 54. Winkler, J. R., Nocera, D. G., Yocom, K. M., Gray, H. B., J. Am. Chern. Soc., 104, 5798 (1982). 55. Yocom, K. M., Shelton., J. B., Shelton, Worosila, G., Isied, S. S., Bordignon, Proc. Nat/. Acad. Sci. USA , 79, 7052 (1982). 56. Toi, H., La Mar, G. N., Margalit, R., B., J. Am. Chern . Soc., 106, 6213 (1984). 57. Beitz, J. (1979). 58. Che, C.-M., Margalit, R., Chim. Acta, 135, 33 (1987). V., and and 1., G., Gray, and Miller, J. Chiang, H. Atherton, R., Bordignon, E., and Am. J. H.-J., Chern. J. Am. Soc., Chern. Bordignon, E., and J. R., Schroeder, W. E., and Gray, H. Che, C.-M, J. B., J. Am. Chern. and and A., B., Gray, H. Phys. , 71, 4579 Gray, H. B., lnorg. 32 CHAPTER II SPECTROELECTROCHEMISTRY AND THE OTTLE CELL 33 Introduction Elucidation transfer of the thermodynamics of reactions 1-21 is a fundamental biochemistry. 22 of redox centers of of the most electron transfer temperature of electron importance in The accurate measurement of the formal reduction po- tentials one matter metalloprotein in metalloproteins important reactions, coefficient parameters such derived it, and inner-sphere terpretation these of in driving reduction questions, Based the (d£ 0 '/dT), potentials, entropy terms can be the effects of be answered by the in- properties of The redox niques, such as cyclic voltammetry, due to the problem of slow elecbetween metalloprotein is why many measurements ten tiometr ic ti tra tion 23 -25 ' rent since errors, sample volume even other and the by conventional electrode tech- surface. This of formal reduction potentials rely on po- successive and properly on cannot exchange studied long-range mettalloproteins tron be data. determination biological concerning reorganization, can thermodynamic the force. as enthalpy and important solvation and the formal thermodynamic from parameters namely of allows though addition properties such of of experiments redox solution. titrants The have in he- change the redox potential data based on these techniques are therefore by no means accurate. It as a known redox proteins of is redox and that mediator or electrodes mediator or by introducing certain promoter, 26 - 30 the can be promoter, small molecules to electron exchange between proper selection achieved. With the of range the possible applications act of 34 electrochemical techniques in the study of biological molecules has been greatly enhanced. Spectroelectrochemistry stry The combination of and spectroscopy, has studying the molecules. tion or ments, used that Oxidation states are electrons at the as of solution of adjacent different techniques, electrochemi- to effective approach be changed an to process. Thus, the in the consequences of solution and offer redox a potentials made phenomena convenient also si- spectroscopy electro I ysis. and addi- measure- thus electrochemical undergoing techniques 31 - 39 by spectral are of spectra and for biological electrochemically electrode, probe both and the electrogeneration spectroelectrochemical organic, electrode the a an inorganic, with occur obtaining proved chemistry multaneously is quite redox removal on two Such means for for observing subsequent chemical reactions of the electrogenerated species. A number trochemistry are technique frared is region. of optical methods I. 34 The spectroscopy in the in Transmission involves transparent or beam directly through and the adjacent solution. 38 - 40 the beam is through surface back through the a most spectroscopy cal passed been coupled Figure summarized absorption that have In solution with elec- frequently used ultra violet-visible-inpassing the semi-transparent specular reflectance and reflected solution. 41 , 42 Internal from opti- electrode spectroscopy the electrode reflectance spec- 35 Figure 1. Block diagram techniques. representation of spectroelectrochemical ...... ~ CONVENTIONAL CELL THIN-LAYER CELL ~ ,.... UV,VIS,IR NMR ESR INTERNAL REFLECTANCE SPECTROSCOPY SPECULAR REFLECTANCE SPECTROSCOPY REFLECTANCE SPECTROSCOPY ABSORPTION SPECTROSCOPY TRANSMISSION SPECTROSCOPY CD RAMAN OR RESONANCE RAMAN SPECTROSCOPY FLUORESCENCE SPECTROSCOPY LUMINESCENCE AND SCATTERING SPECTROSCOPIES w 0\ 37 troscopy of a gle involves introducing the optical beam through transparent electrode an angle greater than is totally reflected. 3B- 4 0, 4 3 at which enhancement the for at beam both types the the back side critical an- Sensitivity of reflectance spectroscopies scattering spectroscopic techniques can be achieved by multiple reflections. Luminescence with coupled and electrochemistry. the spectroelectrochemistry through rected ing is solution observed. 44 which The excitation beam and the a electrode, and the Raman back-sea tter- important aspect of in can structural the is contained in be passed through fluorescence from the served. 45 A variety of emission of light. Such an by particularly species of done A electrogenerated decay is an lies from and at electrode excited resonance been Raman excitation spectroelectrochemistry resulting In have laser Raman on the spectrum. an are Raman accompanied state the solution cell, species chemiluminescence by the electrochemical electrogenerated formed di- beam information electrogenerated reactions Raman ob- by the results reaction of electrogenera ted cations and anions. 46 Electrochemical cells have also been placed in the sample cavi- ties of ESR and NMR spectrometers to record the absorption spectra of electrogenera ted species. 4 7,48 OTTLE (Optically Transparent Thin-Layer Electrode) Cells There are two types of cell geometries which are commonly used 38 in conjunction with the various is analogous to a conventional in contact electrode is than the diffusion layer thin-layer cell 49 - 52 confines adjacent to the with optical electrochemical an adjacent a techniques. electrolyte electrode. thin (0.1 0.5 most The rapidity with which this layer path length of 0.2 cell in which the solution the The normal cell to electrode. The much By mm) significant thicker contrast, solution virtues the layer of thin- species within layer cells are: (I) can be electroactive completely electrolyzed (for cell mm, the time needed for electroly- tic equilibrium is ca. 20 - 120 seconds). (2) The very small amount of sample is needed (typically 700 - 1000 ~1). Both of these virtues, in particular the latter, benefit the study of metalloenzymes, because the proteins are usually expensive and easily spectroelectrochemistry has been transparent elec- denatured. Progress stimulated by in the the methodology development trodes38 that enable the light electrode and and scattering trodes, of of suitable optically beam to be passed directly through the adjacent solution. Although techniques can implemented transparency is be necessary for reflectance, with luminescence, conventional transmission and internal consists of a thin elecre- flectance spectroelectrochemistry. One of type conducting of transparent material, such elecrtrode as platinum, gold, tin very oxide, or film carbon, 39 that is quartz the deposited on a transparent (ultraviolet-visible), spectral region of substrate or germanium interest. The such as glass (infrared), (visible), depending on (20 85%) of minigrid electrode transparency these electrodes is due to the thinness of the conducting film. A which second type consists of micromesh of of a I 00 - transparent metal (gold, silver, 2000 wires per ency (20 - 80%) is due to ture. minigrid The electrode has the been is the copper, inch. or platinum) In this case, the transpar- physical holes in used primarily in nickel the minigrid structhe thin-layer cell configuration in conjunction with transmission spectroscopy. Mercury transparent electrodes have been a thin film of onto platinum electrodes and gold and nickel trodepositing mercury minigrids. prepared and by elec- carbon film This allows use of the ex- cellent negative potential range of mercury. Fabrication of OTTLE Cells The step by step procedure for the fabrication of an OTTLE cell is described below: (I) The cell (methyl in cept body was machined methacrylate). Figure 2. methanol, from clear The dimensions with non-aqueous For use it softens epoxy), a lucite are plastic illustrated solvents (ex- Kel-F (teflon) mm x 21 mm x cell body is recommended. (2) Carefully cleaned quartz windows (1.5 40 Figure 2. Detailed view of OTTLE (Optically body. (A) Front the dimension Transparent view, specifications Thin-Layer (B) side thermocouple plug. All dimensions are in mm. for the Electrode) cell view, and (C) 41 :---12.16_____.. 1 I 5.87 I : ,_ .. 1 7. 1 I I I 1 1 1 1 1 I I I 7/ 25 STD. TAPER I I I I I I I I I I I I I I I I I I I I I I I I r-, l ____ _J I I I I I r-, I I I I I I I I I I I I I I I I 5.11mm DIA. 714 57. 15 2 .58 mm DIA. 6.35 . L A-.-L-L--L.L--1- ].59 4.75 THERMOCOUPLE PORT 2 .78rrm Dl I I I I [J----r1 : __'f_ 0 .76 I I - ... I I B. ~10. 1~ 3.81 '--12 . 7~ •'-----25.4 ..,.j A · 6.87 2.67 4 .76 mm I 1.02 mm. DIA. KJLE Dl~~c¥~~~~2. 64mm DIA. C. THERMOCOUPLE PLUG 42 2lmm, transparency to 170 nm) were used. A thin layer of epoxy was applied to the OTTLE portion of the lucite block and one of the quartz windows was carefully glued in. A small vial filled with mercury provided a sufficient weight to set on top spacer was of the window while If thick- the epoxy set. (3) A tape 53 teflon cut out. two nesses were used, a cell path length of ca. 0.5 mm resulted. A lodged against electrode small, the port lucite openings), window only at second, trapezoid-shaped larger body and piece of (centered epoxied spacer was between the to the quartz the end near the lucite cell body. piece of teflon spacer was cut A approxi- mately 20 mm x 20 mm square and then trimmed to a "U" shape. The interior opening was larger than the open- ing cut in the cell body. (4) minigrid 54 A gold cell is 500 wires / inch with scissors desired, was then two minigrid while fabricated. minigrids is If a thick are required. The cut very delicate and sandwiched between mylar The minigrid was not touch with bare hands. OTTLE was sheets. The di- mension of the minigrid was ca. 17 mm wide and 35 mm long. (5) One minigrid was zoidal teflon spacer. carefully inserted The "U" shaped under the teflon was trapethen 43 placed on top second gold of the gold minigrid. minigrid, Finally, the followed second by quartz the win- dow was placed on top and a mercury-filled vial laid on top in place. of the assembly to keep everything Once this was done, the four corners of the OTTLE assembly were tacked down with epoxy. be used sparingly, since once it The epoxy should hits the minigrid(s) it flows quickly and messes up the cell interior. (6) A thin layer of epoxy was carefully applied to all edges of the OTTLE except the end where the gold minigrid protruded. During this process, which should be done one edge at a time, the cell body should be tilted in such a way that the epoxy flows away from the direction ing of the OTTLE interior. minigrid was lifted up and epoxy applied body. between a Lastly the protrud- very thin layer of the minigrid and the lucite cell The minigrid was pressed down with a pair of forceps and left to dry. (7) The end of a 3" piece of 18 gauge copper wire was tinned and then laid across the ends of the gold minigrid and soldered to the grid. It was found best to sandwich the wire between the two minigrids when making a minigrid, thick the pressed on a OTTLE cell. To tip the soldering of portion of the wire avoid iron not in melting the should be contact with 44 the minigrid. (8) The excess minigrid was gently rolled around the cop- per wire, and the wire epoxied at the base of the cell body, just below the quartz windows. A heavy coat of epoxy was then applied to all four edges of the OTTLE cell. (9) The thermocouple plug which was assembly was tip was epoxied machined slightly then inserted into into a clear luci te The plug thermocouple port undersize. the and epoxy was layered around the inserted plug on the outside of the cell body. The OTTLE cell assembly is illustrated in Figure 3. The (10) counter electrode (auxiliary electrode) consisted of a 9" length of platinum wire (0.0020 inch diameter) sealed into a ground glass exposed at tube of joint, leaving the compartment joint, a ground consisting terminated scription of the by a soft with approximately end. of glass a fine 5/20 outer of wire 1" This was supported in 5/20 inner ground glass porous glass frit. A de- configuration of the non isothermal a reference electrode is given in a later section. Spectroelectrochemistry of Biological Molecules Application of spectroelectrochemistry to biological molecules 45 Figure 3. OTTLE (Optically Transparent assembly. (A) view, Front (B) Thin-Layer Electrode) side (C) view, and (D) enlarged illustration of thin-layer structure. top cell view, 46 Reference electrode port Counter elect rode port Lucite body ' " r - Thermocouple po r t L. v Teflon spacer--I: Th i n layer cavity I Optica I window Tef Ion spacer Copper wire tsoldered tot he gold minigr id) . ' ; , . ' !--... ., ; ~IGold mini grid; working electrode A. Thin layer cavity -+--+--Electrode ports Electrode ports C. Gold /~===--m i nigr ids Quartz plates Teflon spacer D. 47 was first demonstrated exchange electrons by poorly Heineman with an 1975. 55 in electrode, Metalloproteins presumably due often to insu- lation of the redox center from the electrode by the surrounding amino acid residues, however, spectroelectrochemical measurements can be made by adding to the solution a small amount of another redox species, a As mediator. illustrated in Figure 4 mediators indirectly couple the metalloprotein redox species with the electrode. In Eappl., a is typical applied across talloprotein-media tor equilibrium It then solution trode spectroelectrochemical solution [Ox]/[Red] in turn until and value equilibrium the redox cell thin-layer mixture. by homogeneously an all the experiment, The direct a potential, containing mediator the me- adjusts to electrode. electrolysis at the oxidizes or reduces the protein in is reached centers in between Eappl. Thus, solution. at the its the the elec- results are media tor independent. The use of a mediator with an E 0 " too far away from that of the metalloprotein frequently ther too high or be used in spectroelectrochemical a too results low. in Hence, a midpoint the selection experiment potential that is ei- of media tors to important for is the very the precise determination of the redox potentials of metalloproteins. The criteria for the choice of mediators 56 are summarized below: (I) The mediators must be able to readily oxidize and reduce the molecules under study and be able to interact readily with the electrode in order to establish redox stages according to the applied potentials. their 48 Figure 4. Mediator-titrant (MT), talloprotcin the electron exchange outer-sphere electrode in the electrochemical between electron the electrode transfer species besides mediates and via protein mechanism. the media tor-titrant which is reduces/oxidizes the protein. me- experiments, changed, potential reduces/ oxidizes redox When an the electrode in turn 49 ~ E?3 L~ E~ c T R 0 D E e- ( MTox) (PROTEINREO 7 MT RED PROTEINox 50 (2) The mediators must have reduction low them first to potentials which al- span the required potential range approximation an active mediator is from 90% reduction, equivalent to :!: (to a effective 60 mY (n = 1) on either side of the reduction potential). The mediators should not interact with (3) the redox com- ponents so as to modify them chemically. The (4) mediators should not interfere with the measure- must be independent of the ments of the component of interest. The (5) measured values time allowed for measurement. The same (6) results must be obtained by either oxidation be obtained using different or reduction processes. The same results must (7) me- dia tors. Nonisothermal Configuration of the Variable Temperature OTTLE Cells The formal reduction potential, E0 " by sequentially applying a determined across the trol ysis has equilibrium reduced equation: thin-layer as value the forms, of series redox couple of potentials determined by spectroscopically, so that the ratio the concentration of oxidized to as by the [Ox]/[Red] is established maintained defined until is potential of is this Each cell. ceased, for ' elec- Nernst 51 Eappl. = E 0 " + (2.303R T /nF) • log([Ox]/[Red]) The redox state to the which the spectra. couple other is by the corresponding Both formal incrementally series value of of reduction converted applied [Ox]/[Red] from one oxidation potentials, for each of determined from the n be is potentials and the value of can determined from plots of Eappl. vs. log([Ox]/[Red]). Measurements of the temperature coefficient of the e.m.f. from electrochemical cells of the type: 57 Hg I Hg 2 CI 2 (sat.), KCI (sat.) II KCI (sat.) II Mm+, M{m-n)+ I M' T (varied) were normally employed in the earlier studies (Mm+ and M(m-n)+ refer to the of interest, mal oxidized and reduction potentiometrically and reduced halves M" represents respectively, the potentials at using solutions working each of the electrode temperature containing redox couple material). For- were determined equi-molar concentra- tions of the oxidized and reduced halves of the redox couple. Such cell arrangements tions from the reference ficient of the cell An inherent needed for e.m.f. difficulty most contain and entropy contribu- electrode half -cell to the temperature coef- and referred to as "isothermal" cells. procedure lies with common enthalpy are this reference electrodes to in the reach long time temperature 52 equilibrium. It has been demonstrated that a cell arrangement of the type:5 8 Hg I Hg2CI2 (sat.), KCI (sat.) II KCI (sat.) I KCI (sat.) II Mm+, M(m-n)+ I M' T1 (fixed) can be utilized redox reactions tentials are mined. Under entropy, couple of llS~t), is effectively in between or the reduced the proportional thermodynamics of certain thermal junction are constant and be deter- partial molal of redox the and the can difference oxidized electron to the if circumstances (i.e., interest determining solution negligible, favorable directly for aqueous either ionic T2 (varied) halves transfer temperature in the po- reaction entropy, coefficient of the nonisothermal cell:58 where F is the Faraday constant. These two commonly used configurations for the electrochemical cell are illustrated in Figure 5. For isothermal variable temperature configuration arrangement was utilized: in studies our we have employed the OTTLE cells, and following the non- 53 Figure 5. nonisothermal Isothermal and trochemical cells. figuration, the changes with dependence sample, of the that of the formal was temperature isothermal con- of of the reference sample. The reduction potential the therefore elec- case temperature corrected the temperature of the contribution electrode. required that the working and reference electrode compartments be maintained at two different tempera- configuration temperatures. In our of for electrode reference nonisothermal dependence the for the The the of In configurations experiments, the ture of the reference electrode was always held at ambient room temperature. 0 6S,c 0 = 6Si10 + 15.6 eu 0 = flS 110 + 156 eu . = 0 (BY CONVENTION) SH. 0 1/2· H2 OX+ 112· H2 CELL = (S~ed + ~.}- (~x + lj2. S~} H2 0 ~ --loo. -- ~ ---- OX+ E- REFERENCE = 31.2 eu so sred- sox 0 0 flSiso ISOTHERMAL SO....UTla-.1 OF SAMPLE T1 RED+ H+ RED H• + E- 0 0 flS,c = F • (dE /dT) 0 0 = F· (dE /dT} CELL REFERENCE IS HELD FIXED WHILE IS VARIED sred- sox 0 r, T2 T2 NON ISOTHERMAL SOLUTION OF SMf'LE T1 ""' VI 55 Hg I Hg2CI2 (sat), KCI (sat.) II KCI (sat.) I KCI (sat.) II Mm+, M(m-n)+ I Au lj (fixed) where Au represents T2 (varied) the gold minigrid working electrode. The non- isothermal salt bridge consisted of a 5 mm x 25 em glass tube filled with saturated KCl solution. The bottom $7/25 outer ground glass joint with a end was flared to tion, #S-30080-17). ing a specially cell temperatures Inc. precision receive constructed were terminated Pt junction sealed in. Sargent-Welch the variable directly SCE (miniature, 0.2 temperature measured thermocouple, 59 micro to was by a The tip Pt june- The temperature of the OTTLE cell was varied us- i tal Thermometer, ± proximity a end with connected cell an holder Omega to a and the Engineering, Fluke 21754 Dig- °C, situated in the protein solution in close thin-layer cavity. The reference electrode and part of the nonisothermal salt bridge were maintained at ambient room ternperature. squares The fit to Nernst the plot Nernst data were analyzed equation, and using reduction a linear potentials were least re- tained only if the linear correlation coefficients were 0.999. The temperature coefficient of the formal reduction potential, (dE 0 '/dT), was determined to the E 0 ' vs. temperature data. The entropy change for the complete cell (6S 0 ), the reaction the rela tion:60 adjusted as the slope of a to NHE scale linear was least squares fit determined from 56 taking S¥! = (S~ed - S~) - 15.6 eu and to 31.2 eu as zero. 61 adhering the arbitrary convention that 2 S¥!+ be taken Standard free reaction were calculated from the E 0 " and the standard enthalpy changes, corresponding l1G 0 "s and l1S 0 "s: energy the cell (V vs. NHE) values at 25.0 °C l1H 0 "s, were changes for determined from the 57 References R., Jr., and Gray, Crutchley, R. J., Ellis, W. Chem , Soc., 107, 5002 (1985). 2. Margalit, R ., Kostic, N. M, Che, C.-M., Blair, D. F., Chiang, H.-J., Pecht, 1., Shelton, J. B., Shelton, J. R., Schroeder, W. A., and Gray, H. B., Proc. Nat/. Acad. Sci. USA , 84, 6554 (1984). 3. Nocera, D. G., Winkler, J. R., Yocom, K . M, Gray, H. B., J. Am. Chern. Soc., 106, 5145 (1984). 4. Reid, L. S., Taniguchi, V. T., Gray, H. B., and Mauk, A. G., J. Am. Chern. Soc., 104, 7516 (1982). 5. Taniguchi, V. T., Ellis, W. R., Jr., Cammarata, V., Webb, J., Anson, F. C., and Gray, H. B., in Advances in Chemistry Series , No. 201, Electrochemical and Spectrochemical Studies of Biological Redox Components, Kadish, K. M., ed., American Chemical Society, Washington, D. C., 1982, P. 51. 6. Taniguchi, V. T., Malmstrom, B. G., Anson, F. B., Proc. Nat!. Acad. Sci. USA, 79, 3387 (1982). C., and Gray, H. 7. Taniguchi, V. T., Sailasuta-Scott, N., H. B., Pure Appl. Chern. , 52, 2275 (1980). F. 8. Goldberg, M., Ph.D. Rehovot, Israel, 1979. Thesis, Weizmann 9. Sailasuta, N., Anson, F. 101, 455 (1979). C., and Gray, H . B., 10. Wherland, S., and Pecht, 1., Biochemistry, 17, 2585 (1978). 11. Kreishman, G. P., Su, C.-H., Anderson , C. W., Halsall, H. B., Heineman, W. R., in Advances in Chemistry Series , No. 188, Bioelectrochemistry: Ions , Sur faces , Membranes, Blank, M., ed., American Chemical Society, Washington, D. C., 1980, P. 169. 12. Anderson, C. W., Halsall, H. B., Heineman, W. R ., and Kreishman, G. P., Biochem, Biophys. Res. Comm., 76, 339 (1977). 13. Goldberg, M., and Pecht, I., Biochemistry, 15, 4197 (1976). 14. Jacks, C. A., Bennett, L. E., Raymond, W. N., and Lovenberg, W., Proc. Nat!. Acad. Sci. USA , 71, 1118 (1974). Anson, H. B., Am. 1. J. Bordignon, E., and C., Institute J. and of Gray, Science, Am. Chern. Soc. , 58 R., J. 16. Margalit, R., and Schejter, A., FEBS Lett. , 6, 278 (1970). 17. Watt, G. (1969). 18. George, P., Eaton, W. (1968). 19. George, P., Hanania, G. I. H., and Eaton, W. A., in Hemes and Hemoproteins, Chance, B., Estabrook, R. W., and Yonetani, T., eds., Academic Press, New York, 1966, P. 267. 20. Hanania, G. I. H., Ph.D. Cambridge, England, 1953. 21. Ball, E. G., Biochem. Z., 295, 262 (1938). 22. Walz, D., Biochim. Biophys. Acta, 505, 279 (I 979). 23. Wilson, D . F., Dutton, P. L., Erecinska, Sato, N., Ace. Chern. Res., 5, 234 (1972). 24. Clark, W. M., Oxidation-Reduction Williams and Wilkins, Baltimore, 1960. 25. Rodkey, F. L., and Ball, E. G., J. Bioi. Chern. , 182, 17 (1950). 26. Higgins, I. J., and Hill, Volume 21, Marshall, R. Press, London, 1985, P. 119. H. A . 0., in Essays D., and Tipton, K., 27. Allen, P. M., Hill, Chern. , 178, 69 (1984). 0., 28. Eddowes, M. J., and Hill, H. A. 0 ., in Advances in Chemistry Series , No. 201, Electrochemical and Spectrochemical Studies of Biological Redox Components, Kadish, K. M., ed., American Chemical Society, Washington, D. C., 1982, P. 173. 29. Alberg, W. J., Eddowes , M. J., Hill, H. R., J. Am. Chern. Soc. , 103, 3904 (1981). 30. Eddowes, M. J., and Hill, H. A. 0., J. Am. Chern. Soc. , 101, 4461 (1979). 31. Heineman, W. R ., Hawkridge, F. M., and Electroana!ytical Chemistry , Volume 13, Bard, and and A., Biochem., Marga1it, (1973). D., Schejter, Eur. 15. J. Sturtevant, H. A., A. M, Biochemistry, and University M., Potentials Walton, A. 492 8, 4567 Fed. Proc., 27, 526 and Trachtman, M., Thesis, 32, of Cambridge, Lindsay, J. G ., and of Systems, N. 0., Organic in Biochemistry, eds., Academic J., and Blount, A. J ., J. Electroana!. Hillman, A. H. ed., N., in Marcel 59 Dekker, New York, 1984, P. I. 32. Heineman, W. R., J. Chern. Educ., 60, 305 (1983). 33. Heineman, (1980). 34. Heineman, W. R., Anal. Chern., 50, 390A (1978). 35. Heineman, (1978). 36. Muller, R. H., Electrochim. Acta, 22, 951 (1977). 37. Parson, R., Sci. Prog., Oxj., 64, 29 (1977). 38. Kuwana, T., and Heineman, W. R., Ace. Chern. Res., 9, 241 (1976). 39. Kuwana, T., and Winograd, N., in Electroanalytical Chemistry, Volume 7, Bard, A. J., ed., Marcel Dekker, New York, 1974, P. I. 40. Kuwana, T., Ber. Bunsenges. Phys. Chern., 77, 858 (1973). 41. Aylmer-Kelly, A. W. B., Bewick, A., Cantrill, P. Tuxford, A. M., Faraday Discuss. Chern. Soc., No. 56, 96 (1973). 42. Mcintyre, J. D. E., in Advances trochemical Engineering, Volume 9, New York, 1973, P. 61. 43. Hansen, W. N., in Electrochemical Engineering, Wiley, New York, 1973, P. I. 44. Jeanmaire, D. L., Suchanski, M Chern. Soc., 97, 1699 (1975). 45. Hawkridge, F. M., and Ke, B., Anal. Biochem. , 78, 76 (I 977). 46. Faulkner, L. stry, Volume 1977, P. I. 47. McKinney, T. M., in Electroanalytical Chemistry, Bard, A. J., ed., Marcel Dekker, New York, 1977, P. 97. 48. Richards, J. A., and Evans, D. H., Anal. Chern., 47, 964 (I 975). 49. Hubbard, A. T., CRC Crit. Rev. Anal. Chern., 3, 201 (1973). w. w. R., R., F., 10, and and Kissinger, Kissinger, P. P. Advances Volume T., T., Anal. Chern., 52, 138R Anal. Chern., 50, 166R R., and in Electrochemisty and E/ecMuller, R. H., ed., Wiley, in 9, Electrochemistry Muller, R. H., and ed., R., and Van Duyne, R. P., J. Am and Bard, A. J., in Electroanalytical ChemiBard, A. J., ed., Marcel Dekker, New York, Volume 10, 60 50. Hubbard, A. T., and Anson, F. C., in Electroanalytical Chemistry, Volume 4, Bard A. J., ed., Marcel Dekker, New York, 1971, P. 129. 51. Reilley, C. N., Rev. Pure App!. Chern., 18, 137 (1968). 52. Murray, R. W., 39, 1666 (1967). 53. Teflon film for spacers between quartz windows: Specify type DF 1200 2 mil I" x 36 yards. Order from: Dilectrix Corp. 465 Ohio St. Lockport, NY 14094 54. Electroformed gold mesh for the minigrid: 5000 lines/inch, 6" x 6" sheet. Order from: Buckbee-Mears Co. Industrial Products Group 245 E. Sixth St. St. Paul, MN 55101 55 . Heineman, W. 47, 79 (1975). 56. Scott, N. S., Ph.D. Thesis, Pasadena, United States, 1980. 57. Lin, J., and Breck, W. G., Can. J. Chern., 43, 766 (1965). 58. Yee, E. L., Cave, R. J., Guyer, K. L., Tyma, P. M. J., J. Am. Chem. Soc., 101, 1131 (1979). 59. Copper-Constantan thermocouple: specify item TT-T-36-48 in.-PJ Order from: B. J. Wolfe Enterprises 10760 Burbank Blvd. North Hollywood, CA 91601 60. George, P., Hanania, G. 2548 (1959). 61. Latimer, W. M., The Oxidation States of the Elements and Their Potentials in Aqueous Solutions , Second Edition, Prentice-Hall, New York, 1952. W., Anal. Chern., Heineman, W. R., and O'Dom, G. R., Norris, I. B. H., J., and California and J. F., Anal. Institute of Technology, Goelz, Irving, D. Chem., D., and Weaver, H., J. Chern. Soc., 61 CHAPTER III THERMODYNAMIC STUDIES OF THE TYPE-I COPPER IN TREE LACCASE 62 Introduction Copper-containing proteins different spectral, tions copper-proteins of reactions chemical and in functional include the nature, displaying properties. catalysis of The electron functransfer the transport catalysis of the disproportion of the and the catalysis the re- duction of 0 2 to H 2 o 2 (galactose oxidase, amine oxidase) and H 2 0 (cy- molecules superoxide plastocyanin, widely stellacyanin), gen (azurin, occur (hemocyanin), anion tochrome c involved in the the (superoxide oxidase, dismutase), laccase). Furthermore, the of protein transport of copper in mammals. A of oxy- albumin is selection of some important copper proteins is listed in Table 1. Recently, several spectroscopic, of investigations electrochemical, copper-containing have structural proteins. This work been carried and functional has been out on the properties reviewed by sever- al authors. 1- 13 Laccase is tributed in oxidases which a higher blue-copper-containing oxidase which is It belongs to small plants can and utilize fungi. the full oxidizing and reduce it to two molecules of water. proteins ceruloplasmin and a capacity widely dis- group of of dioxygen These other enzymes are the ascorbate oxidase which the complicated have many proper- ties in common with laccase. Laccase is one since it contains three Nevertheless, it has extensively dases of been most types of quite studied by copper-containing oxi- unique ions. a copper number of differ- 63 Table I. Some blue and non-blue copper-containing proteins with the types of copper ions present and their functions. together blue ---+ H o 2 probably involved in iron metabolism oxidase, reducing 0 2 ---+ H 2o and type 3(4) *The number in parentheses designates how many specified type of copper ions per molecule. 2 oxidase, reducing 0 2 ---+ H 20 oxidase, reducing 0 type I (2), type 2( I) and type 3(2) Ceruloplasmin type I (3), type 2(1) type I (I), type 2(1) blue Ascorbate electron carrier type 1(1) blue blue Stellacyanin electron carrier electron carrier Function type 1(1) Laccase blue Plastocya n in type 1(1)• and type 3( 4) blue Azurin Type of coppers oxidase Type of protein Protein .j:>,. 0'1 non-blue Superoxide non-blue non-blue Albumine non-blue Amine oxidase oxidase Galactose oxidase Cytochrome c non-blue non-blue Tyrosinase dismutase non-blue Type of protein Hemocyanin Protein "normal" copper "normal" copper "normal" copper coupled Fe-cu anti-ferromagnetically copper transport oxidase, reducing 0 2 ----+ H 2o 2 oxidase, reducing 0 2 ----+ H 2o 2 terminal oxidase, reducing 0 2 ----+ H 2 o 0 2 detoxification oxidase; catecholase activity coupled Cu pair Cu-Zn imidazole bridge monooxygenase; cresolase activity 0 2 carrier Function an ti-f erromagneticall y coupled Cu pair an ti-f erromagnetica lly Type of coppers 0\ V1 66 ent techniques, and is, therefore, one of the best understood oxi- dases, at least in terms of its catalytic mechanism. Laccase has a rather low specificity with regard Therefore, substrate. readily oxidized. a number of to the reducing quite different substances are many derivatives of Good substrates included (Rhus vernicifera) (p-diphenol: phenol. Tree laccase E.C. 1.10.3.2) from of II 0,000 daltons. The probably consists although 500 it the Japanese lacquer amino of acid residues. No determined. Since the is bohydrate, the enzyme probability that acid a amino tree a molecular sequence is still polypeptide crystal structure glycoprotein, X-ray oxidoreductase, has single a 02 structure weight unknown, chain has ca. of yet been containing 45% car- information will be a vail able in the near future is low. Laccase has four copper ions, and includes examples from each of the three cation was subclasses: Type I, Type 2, and Type originally used to describe the composition containing more acteristic optical center. than one copper center, and spectra and ESR present in 3. This classifi- of proteins was based on parameters for the char- each type of blue-copper-proteins and is 11 13 • Type copper is all characterized by unique spectral and redox properties: (1) A very intense absorption (molar extinction cient- 5000 M-Icm- 1 , for tree laccase, 1cm- 1 ) in the electronic spectrum around € coeffi- 0 600 nm (614 67 nm for tree laccase). A very small A II value in the EPR spectrum (for Cu(II), (2) 0.004 - 0.04 em -l; 0.0043 em -l for tree laccase ). (3) A relatively high reduction generally in the 300 - potential (190 - 500 m V range vs. 800 mY, 150 m V for Cu~~q)). 14-16 These properties are so they have different intrigued from chemists for many analogs in ordinary their years because tetragonal Cu(II) complexes. Recently, a different way of classifying method has focused attention by X-ray crystallography suggested. 17 ' 18 The of structure provided function of molecular highlighted the proteins are those ligands in a coordinated to new that the copper contain a single tetrahedral distorted the ions in copper via sulfur copper the and nitrogen on the proteins. copper geometry. proteins was details and has Group-! ion having four The ligands are donor atoms, with two nitrogens and two sulfur atoms being the most common arrangement. potential, are coordination environment, par- of ligands. Group-II at the catalytic by nitrogen Important physical properties, including modulated by details the the ticularly by the number copper proteins also center, although in and contain this case of nature the the a single the ligands redox sulfur copper are ion coordinated and oxygen donor atoms and the geometry tends to be a square-planar arrangement around arrangement is the that copper there ion. is at An least important one feature readily of this accessible 68 coordination site, either that binds substrate acid properties itant redox of copper mainly of nitrogen protein, two ligand thereby allowing the that centers classification the other plane or along the ligands. In these proteins, employed with, or proteins contain are Group-III centers of the copper(II) uclear and or activity. coordination in with donor atoms a coordination atoms. may dioxygen. can classification of or copper(I) dominate the coordination to fulfill its Group-IV proteins contain I concombin- consisting form of the environment i.e., the two or more of function, to III. Thus the characterize laccase as group number of copper proteins is IV I II III• ' ' given 10 the single blue copper proteins stellacyanin have done much toward a groups Lewis more environment the characterize method one axis, the without, In protein the tetragonal new Table 2. Several azurin, recent experiments plastocyanin, elucidating the and structure of on the blue copper site. studies 19 have shown that the absorption spectrum can tetragonall y-distorted the large charge analysis of transf cr. This has been crystal proteins, i.e., plastocyanin 20 •21 azurin. 22 •23 The X-ray crystallographic In both tetrahedral cases the environment is italica) a tom is by 6-nitrogen in coordinated atoms of structure of poplar Figures and structure shown copper two where by copper var. environment, confirmed blue nigra using a to s-(Cys) ---+ single (Populus be fit been assigned two plastocyanin 2. coordination absorbance near 610 nm has Cu(II) and tetrahedral Spectroscopic in a distorted the imidazole 69 Table 2. AI tern a ti ve class if ica tion for copper proteins. 70 Role Electron transfer Calssifica tion Examples Group I Azurin, plastocyanin, rusticyanin, stellacyanin Substrate oxidation Group II with hydrogen peroxide Galactose oxidase Benzylamine oxidase formation Superoxide dismutation Group II Superoxide dismutase Monooxygena tion Group II Dopamine ~-mono­ oxygenase Group III Tyrosinase Oxygen transport Group III Hemocyanin Terminal oxidase Group IV Laccase Ceruloplasmin Ascorbate oxidase 71 Figure I. An X-ray backbone crystallographic of poplar structure (Populus plastocyanin in the oxidized form. of nigra the polypeptide var. italica) 72 73 Figure 2. The copper-binding site of poplar italica) plastocyanin in the oxidized state. (Populus nigra var. 74 75 rings of histidine residues (His-37 and His-87) and by two sulfur at- oms, one of which is a thioether sulfur from methionine (Met-92), other a thiol ture of plastocyanin appeared per(II) the in the protein: one of whereas the distance; involved in the Cu(II) sites and azurin, the are Cu(l) residue believed resembled that of had moved sulfur atom the high in the to resemble slightly away from was a at somewhat rearrangement in inner-sphere potential, Although stellacyanin is struc- favouring does not acid sequence, 24 all one found in plastocyanin in the the two cop- redox amino It the structural change transition. probably with struc- solved. 6 small explain crystal been has considerable The The also form histidines no (Cys-84). copper methionine can ~ methionine copper cysteine reduced reduction. an a the however, reorganization easy from coordination of tural have atom that copper, closer sulfur the cysteines type case of stellacyanin. 25 Type times 2 called copper exhibits "colorless normal copper", spectral because properties the d-d and is transitions someof the Cu(II) ion are hidden under the strong 600 nm absorption of the type is copper. EPR studies have shown that the type 2 copper of laccase almost certainly arrangement, 26 coordinated and displays four to an EPR nitrogens spectrum in a typical square planar of an axial strongly anti- Cu(II) complex. 7' 11 Type 3 copper consists of f erromagnetically coupled. 2 7,28 borg's type group, the 3 site two coppers Prior to the believed to was which are report be of the EPR-silent Gotein both 76 the resting and gen-carrying the type states. 29 active protein By hemocyanin, 3 site is considered analogy which to be also with the has such the site of molluscan oxy- a pair, copper oxygen binding, and it probably contains no sulfur ligands. There electron have been several attempts transfer from The interpretation of reducing the data to elucidate substrates is a to complex the mechanism dioxygen and by difficult of laccase. task. The experiments involve: (l) Reoxidation of fully reduced laccase. (2) The steady state reaction of laccase with oxygen and reduction of laccase with various re- experiments are consistent reductant. (3) The anaerobic ductants. The results that laccase can one active and from these exist m the other at least two inactive. 30 •31 verted to an inactive form at high pH. with the catalytically different The enzyme active It has view forms, is con- been shown that the enzyme becomes inhibited above pH 6.5 and that the inhibition depends on the formation of a type-2 Cu(II)-OH- complex at high pH values. The reduction mechanism based on studies at high and low pH is shown in Scheme of Figure 3. Since the structure of the enzyme is not copper known the protein is represented by a box. Type-1 and type-2 ions left and are represented placed by at a the pair upper which enzyme can be in two forms. and can lower accept two part electrons. type-3 The is resting One of them has an OH- ion bound to 77 Figrue 3. Scheme pH, 1, reduction and laccase. scheme 2, mechanism of laccase at high or low reoxidation reaction mechanism for 78 - - - •l'- •e- SCHEME I. cu• cu22• cu· ..5. cu• Cu (Cu 0 )2• + 2 2 -H 0 ___1_. 2 Cu • 3 (Cupl • cu· SCHEME 2 . - 2 cu· • e- (Cup> cu• 3· -H 0 Cu • -4 cu· c 1.· u2 79 type-2 Cu(II) and corresponds to the inactive form. is active and probably has water instead of OH- The bound other form to this metal site. At 6.5 both ultaneously. This simultaneous formational interaction tron is pH from reduced type-1 and type-2 Cu(II) are reduced sim- reduction is probably due to a con- between these metal sites. The first elec- a substrate molecule reduces type-1 Cu(II). When this site it the type-2 Cu(II) which seems to affects reduction of be reduced by a second substrate molecule and not by electron fer from type- I coordinated cysteine case, to the which the Cu(I). is two a reduction alteration of the It has been histidine suggested residues that on each proposed type-1 copper ligand. of Cu(II) might cause type-! type-2 Cu(II) site which could type-2 a The reduced type-1 and type-2 ultaneously donate one electron each to the Thereafter, the reoxidized type- I and type-2 coppers completely reduced. reduced and the enzyme becomes is of the is the this conformational affect of this metal site. copper side If trans- the reduction coppers then type-3 are sim- copper pair. once more This reaction sequence is shown in the upper part of Scheme I in Figure 3. Materials and Methods Tree laccase was isolated from an acetone powder extract of the latex of the modification Japanese of the lacquer published tree Rhus procedure. 33 vernicijera, 32 Purity with was a slight checked by 80 the ratio very satisfactory value (ca. and a A28o/A614 tained, as shown in Figure 4. 15.3) was ob- The laccase was then stored at - 60 oc in aliquots. The potassium f erricyanide spectroelectrochemical studies, ckrodt. 6.0, Sodium was buffer phosphate prepared solutions as the obtained as AR grade of ionic strength 100 mM and pH grade reagents. All protein and buffer, from were was used analytical prepared using deionized mediator water in from the Mallin- purified by a Barnstead NANOpure water purifier. Formal reduction copper center layer potentials were determined electrode (OTTLE) at different by using an cell in a temperatures employing gold minigrid as the type-1 optically transparent nonisothermal working thin- configuration. description of this technique is summarized in Chapter II. cell for electrode A The OTTLE material. The optical path length of 0.47 mm was measured by using the molar ex- tinction coefficient nm Potentials were ton Applied at 420 of [Fe(CN) 6 ]3- applied across the Research model l74A accurately measured multimeter. The with cell a thin-layer cell by polarographic Keithley temperature solution was as a using analyzer standard. a Prince- and were model 177 microvolt varied by using a digital variable temperature cell holder and measured directly with an Omega Engineering precision thermometer. m close microthermocouple and a The was situated proximity thermocouple to the thin-layer Fluke model 2175A digital in the protein solution cavity. All visible spectra were obtained with a Cary 219 recording spectrophotometer. 81 Figure 4. UV-VIS absorption spectrum of tree Ia cease. ratio is shown. The A 280 ; A 614 w z 0.6 (f) o.otlllll 0.4 m <! 0.3 0.2 0. I 0 m 0::: 0.5 <[ I I I I I I I I soo I I I I" I . WAVELENGTH (nm) 4oo I I I I 1: I O.OOIA I Goo T I 0.059 Azsol A514 = 15.288 A614 =0.059 Azso= 0.92 0.8 u 0.7 La cease (tree) I .0 ~----=~--==-=---~-0.9~ 0.93 7oo 00 IV 83 Formal applying reduction a series Each trod e. that the to reduced of potentials were potentials, Eappl.• potential equilibrium was determined to the ratio of concentrations couples in solution, of all redox as defined by the incrementally converted from one oxidation potentials, for which series of applied [Ox]/[Red] was determined tials n values and Nernst from were minigrid until of established gold sequentially maintained value forms the by the electrolysis equation. state each spectra. determined ceased of so oxidized [Ox]/[Red], Redox couples to other the corresponding Formal from elec- of were by the value of reduction plots was poten- Eappl. vs. log([Ox]/[Red]). All cycling solution on a deoxygenated vacuum/purified the OTTLE The platinum containing were cell by wire using argon rubber auxiliary deoxygenated prior to double septum electrode by manifold caps was mediator-titrant use and and loaded syringe situated solution vacuum/argon in a that into techniques. compartment was isolated mediator couple from protein solution by a porous glass frit. Results and Discussion The formal reduction potential of the redox [Fe(CN) 6 ]3 -/ 4 -, was determined to be 425 mY vs. NHE. 34 tion coefficients at 420 nm for ferricyanide were calculated to be 1100 and 4.7 overlay UV- VIS spectra of [Fe(CN) 6 ] 3 - and M -1 em -1 , and Molar extinc- ferrocyanide 35 •36 respectively. [Fe(CN) 6 ]4 - are shown The in 84 Figure 5. A typical thin layer 450 nm region is due mainly to ferricyanide, which was present in five-fold nm excess are are over transparent due The 6. protein. above entirely additional to 500 the absorbance tree 300 - 614 Figure with is [Fe(CN) 6 ]4 - in experiment laccase a illustrated spectroelectrochemical both nm, absorbance blue copper the [Fe(CN) 6 ]3- Since all in and change chromophore at in tree (Eappl. vs. laccase 11 and can be analyzed direct! y. A least squares analysis log([Ox]/[Red]) is given from data 0.999. Equilibration tial was that the 614 in Figure 7. yielded of determined of linear the from nm Nernst plot All E 0 ' values were determined correlation blue copper the coefficients center stabilization at of each the of at least applied poten- absorbance at 614 nm. The enzyme could be reversibly cycled between its fully and fully reduced forms, and the value of E 0 ' copper gives center the results determined perature was by independent of three dependence E0 ' at of 25 the calculated for the blue direction of cycling. Table 3 °C for blue copper in tree laccase different methods of formal the oxidized including reduction this work. potential of The tem- the blue copper center in tree laccase is shown in Figure 8. A detailed com- pilation systems of data in Table 4. It for this and other is clearly shown that metalloprotein is listed there are two different thermo- dynamic behaviors adapted by this enzyme. A least squares fit of the data within the temperature range of 20.2 - 42.1 °C gives an E 0 ' at 85 Figure 5. UV- VIS overlay spectra of [Fe(CN) 6 ]3- and of [Fe(CN) ]4 -. 6 86 0 0 z u lO - oc E "'0 Q) N - ~-­ "'0 oLDI ~ l9 z w _j oW 0~ ~s 0 0 r0 0 • (}) CO I'- <.0 lD ~ r0 N - 0 • • • • • • • • • • 0000000000 3JNtf8tjOS8\1 87 Figure 6. Thin-layer spectroelectrochemistry vernicifera) laccase. Solution 100 mM, phosphate 0.27 mM. Redox mediator: spectra and buffer, absorbance of native conditions: 24.8 °C. pH Protein K 3[Fe(CN) 6 ], 1.35 changes applied potentials in m V vs. SCE. at different tree (Rhus 5.84, 1.1 concentration: mM. Overlay values of the u I 300 o.oo. 0.02 0.04 <! 0.06 0 ~ 0.08 ~ 0.10 <! z 0.12 I I I I I I I I I 400 I I I I I I I I I 500 I I I I I I I WAVELENGTH (nm) I 230mV 210mV 190mV 170mV -130mV I I I 600 I I I I I I mediator: K 3 [ Fe (CN) 6 ] 0.16 w 0.14 [mediator] I [ laccase] =4 0.18 Laccase (tree) I I I 700 I 0.20~--------------------------------------------~ 00 00 89 Figure 7. Nernst plot of the absorbance changes at 614 nm during a spectroelectrochemical center in represent squares tree fit of = the points, Eappl. the type-1 copper The circles vernicijera. and vs. the line is log([Ox]/[Red]). 452 m V vs. NHE and Nerst slope conditions: buffer, 25.0 of Rhus laccase experimental yielded E 0 " Solution titration pH = 5.84, 1.1 °C, 5 equivalents K 3[Fe(CN)6l· 100 mM, = a least The fit 76 m V. phosphate 90 545~~~~~~~~--~~~~ 527 509 -w 491 I z 473 en > 455 > 5437 . ~ 419 401 383 36~1.9 -1.1 -0.3 0.5 In ( [ 0 x] / [Red]) 1.3 2.1 91 Table 3. Formal reduction potentials Rhus vernicijera (tree) laccase. for the blue copper center m 92 Reaction Conditions Method (mV vs. NHE) 415 1..1 = 0.2 M, pH 7.0, phosphate buffer 394 1..1 = 0.2 M, pH 7.5, Chemical titration with [Fe(CN) 6 ] 3-/ 4 - as titrants 47 Potentiometric titration; phosphate buffer ascorbate, quinol, and 0 2 mediator: [Fe(CN) 6 ] 3 - were used as titrants 4 8 [med ia tor]/[1accase] = 1/3 434 1..1 = 0.2 M, pH 7.5 Same as above phosphate buffer mediator: [Fe(CN) 6 ] 3 [mediator]/[laccase] = 3 452 1..1 = 0.1 M, pH 5.8 :!: 0.1, phosphate buffer media tor: [Fe( CN) 6 ] 3 [mediator]/[laccase] = 5 •This thesis work. Spectroelectrochemistry • 93 Table 4. Temperature E o ", dependence for laccase, the blue using trochemistry. the formal copper · ton · m nonisothcrmal Solution phosphate buffer. of conditions: reduction Rh us · ·1era vermcz thin-layer pH 5.8, potentials, J..1 (t ree ) spectroelec= 100 mM, 94 Temperature (° C) * E 0 ' (mV vs. NHE) t 6.4 433 6.8 434 8.4 436 11.8 442 14.0 444 15.8 447 18.1 449 20.2 450 22.2 449 25.0 452 25.0 453 27.2 452 29.1 456 30.5 453 32.9 457 34.9 455 38.8 459 40.6 457 42.1 459 * ± 0.2 °c t ± 2 mV 95 Figure 8. Temperature E0 ', for dependence the type of copper vernicifera, using trochemistry. Solution center °C. least squares fit in reduction tree pH the 5.8 °C gave an E data 0 ' in :!: °C and (dE 0 '/dT) 25 o C = 4.18 x I0- 4 V /° C. 0.1, 6.4 the = 452 :!: Rhus spectroelec- Temperature range: of potential, laccase thin-layer conditions: phosphate buffer. range of 20.2 - 42.1 formal nonisothermal 100 mM, A the ll - 42.1 temperature 2 mY at 25.0 w 425 -0--- 435 E > > 445 (f) 5 I ~ 455~ 465 . 10 15 .l 40 ?-~~-~~ 20 25 30 35 TEMPERATURE (oC) (y;" T Lace a se (tree) 45 I '0 0\ 97 25 ° C of 452 mV and (dE 0 "/dT) 25 o C = 4.18 x 10- 4 V ;° C. molal ionic halves of the reaction entropy, the entropy literature The partial difference between the reduced redox couple of interest (i.e., the liS~t; liS~c' the reaction entropy, has been used in same meaning) directly proportional to the with the is and oxidized electron transfer temperature coefficient of the nonisothermal cell:3?-39 liS~t = S~ed - S~x = F • (dE /dT) 25 o C 0 The value of lis~; for the blue cop- where F is the Faraday constant. per center in tree laccase is, therefore, calculated to be 9.6 1.2 ± eu. The transformation of the results from E0 " temperature ( 0 C) vs. in Figure 8 to fiG" vs. temperature (° K) is shown in Figure 9, and the va 1ue of uAso" et be can directly determined from the gradient of the straight-line plot. The entropy for the complete cell reaction !1S 0 ", adjusted to the NHE scale, is -6.0 ± liS~t corresponding The (NHE). 1.2 eu when a value of -15.6 reaction enthalpy, eu is assumed for !1H 0 " ' is -12.2 ± °C) = 452 ± 2 m V. In a similar manner, the 25 °C values of the thermodynamic 0.4 kcal/mol, with E 0 " (25.0 parameters can be calculated range 6.4 - 18.1 °C, giving !1H by 0 " extrapolation = -5.6 the temperature ± 0.4 kcal / mol, !1S 0 " 1.2 eu, add lis~; = 32.5 ± 1.2 eu with E 0 " 459 ± 2 mV. from = 16.9 :!: (extrapolated to 25 °C) 98 Figure 9. Temperature dependence of the Gibbs free energy change for the complete cell reaction of tree laccase. result of transformating the data in Figure 8. This is the .=. '-----L-----1----L-.1-----l.....----L-.I.---L-----L------L-...L--__i.__-L---ll.___-'----l rt-t-4-~--L~ -9.76 . . - - - - - - - - - - - - - - - - - - - - - - - - - - - , ..- -9.86 La cease (tree) 0 E -9 .96 "' -10.06 3 -10. I 6 -10.26 - -10.36 <.9 <J -10.46 -10.56 -10.66 -I 0. 76 318 278 283 288 293 298 303 308 313 TEMPERATURE (°K) \C) \C) 100 value of t1s~; for The is the highest one known copper center in tree laccase the type in the world of metalloenzymes. Hence, as shown in Tables 5, 6 and Figure 8, both the high value of t1s~;, and also the two distinct thermodynamic behaviors shown by this blue copper site in tion. There ASO / u et va I ues, different temperature ranges, are two other examples these values only and are worthy of of systems having positive 0.5 eu are small, i.e. closer Rhodospirillum rubrum cytochrome c"', and 1.7 eu for type atten- for I copper of fungal laccase. has Fungal laccase received attention center of this stry.51 Table trochemical fungal is another and laccase, copper comparable to has been protein 7, blue Figure also 10 as well thermodynamic respectively. tree containing laccase. The oxidase which type-1 copper studied by Table 5 summarize the as spectroelectrochemi- properties of blue copper Apparently, the most elec- center distinct in differ- ence between these two metalloenzymes is the huge difference of their redox potentials, as shown in Figure 11. However, there is no satisfactory explanation on this dramatic phenomenon. In tion, and change data general, structural from oxidized on for more various to multisite subtle foreign about changes reorganization However, adequate ing information reduced this of states, kind m solvation con forma- the proteins, could be extracted of approach following remains from the far from enzymes. Many additional experiments, manipulations, such as site ligands, and site-depleted active techniques, the involv- binding would be with re- 101 Table 5. Thermodynamic parameters for metalloproteins. 102 650' Metalloprotein Cytochrome c38 (mV vs. NHE) (kcal/mol) (eu) (eu) 260 -14.5 -28.5 -12.9 270 -13.9 -25.9 -10.3 39 -5.4 -15.1 +0.5 95 -8 .6 -21.1 -6 .0 324 -15.0 -25.2 -9.6 276 -15 .9 -31.8 -16 .2 (Horse Heart) Cytochrome c49 •50 (Tuna) Cytochrome c' 37 (Rhodospirillum rub rum) Cytochrome c' 3 7 ( Rhod o pseudomonas Palustris) Cytochrome c2 38 ( Rhodospirillum rubrum) Cytochrome c551 38 (Pseudomonas aeruginosa ) 103 Azurin 38 308 -16.6 -31.7 -16.1 360 -13.7 -18 .0 -2.4 191 -10.3 -19.8 -4.2 780 -22.1 -13.9 +1.7 452 -12.2 -6.0 +9.6 352 -15.8 -25.5 -10.0 60 -13.0 -39.2 -23 .6 160 -15 .0 -38 .0 -22 (Pseudomonas aeruginosa) P1astocyanin 38 ( Phaseolus vulgaris) Stellacyanin 38 (Rhus vernicijera) Laccase 51 (Poly porus versicolor) La cease* (Rhus vernicijera) HiPIP 38 (Chromatium vinosum) Myoglobin 52 (Sperm Whale) 54 Hemoglobin 53 • • This thesis work. 104 Table 6. Comparison other proteins. of spectral, thermodynamic formal properties reduction of some potential, and blue copper 610 614 604 597 4900 5700 4090 4500 5700 625 color (nm) the number in parentheses is the one extrapolated to 25 °C. •For tree laccase in the temperature range of 6.4 - 18.1 °·C; (Poly porus versicolor) La cease (Rhus vernicifera) La cease (Rhus vernicifera) Stcllacyanin ( Phaseolus vulgaris) Plastocyanin aeruginosa) (Pseudomonas Azurin Metalloprotelns E).max (M- 1cm- 1 ) ).max or blue 780 t 3 452 t 2 191 t 2 360 t 2 Unknown Unknown [Cu(N(His)) 2(S(Cys))2) Probably [Cu(N(His)) 2S(Met)S(Cys)) [Cu(N(His)) 2 S(Met)S(Cys)) blue copper site (mV n . NHE) 308 t 2 structure or E 0 'at25°C Coordination -22.1 t 0.5 ( -5.6 t 0.4) (32.5 t 1.2)• 1.7 t 2 - 12.2 t 0.4 -10.3 ! 0.4 -13.7 t 0.4 -16.6 t 0.4 • AH 0 '(kcal/mol) 9.6 t 1.2 -4.2 t 1.2 -2.4 t 1.2 -16.1 t 1.2 65 0' et (eu) Vt 0 - 106 Table 7. Temperature dependence of E o ", the copper for (fungal) bl ue laccase, spectrochemistry. phosphate buffer. Solution using the formal · IOn · m reduction potentials, Po I yporus · l or versrco nonisothermal conditions: pH = thin-layer 5.4, 200 mM, 107 Temperature (° C) * E 0 ' (mV vs. NHE) t 7.2 778 12.6 780 17.0 779 20.8 781 25.4 781 30.6 781 35.2 778 40.8 782 * :!: 0.2 °c t:!: 2 mV 108 Figure 10. Temperature dependence Eo, , the for Poly porus type-1 versicolor, spectroelectrochemistry. = 200 40.8 mM, °C. phosphate of the formal reduction potentials, copper center in using nonisothermal Solution conditions: pH 5.4, buffer. Temperature range: 7.2 fungal laccase thin-layer A least squares fit of the data gave an E 0 ' 780 ± 2 mV at 25 °C and (dE 0 '/dT) 25 o C = 6.13 x I0- 5 V;° C. J.l 109 800 I I I I 796 - - 792 f - - w 788 - - z 784 - - I . (/) > > --w E 780 - ' " ~ - I [' 776 - - 0 772 f - - 768 f - - 764 - I I I I 9 19 29 39 TEMPERATURE (°C) 49 110 Figure 11. Temperature E o", dependence for the type-1 vernicifera (!::. and versicolor (X). of the copper 0) and formal cen t ers in reduction · m fungal t ree potentials, 1accase laccase Rh us Polyporus Ill 958 888 -w 818 748 I z 678 CJ) > 608 > E 538 --w 0 468 328 10 20 30 TEMPERATURE (°C) 40 50 112 quired in order mechanism of to extract cooperation further crucial information concerning the among different centers in multisite me- the blue copper center the enzyme, 40 hence talloenzymes. There are two points I can make: (1) Kinetic in evidence suggests that tree laccase is buried deep in protein solvation effects play a much less important role than that of conformational change. (2) The conformation of the polypeptide backbone of tree laccase is probably very much temperature dependent. There are several experiments which would need to be formal done to make the whole picture more complete: (1) A mediator potential dependence study of the the copper of tree of blue reduction Ferri- laccase. cyanide has been used intensively in the thermodynamic as well as kinetic evidence 11 some ligand of studies however this complex copper ions. will 3 these redox this media tor. According the mediator for ricyanide is least for the ions. The will tree which type sites of bind the the by to criteria the choice studies of type couple for 2 and the type cyano and properties of the presence of this fer- purpose, type is of choosing studies, [Co(phen) 3]3+/ 2+ is 2 spectroelectrochemical good redox to There that changed a not shows Obviously, be laccase. at 3 copper a poten- 113 tial candidate reduction for replacing potential ferricyanide. [Co(phen) 3]3+/ 2+ of The ca. is formal 401 mY vs. NH£ 41 and the complex is known not to bind to the enzyme. (2) A study of the temperature dependence of the blue copper redox potential laccase. The typical of sible to type of the type 2 depleted 2 copper displays an ESR spectrum is an axial Cu(II) complex, 7' 11 and solvent and small anions. number A tree accesof re- ports on the type 2 depleted enzyme have also appeared in literature of tree laccase 42 - 45 ' mod if ica tion of the method the slight in and using Gray's a laboratory has resulted in greater than 95% removal of the type 2 tree laccase. 46 copper in dica te that structure, blue so copper type it 2 copper would redox Recent be NMR stabilizes of experiments inthe interest thermodynamics of tree to type laccase study 2 the depleted tree laccase. (3) Determination of the thermodynamic properties and mal reduction potential of type 3 copper pair. for- 114 References 1. Adman, E. T., in Topics in Molecular and Structural Biology, Volume 6, Metalloproteins, Part 1: Metal Proteins with Redox Roles, Harrison, P. M, ed., MacMillan Press, London, 1985, P. 1. 2. Reinhammar, B., in Copper Proteins and Copper Enzymes, III, Lontie, R., ed., CRC Press, Boca Raton, 1984, P. 1. 3. Gray, H. Volume 3, 1981, P. 1. 4. Lappin, A. G., Copper Proteins, P. 15. 5. Reinhammar, B., and Malmstrom, B. G., in Metal Ions in Biology, Volume 3, Copper Proteins, Spiro, T. G., ed., Wiley, New York, 1981, P. 109. 6. Beinert, H., Coord. Chern. Rev., 33, 55 (1980). 7. Reinhammar, B., in Advances in Inorganic Biochemistry, Volume I, Eichhorn, G. L., and Marzilli, L. G., eds., Elsevier, New York, 1979, P. 91. 8. Malmstrom, B. G., in New Trends in Bio-inorganic Chemistry, Williams, R. J. P., and Da Silva, J. R. R. F., eds., Academic Press, New York, 1978, p. 59. 9. Beinert, H., Coord. Chern. Rev., 23, 119 (1977). 10. Holwerda, R. A., Wherland, A., and Gray, H. B., in Annual Review of Biophysics and Bioengineering, Volume 5, Mullins, L. J., ed ., Annual Reviews Inc., Palo Alto, 1976, P. 363. 11. Fee, J. A., in Structure and Bonding, Volume 23, Dunitz, J. D., Hemmerich, P., Holm, R. H., Ibers, J. A., J¢rgensen, C. K., Neilands, J. B., Reinen, D., and Williams, J. P., eds., SpringerVerlag, Berlin, 1975, P. I. 12. Osterberg, R., Coord. Chern., Rei'., 12, 309 (1974). 13. Malkin, R., and Malmstrom, B. G., in Advances in Enzymology and Related Areas of Molecular Biology, Volume 33, Nord, F. F., ed., Wiley, New York, 1970, P. 177. B., and Solomon, E. Copper Proteins, Spiro, in Metal Ions Sigel, H., ed., 1., T. in G., Metal Ions ed., Wiley, Volume in Biology, New York, in Biological Systems, Volume 13, Marcel Dekker, New York, 1981, 115 14. Gray, H. B., in Advances in Inorganic Biochemistry, Volume 2, for Determining Metal Ion Environments in Proteins: Methods Structure and Function of Metallo proteins, Darnall, D. w., and Wilkins, R. G., eds., Elsevier, New York, 1980, P. I. 15. Malmstrom, B. G., Pure Appl. Chern. , 24, 393 (1970). 16. Malmstrom, (I 960). 17. Cass, A. E. G., Copper Enzymes, Raton, 1984, P. 63. 18. Cass, A. E. G., and Hill, H. A. 0., in Ciba Foundation Symposium 79, Biological Roles of Copper, Excerpta Medica, Amsterdam, 1980, P. 71. 19. Solomon, E. 1., Hare, J. Sci. USA, 73, 1389 (1976). 20. Guss, J. M., and Freeman, H. C., J. Mol. Bioi., 169, 521 (1983). 21. Colman, P. M., Freeman, H. C., Guss, J. M., V. A., Ramshaw, J. A. M., and Venkatappa, 319 (1978). 22. Adman, E. T ., and Jensen, L. H ., Isr. J. Chern., 21, 8 (1981). 23. B. G., and Vannga rd, T., Mol. J. Bioi., 2, 118 and Hill, H. A. 0., in Copper Proteins and Volume I, Lontie, R ., ed., CRC Press, Boca W., and Gray, H. Proc. B., Nat!. Acad. Murata, M, Norris, M. P., Nature, 272, Sieker, L. C., and Jensen, L. H., Adman, E. T., Stenkamp, R. E., J. Mol. Bioi. , 123, 35 (1978). 24. 25. Peisach, J ., Levine, 242, 2874 (1967). Hill, H. A. 0., W. G., and Blumberg, and Lee, W. K., and Vannga rd, J. W. E., Inorg. J. Bioi. Chern., Biochem., 11, 101 (I 979). 26. Branden, R., Malmstrom, 36, 195 (1973). Eur. J. Biochem., 27. Dooley, D. M., Scott, R. A., Ellinghans, J., Solomon, Gray, H. B., Proc. Nat!. Acad. Sci. USA , 75, 3019 (1978). E. 1., 28. Petersson, L., Angstrom, Acta, 526, 311 (1978). 29. Reinhammar, B., Malkin, R ., Jensen, P., asson, L.-E., Aasa, R., Vannga rd, T., and Bioi. Chern. , 255, 5000 (1980). B., J., Ehrenberg, T., A., Biochim. and Biophys. Karlsson, B., AndreMalmstrom, B. G., J. 116 30. Andreasson, L.-E., 568, 145 ( 1979). and Reinhammar, B., Biochim. Biophys. Acta, 31. Andreasson, L.-E., 445, 579 (1976). and Reinhammar, B., Biochim. Biophys. Acta, 32. The acetone powder extract of the latex of the Japanese lacquer tree can be ordered from: Saito and Co., Ltd. No. 9, Hommachi 1-chome Higashi-Ku, Osaka Japan 33. Reinhammar, B., Biochim. Biophys. Acta, 205, 35 (1970). 34. Kalthoff, (1935). I. M., and Tomsicek, W. J., J. 35. Alexander, J. (1968). A., and Gray, H. B., J. Am. Chem., Soc., 90, 4260 36. Gray, H. (1963). and J. Am. Soc., 37. Taniguchi, V. T., Ellis, W. R., Jr., Cammarata, V., Webb, J., Anson, F. C., and Gray, H. B., in Advances in Chemistry Series, No. 201, Electrochemical and Spectrochemical Studies of Biological Redox Components, Kadish, K. M., ed., American Chemical Society, Washington, D. C., 1982, P. 51. 38. Taniguchi, V. T., Sailasuta-Scott, N., Anson, H. B., Pure and Appl. Chern., 52, 2275 (1980). 39. Yee, E. L., Cave, R. J., Guyer, K. L., Tyma, P. D., and Weaver, M. J., J. Am. Chem. Soc .. 101,1131 (1979). 40. Holwerda, R. (1975). 41. Farina R., and Wilkins, R. G., 1norg. Chern .. 7, 514 (1968). 42. Graziani, M. T., Lett., 70, 87 (1976). 43. Morpurgo, L., Desideri, Lett., 113, 153 (I 980). 44. Morpurgo, L., Graziani, M. T., Agro, Mondovi, B., Biochem.5 J. , 187, 367 (1980). B., A., Beach, and N. A., Gray, H. Morpurgo, A., L., B., J. Chern., Chem. F. C., Am. Chem. Rotilio, Rotilio, Phys. 85, and 945 2922 Gray, Soc., 97, 6063 Mondovi, B., FEBS Mondovi, B., FEBS Rotilio, G., G., G., 39, A. F., 117 45. Reinhammar, B., and Oda, Y., J. Inorg. Biochem., 11, 115 (1979). 46. Dooley, D. M., unpublished results. 47. Nakamura, T., Biochim. Biophys. Acta, 50, 44 (1958). 48 , Reinhammar, B. R. M, Biochim. Biophys. Acta, 275, 245 (1972). 49. Margalit, (1973). 50. Margalit, R., and Schejter, A., FEBS Lett., 6, 278 (1970). 51. Taniguchi, V. T., Malmstrom, B. G., Anson, Proc. Nat/. Acad. Soc. USA, 79, 3387 (1982). 52. Mayo, S. L., Ellis, W. R., B., Science, 233, 948 (1986). 53. George, P., Hanania, G. I. H., and Eaton, W. A., in Hemes and Hemoproteins, Chance, B., Estabrook, R. W., and Yonetani, T., eds., Academic Press, New York, 1966, P. 267. 54. Hanania, G. I. H., Ph.D. Cambridge, England, 1953. R., and Schejter, A., Jr., Eur. Crutchley, Thesis, Biochem., J. F. R. 32, C., Gray, J., and University of 492 H. B., Gray, H. Cambridge, 118 CHAPTER IV REDOX CHEMISTRY OF NATIVE, SINGLY, AND DOUBLY PENT AAMMINERUTHENIUM-MODIFIED BACTERIAL AZURIN 119 Introduction Blue copper center, and a molar absorption are small value their EPR di-valent blue are proteins containing also coefficient paramagnetic of the in inorganic copper Cu(II) the oxidized in Both this of is these of form, I copper properties protein and copper center values similar to of main hyperfine are The absorption some times larger an unusually A II• in to the atypical of constant, are complexes. 100 10 4 M- 1 cm- 1. l,2 with splitting simple copper amino acid or peptide complexes. the type 10 3 to of hyperfine class Cu(II) center per parallel spectra. 1•2 copper the blue a have a characteristic in tense absorption near 600 nm with They simple proteins special quite coefficient than that of the found for The EPR spectrum of in proteins is also characterized by having g- of with a value approximately one half those simple splitting Cu(II) chelates, constant, AJ., but of that found for the simple copper complexes. Azurin, renamed by Sutherland and Wilkinson, 3 was originally reported as Pseudomonas blue protein 4 and It can be isolated from a bacterial transfer agent in the bacterial intense study by Gray and scopy,10-12 tended X-ray Absorption the reports of solid foundation Fine their crystal toward the source, respiratory co-workers in addition to NMR, 13 • 14 isolated by Horio. 5 was first on and acts as chain. 6 -9 A its an electron long electronic and spectro- EPR, 15 Resonance Raman, 16 Ex- Structure (EXAFS) 17 •18 structures 19 •20 ' understanding of experiments and have now provided a this single copper me- 120 talloprotein. based Figure on X-ray shows crystallographic with the sole copper shaped that molecule. ion The located copper the protein structure of azurin, studies, has strands of ,6-barre1 toward one end coordination tetrahedral, and from their known have identified. Two histidine been amino 8 of the cylindrically environment acid is sequences nitrogens and a close to four ligands cystein sulfur have normal bond lengths and a fourth ligand appears to be a sulfurcoordinated methionine. an ~ S-(Cys) The Cu(II) intense charge blue color transfer is accounted m transition for this by protein, whereas the unusual EPR properties are explained by the distortion of the Cu(II) ligand environment away from planarity. 21 In recent years, electron of approach there been much interest in the metalloproteins. 22 - 33 involving transfer to has this study consists of modifying talloproteins with redox-active kinetic and thermodynamic transfer between the inorganic label and inorganic parameters or complexes for mechanism A novel labelling me- and measuring intramolecular electron the native metal site in the protein interior. Pseudomonas aeruginosa azurin, 6 -9 a the main focus for long-range electron ing blue copper. amino daltons. It these bacterial consists of a a molecular acid residues and has It contains, in total, four single protein, studies involv- polypeptide chain of weight around 14,600 His-35, His-46, of histidines and His-117 - - in which His-46 and His-117 to copper in center. His-35 is buried been transfer His-83, the has the 128 are coordinated protein interior near 121 Figure I. The folding of the the copper ion, coordinating sidues from indicated. computer polypeptide This plots of ligands, ribbon the backbone drawing atomic based on X-ray crystallographic results. in and azurin with histidine re- was coordinates constructed in azurin, 122 123 the blue On the copper other site, hand, and appears His-83 to is exposed be at inaccessible to labelling. provid- the protein surface, with azurin 27 is ing an attractive site for modifier binding. The reaction 1, 24 Scheme [Ru(NH 3) 5(0H 2 )] 2 + of and produces two ruthenated (a 5 Ru) 2 -Az where "a" stands for NH 3 . acterized, by products, (a 5 Ru)-Az The (a 5 Ru)-Az experiments (scheme [Ru(NH 3 ) 5(His-83)]-azurin, a which been at His-83. the fully imidazole ring of characterized. The of an complex. Figure has However, absorption [Ru(NH 3 ) 5 (His-83:peptide)] 3+ spectrum protein fragment and 2),24 as singly-modified (a 5 Ru) 2 -Az has not been spectrum of the isolated virtually the same as is independently characterized 2 the illustrates in has been char- mapping peptide outlined [Ru(NH 3 ) 5 (His)] 3+ absorption spectra of the model these two species, respectively. Figure 3 shows the cross-sectional The two electron the blue copper [a 5 Ru(His-83)] 3+ for a of the protein and edge-to-edge distance and spectroscopic data, respectively, 27 which site perturbed is not The electron ruthenated transfer concentrations, Scheme 3 the list the show that the presence protein is ideally structured Selective reduction experiment. If the experiments then the electron the 2 of by group illustrates parts between Tables is surface-accessible molecular. selected A sites group. long-range along 11.8 tansfer thermodynamic view are kinetic will done transfer of at should results, the produce very low be intra- from flash 124 Scheme 1. Preparation and purification nium modified azurins. scheme of pentaammineruthe- 125 Excess (50 X) [asRu(OH 2 )] 2 + + Az (Cu 2 +) 1 pH7 3 hr dialysis !t\~excess [ ] a 5 Ru(OH2) oxidation [Co ( EDTA)]- CM-52 pH4 yield (0/o) 35 31 12 pi 5.4 7.2 8.2 2+ 126 Scheme 2. Peptide mapping of [Ru 111 (NH 3 ) 5(His-83)]-Az(Cu 11 ). 127 denature l trypsin digest monitor 220nm monitor 220nm HPLC Identify peptides HPLC Identify peptides containing [a 5 Ru(His)]3+ amino acid analysis 80 83 Val-lie-Ala-His-Thr-Lys I a 5 Ru3+ 128 Figure 2. Absorption spectra 83, i.e., of (A) the hexapeptide Vai-Ile-Aia-His-Thr-Lys, containing obtained by His- tryptic hydrolysis of the modified azurin and HPLC separation of the resulting peptide, and of [Ru(NH 3) 5 (His)]CI 3. (B) a 0.3 mM aqueous solution 129 (A) 83 Vol-lle-Aio-His- Ttw"-Lys w u z <I CD ~ (8) CD <I 400 WAVELENGTH (NM ) 500 130 Figure 3. View of selected azurin, with parts of a 5Ru 111 -bonded the to molecular the imidazole The copper ligands are His-46, Cys-112, 121. The edge-to-edge distance between transfer units, i.e., [a 5Ru(His-83)] 3+ copper center, is 11.8 A The skeleton of of His-83. His-117, and Metthe two electron group and the distance was calculated from N1 of the imidazole of His-83 to S of Cys-112. blue 132 Table 1. Thermodynamic [a 5Ru(His)] 3+ parameters and the pentaammineruthenium for the reduction blue-copper, Cu 11 , in modified azurins. tions: pH 7.0, J.l = 100 mM, phosphate buffer. native Solution of and condi- (kcal/mol) 6nD' (kcaljmol) 6G0 ' (eu) 6S 0ct' (cu) !!.So, (mY vs. NHE) Eo, Thermodynamic parameter ± 1.2 ± 1.2 ± 2 -16.6 :!: 0.4 -7.10 ± 0.05 -16.1 -31.7 308 Native Az(Cu2+/l+) :!: :!: 1.2 1.2 ± 2 -15.4 ± 0.4 -7.39 :!: 0.05 -11.2 -26.8 320 fa Ru(His-83) 3 +-Az(Cu2+/+) 5 5 :!: :!: 2 5 -3.0 ± 0.8 -1.96 ± 0.12 12.2 ± 2 -3.4 80 (a Ru(His)l3+/2+ ...... w w 134 Table 2. Spectroscopic properties of the blue-copper native and pentaammineruthenium modified azurins. sites in 135 Spectroscopy Parameter Native Az [a 5 Ru 111 (his-83) lAz(Cu11) EPR 8y Resonance v(cm· 1 ) Raman [CuN 2 SS *] 2.271 2.271 57 57 2.065 2.065 2.043 2.044 369 370 404 404 424 425 136 Scheme 3. The scheme study of the of flash kinetic intramolecular spectrometry long-range reaction in [a 5 Ru 111 (His-83)]-Az(Cu 11 ). used electron in the transfer 137 2• Ru(bpy) 3 flash Ru(bpy~· • 2• 2• 3• Ru(bpy) + Az -Ru 3 't:r • 3• + Ru(bpy)3 138 experiments, photolysis of the following intramolecular electron transfer reaction: Strict 1.9 first-order s- 1 that kinetics is are observed, yielding temperature-independent from a rate -8 to constant of °c, 29 as 53 demonstrated in Figure 4. long-range intramolecular The pentaammineruthenium modified electron transfer kinetics of have been investigated as azurins also a function of pH ranging from 5.0 to 9.0. Figures 5 and 6 show the pH-dependence rate of the electron (a 5 Ru)-Az and (a 5 Ru) 2 -Az, i.e., (a 5 Ru)-Az, However, ket respectively. [a 5 Ru(His-83)]-Az, of transfer (a 5 Ru) 2 -Az has constant, ket• for As shown in Figure 5, ket of been demonstrates found to vary pH-independent with pH. behavior, as depicted in Figure 6. Materials and Methods All the native as well as the singly and doubly modified azurins were prepared by Dr. Nenad M Kostic. Pseudomonas aeruginosa azurin, obtained from the Public Health Laboratory Service in England, has an A 280 ; A 625 ratio phenylethyl chloromcthyl from Worthington of and 1.72. Trypsin, treated ketone, and cyanogen Pierce Chemical Co., with L-1-tosylamido-2- bromide were respectively. obtained Ampholine 139 Figure 4. (A) First-order flash-generated kinetic plot for the reduction range Ru 11 ~ c}I electron transfer. Cu 11 in 23 °C, and rate constant for long- [a 5Ru 11 (His-83)]-Az(Cu 11 ) (B) temperature dependence of the of at 140 (a ) 4~ ·~ 3 ."• 2 3.0 ( b) 2.5 I 2.0 V> 1i; 1.5 1.0 0 .5 0~~--~--~--~~--~--~--~--~ 141 Figure 5. The pH-dependence Ru 11 ~ Cu 11 of the rate intramolecular in [a 5 Ruii(His-83)]-Az(Cu 11 ) at 23 constant electron °C. for long-range transfer reaction 142 6~--~----~--~----~--~~--~ 5 4 ~ ~ I ~ ~ 3 ~ m ~ 2 1 0 5 6 7 8 pH 9 143 Figure 6. The pH-dependence Run ~ Cu 11 of the rate intramolecular in (a 5 Ru 11 )2-Az(Cu II) at 23 °C. constant electron for long-range transfer reaction 144 6r----.----~--~~--~----~--~ 5 4 ~ I ~ ~ 3 ~ m ~ I 2 - 0 5 6 7 pH 8 9 145 plates for isoelectric focusing obtained from LKB. (IEF) in the pH range 3.5 - 9.5 were Column chromatography of the modified azurin was [a 5Ru(OH 2 )](PF 6 )2 was pre- carried out with Whatman CM-52 cellulose. pared according procedure 35 cation (Na 2 co3 was used instead of [a 5Ru(py)](Cl0 4 ) 3 , was prepared as described to a published with one Baco 3). 36 slight modifi- The complex previously. 3 7 Deionized water from a Barnstead NANOpure water purifier was employed in the preparation of all protein and performed with Amicon YM-5 ring at 5 buffer solutions. Ultrafiltration was membranes, under argon, with gentle stir- °C. Azurin prepared was allowed to [a 5Ru(OH 2 )](PF 6 )2, react with a 50-fold excess under argon, at room temperature, mM tris • HCl buffer (pH 7.2) for 3 hours. deaerated solution of 27 mg of the of freshly in 50 In a typical experiment, a ruthenium reagent in tris buffer was added anaerobically to a deaerated solution of 14 mg of azurin in the same buffer. The reaction evacuation and left under argon was not permitted gas The total volume was a mixture was deaerated constant pressure of bubble directly through to reagent ratio, affect the properties of only slightly. removed by To concentrations, the stop repeated argon, by although the solution. 10 ml, so that the concentration of the protein was 0.1 mM and of the ruthenium reagent, 5 mM. the briefly the pH, modified reaction protein reaction, ultrafiltration or of water or, 50 mM NH 40Ac buffer (pH 4.0). Small variations in the the and time affected did not the yields ruthenium reagent was reaction mixture into A solution of ca. 15 mg of 146 Na[Co(EDT A)] ca. ca. ml of 15-fold either excess with water or the respect to both Cu and buffer, ca. NH 4 0Ac Ru, in 15-fold excess with respect to both Cu and Ru, was added to oxidize the concentrated protein. the The oxidation unbuffered oxidant was was allowed or overnight solution, then removed by to proceed in the ultrafiltration for several NH 4 0Ac into the hours buffer. pH 4.0 in The acetate buffer. Recovery of the protein from the reaction was 98 - 100%. All efficiently three components of by cation-exchange the reaction mixture were chromatography on a cellulose, previously equilibrated with pH 4.0, 50 mM NH 40Ac buffer. The separation rate was ca. was 100 carried ml/hr. out at The room first column separated temperature fraction was and eluted of CM-52 the elution quickly with 50 mM buffer and elution with this buffer continued until the second band reached the bottom of the column. This second fraction was then eluted with pH 4.0, 75 mM NH 4 0Ac buffer. The third fraction, which had remained near the top of the column, was then eluted with 150 mM NaCl solution in pH 4.0, 50 mM NH 40Ac buffer. The average yields are follows: fraction, first fraction, 34%, and third i.e., pi values in second fraction, 7.2, change after ling reaction third fraction 4.0 fraction, pH mg or 27%, 2.7 mg or units, are the and 18%, third fraction, exhaustive dialysis by was allowed to proceed increased to ca. fraction decreased to ca. 22%. 28%, second and the first 8.1. pi ultrafiltration. for 5 - whereas values When 5.4, did the of not label- the yield yield or points, fraction, 7 hours, the mg isoelectric following: The 4.9 of second 147 Procedures peptides by for tr yptic reversed-phase hydrolysis, HPLC, and separation amino of the ana1ysis 27 •3 3 acid used with minor modifications. A sample containing 5 - second fraction, water, sample in boiling water for 4 linear gradient for the HPLC The in 2 ml of was denatured minutes prior separation to of resulting 6 mg of the by placing tryptic the were the hydrolysis. tryptic peptides consisted of 45 ml each of phosphate buffer (49 mM KH 2 Po 4;5.4 mM H 3 Po 4 , pH in- creasing from absorbance and at 2.85) and 0% to acetonitrile, the 45%, the concentration elution rate nm, values were recorded at nm, which is the absorption 300 near 220 of the was i.e., the latter ml / min. The peptide bonds, maximum, 303 nm, of parallel on hydrolyzed by [a 5 Ru(His)] 3 +. 38 All third peptide fraction mapping and experiments native azurin. were performed Protein samples were trypsin or by cyanogen bromide, CNBr. The solution of the denatured protein was 25 mM in NH 4 Hco 3 and had a pH of 7.8. of the digestion in At the beginning 5 hours, 50 ml portions of a solu- and again after tion containing 2 mg trypsin per ml were added. For hydrolysis by CNBr, 3 mg of the third fraction in 1.5 ml of water, were lyophilized of 70% HCOOH. to dryness and the residue dissolved in ml A small lump of CNBr was added and the reaction allowed to proceed at room temperature in the dark for 16 hours. sample was then diluted with 10 dryness. Homoserin'e lactone was published procedure. 39 A mg 1.8 ml of water converted sample of and The lyophilized to by a to homoserine the protein, which had 148 been treated with CNBr, was dissolved in 0.5 ml of a I 00 mM solution of pyridine in acetic acid at pH 6.48. The solution was heated in a oc for one hour, the solvent then removed by an air stream at 40 oc and the residue dissolved in 0.1 sealed evacuated tube at 100 ' aqueous DF 3 COOH. ml of 0.1% The HPLC separation of peptide was carried out in a similar manner to that of the second fraction. The phosphate 0.1% buffer and the CH 3CN solutions aqueous CF 3 COOH instead of pure water. were prepared using Absorbance of the eluent was monitored at 220, 270, or 300 nm in different separations. Formal native as reduction well as azurins, were tically transparent potentials singly determined at configuration. described in Chapter pH added all the The II. protein a gold minigrid path length was 0.48 mm. temperatures, modified 6.0, an op- a non- have been [a 5 Ru(py)](Cl0 4 ) 3 was by using electrode (OTTLE) cell in details this and of equivalents as as the Potentials Princeton for pentaammineruthenium 5.0 solutions employed by using a 7.0, Five cell layer cell different doubly thin-layer isothermal to and at methodology of mediator-titrant. The OTTLE working and optical were Applied electrode, applied Research across model the 174A thinpolaro- graphic analyzer, and were accurately measured with a Keithley model 177 was varied by directly with microvolt using a digital variable multimeter. temperature The cell cell holder, temperature and measured an Omega Engineering precision microthermocouple connected to a Fluke model 2175A placed in the digital thermometer. protein solution The m close end of the proximity thermocouple to the was thin-layer 149 cavity. All visible spectra were obtained potentials were potentials, Eappl., with a Cary 219 recording spectrophotometer. Formal applying reduction a series Each trod e. that the to reduced of potential was maintained to value of forms of all redox as defined by the incrementally converted from one oxidation potentials, for which series of applied [Ox]/[Red] was determined tials n values and were by gold electrolysis of concentrations couples in solution, the equation. state each of so oxidized was [Ox]/[Red], Redox couples to other the Formal from elec- ceased, corresponding spectra. determined sequentially minigrid ratio Nernst from the until equilibrium established the determined by the value of reduction plots of were poten- Eappl. vs. log([Ox] / [Red]). All cycling solutions on a deoxygenated vacuum / purified-argon the OTTLE The platinum containing were cell by wire using rubber auxiliary deoxygenated prior double septum electrode use by manifold, caps was media tor-titrant to and and loaded syringe situated solution vacuum/argon in a that into techniques. compartment was isolated from protein solution by a porous glass frit. Results and Discussion Pseudomonas methionines per coordinated to ae ruginosa molecule. the copper azurin contains four Because His-46, His-117, atom, two histidines histidines and and five and six Met-121 are methionines 150 are potential sites of covalent binding to the a 5 Ru-group. In the eluted case of the second 12 ml in the ca. at significantly at contains 300 nm, HPLC labelling takes place at protein fraction. The amino fragment to onine, 85 and of contain lysine, of we concluded [a 5 Ru(His)] 3+ unit, an fraction, as a the shown acid valine, a composition Pseudomonas aeruginosa This Ru-N denaturation the sample at this Figure analysis that fits exactly azurin. 39 We the this showed alanine, that absorbs indicating in also peptide peptide fragment 2, isoleucine, to the hydrolyzate residue bond in [a 5 Ru(His-83)]-Az of only in histidine only trypic that pentaammineruthenium group is attached 83. since that particular this peptide histidine, thre- the segment conclude 80 that imidazole ring of a His- is evidently not affected by °C, nor by the conditions under 100 which tryptic hydrolysis is carried out. The structure that the imidazole of His-83 and protrudes imidazole of into easily Several aspartic vicinity of 19 •20 azurin, acid His-83, shown is located on the surrounding accessible as to This labelling and glutamic acid their negative charges attack of [a 5 Ru(OH 2 )] 2+ at His-83. Figure the surface of medium. the In are presumably A apart. the skeleton of selected makes in the solution. found in the facilitate the The N 1 atom in the imidazole ring of His-83 and the S atom of Cys-112 are 11.8 view reveals the protein exposure reagent residues 1, parts of the molecular the third fraction, Figure 3 shows of this modified azurin. In the case of the tryptic peptide whose 151 composition corresponded aerugionsa azurin istic the of to the exhibited segment strong [a 5Ru(His)] 3+ absorbance The peptide whose of the above. 27 300 nm. at 35, the only the copper labelled, atom. as located segment 28-41 This evidence histidine other The evidenced nm, as described absorbance 300 His-83 modified the Pseudomonas at is to of This a 5Ru-label corresponded at 85 chromophore. imidazole-bound azurin 80 establishes in protein rules out that the not singly- ex hi bi ted a 5Ru-binding is the composition to His-83 that two histidines bound to copper the UV-VIS, EPR, and resonance by than character- no His- coordinated are to not Raman spectra of the blue copper site. Attempts to chain were side absorb than directly I 000 ' (£ nm 41 ' where overwhelming (£ ruthenium unsuccessful. 40 appreciably 290 detect ~ the bonded to a methionine The M- 1 cm- 1 ) only at wavelengths due to the protein For this absorbance 10 000 M-l ' lower itself is reason, chromatograms of the corresponding tryptic peptides from native azurin and from the third fraction to CNBr, a are very similar methionine-specific to each other. Next we hydrolysis reagent. We first under conditions used that [a 5RuS(CH 3)2] is stable and HPLC separation. Experiments with CNBr were pectation that the ruthenium methionine to which it is bound . the methionine residues in the label should retard turned established for hydrolysis based on the excleavage at the Although the CNBr did cleave at all both native this cleavage was less than complete for azurin and the third fraction, both proteins so that no re- 152 tardation could labelling of experiments. A methionine re- sidues, one of which, Met-112, is coordinated to the copper atom. Of the located methionine molecule be could of be are five, copper in four ture dependence, from are the Pseudomonas were electrochemical duction data potentials possible peptide-mapping azurin contains near the The six protein surface and reduction potential of aeruginosa azurin, investigated 100 mM phosphate buffer. 42 7.0, about [a 5Ru(OH 2 )] 2+. to native conclusion aeruginosa accessible blue No drawn Pseudomonas remaining probably found. and its tempera- spectroelectrochemically in pH Table 3 and Figure 7 summarize the of this experiment. In this chapter, and their temperature dependence of formal native re- azurin at pH 6.0 (Table 4, Figure 8), and at pH 5.0, (Table 5, Figure 9); of [a 5Ru(His-83)]-Az at (Table 8, Figure 12); as Figure The 14) and at phosphate acetate buffer pH 7.0 was was used 7, Figure well as of (a 5Ru) 2 -Az pH 5.0 buffer (Table (Table 11, Figure 11), at and pH 15) have used at pH 7.0 and at instead at pH 5.0. In every at 7.0 pH 5.0 (Table 10, been presented. pH 6.0, however, case, the ionic of native 13, Table strength was maintained at 100 mM. The azurin redox (Figure potentials 10, Table 9), and of (a 5Ru) 2 -Az have also plots, we been can and thermodynamic 6), [a 5 Ru(His-83)]-Az (Figure summarized, observe that the of 16, Table 12) at pH respectively. redox parameters From potentials (Figure 7.0, 6.0, these become and 5.0 comparative more positive as the pH values of protein solutions get lower. This phenomenon can be When understood in terms of simple electrostatics. the acidity of 153 Table 3. Temperature Eo " , azurin, stry. buffer. for dependence the using Solution of the formal reduction potentials, bl ue-copper ion in Pseudomonas aeruginosa nonisothermal thin-layer spectroelectrochemi- conditions: 7.0, 100 pH 1..1 = mM, phosphate 154 Temperature (° C) * E 0 ' (mV l'S. NHE) t 6.8 320 10.5 319 10.9 319 15.1 318 16.1 313 20.0 312 24.9 309 25.0 309 30.3 303 35.2 300 40.8 299 * :!: 0.2 °c t:!: 2 mV 155 Figure 7. Temperature dependence E 0 ' , for the type- I osa azurin, trochemistry. using Solution mM, phosphate buffer. of the formal reduction copper center in Pseudomonas aeruginnonisothermal thin-layer = 7.0, Temperature range: 6.8 conditions: pH A least squares fit of the data gave an Eo, at 25.0 potential, spectroelec~ 100 - 40.8 0 c. = 308 ± 2 mY °C and (dE 0 '/dT) 25 o C = -7.00 x 10- 4 V /° C. 156 330 326 -w I z 322 I"- 318 . 314 (j) > 310 .... 0 w 302 290~----~----~----~----~----~ I 10 19 28 TEMPERATURE ( °C) 37 46 157 Table 4. Temperature E o ', for dependence the azurin, using stry. Solution buffer. of the formal reduction potentials, blue-copper · ton m P seu d omonas · aerugmosa nonisothermal thin-layer spectroelectrochemi- conditions: 6.0, 100 pH 1..1 mM, phosphate 158 Temperature (° C) * E 0 ' (mV vs. NHE) t 3.9 333 9.7 329 15.2 327 20.2 324 25.0 325 30.2 320 34.2 318 41.6 316 * :!: 0.2 °c t:!: 2 mV 159 Figure 8. Temperature dependence E 0 ", for type-1 copper center in using nonisothermal thin-layer osa the azurin, trochemistry. phosphate of the Solution conditions: buffer. Temperature formal pH range: reduction Pseudomonas aerugin- 6.0, = °C and (dE 0 , /dT) 25 o C = -4.44 x 10- 1-1 3.9 least squares fit of the data gave an E 0 " 25.0 potential, spectroelec- = 100 41.6 = °C. mM, A 323 ± 2 mV at 4 V j° C. 160 345 341 --w 337 333 I z 329 (/) > 325 > --w E 321 0 317 313 309 30~2 8 18 28 TEMPERATURE(°C) 38 48 161 Table 5. Temperature E o ', for dependence the azurin, using stry. Solution buffer. of the formal reduction potentials, blue-copper · ton · tn Pseu d omonas · aerugznosa nonisothermal thin-layer conditions: 5.0, pH 1..1 spectroelectrochemi= 100 mM, acetate 162 Temperature (° C) * E 0 ' (mY Ys. NHE) t 3.3 363 9.6 365 10.6 359 15.3 362 17.8 368 19.8 364 22.8 369 25.4 372 28.8 372 32.3 374 35.2 376 40.2 376 * ± 0.2 °c t ± 2 mV 163 Figure 9. Temperature E 0 ", for osa azurin, the trochemistry. acetate dependence of the type-1 copper center using nonisothermal Solution conditions: buffer. formal Temperature in pH range: reduction potential, Pseudomonas aerugin- thin-layer = 5.0, 3.3 spectroelec- 1.1 100 mM, 40.2 °c. A . o f t h e d ata gave an E 0 " = 370 -+ 2 mV at Ieast squares f 1t 25.0 ° C and (dE 0 "/dT) 25 0 C = 4.66 x 10- 4 V /° C. 164 388 384 - 380 w 376 I z I 372 (/) > > E -w 0 368 I I I I 364 360 356 352 34~3 7 17 27 TEMPERATURE (oC) 37 47 165 Figure 10. Temperature E0 ', for osa azurin (X). dependence the the type- I copper at pH (.1), Solution (pH 6.0, of pH 7.0 conditions: 7.0) or acetate [Ru(NH 3) 5(py)](Cl0 4 )3 . center at IJ formal potential, Pseudomonas aerugin- ( •), pH in pH 6.0 100 reduction mM, buffer (pH and at phosphate 5.0), 5.0 buffer 5 equivalents 166 398 383 -368 w 2 353 ({) > 338 > -E .0 323 w 308 293 278 263~----~----~----~------~--~ -7 5 17 29 TEMPERATURE (°C) 41 53 167 Table 6. Thermodynamic blue-copper at three phosphate 25 °C. parameters center pH in values. buffer for the reduction native Pseudomonas Solution conditions: (pH 6.0, pH 7.0), acetate of the type-1 aeruginosa azurin ~ = 100 mM, buffer (pH 5.0), 168 E 0 " at 25 °C Native Az Native Az Native Az pH 7.0, 0.1 M pH 6.0, 0.1 M pH 5.0, 0.1 M Phosphate Phosphate Acetate 308 323 370 -31.7 -25.8 -4.9 16.1 -10.2 10.7 -7.10 -7.45 -8 .53 -16.6 -15 .2 -10.0 (mY vs. NHE) (eu) (eu) (kca1 / mo1) (kca1 / mo1) 169 Table 7. Temperature dependence of the formal bl ue-copper ion in Eo ' , for from Pseudomonas the aeruginosa, spectroelectrochemistry. 100 mM, phosphate buffer. using Solution reduction potentials, [Ru(NH 3 )5 (His-83)]-azurin nonisothermal conditions: pH thin-layer 7.0, 1..1 170 Temperature (° C) * E 0 ' (mV vs. NHE) t 6.6 330 9.8 330 15.5 322 20.7 322 25.0 315 30.0 323 34.7 318 40.2 311 * :t 0.2 °c t :t 2 mV 171 Figure 11. Temperature E 0 ", for azurin dependence the type-1 of copper Pseudomonas from the formal center aeruginosa, thin-layer spectroelectrochemistry. pH = 7.0, 1J range: 6.6 - 40.2 = 320 :!: 2 mV at 25.0 gave an E 0 " 100 4.86 x 10- 4 V / 0 C. °C. mM, A phosphate least reduction in potential, [Ru(NH 3) 5(His-83)]using nonisothermal Solution conditions: buffer. Temperature squares fit of the data °C and (dE "/dT) 25 o C = 0 172 341 337 --w I z CJ) > > 333 329 I 325 I 321 E --w 317 I 0 313 I ~ 309 I 305 3011 10 19 28 TEMPERATURE (°C) 37 46 173 Table 8. Temperature dependence of the formal bl ue-copper ion in Eo " , for from Pseudomonas the aeruginosa, spectroelectrochemistry. 100 mM, acetate buffer. using Solution reduction potentials, [Ru(NH 3 )5 (His-83)]-azurin nonisothermal conditions: pH thin-layer 5.0, 1.1 174 Temperature (° C) * E 0 ' (mV vs. NHE) t 9.8 356 13.2 356 19.4 354 21.0 356 26.0 358 29.8 354 35.6 356 * ± 0.2 °c t ± 2 mV 175 Figure 12. Temperature E 0 ", for azurin dependence the from thin-layer pH 5.0, 35.6 type-1 of the copper Pseudomonas formal center aeruginosa, spectroelectrochemistry. IJ °C. in reduction potential, [Ru(NH 3) 5(His-83)]using nonisothermal Solution conditions: 100 mM, acetate buffer. Temperature range: A least squares fit data gave of the an 9.8 - E0 " = 356 :!: 2 mY at 25.0 °C and (dE 0 "/dT) 25 o C = -4.37 x 10- 6 V /° C. 176 376 I I I I 372 - - 368 - - -364 - - z 360 - - w :r: I (f) > 356 > -E --. 352 - - w 348 - - 344 - - - - ... 0 I I I l 9 18 27 36 TEMPERATURE (°C) 45 177 Figure 13. Temperature E0 ', for dependence the type-1 of the copper formal center in reduction potential, [Ru(NH 3) 5(His-83)]- azurin from Pseudomonas aeruginosa at pH 7.0 (ll) and at pH 5.0 (X). buffer (pH Solution conditions: 7.0) acetate or [R u(NH 3) 5(py )](ClO 4 )3. 100 1.1 buffer (pH mM, 5.0), phosphate 5 equivalents 178 383 371 -359 w :r: ~-----4~~~~--~----~------~ z 347 (/') > 335 > -E 323 w 311 ... 0 299 287 8 18 28 TEMPERATURE (°C) 38 48 179 Table 9. Thermodynamic parameters blue-copper center in Pseudomonas aeruginosa at tions: 1.J 100 buffer (pH 5.0), 25 mM, °C. for the the type-1 [R u(NH 3 )5 (His-83 )]-azurin from two phostate pH reduction values. buffer of Solution (pH 7.0), condiacetate 180 (a 5 Ru)-Az (a 5 Ru)-Az pH 7.0, 0.1 M pH 5.0, 0.1 M Phosphate Acetate 320 356 -26.8 -15.7 -11.2 -0.1 -7.39 -8.20 -15.4 -12.9 (mV vs. NHE) (eu) (eu) (kcal/mol) (kcal/mol) 181 Table 10. Temperature dependence of the formal reduction potentials, E 0 ", for the blue-copper ion in doubly pentaammineruthenium modified thin-layer Pseudomonas aeruginosa azurin, spectroelectrochemis try. pH 7.0, 1.1 = 100 mM, phosphate buffer. using nonisothermal Solution conditions: 182 Temperature (° C) * E 0 ' (mV vs. NHE) t 4.1 349 8.6 349 14.0 340 16.2 342 20.6 340 25.0 335 31.0 324 34.0 317 34.4 317 39.3 309 * :!: 0.2 °c t:!: 2 mV 183 Figure 14. Temperature dependence E 0 ", for the type-1 thenium modified isothermal of the formal reduction potential, copper center in doubly pentaammineru- Pseudomonas aeruginosa azurin, using thin-layer spectroelectrochemistry. non- Solution = 7.0, 1J = 100 mM, Temperature range: 4.1 - 39.3 °C. A least squares fit of the E O" 330 ± conditions: d ata pH gave an = (dE 0" /dT)25 0 c = -1.17 X 10 -3 vI 0 c. 2 mY phosphate at 25.0 buffer. °C and 184 365 -w I z 357 349 . 341 (/) > > 333 E -325 - 0 w 317 309 301 44 185 Table 11. Temperature dependence of the formal reduction potentials, E 0 ' , for the blue-copper ion in doubly pentaammineruthenium modified thin-layer Pseudomonas aeruginosa azurin, spectroelectrochemistry. pH 5.0, IJ = 100 mM, acetate buffer. using nonisothermal Solution conditions: 186 Temperature (° C) * E 0 ' (mY vs. NHE) t 4.0 377 11.2 371 15.1 370 20.0 373 25.2 371 30.6 365 * ± 0.2 °c t ± 2 mY 187 Figure 15. Temperature dependence E 0 ", for the type-1 thenium modified of the formal reduction copper center in doubly pentaammineru- Pseudomonas aeruginosa azurin, using non- isothermal thin-layer spectroelectrochemistry. conditions: pH 5.0, 1-1 Temperature range: 4.0 - 30.6 °C. the E O" = 369 ± -4 vI 0 c. (dE d ata 0 , gave an potential, /dT)25 0 c = -3 .28 X 10 = 100 mM, acetate Solution buffer. A least squares fit 2 mY at 25.0 °C of and 188 386 383 - 380 w 377 I z en > > --w I I 374 371 E 368 0 365 362 359 3560 7 14 21 TEMPERATURE (oC) 28 35 189 Figure 16. Temperature E 0 ", for dependence the type-1 of the copper center ruthernium modified Pseudomonas 7.0 at 5.0 mM, (6) and phosphate pH (X). buffer (pH formal in doubly aeruginosa Solution 7.0) reduction 5 equivalents [Ru(NH 3 ) 5(py}](Cl0 4 )3. pentaammineazurin conditions: or acetate potential, at 1..1 buffer (pH pH 100 5.0), 190 402 388 (f) > 346 > -E 332 w 318 ... 0 304 290 27 ~3 7 17 27 TEMPERATURE (°C) 37 191 Table 12. Thermodynamic parameters for the reduction of the type-1 blue-copper center in doubly pentaammineruthenium modified Pseudomonas aeruginosa azurin at Solutions conditions: ~ = buffer (pH 5.0), 25 100 mM, °C. two pH values. phosphate buffer (pH 7.0), acetate 192 (a 5 Ru)rAz (a 5 Ru)rAz pH 7.0, 0.1 M pH 5.0, 0.1 M Phosphate Acetate 330 369 -42.5 -23.2 -26.9 -7.6 -7.60 -8.50 -20.3 -15.4 (mY vs. NHE) (eu) (eu) (kca1/mol) (kcal/mol) 193 the protein positively tron charged, by the in redox than environment and redox this center. properties caused that in far as some amino acid residues become facilitates the acceptance of an native azurin, however, the difference In by the change [a 5 Ru(His-83)]-Az pentaammineruthemium As increases, labelling elec- in acidity is more drama tic (a 5Ru) 2 -Az. The and in on proteins effect of is a intriguing subject. spectra- the protein structure is concerned, the available scopic evidence suggests that conformations of proteins have not been perturbed significantly. 2 7,33 be interesting to see the electrochemical and whether or not the same the can It be would said for thermodynamic properties of the proteins upon Ia belling. The formal native, as well azurins, have (Figure 17, Table reduction potentials and as and singly been compared 13) and doubly in pH thermodynamic parameters of pentaammineruthenium 7.0, in pH 5.0, 100 mM 100 mM modified phosphate acetate buffer buffer (Figure 18, Table 14). At pH 7.0, tials and azurin is thermodynamic quite a 5 Ru-label properties native the effect of a 5 Ru-labeling small. However, produces quite azurin. The of azurin, 0.0 properties eu a for phenomenon on either the influencing electron transfer of the blue-copper pH 5.0, the dramatic reaction This effect at in 0' !!.Set• [a 5 R u(His-83 )]-Az, that conformational, the blue copper of in an thermodynamic are 10.7 eu for and -7.6 eu for a key protonation or center introduction change entropies, indicates to on both the redox poten- has electrostatic factors, The peptide site. 194 Figure 17. Temperature E 0 ", for dependence the type-1 of the formal reduction potential, copper center m (.tJ.), [Ru(NH 3) 5(His-83)]-modified ( • ), and -ruthenium Solution buffer. in doubly modified (X) Pseudomonas conditions: pH 7.0, 1..1 native = pentaammine aeruginosa 100 in mM, azurins. phosphate 195 375~----------~----~------~--~ 365 -w 355 345 I z 335 (J) > 325 > E 315 -CCu 305 295 275_7 5 17 29 TEMPERATURE (°C) 41 53 196 Table 13. Thermodynamic blue-copper fied, and parameters centers in in doubly for the native, in reduction of the type-1 [Ru(NH 3 )5 (His-83)]-modi- pentaammineruthenium modified Pseudomonas aeruginosa azurins at pH 7.0, 1.1 = 100 mM, phosphate buffer, 25 °C. 197 Nathe Az (a 5 Ru)-Az (a 5 Ru)rAz pH 7.0, 0.1 M pH 7.0, 0.1 M pH 7.0, 0.1 M Phosphate Phosphate Phosphate 308 320 330 -31.7 -26.8 -42.5 -16.1 -11.2 -26.9 -7.10 -7.39 -7.60 -16.6 -15.4 -20.3 (mY vs. NHE) (eu) (eu) (kcal / mol) (kcal j mol) 198 Figure 18. Temperature Eo ' , f or dependence the type- 1 of the formal reduction potential, copper center · m ( • ), na t1· ve m [Ru(NH 3) 5(His-83)]-modified (X), and in doubly pentaammine -ruthenium Pseudomonas modified (h.) aeruginosa Solution conditions: pH 5.0, 1..1 = 100 mM, acetate buffer. azurins. 199 385 381 -w 377 I z (/) > > --- E 361 0 w 357 353 349 34~3 7 17 27 TEMPERATURE (°C) 37 47 200 Table 14. Thermodynamic blue-copper fied, and Pseudomonas parameters centers in in doubly aeruginosa acetate buffer, 25 °C. for the native, in reduction of at type-1 [Ru(NH 3 ) 5 (His-83)]-modi- pen taammineru theni urn azurins the pH 5.0, 1.1 modified 100 mM, 201 (Rua 5 )-Az (RuA 5 )rAz pH 5.0, 0.1 M pH 5.0, 0.2 M pH 5.0, 0.1 M Acetate Acetate Acetate 370 356 369 -4.9 -15.7 23.2 10.7 -0.1 -7.6 -8.53 -8.20 -8.50 -10.0 -12.9 -15.4 Native Az (mY vs. ' NHE) (eu) (eu) (kcaljmol) (kcaljmol) 202 mapping analysis of and an (a 5 Ru) 2 -Az unconfirmed It instead . is indicates methionine difficult to that His-35 modification draw a IS not modified, a 5 Ru- is suggested conclusion in the by definite of doubly modified azurin, however, if an a 5 Ru83, which is 11.8 A. from eu, is reasonable then it distant from the copper order of eu, 7.6 on modification on His- the copper site, can decrease !).So, et by that center, the - II near or equivalent effect of the a second a 5 R u-modifica tion, can have an reaction entropy case along the same transition direction. °C for The pH-dependence of the formal reduction potential at 25 the blue-copper in doubly azurins is center in native, in in Figure 19. Pseudomonas aeruginosa It is clearly shown that the in native azurin has potential of the blue-copper center change while pH value the protein notice that the pH-dependent of the blue-copper 5.0. It formal is interesting reduction of to potentials [Ru(NH 3 ) 5(His-83)]-modified fied azurins are the pH-dependent Cu 11 quite and similar. electron The dramatic from 6.0 to phenomena of center in pentaammineruthenium modi- solution doubly transfer modified and in doubly is shown in Figures 5 and 6. leads to conclude that us in changed observation reaction pentaammineruthenium the By the reason is in in conflict with Ru 11 ~ [Ru(NH 3) 5(His-83)]- modified comparison of for redox a behaviors of rate constant for long-range intramolecular and modified pen taammineru theni urn shown [Ru(NH 3) 5(His-83)]-modified, azurins, which these data thus pH-inpendence of electron transfer rate constant for Ru 11 ~ Cu 11 in doubly pentaammineruthe- 203 Figure 19. The pH-dependence formal reduction center in [Ru(NH 3) 5(His-83)]-modified (0), and in doubly eruthenium Pseudomonas for the blue-copper modified Solution condition: and 7.0) pH of the or 1..1 (X) = acetate [Ru(NH 3) 5(py)](Cl0 4 )3 . I OOmM, buffer (6), native 5.0), in pentaammin- aeruginosa phosphate (pH potential, buffer 5 azurins. (pH 6.0, equivalents 204 404~----------~------r-----l 391 -w I z . (/) > > --w E 0 274L_ ____~------~ 6------~7~----Es 4 5 pH 205 nium modified azurin in the domain ranging from pH 9.0 to pH 5.0 is not due to the factor of driving The force. increase in driving force causing by the solvent acidification must be compensated out by some time. other factor Since which this [Ru(NH 3) 5(His-83)]-Az, we are not situation is the second able to identify at not present in singly a 5Ru- modifier must to pH-independent electron transfer kinetics in (a 5Ru) 2 -Az. be the present modified responsible 206 References I. Fee, J. A., in Structure and Bonding, Volume 23, Dunitz, J. D., Hemmerich, P., Holm, R. H., lbers, J. A., J¢rgensen, C. K., Nei1ands, J. B., Reinen, D., and Williams, J. P., eds., SpringerVerlag, Berlin, 1975, P. I. 2. Malkin, R., and Malmstrom, B. G., in Advances in Enzymology and Related Areas of Molecular Biology, Volume 33, Nord, F. F., ed., Wiley, New York, 1970, P. 177. 3. Sutherland, 105 (1963). 4. Horio, T., J. Biochem. {Tokyo), 45, 195 (1958). 5. Verhoeven, W., and Takeda, Y., in McElroy, W. D., and Glass, B., Baltimore, 1956, P. 159. 6. Adman, E. T., in Topics in Molecular and Structural Biology, 6, Metalloproteins, Part 1: Metal Proteins with Redox Volume Roles, Harrison, P. M., ed., MacMillan Press, London, 1985, P. I. W., and Wilkinson, J. J. F., Gen. Microbial., 30, Inorganic Nitrogen Metabolism, eds., Johns Hopkins Press, I. 7. Ryden, L., in Copper Proteins and Copper Enzymes, Lontie, R., ed., CRC Press, Boca Raton, 1984, P. 157. 8. Farver, 0., and Pecht, 1., in Copper Proteins, Spiro, T. G., 151. 9. Lappin, A. G., Copper Proteins, P. 15. 10. Gray, H . Volume 3, 1981, P. I. 11. Solomon, Stephens, (1980). in Metal Sigel, H., Ions ed., Volume I, Metal Ions in Biology, Volume ed., Wiley, New York, 1981' 3, P. in Biological Systems, Volume 13, Marcel Dekker, New York, 1981, B., and Solomon, E. Copper Proteins, Spiro, E. P. 1., J., Hare, J. W., and Gray, H . 12. Solomon, E. 1., Hare, J. Sci. USA , 73, 1389 (1976). 13. Cass, A. E. G., Copper Enzymes, W., 1., T. in G., Metal Ions ed., Wiley, Dooley, D. B., J. Am. and Gray, H. M., Chern. B., in Biology, New York, Dawson, J. Soc. , 102, Proc. Nat/. H., 168 Acad. and Hill, H. A. 0., in Copper Proteins and Volume I, Lontie, R., ed., CRC Press, Boca 207 Raton, 1984, P. 63. 14. Markley, J. L., Ulrich, E. L., Berg, S. P., Biochemistry, 14, 4428 (1975). 15. Boas, J. F., in Copper Proteins and Copper Lontie, R., ed., CRC Press, Boca Raton, 1984, P. 5. 16. Loehr, T. M, and Sanders-Loehr, J., in Copper Proteins and Copper Enzymes, Volume I, Lontie, R., ed., CRC Press, Boca Raton, 1984,P. 115. 17. S., and Hodgson, K. 0., in Copper Proteins and Copper Co, M Enzymes, Volume I, Lontie, R., ed., CRC Press, Boca Raton, 1984, P. 93. 18. Tullius, T. D., Frank, P., Sci. USA , 75, 4069 (1978). 19. Adman, E. T., and Jensen, L. H ., lsr. J. Chern. , 21, 8 (1981). 20. and Hodgson, and Krogmann, D. Enzymes, 0., K. W., Volume Proc. Nat!. I, Acad. Adman, E . T., Stenkamp, R. E., Sieker, L. C., and Jensen, L. H., J. Mol. Bioi .. 123, 35 (1978). and Blumberg, Biophys., 165, 22. Crutchley, R. J., Ellis, W. R., Jr., and Gray, H . B., in Frontiers in Bioinorganic Chemistry, Xavier, A. V., ed., VCH erlagsgesellschaft, Weinheim, F. R. G., 1986, P. 679. 23. Mayo, S. L., Ellis, W. R., B., Science, 233, 948 (1986). 24. Gray, H. B., Chern. Soc. Rev., 15, 17 (1986). 25. Crutchley, R . J., Ellis, W. Chern. Soc. , 107, 5002 (1985). 26. Isied, S. S., Kuehn, 106, 1722 (1984). 27. Margalit, R., Kostic, N. M., Che, C.-M., Blair, D. F., Chiang, H.-J., Pecht, 1., Shelton, J. B., Shelton, J. R., Schroeder, W. A., and Gray, H. B., Proc. Nat!. Acad. Sci. USA , 81, 6554 (1984). 28. Nocera, D. G., Winkler, J. R., Yocom, K. M., Gray, H. B., J. Am. Chern. Soc. , 106, 5145 (1984). Jr., R., and E., Biochem. Peisach, J., 691 (1974). C., W. Arch. 21. Crutchley, Jr., and Worosila, R. J., and Gray, H. Gray, H. B., J. Am. J. Am. Chern. Soc. , G., Bordignon, E., and 208 29. Kostic, N. M., Margalit, R., Che, C.-M., Chern. Soc. , 105, 7765 (I 983). 30. Yocom, K. M., Winkler, J. R., Nocera, D. Gray, H. B., Chern. Scr. , 21, 29 (1983). G., Bordignon, E., and 31. Isied, S. S., Worosila, Soc. , 104, 7659 (I 982). S. 32. Winkler, J. R., Nocera, D. G., Yocom, K. M., Gray, H. B., J. Am. Chern. Soc. , 104, 5798 (1982). 33. Yocom, K . M., Shelton., J. B., Shelton, J. R., Schroeder, W. H. Worosila, G., Isied, S. S., Bordignon, E., and Gray, Proc. Nat!. A cad. Sci. USA, 79, 7052 (I 982). 34. Kostic, N. M., unpublished results. 35. Curtis, J. C., 22, 224 (1983). 36. Kirschner, S., in Inorganic Syntheses, ed., McGraw-Hill, New York, 1957, P. 186. 37. Cummins, D., Sullivan, and G., B. Gray, and P., H. and Gray, H. B., J. Am. Atherton, and B., J. Meyer, J., Am. J. Chern. Bordignon, E., and T. J., Inorg. Volume 5, Moeller, Am. Chern. Soc. , A., B., Chern. , T., 5158 99, (I 977). 38. Sundberg, R. J., and Gupta, G., Bioi nor g. Chern., 3, 39 (I 973). 39. Ambler, R. P., and Brown, L. H., Biochem. J., 104, 784 (1967). 40. Kostic, N. M., Shelton, J. B., Shelton, J. R., Schroeder, Blair, D. F., Chiang, H.-J., and Gray, H. B., in Preparation. 41. Stein, C. A., and Taube, H., Inorg. Ch ern. , 18, 1168 (1979). 42. Taniguchi, V. T., Sai1asuta-Scott, N., H. B., Pure Appl. Chern. , 52, 2275 (I 980). Anson, F. C., and W. A., Gray, 209 CHAPTER V RUTHENIUM-MODIFIED PROTEINS. REACTIONS OF cis-[Ru(NH 3 ) 4 (0H 2 ) 2 J2 + AND cis-[Ru(en) 2 (0H 2 ) 2J2+ WITH AZURIN, MYOGLOBIN, AND CYTOCHROME c 210 Introduction The electron es necessary of consecutive which have order to fer through for the evolved the of what as these reactions factors in function pairs process- as a series of proteins for energy conservation. the rate electron control chains, involving proteins artificial substrates. 1- 12 inorganic metabolic between polypeptide protein - the organisms, couple efficiently intervening complexes involved all transfer to reactions of many of chains survival electron understand vestigated metal transport and of scientists complex electron trans- have redox-active However, In in- transition the kinetics transfer reac- tions were found to be second order and bimolecular mechanisms were proposed. 6•13 Among the various factors kinetics, the distance between important. 14 In an effort to investigate long-range electron transfer, a novel approach modification of metalloproteins with redox-active fer es, and con trolling long-range redox is measurement of the kinetic and intramolecular electron transfer between centers the one distance has electron trans- of most the dependence consisted inorganic of of the complex- thermodynamic parameters for the label inorganic and the native metal site in the protein interior. 15 - 2 5 Recent of proteins horse heart experiments react rapidly have demonstrated with cytochrome sperm whale myoglobin 15 •16 •21 that the [Ru(NH 3 ) 5(0H 2 )] 2 +, Pseudomonas surface and aeruginosa have been characterized. histidines derivatives of azurin 18 ' and We have begun 211 an investigation of with proteins, because with variations in are is amines, or worked tions of horse heart of the the of need reaction two other ruthenium-amine for electron driving force. hi-dentate attractive m out. 26 , 27 In particularly well binding transfer complexes experiments Complexes of trans-[Ru 11 L 4 (0H 2 )2 ]2 + in which L 4 denotes four types cis- and dentate the horse heart this c chapter, and parameters a tetra-dentate because their monoamine, chemistry the products of the reac- Pseudomonas aeruginosa azurin, and described. 28 are or context, with [Ru(NH3)4(0H2)2]2+ thermodynamic this myoglobin, cytochrome amines, the of cis-[Ru(NH 3 )4 (0H 2 ) 2 ]2 + and The potential formal reduction cis-[Ru(en) 2 (0H)(His-33)]-horse cis- heart cytochrome c are also investigated by spectroelectrochemistry. Materials and Methods Sperm whale and horse heart myoglobins (Sigma Chemical Co.) were purified before use by ion exchange chromatography on Whatman CM-52 cellulose and eluted with pH 7.8, 50 mM tris•HCI buffer at 4 °C. The major myoglobin components were the last to come off the column and had pi ca. Public Health 8.1. Pseudomonas Laboratory aeruginosa Service in azurin England, obtained has an from the A 280 ; A 625 ratio of 1.72. Trypsin ketone was ace toni tr ile treated obtained was L-1-tosylamido-2-phenyleth yl with used from for Worthington. HPLC. chloromethyl reagent grade cis-[Ru(NH 3 ) 4 Cl 2]+, 26 cis- Baker 212 [Ru(en) 2 Cl 2 ]+, 27 [Ru(NH 3) 5(py)](CI0 4) / 9 and to published procedures. water purifier was were Deionized water from used throughout. prepared according a Barnstead NANOpure Ultrafiltration was performed with Amicon YM-5 membranes under argon with gentle stirring at 5 A solution cis-[Ru(NH 3)4(0H 2 )2 ]2+ of (OH 2 )2 ]2 + (2) was generated or cis-[Ru(en) 2 Cl 2 ]+ an inert in atmosphere or (1) °C. cis-[R u(en) 2 by Zn(Hg) reduction of cis-[Ru(NH 3 )4 Cl 2 ]+ tris•HCI or phosphate for hour. 27 buffer (pH 6.5) under cis-[Ru(en) 2 (0H)(His-81 )]-horse heart myoglobin (A) was prepared by mixing 1, which was generated by Zn(Hg) reduction phosphate, and 50 of 50 mg horse [Ru(en) 2 (0H)(His)]) 2-horse mixing 1, which was mg heart heart and 50 generated mg horse myoglobin cytochrome 20 myoglobin by ml c Zn(Hg) tris • HCl, heart 15 20 ml for 4 - 5 hours. (cis- (C) were synthesized by reduction of 50 cis- and mg horse 50 c, cytochrome cis-[R u(en) 2 (0H)(His-83 )]-Pseudomonas hours. m cis-[Ru(en) 2Cl 2 ]Cl0 4 respectively, aeruginosa mg heart for azurin 24 (D) was made by mixing 1, which was generated by Zn(Hg) reduction of 7 mg cis-[Ru(en) 2 Cl 2 ]Cl0 4 in azurin aeruginosa 10 for ml tris•HCl, and 12 mg 4 hours. cis-[Ru(NH 3 ) 4 (0H)(His-83)]- Pseudomonas Pseudomonas aeruginosa azurin (E) was obtained by mixing 2, which was generated by Zn(Hg) reduction of 7 mg cis-[Ru(NH 3)4 C1 2 ]Cl in 10 ml tris•HCl, and 10 mg of Pseudomonas aeruginosa azurin for 4 hours. The procedures for tryptic hydrolysis used peptides experiments, separation of the resulting HPLC, amino analysis, have and acid been in peptide mapping by reversed-phase described previously. 18 •25 213 A sample containing 5 - 6 mg of modified proteins in 2 ml of water, was 4 minutes m linear gradient, for denatured for The hydrolysis. boiling water the prior HPLC the latter tryptic separation tryptic peptides, consisted of 45 ml each of phosphate KH 2 P0 4 /5.4 to of the buffer (49 mM mM H 3 Po 4 , pH 2.85) and acetonitrile, the concentration of increasing from 0% to 45%, and the ml/min. Absorbance measurements were recorded peptide bonds A, and E; in B, m [Ru(en) 2 (0H)(His)] D, B, A, and D; experiments, a elution rate at nm 220 was for the ca. 450 nm for cis- and 370 nm for cis- [Ru(NH 3 )4 (0H)(His)] in E. In model cyclic 174A voltammetry polarographic Hewlett-Packard digital to a 7004B multimeter scan. all-glass by a sintered for degassing disk measuring A coil of platinum wire. calomel electrode (SSCE). NaCl0 4 in to cell glass disk. sample. 3 mm pH recorder. used ated the was X-Y was The analyzer 7.0, in The 100 used consisted in Keithley confirm the of Applied Research conjunction with model starting two 177 a microvolt potential prior compartments, separ- It was also equipped with a side arm working electrode of diameter, The Princeton and reference the auxiliary electrode The supporting mM phosphate consisted was electrode a electrolyte buffer. a All gold was a saturated NaCl was 100 mM solutions were deoxygenated by bubbling with argon for several minutes prior to use. Formal reduction [R u( en) 2 (0H)(His-33 )]-horse determined at different potentials heart for the heme center c, at pH using an cytochrome temperatures by in cis- 7.0, were optically transpa- 214 rent thin-layer electrode tion. The details of (OTTLE) cell in a this methodology are given [Ru(NH 3) 5(py)](Cl0 4 ) 3 equivalents of solution as mediator-titrant. grid as the working mm. Potentials Princeton and the applied across the Research were accurately measured multimeter. The cell temperature cell holder, eering precision digital thermometer. solution in model and measured microthermocouple The close was optical path thin-layer proximity model directly with a connected to the to was gold mini- was cell using by 0.48 analyzer, a and microvolt digital using a variable an Omega Engin- Fluke model 2175A in the protein cavity. All visible situated thin-layer protein length 177 by Five the a polarographic varied thermocouple to employed with a Keithley configura- Chapter II. cell 174A temperature in introduced OTTLE electrode, were Applied The were nonisothermal spectra were obtained with a Cary 219 recording spectrophotometer. Formal applying reduction a series Each trod e. that the to reduced of potential were potentials, Eappl., was of of all redox as defined by the incrementally converted from series of forms applied potentials, [Ox]/[Red] was determined tials n values and log([Ox]/[Red]). the to until gold electrolysis concentrations couples in solution, which the equation. state each spectra. determined sequentially minigrid of one oxidation for the by ratio Nernst from were determined maintained value established equilibrium potentials from ceased, of [Ox]/ [Red], couples to other the corresponding plots so oxidized Redox Formal elec- were by the value of reduction of was poten- Eappl. vs. 215 The protein was solution deoxygenated prior to use experiments by vacuum/argon cycling on a vacuum/purified-argon in double manifold, and loaded into the OTTLE cell by using rubber septum caps and syringe situated in solution that techniques. a The compartment was isolated platinum wire containing deoxygenated from the auxiliary protein electrode was mediator-titrant solution by a porous the ruthenated glass frit. Results and Discussion The modification proteins, are reactions, summarized in and Table the properties In 1. most of preparations, the reac- tion was quenched by applying the solution to a Sephadex G-25 column, and the ruthenated proteins were then purified by standard procedures. 18 •25 both (A) and (B) 1:1 analyses ruthenated 83 for by For horse derivatives were isolated. that the sites of proteins are His-81 for showed 1:1 oxidized 2:1 conclusively Pseudomonas aeruginosa [Co(dipic) 2 heart r and myoglobin, Tryptic modification peptide m the horse heart myoglobin and His- azurin. In product C, the absence of the His-33 C-2 proton peak at & = 8.84 ppm in the high-field 1H NMR spectrum, as shown in Figure I, strongly indicates that His-33 of indicate that significantly by horse heart cytochrome c is labelled with cis-[Ru(en) 2 (0H 2 )2 )2 +. Preliminary UV- VIS the conformations of the ruthenium the complexes, circular dichroism proteins a finding are measurements not that is perturbed in accord with previous 216 Table 1. Preparation and characterization of ruthenated proteins. 217 Reaction a Product pi E; (V !e.· NHE)b 1 and 50 mg Mbh, 4-5 h cis-[Ru(en)2(0H)](His-81)Mbh (A) 8.2 -0 . 12 1 and 50 mg Mbh, 24 h cis-[Ru(enl2(0Hll2Mbh (B) 9.2 -0 . 13 1 and 50 mg cyt E· 24 h cis-[Ru(en)2(0H)](His-33)cyt £(C) c 0.265d 1 and 12 mg Az, 4h cis-[Ru(enl2(0H)](His-83)Az (D) 7.2 -0.09 2 and 10 mg Az, 4h cis-[Ru(NH3l4(0H)](His-83)Az (E) 7.2 -0.10 0.3oe a1 was generated by Zn(Hgl reduction of 50 mg (A,B,C) or 7 mg (D ) cis-[Ru(en)2CJ2]CI04 in 15-20 mL phosphate (A) or 15-20 mL tris·HCI (B,C) or 10 mL tris·HCI (D); 2 was generated by Zn(Hg) reduction of 7 mg cis-[Ru(NH3l4Cl2]Cl in 10 mL tris·HCl. bRu(ill) /(II) at pH 7.2 and 25 ° C unless noted otherwise. CNot determined. dcyt E [Fe(ill )/ (ll)]. eAz[Cu(ll)/ (1)]. 218 Figure 1. Proton NMR spectra cytochrome c and cytochrome c in (Bruker (B) the horse heart cis-[Ru(en) 2 (0H)(His-33)]-horse heart region WH of 500) of imidazole (A) 1H Solution conditions: pH 5.5, D 2 0 / DCI; internal DSS). resonances. 219 His-33 (A) 9.5 9.0 8.0 8.5 (B) 9.5 9.0 8.5 ppm 8.0 220 work R u(NH3)5-proteins. 15,16,18,21 ,25,30 on measurements on C show that the structure Spectroelectrochemical of the redox-active metal site (heme c) is intact in the modified protein. At pH 7.2, and in tris•HCI [Ru(en)2(0H)(His))2+/+ in products A NHE; these E 0 ' V vs. 0.13 cis-[Ru(NH 3 ) 4 (0H)(His)) 2 +/+ electrochemical binding in results A products values are vs. suggest that the - are similar. (0.12 V) 26 E V) to trans-[Ru(NH 3 )4 (Im)(SO 4 ))+ creasing charge on the Ru(III)-amine dependence of the E 0 ' V) of the cis- are in the range -0.07 to - the comparable V (-0.10 trans-[Ru(NH ) 3(Im)(OH 2 )) 3 + 3 ( -0.10 D E0 ' of buffer, NHE) The to to in sites values product of decrease V) 31 ( -0.3 7 is E. The E0 ' in from cis-[Ru(NH 3) 4 (0H)(His)) 2+ complexes. derivatives, for ruthenium-amine parallels the de- Thus the observed pH values (e.g., C: pH 7.2, -0.13 ruthenium-amine that found V; pH 4.0, consistent with the 0.013 formu- lation cis-[Ru(en) 2 (0H)(His)) 2 + at pH 7.2. The UV- VIS absorption spectra of D and E in show the blue copper signature; the probably due (His)] 2+ respectively. A similar for nm). Virtually the same UV- VIS UV- VIS cis-[Ru(en) 2(0H)(His-81)]-horse product of 390 and and to found the bands at - Figure 2, clearly the reaction of absorption heart 430 nm are cis-[Ru(en) 2 (0H) spectrum myoglobin has (A.max been - absorption spectrum is obtained cis-[Ru(en) 2 (0H 2 )2 ]2 + with 450 from imidazole, under similar conditions. It appears [Ru(NH 3) 5 (0H 2 )) 2 + is that as a protein modification very reagent. similar With to horse 221 Figure 2. UV-VIS 83)]-azurin absorption spectra from Pseudomonas [Ru(NH3)4(0H)(His-83)]-azurin Solution conditions: pH 7.2, 25 of (A) cis-[Ru(en) 2 (0H)(His- aeruginosa from and Pseudomonas °C, tris•HCI buffer. (B) cis- aeruginosa. 222 (A) 0.5 ~ E :::> >.... 0 :E 1.0 (8 ) D <:[ .... <ll u c:: 0 D .... .§ <:[ x2- 0 .5 300 400 nm 223 heart cytochrome histidine and c (His-33 and Pseudomonas His-83, respectively) horse heart myoglobin, His-81, the reaction 15 •16· 21 azurin, the same labelled; and, with aeruginosa are which is one of the major products of of [Ru(NH 3) 5(0H 2 )] 2 + with sperm whale myoglobin, is readily modified. The formal reduction potential [Ru(en) 2 (0H)(His-33)]-horse heart spectroelectrochemistry in pH 7.0, shows the overlay spectra and of the heme c, was cytochrome 100 mM phosphate absorbance changes center in cis- determined by buffer. at Figure 3 different values of the applied potentials. The value of 265 :t 2 mY vs. NHE at 25 xs formal very similar to the reduction heart cytochrome c, which is 260 :t The results for the temperature potential dependence of the of cis-[R u(en) 2 (0H)(His-33)]-horse heart presented in Table Figure and illustrated in :!: 1.2 eu; l!..G 0 , :!: 0.05 Figure 6 , -6.11 kcal/mol. Table 3 and of native horse heart the electrochemical cytochrome and [Ru(en) 2 (0H)(His-33)]-modified horse horse present c. 32 the reduction cytochrome kcaljmol; and thermodynamic formal 5. l!..S 0 ' , -24.4 thermodynamic parameters are as follows: -8 .8 native 2 m V under identical conditions. 32 potential 2 of °C The :t are c derived 0' 1.2 eu; !!..Set• l!..H 0 ' , -13.4 electrochemical :t 0.4 results Comparative summarization of properties native and cis- are shown in xn the cis- than those of heart of cytochrome c Table 4 and in Figure 7. The formal reduction potentials roughly of the 0.25 Ru V sites [Ru(en)2(0H)(His)]2+-proteins are lower the Ru(NH 3 ) 5 3+ analogues. In line with this finding, neither A nor B 224 Figure 3. Thin-layer s pectroel ec troche mist r y -33)]-horse heart 7.0, 1-1 cytochrome c. Solution 0.26 mM. (Cl0 4 ) 3, mM. Overlay spectra values of applied 1.30 different SCE. Redox the conditions: °C. 100 mM, phosphate buffer, 16.6 = centra tion: at cis-[Ru(en) 2 (0H)(His of pH Protein con- media tor: and absorbance potentials in changes mV vs. 225 -407.6 mV -49. 5mV -29. 7mV 0.05A -9.4 mV 10.4 mV w u 50.4 mV z <l: 0) 0:: 0 (f) 0) <l: 490 500 510 520 530 540 550 560 570 580 590 600 WAVELENGTH (nm) 610 226 Figure 4. Nernst plot of the absorbance changes at 549 nm during a spectroelectrochemical titration of the iron cis-[R u( en) 2(0H)(His-33 )]-horse heart cytochrome circles points, and least represent squares fit yielded £ 0 ' Solution experimental fit the vs. Eappl. m c. The line log([Ox]/[Red]). is a The = 265 mY vs. NHE and Nernst slope = 61 mY. conditions: buffer, 25.0 of the center pH = 7.0, 1.1 I 00 mM, °C, 5 equivalents [Ru(NH3) 5(py)](Cl04)3. phosphate 227 382~~~--~~~--~~~--~~ 360 338 -w 316 I z 294 (/) > 272 > ~ 250 ' cru 228 206 184 16 ?3.0 -1.6 -0.2 1.2 In ([ox]/ [Red]) 2.6 4.0 228 Table 2. Temperature Eo " , heart for dependence th e h erne cytochrome of the formal cen t er in cis-[Ru(en) 2 (0H)(His-33)]-horse c, spectroelectrochemistry. 100 mM, phosphate buffer. using Solution reduction nonisothermal conditions: pH potentials, thin-layer 7.0, ll 229 Temperature (° C) * E 0 ' (mY vs. NHE) t 9.2 271 16.6 269 25.0 265 * :!: 0.2 °c t:!: 2 mY 230 Figure 5. Temperature dependence of E0 ', for the heme center m horse heart cytochrome c, using spectroelectrochemistry. = 100 mM, 25.0 °C. 265 :!: v; 0 c. phosphate the formal reduction potentials, cis-[Ru(en) 2(0H)(His-33)]nonisothermal thin-layer Solution conditions: pH 7.0, buffer. Temperature range: 9.2 ~ A least squares fit of the data gave an E 0 ' 2 mY at 25 °C and (dE '/dT) 25 o C = -3.82 x 10-4 0 231 288~----~----~----~----~------ 284 -w:r:: z . V) > > 268 E -' 0 w 260 256 252 248~----~----~----~----~----~ 2 8 14 20 TEMPERATURE ( oc) 26 32 232 Table 3. Temperature E 0 ", for c, using dependence the of heme center nonisothermal the in formal native thin-layer reduction horse potentials, heart cytochrome spectroelectrochemistry. Solution conditions: pH 7.0, 1..1 = 100 mM, phosphate buffer. 233 Temperature (° C) * E 0 ' (mV YS. NHE) t 8.6 269 9.2 270 15.5 267 16.8 265 19.8 262 20.2 263 24.4 262 29.8 257 30.6 258 35.2 254 39.4 253 * :!: 0.2 °c t:!: 2 mV 234 Figure 6. Temperature dependence of the formal reduction potential, E 0 ' , for the heme center in native horse heart cytochrome c, using nonisothermal Solution conditions: buffer. Temperature thin-layer pH 7.0, range: 1.1 = 8.6 squares fit of the data gave an E 0 ' °C and (dE 0 , /dT)25 OC = -5.61 spectroelectrochemistry. X = 100 mM, phosphate 39.4 °C. A 260 10- 4 V/°C. :!:: least 2 mY at 25.0 235 281~----~----~----------~------ -w I z . (J) > > E -' 261 257 0 w 253 249 245 241 I 10 19 28 TEMPERATURE ( oc) 37 46 236 Figure 7. Temperature the formal centers in native (0) [Ru(en)2(0H)(His-33)]-modified (X) horse heart c. 7.0, E0 ', for Solution buffer. dependence the heme conditions: of pH 1..1 reduction 100 potential, and mM, m cis- cytochrome phosphate 237 293 287 -w 281 J: z 275 . (/) > 269 > 263 E - ...... 257 0 w 251 245 233------~----~----~----~----~ I 10 19 28 37 TEMPERATURE ( oc ) 46 238 Table 4. Thermodynamic centers in parameters native and in for the reduction °C. the heme cis-[Ru(en) 2 (0H)(His-33)]-modified horse heart cytochrome c at pH 7.0, 1.1 buffer, 25 of = 100 mM, phosphate 239 Native Cyt. c cis-[Ru(en) 2 (0H) (His-33))-Cyt. c pH 7.0, 0.1 M pH 7.0, 0.1 M Phosphate Phosphate 260 265 -28.5 -24.4 -12.9 -8.8 -6.00 -6.11 -14.5 -13.4 (mY vs. NHE) (eu) (eu) (kcaljmol) (kcal/mol) 240 catalyzes in the aerial the low potential ascorbate to Ru(III) oxidation of derivatives plays a key ascorbate. This lack of reactivity suggests that electron transfer role the observed 21 catalytic in tivity of (Ru(NH 3 )5 )3 -sperm whale myoglobin. from ac- 241 References I. Brunschwig, B. S., DeLaive, P. J., English, A. M, Goldberg, M., Gray, H. B., Mayo, S. L., and Sutin, N., Inorg. Chern., 24, 3742 (1985). 2. English, A. M, Lum, V. R., DeLaive, P. Am. Chern. Soc. , 104, 870 (1982). 3. Mauk, A. G., Bordignon, E., and Gray, H. B., 104, 7654 ( 1982). 4. Gray, H. B., Coyle, C. L., Dooley, D. M, Grunthaner, P. J., Hare, J. W., Holwerda, R. A., McArdle, J. V., McMillin, D. R., Rawlings, J., Rosenberg, R. C., Sailasuta, N., Solomon, E. 1., Stephens, P. J., Wherland, S., and Wurzbach, J. A., in Advances in Chemistry Series, No. 162, Bioinorganic Chemistry-//, Raymond, K. N., ed., American Chemical Society, Washington, D. C., 1977, P. 145. 5. McArdle, J. V., Coyle, C. L., Gray, H. B., Yoneda, Holwerda, R. A., J. Am. Chern. Soc., 99, 2483 (1977). 6. Holwerda, R. A., Wherland, S., and Gray, H. of Biophysics and Bioengineering, Volume ed., Annual Reviews Inc., Palo Alto, 1976, P. 363. 7. Cummins, D., and Gray, H. B., Inorg. Chern., 20, 3712 (1981). 8. Holwerda, R. A., Knaff, D. B., Gray, H. B., Clemmer, J. D., Crowley, R., Smith, J. M., and Mauk, A. G., J. Am. Chern. Soc., 102, 1142 (1980). 9. McArdle, J. V., 99, 4141 (1977). Yocum, K., and 10. Coyle, C., and 1122 (I 976). Gray, 11. Wherland, S., 2950 (1976). 12. McArdle, J. V., Gray, H. Chern. Soc. , 96, 5737 (1974). J. Am. 13. Wherland, S., and Gray, H. B., in Biological Aspects Inorganic Chemistry, Addison, A. W., Cullen, W. R., Dolphin, and James, B. R., eds., Wiley, New York, 1977, P. 289. of D., and H. Gray, B., H. B., Gray, H. Biochem. B., J., Proc. Creutz, and Gray, H. Am. Chern. Soc., J. J. Biophys. C., J. G. S., and B., in Annual Review 5, Mullins, L. J., B., Nat!. B., Am. Chern. Res. Acad. and Sci. Sutin, Soc. , Comm., 73, USA, 73, N., 242 14. Hush, N. S., Coord. Chem. Rev., 64, 135 (1985). 15. Crutchley, R. J., Ellis, W. R ., Jr., and Gray, H. B., in Frontiers in Bioinorganic Chemistry, Xavier, A. V., ed., VCH erlagsgesellschaft, Weinheim, F . R. G., 1986, P. 679. 16. Crutchley, R. J., Ellis, W. Chern. Soc. , 107, 5002 (1985). 17. Isied, S. S., Kuehn, 106, 1722 (1984). 18. Marga lit, R., Kostic, N. M., Che, C.-M., Blair, D. F., Chiang, H.-J., Pecht, 1., Shelton, J. B., Shelton, J. R., Schroeder, w. A., and Gray, H. B., Proc. Nat!. A cad. Sci. USA , 81, 6554 (1984). 19. Nocera, D. G., Winkler, J. R., Yocom, K. M, Gray, H. B., J. Am. Chem. Soc. , 106, 5145 (1984). 20. Kostic, N. M, Margalit, R., Che, C.-M., Chern. Soc., 105, 7765 ( 1983). and Gray, H. B., J. Am. 21. Margalit, R., Pecht, 105, 301 (1983). B., 22. Yocom, K . M., Winkler, J. R., Nocera, D. G., Gray, H. B., Chem. Scr. , 21, 29 (1983). Bordignon, E., and 23. Isied, S. S., Worosila, Soc., 104, 7659 (1982). J., 24. Winkler, J. R., Nocera, D. G., Yocom, K. M, Gray, H. B., J. Am. Chem. Soc., 104, 5798 (1982). Bordignon, E., and 25. Yocom, K. M., Shelton., J. B., Shelton, J. R., Schroeder, W. Worosila, G., Isied, S. S., Bordignon, E., and Gray, H. Proc. Nat!. Acad. Sci. USA, 79, 7052 (1982). A., B., 26. Tweedle, M. F., and Taube, H., Inorg. Chem., 21, 3361 (1982). 27. Poon, C. K., Che, Trans ., 128 (1980). 28. Che, C.-M., Margalit, R ., Chim. Acta, 135, 33 (1987). Chiang, H.-J., 29. Cummins, (1977). H. J. D., and C., R ., and and 1., G., C.-M., Gray, Jr., Worosila, Gray, and and and Gray, G., H. Atherton, Kan , Y. B., J. S. J. and Am. B., J. Am. Am. Chem. Soc., Bordignon, E., and Am. J. P., H. Chem. Chern. Gray, Chem. Am. J. Soc. , Chem. Soc., Dalton H. B., Inorg. Soc., 99, 5158 243 30. Toi, H., La Mar, G. N., Margalit, B., J. Am. Chern. Soc., 106, 6213 (1984). R., 31. Isied, S. (1978). J. 32. Taniguchi, V. T., Ellis, W. R., Jr., Cammarata, V., Webb, J., Anson, F. C., and Gray, H. B., in Advances in Chemistry Series, No. 201, Electrochemical and Spectrochemical Studies of Biological Redox Components, Kadish, K. M., ed., American Chemical Society, Washington, D. C., 1982, P. 51. S., and Kuehn, C. G., Che, C.-M., Am. Chern. and Gray, H. Soc., 100, 6754 244 CHAPTER VI SYNTHESIS AND SPECTROELECTROCHEMISTRY OF [Ru(MpiX)(DMSO) dicarboxylic acid]RECONSTITUTED MYOGLOBIN 245 Introduction In long-range two different metalloenzymes, for of studies investigation, viz., electron kinds of horse heart transfer reactions of hemoproteins have been chosen c 1-5 and sperm cytochrome whale myoglobin. 6•7 The prosthetic groups of c and myoglobin are identical in each case, i.e., iron-protoporphyrin IX, commonly called heme, and the molecular c is cytochrome structure of the basic heme unit is shown in Figure I. Cytochrome mitochondrial essentially respiratory constant the respiratory c reductase chains, for process complex, present at in all its conformation has remained years. 8 It's role and least one billion organisms is to carry electrons from the second energy-conserving possessing in the QH 2-cytochrome site, to cytochrome c oxidase, the third energy-conserving site. 9 •10 Cytochrome c 11 - 15 amino acid residues daltons. The from tuna is and no and has a a single polypeptide molecular weight three-dimensional structure of shown ~-pleated diameter of 34 consists of A of reduced chain of around I 04 12,500 c cytochrome m Figure 2. 16 It contains very sheet. The protein is roughly spherical, little ex-helix with a The heme of cytochrome c is covalently attached to the protein by two thioether linkages between the vinyl groups of the heme and cysteine-14 and cysteine-17 residues of the protein. The iron atom is also axially ligated by the sulfur atom of a methionineSO residue and by the nitrogen atom of a histidine-18 residue. 246 Figure I. Molecular structure of iron protoporphyrin IX, the prosthetic group in cytochrome c and in myoglobin. which is 247 248 Figure 2. X-ray crystallographic structure of the bone of tuna cytochrome c in the reduced form. polypeptide back- 249 250 Myoglobin shows vertebrates, myoglobin and as serves a bines reversibly no structural facilitates reserve with similarity to cytochrome c. transport of oxygen muscle the tissue. 17 the supply of oxygen molecular dioxygen, in which it blood and passes on to mitochondria in muscle. remains ferrous, but it vitro in takes in up In It com- from the vivo its iron atom In a utoxidizes the to ferric metmyoglobin in which the sixth ligand at the iron is water. The elucidation C. structural model graphic studies 19 - 21 ' tural studies on strated the details tains a of three-dimensional was a landmark on these is in Figure 3.22 illustrated of the mechanism chain around of 17,800 and in 153 amino structural feature total lack of symmetry. It is extremely sions are ca. 45 x 25 A, water molecules. helical polar x 35 About conformation. residues, myoglobin the histidines, viz., coordination ligand a 75% The only of with interior polar proximal for lies the the acid The in X-ray its very main struc- have demonrela- Myoglobin residues, acid con- with a sequence The most striking the little and overall its dimen- interior room for is folded in a- almost entirely inside histidine-93, a tom, crystallo- irregularity, chain consists a Further amino compact, residues iron biology, and myoglobin. daltons. myoglobin structure-function of sperm whale myoglobin 24 is shown in Figure 4. 6 of of deoxymyoglobin 23 and binding process polypeptide of molecular based oxymyoglobin 20 weight in structure metmyoglobin, the oxygen single molecular the Kendrew, 18 by John tionship of of and myoglobin which a is distal an of non- being two the fifth histidine-64, 251 Figure 3. X-ray crystallographic structure of the polypeptide bone of sperm whale myoglobin in the oxidized form. amino code. acid residues are identified by the backThe single-letter 252 <> .. YAit tANT "" ALL Oflt lltOST 0' TH£ K"ttWN V(ltT[IJtAT[ &LO.INS 0 ~~~~~.~rso~~ (i) :::;~~ ~~~Ud 253 Figure 4. The amino acid sequence of sperm whale myoglobin. Tl through T23 were obtained with trypsin. Peptide after digestion of myoglobin T4 T5 ,. .,. T6 ~- l l T14 I •· T3 ILys I ~T2J-"' -01 I T21 Asp-lie- Alo-Aio-Lys • T22:_::j j Tyr-Lysl T18 1... T23 •· 1531 Glu- Leu-Giy-Tyr -Gin-GI~ ,.. ,. I T19 - - - - His- Pro- Gly- Asn- Phe-Giy- Alo-Asp-Aio-Gin- Gly-Aio- Met-Asn-~~; J Ala- Leu-Giu-Leu- Phe -Arg l~ic._ Lysj Ile- Pro-IIe -Lys jryr- Leu-Giu- Phe- Ile- Ser-Giu -Aio-lle -lie -His-~1- Leu- ~~s- Ser- Arg} ~T15 -• T16 .,.,. T17------------~~ -• l m ~ -Ala- Thr- Lys Gly-His-His-Glu-Alo-Giu-Leu-LysPro-l.eu-Aio -Gin- Ser- His ~~s-Gly-Vol- Thr- Vol- Leu- Thr-~11?- Leu-Gly-Ala-lle- Leu-Lys lt,il~~ ·• T7 I~s-Leu-LysJ ,.. " Phe- Asp- Arg j Phe- Lys I ., . .,. T2 Thr- Glu- Ala- Glu- Met-Lys} R~-Ser-Giu -Asp- Leu- Lys Lvs I 18 ~. T9 ;Jt- T10~ ... I I Ser- ~s- Pro- Glu- Thr-Leu- Glu- Lys ·• !val- Glu- Ala- Asp- Vol- Ala- Gly- His-Giy- Gin- Asp- Ile- Leu- Ile- Arg} Leu- Phe-Ly~ 20 ~ol- Leu- Ser -Giu- Gly -Giu- Trp -~n- Leu- Val- Leu- His- Vol- Trp -Ala- Ly~ IV ~ Vl 255 which is close iron atom. to the position The sixth coordination binding site for oxygen. of the sixth site coordination site of the the unit is the of heme The heme iron in myoglobin is high-spin both ferric and ferrous oxidation states, heme iron in cytochrome c,25,26 and the spin in the control contrast of state low-spin the to the heme certainly plays an important transfer kinetics of heme-containing oxidase and oxygenase enzymes. 27 Although the will probably not determine the in heme geometry transfer. 28 •29 Tables 12,7 kinetic data for intramolecular horse heart kinetics of spin slow itself state heme a result in pare thermodynamic role in in site, electron change a and transfer reaction in ruthenated whale myglobin. It shows that the the of is reorganization myoglobin high spin heme is much larger than 21,4,30 c to com- electron and energy that for redox expected and cytochrome electron the sperm of the the low spin heme in cytochrome c. Ruthenium Figure 5, is therefore, and the low-spin that would allow substituted in heme, both su bsti tu ting study of reorganization the +3 myoglobin the effects of energy, whose on structure and with changing the +2 is depicted states. It ruthenium both the intramolecular in appeared porphyrin, driving force electron trans- fer rate. Materials and Methods Ruthenium porphyrin 31 compounds were synthesized by Dr. Chi-Ming 256 Table 1. Thermodynamic parameters for the reduction of the heme centers in native and in (NH3)5Ru3+/2+ modified horse heart cytochrome c as well as in native and (NH3)5Ru3+/2+ modified sperm 1-1 = 100 mM, whale myoglobin. phosphate buffer, 25 Solution °C. conditions: pH 7.0, AH "(kcal/mol) 0 AS "(cal/deg•mol) 0 AG "(kcal/mol) 0 -14.9 -29.2 0.8 t 0.3 :!: -6.22 :!: 0.05 2 t E0 "(mY vs. NHE) 270 a 5 Ru(His-33)Cyt c Measure t 2 t 1.2 -14.5 t 0.4 -28.5 -6.00 :!: 0.05 260 Native Cyt c -13.0 -39.2 :!: 0.4 t 1.2 -1 .36 t 0.05 60 t 2 Fe3+/2+ Native Mb -12.7 -37.6 -1.51 0.05 :!: 0.4 t 1.2 :!: 65 t 2 Fe3+/2+ Modified Mb -0.7 4.2 :!: :!: 0.4 1.2 -1.98 t 0.05 86 t 2 a Ru 3 +/l+ 5 N -.J Vl 258 Table 2. Intramolecular long-range electron transfer kinetics m (NH 3 ) 5Ru 3+/ 2+ modified horse heart cytochrome c and sperm whale myoglobin. 259 Edge-to-edge distance between (a 5 Ru(His) 3 +/ 2 + and 1 heme (Fe 3 + 2 +) (A) [a 5 Ru(His-33)]-Cyt. c 12.1 30 * 50 t [a 5R u(His-48)]-Mb 14.6 *Flash photolysis. tPulse radiolysis. +Backward rate constant k_ 1 is 0.041 sec- 1 . 260 Figure 5. Molecular structure of ruthenium mesoporphyrin IX. 261 262 Che. Equal Chemical Co.) weights Co.) were and of mesoporphyrin refluxed under toluene was removed solved in for additional an over 50:50 neutral dodecacarbonyl IX dimethyl nitrogen, by under in reduced 30 minutes. using was for 24 Chemical hours. The the residue and under The solid removed (Sigma Strem pressure, and refluxed residue was dichloromethane again (Ru 3 (C0) 12 , ester toluene ethanol/dichloromethane alumina dichloromethane ruthenium as under nitrogen chromatographed eluting reduced dis- solvent. pressure, The and the solid residue was washed with petroleum ether and dried in vacuum to give ruthenium(II)(CO) nium(II)(CO) fluxing ester. dimethyl IX ester at The ethanol was removed recrystallized ruthenium(II)(CO) dimethyl derivative for was IX dicarboxylic hydroxide residue of mesoporphyrin the potassium mesoporphyrin least from six acid in to IX The prepared re- containing 2% remove The dicarboxylic rutheby evaporation, acetone/water. mesoporphyrin was ethanol hours by rotary ester. the dimethyl and the visible acid solid spectrum in dimethyl sulfoxide (DMSO) is shown in Figure 6. Ru 11 (MpiX)(DMS0) 2 dicarboxylic acid was prepared by degassing a dimethyl sulfoxide through five tungsten lamp. filtered was solution freeze-thaw cycles The light of out. After every nine degassed again through monitored spectroscopically Ru 11 (MpiX)(CO) dicarboxylic as Ru 11 (MpiX)(CO) dicarboxylic and with a I ,000-wa tt than 300 nm of irradiating wavelength five to ten hours freeze-thaw shown acid shorter in acid irradiation, the cycles. result in Figure 20 ml 7. The For dimethyl 32.5 was sample was mg of sulfoxide, the 263 Figure 6. Absorption spectra of ruthenium(II)(CO) dicarboxylic acid in dimethyl mesoporphyrin sulfoxide (DMSO). wavelengths of peaks are 548, 516, 399, and 341 nm. IX The 264 x7.5 I 0.25A w u z <1: m ~ 0 Cf) m <1: xiO 300 400 500 WAVELENGTH (nm) 600 700 265 Figure 7. The overlay dicarboxylic spectra acid in time. After every sample was degassed of photolysis dimethyl sulfoxide a function of ten hours, the five freeze-thaw cycles. The hr corresponds to final irradiation through spectrum marked with 195.5 product, Ru 11 (MpiX)(DMS0) 2 Ru 11 (MpiX)(CO) of of ca. as dicarboxylic the acid. wavelengths of peaks before photolysis are 548 and 518 nm. The 266 0.0 hr 9. 5 hr 18.5 hr 28.0 hr 38.0 hr 48.0 hr 57.0 hr 65.0 hr 195.5 hr LJJ u z <( ro 0::: 0 (J) ro <( I 0. 2A I I I I I I I I I I I I I I I I I I I 450 470 490 510 530 550 570 590 610 630 WAVELENGTH ( nm) 267 total irradiation time is around 196 hours. Sperm whale myoglobin (Sigma Chemical Co.) was purified before use by ion exchange chromatography on Whatman CM-52 eluted with pH 7.8, mM 50 tris•HCI buffer at cellulose, and °C. 4 The major myoglobin component was the last to come off the column and had pi 8.11. Ultrodex granulated gel and ampholytes (pH 7 - were purchased from LKB. The described previously. 32 The solutions employed deionized synthesis of 9 and 9 - 11) K 4[W(CN) 8]•2H 2 0 was preparation of water Barnstead from = all protein and buffer NANOpure water purifier. apo-myoglobin, 33 In the preparation removed by ultrafiltration into with stirring under argon of gentle water at the tris•HCl using Amicon °C. The 5 low ionic strength was adjusted to pH 2.3 - buffer YM-5 was membranes protein solution of 2.5 by dropwise additions of 0.1 M HCl and immediately mixed with an equal volume of ice-cold 2-butanone. The mixture allowed to stand at upper layer of separated tained with from the butanone phase the colorless and butanone. shaken which contained for aqueous phase dialyzed against two Then the lower layer of aqueous The bicarbonate solution followed dissolved vigorously minute and °C for 30 minutes until the deeply colored 4 apo-protein. 2-butanone was was changes heme phase treated of was well which contwice 10 mM more sodium by three changes of water to remove the the apo-protein solution was dialyzed RuMb 35 have been three times against tris • HCI buffer of pH 7.2, 50 mM. The syntheses of Ru(CO)Mb 34 and described 268 elsewhere. The the of cavity solvent used apo-myoglobin (DMF) or pyridine. to introduce was DMSO and the stoichiometric acid was added process of mixing was glove box. After mixing The atmosphere free was passed through with protein desalted then aqueous free protein amount dropwise a column viously equilibrated was of into dimethylformamide The protein concentration half dicarboxylic stirring. instead porphyrin The freshly prepared apo-myoglobin was mixed with Ruli(MpiX)(DMS0) 2 dicarboxylic acid. mM, ruthenium to of 50 mM tris•HCI by 1 Ruli(MpiX)(DMS0) 2 apo-myoglobin carried out for hour, one of Sephadex G-25 was with gentle an inert in the solution which had been pre- pH The buffer 7.2. ultrafiltration into then further purified by preparative flat-bed isoelectric focusing has ampholyte solutions used solution water. eluted The salt isoelectric focusing (IEF). The been methodology described in to make the pH tion to get rid great 2 ml near water, the of 36 37 • any high dicarboxylic was anode. detail. The 2% - 10 gradient gels were pretreated by ultrafiltra- 7 [Ru 11 (MpiX)(DMSO) in of pipetted After molecular acid]-reconstituted onto running weight the for gel myoglobin, surface hours 20 power, and with the temperature maintained at 10 component. at as a 8 watts The 0.3 narrow g zone constant °C, IEF purification was complete. Two very closely-running narrow bands were resolved in the Both were collected although band to IEF gel. bands i.e., higher pi value, the gated by spectroelectrochemistry. closer Extensive the only the band cathode, ultrafiltration was with investi- was used 269 to remove ampholyte from the protein solution, which was removed from the IEF gel with tris buffer. The protein was again exchange chromatography on CM-52 purified by ion cellulose, and eluted with pH 7.8, 50 mM tris•HCI buffer at 4 °C. The protein eluent was then dialyzed extensively against 100 spectrum of myoglobin is pH 7.0, mM phosphate [Ru 11 (MpiX)(DMSO) shown in Figure buffer. dicarboxylic 8. The protein The UV-VIS acid]-reconsti tu ted concentration used m spectroelectrochemical experiments was 1.25 mM, which was based on a calculation using coefficient at M -1 em -1 . One equivalent of protein a solution molar as extinction 280 nm of 35,000 K 4[W(CN) 8 ]•2H 2o was introduced The mediator-titrant. absorption to the spectrum of K 4[W(CN) 8] •2H 20 is shown in Figure 9. Formal [Ru(MpiX)(DMSO) determined potentials reduction dicarboxylic by using an different at acid ]-reconstituted optically transparent temperatures, for myoglobin, were thin-layer electrode (OTTLE) cell in a nonisothermal configuration. A summarized descrip- tion II. of this employed optical a technique gold path thin-layer given minigrid length cell is by as the was 0.49 mm. using a and model 177 microvolt digital varied by using directly with an connected to a electrode Potentials were Applied OTTLE cell material, and the applied the Research across model with multimeter. The cell temperature variable temperature cell holder, Omega Engineering precision model accurately The measured Fluke were Chapter working Princeton polarographic analyzer, a in 2175A digital and a 174A Keithley was measured microthermocouple thermometer. The 270 Figure 8. The UV-VIS dicarboxylic pH 7.0, 1-1 absorption spectrum acid]-reconstituted = of sperm 100 mM, phosphate buffer. peaks are 522, 400, 381, 351, and 274 nm. [Ruii(MpiX)(DMSO) whale myoglobin in The wavelengths of 271 I 0.2A X 5.71 w u z <l: CD a:: 0 (/) CD <l: 250 300 400 500 WAVELENGTH (nm) 600 700 272 Figure 9. The absorption spectrum of K 4[W(CN)g] • 2H 2 0 100 mM, phosphate 429 and 370 nm. buffer. The wavelengths in pH 7.0, of peaks 1..1 = are 273 w 0 z <r a::l a::: 0 (f) a::l <r 300 400 500 WAVELENGTH (nm) 600 274 thermocouple to the was situated in the thin-layer cavity. All protein visible solution spectra in were close proximity obtained with a Cary 2 I 9 recording spectrophotometer. Formal applying trod e. so reduction a series Each of potentials were potentials, Eappl.• potential was that the equilibrium oxidized to reduced [Ox]/[Red], was couples other were by the forms incrementally series of as of the of of couples equation. Redox oxidation potentials, for which were determined of solution, one values Nernst the ceased, in from from had elec- concentrations the duction n electrolysis redox was determined sequentially minigrid by converted applied by gold ratio all defined the until ing value of [Ox]/[Red] potentials and to maintained value established determined state each spectra. from to the correspondFormal plots of re- Eappl. vs. log([Ox]/[Red]). All cycling on solution a deoxygenated vacuum/purified the OTTLE cell The platinum containing were by wire using double to electrode was mediator-titrant use by manifold, rubber septum caps auxiliary deoxygenated argon prior and and loaded into syringe techniques. situated solution vacuum/argon in that a compartment was isolated from the protein solution by a porous glass frit. Results and Discussion A typical [Ruii(MpiX)(DMSO) thin-layer spectroelectrochemical dicarboxylic acid ]-reconstituted experiment with sperm whale 275 myoglobin is [W(CN)g] 4 - absorb quite at 523 center shown in nm are mainly and be can Figure weak due above to analyzed Because 10. nm, 38 500 all the absorption directly. In [W(CN) 8]3- both of absorbance and changes ruthenium porphyrin Figure 11, a least squares analysis of the 523 nm Nernst plot gives a midpoint potential of 540 mY vs. NHE and a slope of 61 mY at 13.0 The protein could be °C. reversibly cycled between its fully oxidized and fully reduced forms, and the value of E 0 ' calculated for the of the ruthenium center was independent of the direction experi- ment. The the temperature dependence ruthenium center is shown of the formal reduction in Table 3 Figure squares fit of these data gives an E 0 ' , at 25 and (dE 0 '/dT) 25 o C = -2.06 difference couple ~s~;. between of is the interest, directly mY ; 0 C. reduced i.e., the and to the the constant. 12. A of least °C, of 514 mY vs. NHE, The partial molal ionic entropy oxidized electron proportional and potential halves transfer temperature of the redox reaction entropy, coefficient of the nonisothermal cell: m which F 1s Faraday center is therefore calculated the ruthenium to be -47.6 ± 1.2 eu. ~S 0 ' , adjusted to the NHE scale, is -63.2 1.2 eu, when a value of -15.6 eu is assumed for ~S~t (NHE). 39 The the complete cell ± The reaction, The entropy for 276 Figure I 0. Thin-layer spectroelectrochemistry dicarboxylic acid )-reconstituted Solution buffer, conditions: 3.0 7.0, Protein absorbance potentials changes in out ~Rulli. mY on at whale I 00 1.1 1.20 mM. different vs. SCE. oxidation sperm This of the myoglobin. mM, 1.25 concentration: K 4[W(CN) 8] •2H 2 0, mediator: carried °C. pH [R u(MpiX)(DMSO) of phosphate mM. Redox Overlay spectra and values particular protein, of the applies experiment was i.e., Ru 11 from 277 -14.10 mV 285.40 mV 305.25 325.20 mV 345.10 mV 365. 55 mV 385.90 mV 554.70 mV I 0.05 A w (.) z <X: Q) a:: 0 (/) Q) <X: 480 490 500 510 520 530 540 550 560 WAVELENGTH (nm) 278 Figure 11. Nernst plot of the absorbance changes at 523 nm during a spectroelectrochemical [Ru(MpiX)(DMSO) titration dicarboxylic whale myoglobin. The points, and is the line vs. log([Ox]/[Red]). circles a 100 mM, ru theni urn represent The fit yielded E 0 ' phosphate center acid]-reconsti tu ted least squares and Nernst slope = 61 mY. = of fit = 13.0 sperm experimental of the Eappl. 540 m V vs. NHE Solution conditions: buffer, in I pH 7.0, 1..1 equivalent 279 650~~~--~~~--~~~--~~ 630 610 -590 w I z 570 (/) > 550 > ~530 ' ~ 510 490 470 45 92.6 -1.4 -0.2 1.0 In ([ox] / [Red]) 2.2 3.4 280 Table 3. Temperature dependence E o", [Ru(MpiX)(DMSO) sperm for whale of myoglobin, spectroelectrochemistry. 100 mM, phosphate buffer. the formal reduction d.1car b oxy 1·1c using Solution act"d] -recons t"t 1 u t ed nonisothermal conditions: potentials, pH thin-layer 7.0, ~ 281 Temperature (° C) * E 0 ' (mV vs. NHE) t 3.0 565 8.0 536 13.0 540 18.0 533 23.0 518 28.0 519 33.0 491 38.8 483 * ± 0.2 °c t ± 2 mV 282 Figure 12. Temperature E0 ', for sperm dependence [Ru(MpiX)(DMSO) whale myoglobin, spectroelectrochemistry. 100 38.8 mM, °C. 514 ~ v;D c. of phosphate the formal reduction dicarboxylic using potentials, acid]-reconstituted nonisothermal thin-layer Solution conditions: pH buffer. Temperature range: 7.0, 3.0 A least squares fit of the data gave an E 0 ' 2 mY at 25 °C and (dE '/dT) 25 o C = -2.06 x 10- 3 0 283 596 578 560 w I z 542 (f) > 524 > -E ... 506 0 w 488 470 452 434_· 4~--------~--------~--------~--------~~----~46 6 16 26 TEMPERATURE (°C) 36 284 corresponding reaction ~H 0 ", enthalpy, is -30.7 0.4 :!: kcal/mol, and reaction free energy change, ~G 0 ", is -11.85 :!: 0.05 kcaljmol. The most experiment, formal interesting observation in relatively lies reduction the potential, the reaction entropy, ~s~;. indication ion. of the nature According complexes those and one sulfur are it possible quite high the spectroelectrochemical value determined very negative value sphere around investigations model of ring of the ruthenium sulfur ligands, 40 coordination sphere consists of likely to have their ion histidine one sulfur from dimethyl sulfoxide. redox five poten- With reference to this finding, ruthenium proximal for ammineruthenium is coordinated four pyrrolic nitrogens from the porphyrin, one nitrogen idazole found related most the the and sulfide the for value of 514 vs. NHE, gives some 700 mY vs. NHE. that the the coordination hydrogen which 500 - is of in tials around in The E 0 " previous containing systems nitrogens to and from i.e., F8, by the from the im- histidine-93, However, the formal and redox poten- tial of [Ru(NH 3 ) 5(DMS0)] 3+/ 2+ is 1,000 mY vs. NHE in 0.1 M HCl solution. 40 In some Figure transition metalloproteins, metal complexes, 13, the reaction entropies metal complexes, are summarized. 6, 7,4 1-45 the reaction in for addition entropies to In of the redox native contrast a large couples of and modified to transition number of me- talloproteins are found to range from 0 eu to -16 eu. In the case of [R u(MpiX)(DMSO) sperm dicarboxylic acid ]-reconstituted myoglobin, the value of ~s~; is exceptionally negative, -47.6 eu. whale 285 Figure 13. Reaction tion entropies metal talloproteins. of complexes redox as well couples as for native and selected transi- modified me- 286 REACTION ENTROPY (eu) 50 40 30 (Ru ( NH3 ls p+; 2+ 20 [Ru (NH3)4 (phen))3+f2+ ~ LACCASE ( R. vernicifera) (Ru ( NH3)2 (bpy))3+f2+ [os ( bpyhJ3+f2+ [Fe (ph en )3 p+/2+ [Fe (bpy) 3 ]3+ /2+ ______j LACCASE (P. versicolor) 1 - o - - CYTOCHROME £ (R.rubrum) PLASTOCYANIN (R. vulgaris) STELLACYANIN ( R. vernicifera) ___I - 1 o - - CYTOCHROME .£ 2 (R. rubrum) CYTOCHROME.£ (Horse Heart) [Fe(4, 7 -dihydroxyphenhp-;4 - ~ CYTOCHROME .£ AZURIN (P. aeruginosa) 551 (P. aeruginosa) MYOGLOBIN (Sperm Whale) -30 (Fe (CN)4 ( bpy)] -/2-40 (Fe (CNls] 3 -;4 - -50 (Ru (MpiX) (DMSO) H2 ) -MYOGLOBIN (Sperm Whale) 287 According myoglobin, to the spectroelectrochemistry which has been investigated native myoglobin has been found entropy, eu. 6 •7 Since measurement was -23.6 carrier, tions, of this in the order to protein. prevent The to native Dr. Walther possess quite a myoglobin made of reduction sperm R. Ellis, oxygen strictly anaerobic condi- oxymyoglobin upon and its reduction temperature native sperm whale myoglobin, are presented in 4 14. 45 The comparative plot the formal reduction potentials Figure dependence of [Ru(MpiX)(DMSO) in Figure native 15. and dicarboxylic The histidine-48 [Ru(MplX)(DMSO) of acid]-reconstituted electrochemical and Table the temperature of native myoglobins thermodynamic pentaammineruthenium-modified, dicarboxylic Jr., an potential, as dependence, for and whale negative reaction functions under formation formal by of acid ]-reconstituted, is and shown parameters of in to addition sperm whale myoglobins, are summarized in Table 5. The heme heme plane m in iron in myoglobin is both of the available oxidation case. The out-of -plane aquometmyoglobin, 0.55 A each for oxymyoglobin. The axial from iron upon reduction to positive to the lead a significantly distances ligand of from the states, and is highspin are 0.40 and 0.33 deoxymyoglobin, water value displaced metmyoglobin of the protein, and for the reaction this entropy. for A for dissociates process ought However, a quite negative l!.s~; has been observed instead . 45 The results in Table 6 show that aquometmyoglobin to deoxymyoglobin is associated the transition with a from movement of 288 Table 4. Temperature dependence of the formal reduction potentials, E 0 ", for the heme center in native sperm whale myoglobin, using nonisothermal Solution conditions: This experiment formation thin-layer pH 7.0, was done spectroelectrochemistry. 100 mM, phosphate buffer. 1..1 = in anaerobic condition of oxymyoglobin was prevented upon the metmyoglobin. and the reduction of 289 Temperature (° C) * E 0 ' (mV vs. NHE) t 4.9 79 7.5 77 12.5 71 14.9 70 20.0 64 25.0 60 30.0 54 35.0 48 39.8 44 44.8 38 * :!: 0.2 °c t:!: 2 mV 290 Figure 14. Temperature E 0 ", for dependence native sperm spectroe1ectrochemistry. 100 mM, of the formal reduction whale myoglobin, using thin-layer Solution condition: pH 7.0, Temperature range: 4.9 - 44.8 = 59 :!: phosphate buffer. °C. A least squares fit of the data gave an E mY at 25 potentials, 0 " °C and (dE "/dT) 25 o C = -1.03 x 10· 3 V /° C. 0 = ~ 2 291 91 83 -75 w :r:: z 67 u; > 59 > - ..0 E 51 w 43 35 27 8 19 30 TEMPERATURE (°C) 41 52 292 Figure 15. Temperature dependence of the formal reduction potentials, £ 0 ' , for the native sperm whale myoglobin (!::.) and for the [Ru(MpiX)(DMSO) whale myoglobin ( • ). dicarboxylic acid ]-reconstituted sperm 293 800 I I I I - 700 - -w 600 500 -- - . • • • - - 400 - -- 300 - - E 200 - - I z (/) > > -0 w 100 - .. ... r . . r . r . .. 0-100 -200 - -4 .... - I I I I 7 18 29 40 TEMPERATURE (°C) 51 294 Table 5. Thermodynamic parameters for in native and myoglobins, as in [R u(MpiX)(DMSO) the reduction of iron centers [Ru(NH 3 )5(His-48)]-modified well as dicarboxylic of sperm ruthenium . . center acid]-reconstituted whale myoglobin. Solution conditions: phosphate buffer, 25 °C. pH 7.0, 1.1 whale in sperm = 100 mM, (kcal/mol) 111-f, (eu) /1So, et (eu) /1So, (kca1/mol) 11G0 " (mY vs. NHE) Eo, -13.0 -23.6 -39.2 -1.36 59 Fe3+/2+ Nathe S.W. Mb -12.7 -22.0 -37.6 -1.51 65 Fe3+/2+ (a 5 Ru(Hls-48))-S. W. Mb -30.7 -47.6 -63.2 -11.85 514 Ru3+/2+ (Ru(MpiX)(DMSO) dlacld)-S.W. Mb N VI \0 296 Table 6. Heme geometry in myoglobin derivatives. 297 Distance Deoxy-Mb • Aquo-Met-Mb t Oxy-Mb Fe-herne plane 0.55 0.40 0.33 His F8-heme plane 2.6 2.5 2.3 •Reference 17. t Ref ere nee 16. +Reference 20. + 298 the iron atom and plane, of the order proximal histidine of A and 0.15 between the iron center and idazole ring of proximal away F8 0.1 histidine the porphyrin A respectively. The distance nitrogen in the A shorter. coordinating the from F8, becomes 0.05 im- However, this kind of change in the coordination sphere of the metal ion does not seem to be the main factor accounting for the large negative value found planation for the of protein the for the reaction phenomenon structure, entropy. probably leaving lies little The most convincing ex- in dramatic contraction a interior space for water molecules. The question the reaction myoglobin much more to of is reasonable rigid, leading greater [Ru(MpiX)(DMSO) interesting before seems heme of an dimethyl most a is work Ru(III), stituted rationalizing the large negative entropy whale sociation of a satisfactory sulfoxide, unlikely expected problem, in A +2 and both to internal of ordering fallowing reduction of the protein. in in the the will solution occur. lies degree and the explanation stonger dicarboxylic following to value found is found . low-spin +3 solvation at The disRu(II) ru theni um-su bstates. The becoming more interactions, with itself solvent-protein require from oxidation protein acid]-sperm probably transition for the protein surface 299 References C., and Soc., 2. Nocera, D. G., Winkler, J. R., Yocom, K. M., Gray, H. B., J. Am. Chern. Soc., 106, 5145 (1984). Bordignon, E., and 3. Yocom, K. M., Winkler, J. R., Nocera, D. Gray, H. B., Chern. Scr., 21, 29 (1983). G., Bordignon, E., and 4. Winkler, J. R., Nocera, D. 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