i 4-1. . M‘ llllxl Ufa? hiichigan State University This is to certify that the dissertation entitled THE APPLICATION OF OPTICAL ABSORPTION AND RESONANCE RAMAN SPECTROSCOPY TO THE STUDY OF HEME PROTErIeNS mA‘lillgy MODEL COMPOUNDS Robert T. Kean has been accepted towards fulfillment of the requirements for Ph . D . degree in Chemistrz AW 7, MM Major professor 3///X7. 0-12771 _— _._‘__—*—»_._.-~r—§, __ _t—-——‘—— x— ‘— 5» v-—’W~-i_ _.._-_.. . A __ A RETURNING MATERIALS: Place in book drop to‘ remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. MSU i LIBRARIES 2030 Jstzol'm THE APPLICATION OF OPTICAL ABSORPTION AND RESONANCE RAMAN SPECTROSCOPY TO THE STUDY OF HEME PROTEINS AND MODEL COMPOUNDS By Robert T. Kean A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1987 '\ O 0 m m m 3. ABSTRACT THE APPLICATION or OPTICAL ABSORPTION AND RESONANCE RAMAN SPECTROSCOPY TO THE STUDY OF HEME PROTEINS AND MODEL COMPOUNDS By Robert T. Kean A variety of experiments have been performed with the goal of elucidating the reaction pathway for the reduction of oxygen by cytochrome oxidase. Because of the complexity of cytochrome oxidase, much of this work has involved studies of heme compounds in solution and species of simpler heme proteins as models for species in the cytochrome oxidase catalytic cycle. These studies have focused on ferrous oxy (FeII—Oz) and ferryl oxo (FeIV=O) hemes, which have been identified as key intermediates in other heme enzymes. Since ferrous oxy and ferryl oxo hemes are unstable at room temperature, low temperature optical absorption and resonance Raman spectroscopic techniques have been developed for characterization of these species. Low temperature studies of solution species have been used to interpret results from the corresponding protein species. Raman studies with cold trapped cytochrome oxidase species support the hypothesis that both ferrous oxy and ferryl oxo species are active in the catalytic cycle. These studies, in conjunction with studies of other ligand bound heme species, demonstrate that the peripheral porphyrin substituents have little or no affect on the bond strengths of the axial ligands as monitored by their Fe-ligand vibrational frequencies. The chemistry of species such as the ferrous oxy (Fell-02), ferryl oxo (FeIV=O) and ferric cyanide (FeIII-CN‘) seem to be controlled by out-of-plane effects such as trans ligand strength, steric constraints, and hydrogen bonding. However, comparison of the optical absorption spectra of protein and solution hemes suggests direct perturbation of the porphyin ring by specific amino acid residues in the protein species. Studies with a copper chelating heme model species indicate a structure analogous to that of the cytochrome oxidase oxygen reduction site. This model duplicates the six—coordinate high-spin heme geometry of cytochrome oxidase as detected by Raman spectroscopy. EPR studies indicate that the presence of an oxo (0'2) bridge between the two metal centers can produce magnetic exchange coupling like that observed in the resting state of cytochrome oxidase. Optical absorption studies of various ligated states.of this model suggest a possible identification of the 655 nm absorption band observed in resting cytochrome oxidase. To my mother and in memory of my father. ii ACKNOWLEDGMENTS I would like to thank: Keki, Manfred, and Scott (the worlds finest glassblowers) for the fabrication of Dewars and glassware; Deak, Russ, and Dick for equipment fabrication; Marty for electronic design and assistance in electronic trouble-shooting; Ron and Scott for laser and equipment repairi and Tom for assistance with my computer work. Without their skills and expertise, I could not have completed this research. I would like to thank: Dr. Babcock, my graduate adviser, for his guidance and financial support; Dr. Chang for his cooperation and advice; and Dr. Schwendeman and Dr. Ferguson-Miller for being part of my committee. M. S. K00 and Asaad Salehi have graciously provided me with compounds for study; John Manthey and Stephan Witt (from the California Institute of Technology) have worked with me in the joint study of cytochrome oxidase intermediates. Tony and Dwight deserve a special thanks for their technical assistance with Raman spectroscopy and computers respectively. I am grateful to the other members of the Babcock group for their friendship, comradery, and comic relief. I am extremely indebted to Nirmala and Ravishankar, who helped me in my final months by giving me a place to live. Finally, I would like to thank Mary for her love, help, and constant encouragement through my last year of research and the entire preparation of this dissertation. This research was supported in part with a grant from the National Institute of Health. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES CHAPTER 1 INTRODUCTION A. B. . Physical Techniques for Heme Protein Research Overview of Heme Proteins Structure and Reactivity of Molecular Oxygen Peroxidases, Catalases, and Cytochromes P-450 . Cytochrome g Oxidase CHAPTER 2 EQUIPMENT AND TECHNIQUES A. B. CHAPTER 3 RESONANCE RAMAN CHARACTERIZATION OF CYANIDE Resonance Raman Spectroscopy Low Temperature Spectroscopy . Anaerobic Techniques . Electrochemistry BOUND HEME PROTEINS AND MODEL COMPOUNDS . Introduction . Materials and Methods . Results . Discussion 10 23 25 36 41 48 53 57 59 61 66 CHAPTER 4 CHARACTERIZATION OF CYTOCHROME g3 MODEL COMPOUNDS A. Introduction 77 B. Materials and Methods 80 C. Meso-diphenylporphyrin Results 82 D Cytochrome Oxidase Model Compound Results 91 E. Discussion 111 CHAPTER 5 CHARACTERIZATION OF "OXY" AND "OX0" HEMES AS MODELS OF HEME PROTEIN REACTION INTERMEDIATES A. Introduction 124 B. Materials and Methods 128 C. Results 130 D. Discussion 146 CHAPTER 6 RESONANCE RAMAN SPECTROSCOPY OF CYTOCHROME OXIDASE "PSEUDO-INTERMEDIATES" A. Introduction 167 B. Materials and Methods 169 C. Results 171 188 D. Discussion CHAPTER 7 CONCLUSIONS AND FUTURE WORK A. Conclusions B. Future Work APPENDIX 1 LIST OF REFERENCES vi Table Table Table Table Table Table Table Table Table Table Table Table Table Table U! U! 0" 0" LIST OF TABLES Raman Peaks of FeIII Meso-Diphenylporphyrins. Comparison of High-Spin FeIII Meso-Diphenylporphyrin Concentrations as Determined by Optical Absorption and EPR Spectroscopy. Slope and Intercept Values for the Core Size Dependent High Frequency Raman Peaks of FeIII Meso-Diphenylporphyrins. Comparison of the Vibratons of Three Different Porphyrin Types. Optical Absorption Peaks of Ferrous Oxy and Ferryl Oxo Heme Species. Raman Peaks of Iron Octaethylporphyrin Species. Raman Peaks of Iron PPIX Protein and Model Species. Raman Peaks of Various TPP Type Heme Species. Optical Absorption Peaks of Protoheme Containing Ferrous Oxy and Ferryl Oxo Species. Comparison of Iron-Oxygen Stretching Frequencies for Various Ferrous Oxy and Ferryl Oxo Species. Iron-Imidazole Stretching Frequencies of Five-Coordinate ferrous Hemes. Distinguishing Characteristics and Raman Peaks of Frozen Cytochrome Oxidase Species. Raman Peaks of Heme a Model Compounds. Assignment of the Mid- and Low-Frequency Raman Peaks of Resting and 580 nm Species of Cytochrome Oxidase. 90 105 105 132 137 142 148 149 153 163 173 174 189 Figure 1.1 ?igure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 igure 1.7 igure 1.8 igure 1.9 gure 1.10 gure 1.11 gure 1.12 ure 1.13 LIST OF FIGURES The structure of a heme (from Callahan, 1983). The structure of protoheme: a) unmodified, b) thioether-linked to the protein. The structure of heme a. The molecular orbital description of dioxygen (02) (adapted from Jones et al., 1979). The one electron reduction potentials of 02 in solution at different pH values. Potentials are solution versus a normal hydrogen electrode (NHE) (from Sawyer and Nanni, 1981). The interaction of iron out of plane orbitals with the n* orbitals of 02 in FeII "oxy" heme complexes (from Reed, 1978). The optical absorption spectrum of FeII cytochrome C. The 2 HOMO's and 2 LUMO's of porphyrins, within the 4 orbital model (from Longuet-Higgins, 1950). Relative energy levels of porphyrin (_._) and iron (-—-) orbitals for ferric porphyrin complexes (adapted from Zerner et al., 1966). Raman (Stokes and anti-Stokes) and Rayleigh light scattering. A comparison of normal and resonance Raman light scattering (from Ondrias, 1980). Aerobic respiration (adapted from Lehninger, 1975). The shape of cytochrome oxidase (adapted from Henderson, 1977). viii ll 16 18 20 22 27 28 Figure 1.14 The relative locations of the metal centers in cytochrome oxidase (from Blair, 1983). Figure 1.15 Figure 1.16 Figure 1.17 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 igme3.l igueSl gure 3.3 a) The optical absorption spectrum of oxidized and reduced cytochrome oxidase, b) The approximation of the spectral contributions from the individual heme centers (from Vanneste, 1966). Heme a models for the heme centers in cytochrome oxidase (Figure courtesy of G. T. Babcock). A proposed catalytic cycle for oxygen reduction by cytochrome oxidase (Figure courtesy of G. T. Babcock). Computer interfaced scanning Raman spectrometer system. Low temperature Raman backscattering Dewar. Liquid nitrogen temperature backscattering Dewar system. Low temperature Dewar system for optical absorption spectroscopy. Anaerobic/vacuum system. Anaerobic glassware. Electrochemistry cell for small volume anaerobic work. A. Cyclic voltammagram of his 1~methy1- imidazole heme a (FeI ) in CH2C12 (electrolyte TBAP .lM) B. Optical absorption spectrum of his 1-methylimidazole heme a in CHZCIZ, which was reduced at -0.2 V (Vs. pesudo reterence). The structures of iron deuteroporphyrin DME and iron deuterochlorin. Optical absorption spectra of bis-NMI, CN' NMI, and bis-CN' ligated Pal I deuteroporphyrin. The optical absorption spectra ofIbis-NMI, NMI/CN', and bis-CN' ligated Fe deuterochlorin. ix 32 34 35 38 43 45 47 49 51 54 62 gure 3.4 gure 3.5 gure 3.6 gure 3.7 .gure 3.8 .gure 3.9 Lgure 3.10 Lgure 4.1 Raman spectra of bis-NMI, NMI/CH', and bis-CN' ligated FeIII deuteroporphyrin. Raman spectra of bis-NMI, NMI/CN‘, and bis-CN‘ ligated FeII deuterochlorin. Raman spectra of NMI, CN' NMI/CN' ligated FeIII deuteroporphyrin and deuterochlorin with isotope labeled CN‘. The Raman spectra of bis-CN' FeIII deuteroporphyrin and deuterochlorin with isotope labeled CN’. The optical absorption spectra of native and CN' bound horseradish peroxidase. The Raman spectra of native and CN' bound horseradish peroxidase. Raman spectra of CN' bound horseradish peroxidase with isotope labeled CN'. Structures of FeIII meso-diphenylporphyrin species. . Optical absorption spectra of FeIII meso—diphenylporphyrin species. Comparison of the optical absorption spectra of six-coordinate high-spin FeI I meso-diphenylporphyrin species. High frequency Raman spectra of FeIII meso-diphenylporphyrin species. Low frequency Raman spectra of FeIII meso—diphenylporphyrin species. Structures of meso-diphenylporphyrin cytochrome oxidase model compounds. Comparison of the optical absorption spectra of five—coordinate, Cl' ligated FeI meso-diphenylporphyrin species. Comparison of the optical absorption spectra of five-coordinate, OH' ligated/ 0' bridged FeIII meso-diphenylporphyrin species, Comparison of the optical absorption spectra of six-coordinate, Cl‘ ligated Fe I meso-diphenylporphyrin species. 65 67 68 69 7O 71 72 83 84 86 88 89 92 94 95 96 gure 4.10 gure 4.11 gure 4.12 ,gure 4.13 .gure 4.14 Lgure 4.15 igure 4.16 gure 4.17 gure 4.18 ure 4.19 re 5.1 re 5.2 re 5.3 re 5.4 re 5.5 ComParison of the optical absorption spectra 97 of six-coordinate, OH" ligated/ O' bridged FeIII meso-diphenylporphyrin species. Comparison of the high frequency Raman 99 spectra of five-coordinate, Cl' ligated FeIII meso-diphenylporphyrin species. Comparison of the high frequency Raman 100 spectra of five-coordinate, OH' ligated/ O‘2 bridged FeIII meso-diphenylporphyrin species. Comparison of the high frequency Raman 101 spectra of six-coordinate, Cl‘ ligated FeIII meso-diphenylporphyrin species. Comparison of the high frequency Raman 102 spectra of six-coordinate, OH‘ ligated/ 0'2 bridged FeIII meso-diphenylporphyrin species. EPR spectra of Met Mb F' and five-coordinate, 104 61' ligated FeIII meso-diphenyl porphyrin species. EPR spectra of five-coordinate, OH' ligated/ 107 0‘2 bridged FeIII meso-diphenylporphyrin species. EPR spectra of six-coordinate FeIII 103 meso—diphenylporphyrin species. A plot of signal intensity versus l/T (Curie 110 Law) for Met Mb F" and the five-coordinate O'2 bridged cytochrome oxidase model compound. The core size dependence of the high 113 frequency Raman vibrations of FeII meso-diphenylporphyrin species. Optical absorption spectra of Bis-NMI and 131 "oxy" ferrous octaethylheme. Identification of u(FeII-02) for ferrous oxy 133 octaethylheme, protoheme, and heme a. Raman spectra of Bis-NMI and "oxy" ferrous 135 octaethylheme (low frequency region). Raman spectra of Bis-NMI and "oxy" ferrous 135 octaethylheme (high frequency region). Optical absorption spectra of ferryl oxo 138 protoheme, octaethylheme, and tetraphenylheme. xi igure 4.10 igure 4.11 igure 4.12 igure 4.13 igure 4.14 igure 4.15 Igure 4.16 'gure 4.17 gure 4.18 re 4.19 ure 5.1 re 5.2 re 5.3 re 5.4 re 5.5 Comparison of the optical absorption spectra of six-coordinate, OH‘ ligated/ 0’ bridged FeIII meso—diphenylporphyrin species. Comparison of the high frequency Raman spectra of five-coordinate, Cl' ligated FeIII meso-diphenylporphyrin species. Comparison of the high frequency Raman spectra of five—coordinate, OH‘ ligated/ 0'2 bridged Fe II meso—diphenylporphyrin species. Comparison of the high frequency Raman spectra of six—coordinate, Cl" ligated FeIII meso-diphenylporphyrin species. Comparison of the high frequency Raman spectra of six-coordinate, 0H” ligated/ 0'2 bridged FeIII meso-diphenylporphyrin species. EPR spectra of Met Mb F~ and five-coordinate, Cl' ligated FeIII meso-diphenyl porphyrin species. EPR spectra of five-coordinate, 0H" ligated/ 0' bridged FeIII meso-diphenylporphyrin species. EPR spectra of six-coordinate FeIII meso-diphenylporphyrin species. A plot of signal intensity versus 1/T (Curie Law) for Met Mb F' and the five—coordinate 0' bridged cytochrome oxidase model compound. The core size dependence of the high frequency Raman vibrations of Fe11 meso-diphenylporphyrin species. Optical absorption spectra of Bis-NMI and "oxy" ferrous octaethylheme. Identification of v(FeII-02) for ferrous oxy octaethylheme, protoheme, and heme a. Raman spectra of Bis—NMI and "oxy" ferrous octaethylheme (low frequency region). Raman spectra of Bis-NMI and "oxy“ ferrous octaethylheme (high frequency region). Optical absorption spectra of ferryl oxo protoheme, octaethylheme, and tetraphenylheme. xi 97 99 100 101 102 104 107 108 110 113 131 133 135 136 138 gure 5.6 gure 5.7 gure 5.8 gure 5.9 gure 6.1 gure 6.2 gure 6.3 gure 6.4 ure 6.5 ure 6.6 re 6.7 re 6.8 re 6.9 Identification of u(FeIV-0) for ferryl oxo protoheme, octaethylheme, and tetraphenylheme. Raman spectra of ferryl oxo, p-oxo dimer and p-peroxo dimer of protoheme (high frequency region). Raman spectra of ferryl oxo and four-coordinate ferrous octaethylheme (high frequency region). Raman spectrum of ferryl oxo tetraphenylheme (high frequency region). High frequency spectra of photoreduced cytochrome oxidase samples (liquid and frozen). High frequency Raman spectra of resting cytochrome oxidase (liquid and frozen). High frequency Raman spectra of 580 nm species of cytochrome oxidase (liquid and frozen). High frequency Raman spectra of frozen compound C cytochrome oxidase (MVCO + 02 and pulsed plus peroxide) and pulsed cytochrome oxidase. High frequency Raman spectrum of frozen "reoxidized" cytochrome oxidase. High frequency Raman spectrum of CO reacted 580 nm species of cytochrome oxidase. Low frequency Raman spectra of frozen resting and 580 nm ( 6O and10) cytochrome oxidase. Intermediate frequency Raman spectra of frozen resting and 580 nm ( O and 18O) cytochrome oxidase. Upper-intermediate frequency Raman s ectra of frozen resting and 580 nm ( 60 and1 0) cytochrome oxidase. 140 141 145 147 172 176 178 180 183 185 186 187 CHAPTER 1 INTRODUCTION OVERVIEW OF HEME PROTEINS Heme proteins are well known for their role in oxygen transport amoglobin) and oxygen storage (myoglobin). Less well known are the Ltitude of other heme proteins and enzymes which are amazing in their :iations of structure and function and their occurrence in nearly all >logical systems. The basic structure of a heme can be seen in Figure It consists of a central iron ion strongly chelated by the four :role nitrogens of the conjugated porphyrin macrocycle. What :tinguishes one heme from another is the pattern of substitution at labeled peripheral positions. The biochemical function and the sical properties of a heme protein or enzyme are controlled by these substituents, and specific environmental factors produced by the ounding protein. The latter effects include hydrogen bonding, tion (from amino acid residues) to the axial position of the iron, tron transfer pathways, and hydrophobic or hydrophilic "pockets" ssible to exogenous ligands. It is the variability of the above ined effects which allows for the great variety of heme eme proteins and enzymes are generally categorized into groups ding to the heme which they contain or their general functional ties (see Adar 1978). Cytochromes b utilize unmodified protoheme l Figure 1.1 The structure of a heme (from Callahan, 1983). PW inse Chem “mm displ Prote in th 3 (Figure 1.2a) as the prothetic group, with the heme held in the protein 3y axial ligation (normally the nitrogen of a histidine residue). Iytochromes c are protoheme containing globular proteins, in which the >rotoheme is covalently linked to the protein through the thioether inkages to the 2,4 vinyl groups (Figure 1.2b). Although some of the emes in these proteins will bind exogenous ligands (Andersson et al. 986; 0ndrias 1980), the axial ligation of cytochromes b and c does not mange under normal activity. They appear to function primarily as .ectron carriers with the iron undergoing redox changes between the +2 d +3 states. In contrast to these, there exists a large number of me enzymes and proteins in which changes in the axial ligation is herent to normal activity. In these systems, one of the iron axial sitions is accessible to exogenous ligands that can bind to or react :h the heme iron. The chemistry of these heme systems is generally e complicated than that of the cytochromes b and c and owing to it prominence in biological systems, they have been the subjects of ensive research. Included in this group are the globins, cytochromes 50, peroxidases, catalases, and cytochrome oxidase. Hemoglobin and globin bind 02 for transport and storage respectively. Cytochromes ‘use 02 to metabolize various compounds via specific oxygen rtion (Griffin et a1. 1979). Peroxidases use peroxide (H202) as a ical oxidant. They react with a wide variety of substrates and are :ional, for example, in anti-infection defense systems. Catalases re peroxides from biological systems by catalyzing their Oportionation to 02 and H20 (Hewson and Hager 1979). These ins usually contain protoheme, the most commonly occurring heme, 3 active site. Cytochrome oxidases catalyze the exothermic 4 EH2 0) CH CH3 H3C CH=CH2 C‘Hz CIHz cH2 CH2 l l COOH COOH protoheme b scnzcmunpcoou ) CH CH3 £3? H3C cuscu ' $00 CH, H H3C CH3 sz sz CH2 CH2 COOH COOH fhioesfer—linked profoheme Figure 1.2 The structure of protoheme: a) unmodified, b) thioether-linked to the protein. I -‘_"...l“,'1:L.--'.! " ‘fl — h ' Spl PIE que res CHI 50 r The Stat. Stan of t! in 9E 5 duction of 02 to H20, thereby providing the thermodynamic driving rce for the synthesis of ATP (stored chemical energy). Cytochrome idase is unusual and complex in that it utilizes two hemes and two per ions in the catalytic process (Wikstrom, M. et al. 1981). The es of cytochrome oxidase are heme g, the structure of which is seen Figure 1.3. The common factor in all these heme enzymes is that they lize oxygen (02 or H202) as a ligand or reaction substrate. Although se heme systems can bind other small ligands (CO, CN-, NO, etc ), ctivity is predominantly restricted to oxygen. To understand this ific and unusual chemistry, the following questions must be ered: (1) what is the physical nature of the interaction between as and oxygen; (2) what are the specific mechanisms of these erent enzyme reactions; and (3) what factors control the rate and ificity of these reactions? The results of my research will be ented in later chapters and the discussion will address these :ions. The remainder of the introduction will summarize the .ts, tools and ideas which have brought this area of science to its nt state . STRUCTURE AND REACTIVITY OF MOLECULAR OXYGEN > understand why molecular oxygen binds to and reacts with hemes Idily, it is useful to examine the structure of molecular oxygen. 'bital diagram of dioxygen is shown in Figure 1.4. The ground contains a double bond and has two unpaired electrons (triplet Conservation of spin requirements severely limit the reactivity >let species. The lowest singlet state is ~22.3 kcal/mole higher 'gy (Jones et al. 1979). Peroxides, which contain two more 5 duction of 02 to H20, thereby providing the thermodynamic driving rce for the synthesis of ATP (stored chemical energy). Cytochrome idase is unusual and complex in that it utilizes two hemes and two per ions in the catalytic process (Wikstrom, M. et al. 1981). The es of cytochrome oxidase are heme g, the structure of which is seen Figure 1.3. The common factor in all these heme enzymes is that they lize oxygen (02 or H202) as a ligand or reaction substrate. Although se heme systems can bind other small ligands (CO, CN~, NO, etc.), tivity is predominantly restricted to oxygen. To understand this ific and unusual chemistry, the following questions must be ered: (1) what is the physical nature of the interaction between as and oxygen; (2) what are the specific mechanisms of these crent enzyme reactions; and (3) what factors control the rate and ificity of these reactions? The results of my research will be ented in later chapters and the discussion will address these :ions. The remainder of the introduction will summarize the .ts, tools and ideas which have brought this area of science to its nt state. STRUCTURE AND REACTIVITY OF MOLECULAR OXYGEN > understand why molecular oxygen binds to and reacts with hemes Ldily, it is useful to examine the structure of molecular oxygen. 'bital diagram of dioxygen is shown in Figure 1.4. The ground contains a double bond and has two unpaired electrons (triplet Conservation of spin requirements severely limit the reactivity alet species. The lowest singlet state is '22.3 kcal/mole higher 'gy (Jones et al. 1979). Peroxides, which contain two more 6 HO- CH ”SC CH: -—CH2 \ O=C CH3 H CH: H2 COOH COOH heme 9 Figure 1.3 The structure of heme a. 2: Figure 1' Figure 1.4 The molecular orbital description of dioxygen (02) (adapted from Jones et al. , 1979). electronS. are Sin The four electron stage, to water ha solution under con: in Figure 1,5. 58‘” (1) the overall fOL favorable (large PC (2) the reaction be electron reduction high pH). It is the thermodynamically m almost inert in the with a singlet grout high pH), is a much The energies of the oxygen HOMO's (2; °f Plane orbitals arc interactions. These ¢ Weak 0 donor but a adelocalization of e 1977) from the iron t structure of Oxygen b Converged on roughly ”111mm“ pp. 26-: Q0 ' nflgurationsz the f] 0x hen. the Second is 8 trons, are singly bonded and have no unpaired electrons (singlet). four electron electrochemical reduction of 02, through the peroxide e, to water has been studied by Sawyer and Nanni (1981) in aqueous tion under conditions of different pH. These results are reproduced igure 1.5. Several important points can be observed in these data: he overall four electron process is highly thermodynamically able (large positive reduction potential) at all values of the pH; he reaction becomes more favorable at lower pH values; and (3) one ron reduction of 02 is unfavorable at all values of pH (more so at pH). It is the triplet ground state of 02, coupled with the -dynamically unfavorable first reduction step, that makes 02 inert in the absence of a catalyst (Malmstrom 1982). Peroxide, I singlet ground state and exothermic reduction steps (except at IH), is a much more reactive species. a energies of the iron 3d orbitals in hemes are close to those of rgen HOMO’s (2ng). In addition, the symmetries of the iron out 1e orbitals are suitable for both a and N type bonding tions. These can be seen schematically in Figure 1.6. Oxygen is a donor but a stronger n acceptor. The net result of the bond is alization of electron density (ca. 0.1 e', Olafson and Goddard ion the iron to the oxygen. Calculations of the electronic 'e of oxygen bound hemoglobin by a variety of methods have d on roughly the same physical model (see Gubelmann and 1983 pp. 26-27). Oxyhemoglobin is pictured as a mixture of two itions: the first is a low spin Fe+2 (S - 0) bound to singlet :he second is an intermediate spin Fe+2 (S - l) bound to STANDARD REDUCTJ Figul'e 1. 5 “Trap-4...: STANDARD REDUCTION POTENTIALS FOR DIOXYGEN SPECIES IN WATER +L66 If +n.oa I I 1 ' +0595 1 l “763 J +0.70 i use. pH o “M W) +:.20 I! +0.59 1 l +0.28: l l_ +l.349 J 1 +0.29 1 I +0315 pH 7 +055 I —o.03 I l 32 —o.33 0‘2; +0.20 H02- —0.25| HO‘+-0H H.985 40“. L_ —0.065 I 1 +0.86? 1 [_ -o.|3 g +0.40: pH l4 UM OH") Figure 1.5 The one electron reduction potentials of 02 in solution at different pH values. Potentials are solution versus a normal hydrogen electrode (NHE) (from Sawyer and Nanni, 1981). triplet oxygen. T“ 1.5, with each SPe Since the spins co are net S -0 (Sin. transfer from the I calculations but it oxygen can be consi to reaction has bee hemoglobin has serv reactive heme syste1 higher energy anti-l (0-0) bond (Gubelmar fundamental step in 02 Will only bind to also react with oxid Selution) With 02 an given clues as to re. 1978; Chin 1980). m in the analysis of or PHYSICAL TECHNIC In an ideal bioch 1n . terest In a pure f0 10 iplet oxygen. The latter configuration is the one depicted in Figure 6, with each species contributing an electron to the a and « bonds. nce the spins couple in the second case, both these configurations e net S - O (singlet). A third configuration corresponding to eletron ansfer from the iron to the oxygen (Fe+3, 02‘) can be included in the culations but it makes only a minor contribution. The heme bound gen can be considered "activated" in that the triplet state barrier reaction has been removed. This description of oxygen binding to oglobin has served as a model for the initial binding of oxygen to ctive heme systems. Transfer of electrons from the heme to the er energy anti-bonding oxygen orbitals results in cleavage of the ) bond (Gubelmann and Williams 1983 pp. 20-24). This is the amental step in the reaction of heme proteins with oxygen. Although rill only bind to and react with reduced hemes (Fe+2), peroxide will react with oxidized hemes (Fe+3). The reactions of hemes (in tion) with 02 and peroxide have been studied and these results have a clues as to reasonable mechanisms in the protein species (James r Chin 1980). These mechanisms will be discussed in later chapters Le analysis of our experimental results. PHYSICAL TECHNIQUES FOR HEME PROTEIN RESEARCH 1 an ideal biochemical world, we would isolate the enzyme of est in a pure form, perform an elemental analysis (to identify and other non-protein components), sequence all the peptides and a crystal structure. Although enzymes don’t normally function in lline form, this would give us the necessary structural ltion to begin intelligent investigations of the mechanism. It ZIIO‘OIIF lz o l O O 8. 7 O 9. a, b 0‘ 17" 0 The interaction of iron out of plane orbitals with the n* orbitals of 02 in Fe "oxy" heme complexes (from Reed, 1978). Figure 1.6 would be better y‘ intermediates as ‘ studies COUld be e Preferred. With SO hemoglobin, and my extent. Unfortunatl molecules, crystal: it seems to be 111111j species. Many enzle cytochrome oxidase, the isolation and 0 been well character still lacking. In mt most important is ti this is where the im obtain this informat resort to less direc their usefulness, vi The techniques 01 ) Spectroscopies containing Proteins, 381151 is dominated b tec ' hnlques, aqueous s d e . C susceptibility are sh gnal provides specii 12 d be better yet to obtain crystal structures of the reaction rmediates as well. Once the reaction pathway was known, theoretical ies could be employed to try to understand why this mechanism is erred. With some of the simpler heme systems (such as cytochrome c, globin, and myoglobin) these steps have already occurred to a great t. Unfortunately, the biochemical world is still real. For large ules, crystallography can be a very slow process and at present, ems to be limited to small proteins and very stable intermediate es. Many enzymes, especially membrane bound enzymes like rome oxidase, have not been successfully crystallized. Although olation and chemical reactivity of many of the heme enzymes have sell characterized, structural and mechanistic information is lacking. In most heme enzymes, the structural information that is .mportant is that in the immediate vicinity of the heme, since 5 where the important chemistry occurs. Since we cannot always this information directly by crystallographic means, we must to less direct techniques. Some of the possible techniques, and lsefulness, will be discussed below. techniques of infrared (IR) and nuclear magnetic resonance ectroscopies have been of limited use for the study of heme ng proteins, as they are not very specific and the observed 3 dominated by bands from bulk protein. In addition, for both es, aqueous solution systems present technical problems for lection. Electron paramagnetic resonance (EPR) and magnetic bility are useful for the study of heme proteins. The observed ovides specific information about the metal centers because the signals arise Although EPR is 11 utility for the id heme proteins (Pei: especially valuable the heme and the cc under various condi electron nuclear do extensively in heme ENDOR allows the ob: 59115181181. The tecl environment of paran limited by the lack Valuable for the stu al. 1978). Magnetic technique which has . heme proteins (Babcoo direct information al it is of limited use Of S7Fe ( (XAFS) Munck, E. 19 is a technique Systems. Although int “Sh, this method is . Uh der favorable condii The techniques the the ‘ racterization and 13 ignals arise from unpaired electrons on the metal centers. .ugh EPR is limited to odd spin systems, it has been of great ty for the identification of imidazole ligation in a number of proteins (Peisach and Mims 1977). This technique has been an ially valuable tool in the study of cytochrome oxidase since both eme and the copper contain unpaired electrons and can be detected various conditions (Blair et al. 1983). A companion technique, on nuclear double resonance (ENDOR), has not yet been used ively in heme research but shows great promise (Palmer 1979). allows the observation of nuclear transitions via changes in the gnal. The technique gives NMR like resolution in the restricted nment of paramagnetic centers. Magnetic susceptibility is d by the lack of structural detail it provides, but it is Le for the study of both odd and even spin systems (Tweedle et '8). Magnetic circular dichroism (MCD) is another magnetic :ue which has allowed identification of the iron spin state in oteins (Babcock et al. 1976). Mossbauer spectroscopy provides information about the iron environment and oxidation state but f limited use if the system cannot be enriched in the percentage (Munck, E. 1979). X-ray absorption fine structure spectroscopy .5 a technique that has been applied extensively to heme Although interpretation of the results has not been a simple is method is capable of providing direct structural information vorable conditions (Powers et al. 1981, 1982). echniques that have been used extensively for heme ization and structural investigation are optical absorption and resonance R3111" have used "1°“ ext more dEtail than t absorption SPeCtm cm'1) at 7‘00 m 1 bands (e-'5 3° 20 ' bands. These consis transition (0'0), 5 (0-1). 0f the theor Gouterman 1978), th to account best for HOHO’s and 2 LUMO’s asimple Huckel mode schematically in Fig atomic orbitals, cer orbitals. Dashed cir Planes are represent hemes the HOMO’S de respectively; the LU! symmetry. If one am electronic levels shc strong (allowed) high (forbidden) lov energ Predicts that was? ' 14 :esonance Raman spectroscopies. Since these are the techniques I used most extensively in my research, they will be described in detail than the techniques mentioned above. A typical heme 'ption spectrum is seen in Figure 1.7. The strong band (e-"lOO mM'1 at ~400 nm is referred to as the "Soret" or "B" band. The Weaker (e-'5 to 20 mM‘1 cm'l) to the red of the Soret are called the "Q" . These consist of the a band, which is a fundamental electronic ition (O-O), and the fl band, which is the first vibronic overtone , Of the theoretical treatments applied to heme spectra (see rman 1978), the four orbital model (Gouterman 1961 and 1978) seems :ount best for the observed behavior. In this model only the 2 and 2 LUMO's are considered. These orbitals, which are based on 1e Huckel model (Longuet-Higgins et al. 1950), are shown tically in Figure 1.8. The circles represent contributions of the orbitals, centered on the enclosed atoms, to the molecular .s. Dashed circles represent orbitals of opposite sign and nodal are represented by the heavy lines. Under the ~D4h symmetry of he HOMO’s, designated b1 and b2, are of azu and alu symmetry vely; the LUMO's (c1 and c2) are a degenerate set of eg If one assumes that the HOMO’s are nearly degenerate, the ic levels should mix through electron interactions, yielding a allowed) high energy transition (Soret) and a weaker en) low energy band (Q) (Gouterman, M. 1959). The theory that, as this degeneracy is relaxed, the Q band will become in relation to the Soret. In addition, if the degeneracy of Y axes is removed, the Q bands will split into their X and Y .3. These predictions agree with the observed spectra of hemes 2.50 ABSORBANCE 0.00 350.00 I”Si-Ire j 15 0.00 p I i ' ‘L ' ' l l r 350.00 550-00 WAVELENGTH (nm) Figure 1.7 The optical absorption spectrum of Pen cytochrome C. Figure 1_ 8 Nun's" 16 ’r’\ /‘| l \ If v- ’5 “’ I \_ , \ a I c, (e9) b'(02u) Figure 1.8 The 2 HOMO's and 2 LUMO's of porphyrins, within the 4 orbital model (from Longuet-Higgins, 1950) . with asylumetric r substitu‘?nt pertu other. Absorption spe metalloporphyrins orbitals to the HO which results in m specific mixing vai 1966). Spectra of i charge transfer bar region and arise pr 107-114). The charg weaker and at highe observed. Other asps needed in future Chi material, see Calla} Raman spectrosco information, similar molecular vibrations 0f heme proteins is 1 give 17 with asymmetric ring substitutions. These effects result from the substituent perturbation of one of the HOMO's (or LUMO's) but not the other. Absorption spectra of hemes are more complicated than other etalloporphyrins owing to the closeness in energy of the iron d :bitals to the HOMO’s of the porphyrins (and compatible symmetries), rich results in mixing. This can be seen in Figure 1.9 where the ecific mixing varies with different axial ligation (Zerner et al. 66). Spectra of high spin hemes are also complicated by fairly strong arge transfer bands. These are most often seen in the 500 to 700 nm gion and arise primarily from HOMO to dn transitions (Spiro 1983, pp 7-114). The charge transfer bands in other heme states are often rker and at higher energy wavelenghts (near IR) and are typically not erved. Other aspects of heme absorption theory will be discussed as ded in future chapters. For a more detailed discussion of the above erial, see Callahan (1983, Chapter 2). Raman spectroscopy is a technique which provides structural urmation, similar to IR spectroscopy, through the detection of cular vibrations. The advantage of Raman spectroscopy for the study eme proteins is that it can be used in a resonance condition to information specific to the heme vicinity. To understand the :e of this resonance, a brief theoretical discussion is useful. light scatters off a molecule, the scattered frequency will 11y be the same as the incident frequency (Rayleigh scattering). Raman scattering, the electromagnetic oscillations of light couple Energy (eV) Orbi‘l‘Ol I “Suite 1_ 9 18 bmtdxayz) ‘_____-—-—-F—-—-~---.-_-_p_ 99(77)—'_ —"— —--—-- —”_ _.._. o‘g(d22) ~ -. -90 ~-_ ; \ r\ 4— ‘I0.0 egidTr) ", \\\ 529(dxy)-d+ :=___~,___ _,.__ _____ ‘ \ 02 (r)""" “—+ ~~~~~~~ I __ _i ' u _ _-"‘ - ~~ ‘ _— alu(1r)—'— “" " ‘ “ —-..-_.. 6 'H.O 259 6A19 Alg Fe (111) CN Fe(III)OH Fe(]JI)F Figure 1.9 Relative energy levels of porphyrin (--—) and iron (-—) orbitals for ferric porphyrin complexes (adapted from Zerner et al., 1966). with the vibratio a vibrational fre note that Raman Si factor of aPPr°Xir "anti-Stokes" indl frequency, respect temperature or low originates from th Raman scattering i: scattered frequency squared. The polari dipole moment in re Albrecht 1970). A s as the Kramersheisi (a) ‘1 pf\ ag lize (apaigf is the trans scattered polarizati are dipole moment tr. e> l . and if> are the states . and “eg and b etween the subscript 1s a function of the 92.9 3). The integrals e - intensrties of th the ' relationships bets 19 ith the vibrations of the molecule to produce light shifted by + or - vibrational frequency. This is shown schematically in Figure 1.10; ite that Raman scattering is weaker than the Rayleigh scattering by a ctor of approximately 105. The designations'"Stokes" and nti-Stokes" indicate that the scattered light is of lower or higher aquency, respectively, relative to the incident light. At room nperature or lower, the Stokes Raman scattering will dominate, as it .ginates from the ground vibrational state. The intensity of the an scattering is proportional to the incident intensity, the ttered frequency to the fourth power, and the polarizability ared. The polarizability is defined as the change in the molecule’s ale moment in response to an applied electric field (Tang and 'echt 1970). A second order perturbation expression for this, known he Kramers-Heisenberg dispersion formula, is seen below. (“pa)gf -_1_ 2: + h e Veg-V0+1Fe Uef+uo+ire gf is the transition polarizability tensor, with incident and ered polarizations indicated by p and 0 respectively. pp and p0 ipole moment transition operators of polarization p and a; [g>, and lf> are the wave functions for the ground, excited, and final s , and Veg and Vef are the frequencies for the transitions an the subscripted states. Fe is the transition halfwidth, which ’unction of the lifetime of the excited state le> (Spiro 1983, pp. . The integrals in the numerators of the two terms evaluate to tensities of the electronic transitions; the denominators define lationships between the incident frequency and the frequencies of 20 M 'LLJ‘J‘AAJ (Va-'Vv) V0 (Vo‘i'l/v) ' E2 ‘i i 4i i 4i 1 r r E1 Au=i zit/=0 Ay=—1 Stokes Rayleigh Anti—Stokes Figure 1.10 Raman (Stokes and anti-Stokes) and Rayleigh light scattering. the electronic t1 incident frequenc transition ”eg' T consider all the ‘ following behaviOJ incident intensit) a fourth power dep in energy to an al polarizability EXP scattering will be if the excited stai spectroscopy is sin selective scatterir normal Raman is see bands in the visibl at wavelengths CIOSI scattering in the it from the bulk of the heme models in solut enhanced over those theory (Clark and St predictions: (1) r Predominantly the to the Q bands will enhe the polarization of t 11 tilt. is characteris Predictions n conju 21 the electronic transitions. The resonance effect is observed when the incident frequency is very close to the frequency of the electronic transition ”eg' This will cause the first term to dominate. If we ' consider all the variables of Raman scattering together we observe the following behavior: (1) Raman scattering increases linearly with incident intensity; (2) blue light scatters better than red light with a fourth power dependence; and (3) if the incident frequency is close Ln energy to an allowed electronic transition, the first term in the rolarizability expression becomes large and the total observed Raman cattering will be dominated by the resonance contribution; especially f the excited state has a long lifetime (small Fe). Resonance Raman pectroscopy is simply the use of the resonance effect to obtain elective scattering from a light absorbing species. A comparison with 'rmal Raman is seen in Figure 1.11. Since hemes have strong absorption nds in the visible region of the spectrum, the use of incident light wavelengths close to these absorption bands results in strong Raman attering in the immediate heme vicinity with negligible scattering >m the bulk of the protein. This process is also advantageous for ie models in solution, since the heme vibrations will be greatly :anced over those of the solvent. A more detailed analysis of the ory (Clark and Stewart 1979; Spiro 1983) yield the following dictions: (1) resonance with the Soret band will enhance dominantly the totally symmetric heme vibrations; (2) resonance with Q bands will enhance the non-totally symmetric vibrations; and (3) polarization of the scattered light, relative to the incident t, is characteristic of the vibrational symmetry. These ictions, in conjunction with calculations of metalloporphyrin lzgfifffifl'flWVT (SE NO! R01 Figure 1‘ 22 E2 5 E hvo hps - - Normal Resonance Ramon Roman Figure 1.11 A comparison of normal and resonance Raman light scattering (from Ondrias, 1980). normal modes of ‘ have made possibl vibrations. This for the analysiS in heme proteins. found in Callahan D. PEROXIDASES, Peroxidases an share many struc tu these enzymes has ' and Jones (1984). l prothetic group whl ligated by an axial to small ligands. P and the resting for state. The catalyti< Native Enzyme + } Compound I + c°mP°und II + All 2 is a substrate tv 1 “termedlates. The c Native Enzyme + HQC compound I + H20 23 normal modes of vibration (Abe et al. 1978; Gladkov and Solovyov 1986) have made possible the assignment of many of the observed heme vibrations. This has made resonance Raman spectroscopy a powerful tool For the analysis of the heme structure and of the immediate environment .n heme proteins. A more detailed analysis of the above theory can be ound in Callahan (1983, Chapter 2) or Ondrias (1980, Chapter 5). PEROXIDASES, CATALASES, AND CYTOCHROMES P-450 Peroxidases and catalases are often discussed together because they [are many structural and reactive similiarities. The biochemistry of ese enzymes has been summarized by Hewson and Hager (1979), and Frew d Jones (1984). Peroxidases and catalases usually contain a protoheme othetic group which, except for the subgroup chloroperoxidases, are gated by an axial histidine imidazole. The heme pocket is accessible small ligands. Peroxide is the oxidant common to both these systems, .the resting forms of these enzymes have iron in the +3 oxidation te. The catalytic cycle of peroxidases is summarized below: Iative Enzyme + H202 ---> Compound I Compound I + AH2 ---> Compound II + AH Compound II + AH2 -—-> Native Enzyme + AH is a substrate to be oxidized and compounds I and II are reaction rmediates. The catalytic sequence of catalases is similar: :ive Enzyme + H O ---> Compound I + H O 2 2 2 Compound I + H202 ---> 02 + H20 + Native Enzyme In addition, Pet“ The comm“ I a and they are bell u an Few-0 spec porphyrin orbital which are only or“ been positively 1‘ (Hashimoto et al. crystallized (Finz structural changes characterized. The well understood eii characterized of ti to it frequently. Cytochromes P-4 insertion of one no Slibstrate specificii reViev of this fiel< additional recent re 24 In addition, peroxidases can display catalase activity and vice versa. me compounds I are two oxidizing equivalents above the resting enzyme and they are believed to have a ferryl x-cation radical structure. This .s an FeIV-O species in which an electron has also been removed from a vorphyrin orbital to yield a cation radical species. The compounds II, rhich are only one oxidizing equivalent above the resting enzyme, have een positively identified as ferryl species for some peroxidases, Hashimoto et al. 1984). Although some of these enzymes have been rystallized (Finzel et al. 1984), the full catalytic cycle and tructural changes under turnover conditions have not been well haracterized. The factors which control substrate specificity are not all understood either. Horseradish peroxidase is probably the best maracterized of this group of enzymes and future chapters will refer > it frequently. Cytochromes P-450 are a group of heme enzymes which catalyze the ertion of one molecule of oxygen into a substrate. The degree of strate specificity varies with the particular enzyme. A thorough iew of this field can be found in Griffin, et al. (1979) while itional recent results have been discussed by Dolphin, et al. (1931). 01’0"“ (1 catalyzed by this on p.450 + 2e' vhere RH is the St in the (Hi bond. 1 Hi bond, at a $136 amount of attentio contain protoheme occupied by an R3' common histidine i1 mechanisms are stiI that the heme is ir 25 L981), Groves (1985), and Eble and DaWSon (1986). The basic reaction atalyzed by this class of enzymes is as follows: an P-ASO + 2e' + 2m“ + 02 + RH ----- > CYT P-4SO + ROI-i + H20 mere RH is the substrate and ROH is the product with oxygen inserted 1 the C-H bond. It is this ability to activate the normally unreactive H bond, at a specific substrate position, that accounts for the large bunt of attention that cytochromes P—450 have received. These enzymes ntain protoheme in the active site, but the axial position is cupied by an RS' ligand (from a cysteine residue) instead of the more mmon histidine imidazole. Like the other heme enzymes, their :hanisms are still not well understood. The evidence to date suggests it the heme is initially reduced to Fe+2. It can then bind oxygen to rm a species analogous to oxymyoglobin. Later steps are not clear but [re is a general consensus that a ferryl species is involved (Groves 5). Although the chemistry of cytochromes P-ASO are quite different that of the peroxidases and catalases, their reaction rmediates may contain some analogous structures. CYTOCHROME g OXIDASE The enzyme cytochrome g oxidase (cytochrome oxidase) has been a r focus of research in the laboratory of G. T. Babcock and it will scussed in greater detai1.than the previous enzyme systems. It is ps the most complex of the heme enzymes and its dominant role in recess of aerobic respiration has made it the subject of intense '. Several books and extensive reviews have been written on the subjea of cytocl 1979; WSW” at is referred t° th summary below wil function of the e1 an be emphasize‘ Cytochrome oxi functions at the t equivalents (elect increasingly bette: potential) until ti reaction is shown 12 on + oCYT c+ Coupled to oxygen In gradient which is fr biochemical energy). Provided by the redu Imlecules of ATP per anaerobic organisms localized on the inn 4 26 subject of cytochrome oxidase (see King et al. (ed) 1979; Malmstrom 1979; Wikstrom et a1. 1981; and Naqui and Chance 1986) and the reader is referred to them for details of its chemistry or biochemistry. The summary below will provide a brief overview of the structure and function of the enzymes. The role of the metal centers in catalysis will be emphasized. Cytochrome oxidase (labeled as "Cytochrome g3“ in Figure 1.12) functions at the terminus of the electron transport chain. Reducing equivalents (electrons) from the tricarboxylic acid cycle are passed to .ncreasingly better electron acceptors (more positive reduction otential) until they are used to reduce O2 to H20. The overall eaction is shown below, where CYT stands for cytochrome. 4H+ + 4cm: _c_+2 + 02 -—-> 2H20 + acy'r 9+3 upled to oxygen reduction is the generation of a transmembrane pH adient which is functional in the production of ATP (stored chemical energy). Because of the large thermodynamic driving force vided by the reduction of oxygen, aerobic organisms can produce 36 ecules of ATP per molecule of glucose as opposed to only 2 for erobic organisms (Lehninger 1975 p. 517). Cytochrome oxidase is alized on the inner membrane of the mitochondria with portions truding on both sides. The shape and dimensions of the enzyme mer have been reported by Henderson et al. (1977) and can be seen igure 1.13. This result was obtained through the use of electron oscopy of oriented membrane layers. Cytochrome oxidase has been Hobiliralron acetyl- Tha rrrcarboxyi leid cyc] Hcctron transport a °xl¢ltivo MNPhoryHnM Figllre mobilization of acotvl-Coh the tricarboxylic acid cycle Electron transport and oxidativo phosphorylntion Figure 1. 27 Amino acids Glucose Fatty acids Pym-unto he» Citrate Oxaloncauta [cit-Acclaim.) lsocimto CU: a-Kotogluunto Succinnlo ._.. w-COA \Nm/ .5; -.320 Flavoplotain I ridges and magnetically couples the Fea3 to the CuB (Tweedle et al. 978). The identity of this bridging ligand and the ligands to the CuB ave not been determined with any certainty. The distance between these wo metal systems has been estimated at ‘5 angstroms. This is ifficiently close to allow for a wide variety of possible bridging gands; both amino acid residues and exogenous ligands are currently der investigation. The optical absorption spectrum of cytochrome idase in the fully oxidized and fully reduced states is seen in gure 1.15a. This composite spectrum of the two hemes has been onvoluted by Vanneste (1966) into the components of the individual e centers. These results are seen in Figure 1.15b. These spectra of individual heme sites have been closely approximated with the use heme a models (Callahan and Babcock 1981; and Van Steelandt-Frentrup a1. 1981) in terms of a six-coordinate, low-spin heme in cytochrome ———-—-+" 400 i b) _.- a” .. a" W ("ll“) {l mum .- '. (3 v ~09 400 m ..... 3 TE — a 7: I W H“ II.) ~ ,. :". “um: : ‘. ; . ' ' I v v v v ' u. ‘0. I" I“ MVILII‘M I up I Figure 1.15 32 CYTOCHROME OMDASE N 3 I I": 5: ~ 2.0 A (rs) ,., : '. s .1' i ' I g f- o l 1' ‘I E n I I : s . ' l ' In II as ['3 : r. ,1 . ‘ I i I l 500 500 700 REDUCED ----- 2 mm CUVETTE 62 ml OXIDIZED ' nun-as" 'p “13“!” AISOIUK V 'n. . . m In uvtuuvu c a, I a) The optical absorption spectrum of oxidized and reduced cytochrome oxidase, b) The approximation of the spectral contributions from the individual heme centers (from Vanneste, 1966). e. AWMSOV a and a high-Sp oxidized enzyme reproduced in F: the two heme cer Raman spectra of line. Resonance reviewed recentl further in the l; The catalytic great deal of res The scheme presen intermediates the figure only the t1 are shown. The fir oxidation states. bind oxygen to £0 involved as the o cleavage of the 0- ferryl intermediat there is still no exists as drawn. R the possibility of 33 and a high-spin heme in cytochrome a3 which is six-coordinate in the xidized enzyme and five-coordinate in the reduced. These spectra are eproduced in Figure 1.16. Note that the different absorption maxima of me two heme centers make it possible to obtain selective resonance aman spectra of the two centers by careful choice of laser exciting ine. Resonance Raman spectroscopy of cytochrome oxidase has been eviewed recently by Babcock (1986). This topic will be discussed arther in the later chapters in the discussion of my results. The catalytic cycle of cytochrome oxidase has been the subject of a teat deal of research and the source of a great deal of speculation. ne scheme presented in Figure 1.17 is representative of the types of ntermediates that have been proposed (Blair et a1. 1985). In this igure only the two metals of cytochrome g3 and the space between them re shown. The first species contains the metals in their resting ridation states. Upon reduction of the metal centers, the heme may .nd oxygen to form an "oxy" structure. Bridging structures may be volved as the oxygen is reduced to the level of peroxide. The eavage of the 0-0 bond may be associated with the formation of a rryl intermediate. Unfortunately, despite a great deal of research, are is still no definite evidence that any of these intermediates ists as drawn. Results discussed in later chapters will comment on a possibility of these structures being correct. g 2.0 AU. . wUngOmmd MUémgwm/s ABSORBANCE ABSORBANCE 34 c) OXIDIZED heme 33’ (NMeIm)2Cl' ----- heme 93* CI' b) REDUCED 2-0' A heme 92*(N MeIm)2 90.5 B ------ heme g2*(2-MeIm) ‘ 436 ‘ I 587 I . ,442 . -O.25 .1 \ ”—- ‘\\ . e , x ‘|‘ ’tt" ‘\\ \ e’ \ ‘ \ \‘ 1 l t. 500 600 700 WAVELENGTH (nm) Figure 1.16 Heme a models for the heme centers in cytochrome oxidase (Figure courtesy of G. T. Babcock). 2+ CuB L Fe3+ ‘3 (oxidized) (ferryl) 1+ CUB 2e 02 040 l 2 2+ Fed; Fees (oxidized) (reduced) (oxy) 20H- e,H* Cug'r CU? 0' e', H+ /O' -<——-— 4—— .- HO O l 2 . 3+ Pena+ F603 (ferryl) (ferrous peroxy) (ferric per OXY) Figure 1.17 A proposed catalytic cycle for oxygen reduction by cytochrome oxidase (Figure courtesy of G. T. Babcock). A. RESoNANCE I To obtain RE include? (I) ah laser): (2) a sa other chromatic htectOru and (5 laboratory f“ r‘ sources: sample holders monochromat“: detector: data collectior output device I The commercially 11‘ Appendix 1. The ‘73 frequencies througl for resonance Rama! CHAPTER 2 EQUIPMENT AND TECHNIQUES RESONANCE RAMAN SPECTROSCOPY To obtain Raman spectra, several components are necessary. These elude: (1) a high intensity monochromatic light source (usually a ser), (2) a sample holder, (3) a high resolution monochromator or her chromatic dispersing component (prism or grating), (4) a light tector, and (5) a data output device. The components available in our boratory for resonance Raman spectroscopy consist of the following: sources: Kr ion, Ar ion, tunable dye, and He-Cd lasers sample holders: cuvette, capillary, spinning cell, spinning difference cell and low temperature EPR tube monochromator: scanning double grating detector: photomultiplier tube data collection: dedicated computer output device: chart recorder and digital plotter commercially manufactured components of this system are listed in ndix l. The variety of laser sources provide different excitation uencies throughout the visible region of the spectrum. This allows resonance Raman investigation of hemes utilizing both Soret and 36 nvisible" (Q b5] excellent for 51 light or temper‘ nonochromatol’ ar resolution, reje throughput Alth samples, a major was the inabiliti deficiency resuli drive. DifferenCE two different san scan to scan vari problem was solve computer control of the data. The . of a Raman differi collection of RAma aPparatus utilizec Software modificat are described in t Aschematic dr. 2-1. The Digital Er Ramalog spectrometi Was designed and as Atkinson and me. Th 3 p°°trometer steppe 37 "visible" (Q band) excitation. The cuvette and capillary cells are excellent for stable samples, but the alternate cells are necessary for light or temperature unstable samples. The combination of a scanning nonochromator and a PMT produces excellent spectra in terms of :esolution, rejection of the strong Rayleigh scattering, and light ;hroughput. Although a chart recorder output is sufficient for many amples, a major deficiency of an earlier version of the Raman system as the inability to signal average or reformat data. Another ficiency resulted from the mechanical limitations of the grating ive Differences in the peak positions of less than 2 cm‘l, between 0 different samples, could not be assigned with confidence since the an to scan variability of the instrument was ~+/- l cm'l. The first oblem was solved by the construction of an interface which provided mputer control of the instrument and allowed collection and storage the data. The second limitation was alleviated by the construction a Raman difference apparatus, which allows for simultaneous Llection of Raman spectra of two different samples. The difference taratus utilized the interface and required only minor hardware and tware modifications. The design and operation of these two systems described in the following paragraphs. A schematic drawing of the Raman system layout is seen in Figure , The Digital Equipment Corporation (DEC) LSI-ll/2 is linked the the Llog spectrometer through a house built interface. This interface designed and assembled by Martin Rabb with assistance from Thomas nson and me. The interface allows the computer to actuate the trometer stepper motor and drive the gratings. Since the level of KOF‘I _‘.KUQ.D e1 MOI-3t 2...... ‘- .‘ueieefe «880“: V... J“; a .Eoumxm wouoaouuoonm con—om mowccoom poomuuouaw yousasou ad madman SE 530 «:33 ”2:13”. own 33 x 228 . 28 es 2...... I 5&3» ~32 , t I .1 I «was 59.. 58: @ :0. 2o. cutout. “859.: e. E w Sewage!» 92.223» @255 538 «O! xuuw a .003 .0 £029...” 5‘ u .- 4, I '3. Hate 80.52.3555 3 wan—3t 46. 5.1. c a???” gmflflx zwhwzobbot light observed i from the PMT is single ph°t°“' T and then passed an analog Value Y on the ratemeter these ph°t°n ”11 logic level pulsé digital counting over the colmn‘ErCi ratemeter analog accuracy. The S°f amenu fashion f0 parameter are dis photon count valu: and graphics rout: user specifies thi number of scans tc specified time int counts again, etc. scan time, the ope Utilize time more that it allows for impossible before. my experimental woi identifying were e: no' ' rse with only a s 39 light observed for Raman spectroscopy is typically very low, the signal from the PMT is observed as distinct pulses, each corresponding to a single photon. These pulses are shaped by a discriminator/preamplifier and then passed on to a ratemeter where the pulse rate is converted to an analog value which is sent to the chart recorder. A digital output on the ratemeter makes the photon pulses available directly. Since these photon pulses were very similar in shape and size to computer logic level pulses, we connected the raw pulse signal directly to a digital counting chip in the interface. This design was an improvement aver the commercial systems available at the time which redigitized the :atemeter analog signal at the loss of collection speed and data eccuracy. The software is written in FORTRAN and MACRO and operates in 1 menu fashion for user convenience. The data file name and all current parameter are displayed continuously; the spectrometer wavenumber and hoton count values are updated as available. Some data manipulation nd graphics routines are also available directly from the program. The ser specifies the scan limits, point spacing, counting time, and the imber of scans to be averaged. The computer counts photons for the Jecified time interval, moves the spectrometer to the next point and aunts again, etc. Since the computer is in control during the entire tan time, the operator can now do other things while it is running and ilize time more efficiently. The greatest advantage of this system is at it allows for long signal averaging experiments that were possible before. Implementation of this capability was essential for experimental work since many of the peaks I was interested in rntifying were extremely weak and could not be resolved from the se with only a single scan. The Raman c which is divide is spun by a no operation, a 5) motor mount eac (switches from gating of the P the accumulatio different count subtracted from the two spectra result from gra‘ asSiemens of 1 selects the pho: sec0nds, Since 1 the tw° Channel: mount were desig designed the (111 design of this s however the 'use the rePOIted mix 40 The Raman difference apparatus utilizes a short cylindrical cell ch is divided down the center to provide two compartments. This cell spun by a motor drive such that alternate halves are illuminated. In ration, a synchronization pulse is generated from a circuit on the or mount each time the cell rotates through the cell divider itches from one cell half to the other). This pulse activates the ing of the PMT output from one counter to another. The net result is accumulation of data from the two different samples in two erent counters. The scans can be plotted separately or one tracted from the other to accentuate the differences. Collection of two spectra simultaneously eliminates the uncertainty which may alt from grating slippage. Use of this technique allows confident .gnments of peak differences of -1 cm'l. For these spectra, the user :cts the photon counting time in terms of cell rotations rather than nds, since the cell must complete full rotations for the signals on two channels to be equivalent. The electronic hardware and motor t were designed by Martin Rabb and Jose Centeno; Jose and I ed the difference cell, and I wrote the necessary software. The n of this equipment is based on that of Kiefer et al. (1975); er the use of digital counting and switching logic, rather than eported mixture of analog and digital processing, turned the el separation from a difficult task to a nearly trivial one. The are schematics and mechanical details of the instrument can be in Centeno (1987). 5. mil Tm“ Many of the temperatures or intention was t spectr05c°Py’ 1 them in the sam' Possible. it W introduce addit: (outside dime“: qualities: (1) t glass shop; (2) experiments With reagents or diff cylindrical shap vacuum and the f (o) quartz is in transparent in t3 definition they ' temperature rig l for Ranan spectrc equipment (either could be easily a draw~backs of usi events required t 1n the small tube absorption spectr 8amllles. I had t o 41 LOW TEMPERATURE SPECTROSCOPY Many of the compounds I have worked with are stable only at low mperatures or under anaerobic conditions or often both. Since my tention was to study these compounds with different types of ectroscopy, it was desirable to be able to make them and characterize em in the same cell. Even if transfering them to different cells was ssible, it would make the experiments more difficult and possibly troduce additional variables. The standard cell chosen was a 4 mm utside dimension) quartz EPR tube. These displayed many positive alities: (1) they were inexpensive and easily manufactured by our ass shop; (2) their small dimensions made it possible to do periments with small volumes of sample, thus conserving valuable agents or difficult to isolate biological substances; (3) the .indrical shape of tubing makes it structurally able to withstand :uum and the freezing and thawing of aqueous and organic solutions; quartz is inert to esentially all solvent systems and optically nsparent in the UV and visible regions of the spectrum; (5) by inition they could be used for EPR spectroscopy and a low perature rig had already been constructed for the use of these tubes Raman spectroscopy; (6) they could be easily connected to anaerobic meent (either direct seal or heat shrink tubing); and (7) samples .d be easily stored in a liquid nitrogen freezer in them. The only '-backs of using these tubes were: (1) the sophisticated sequence of CS required to produce most of the samples was difficult to perform be small tubes; and (2) we had no practical way to obtain optical rption spectra of samples in these tubes, especially frozen es. I had to learn to live with the first problem but the second was elillliHated optical absorpt the other 1°“ t‘ The origina: was designed by laser bed1m ““91 Prisms, and refl Light scattered collected by the contrast to a tr vertically throu the scattered 111 is held by fricti spun in the mom“ obtain a more hon degradation that to the laser beam gas through coppe inner passage of controllable from thermocouple posi‘ through the dewar impractical to at! Although there are slstem produces ea througlmut its ful 42 was eliminated by the design and construction of a low temperature optical absorption rig for samples in EPR tubes. Details of this and the other low temperature rigs are presented below. The original functional low temperature Raman backscattering system was designed by W. Anthony Oertling and is seen in Figure 2.2. The laser beam enters from below, is horizontally moved by two 90 degree risms, and reflects off the backscattering mirror on to the sample. ight scattered from the sample at -180 degrees to incidence is :ollected by the optics and focused into the monochromator. This is in :ontrast to a traditional Raman experiment in which the laser passes vertically through the sample (in an optical cuvette for example) with :he scattered light collected at 90 degrees to incidence. The EPR tube 3 held by friction against two O-rings in a plastic "spinner" which is pun in the mounting block by tangential air jets. The tube is spun to btain a more homogeneous sampling and to minimize the photo or thermal egradation that may occur if the same region were continuously exposed > the laser beam. Temperature is controlled by the flow of nitrogen rs through copper coils (submerged in liquid nitrogen) and through the rner passage of the dewar. The temperature, which is continuously introllable from room temperature to ‘-130 C, can be monitored by a ermocouple positioned directly below the EPR tube. Thermal leakage rough the dewar walls, transfer tubes, and connections, make it practical to attempt to use this system at lower temperatures. :hough there are reflective losses at each optical surface, this tem produces excellent spectra for both liquid and frozen samples oughout its full temperature range. In addition, for room r— Translator: _V ”0me \ \ La: Figure 2 43 Spinner Samplo Tuba '/\. {—- Translulors Thermocouple Collection Optics Lasor Harv -__._, Figure 2.2 Low temperature Raman backscattering Dewar. temperature wor reflective loss. absorbing 53mph technique over i can be Used’ St] solvent scatteri Although the use, temperature not attainable w liquid nitr°gen' temperature reg‘JI nitrogen interfe] designed and had operates on a flc entry surfaces. N resewoir and dire The sample is 383 of the sample com] are submerged in 3 front wall and th‘ monitored by an in Dewar system, desp or better than the readily controlled C) with warmer tem s . yStem is more dif 44 mperature work, the Dewar flask can be removed to reduce the flective losses. For resonance Raman spectroscopy of strongly sorbing samples in organic solvents, backscattering is the preferred :hnique over 90 degree scattering. Since much higher concentrations 1 be used, stronger resonance scattering relative to the background Lvent scattering is obtained. Although the above mentioned Dewar flask works well and is easy to :, temperatures low enough for some of the planned experiments were attainable with it. We needed to maintain temperatures near that of uid nitrogen. Immersion Dewar systems are effective but offer no perature regulation and the spontaneous bubbling of the liquid rogen interferes with the signal collection. For this reason, I lgned and had constructed the Dewar system in Figure 2.3. It rates on a flowing gas principle but it is designed to minimize heat 7y surfaces. Nitrogen gas flows through the coils in the central :voir and directly out through the tube imbedded in the sidewall. sample is again suspended and spun in this tube. Because the back he sample compartment is the reservoir and all connection points submerged in liquid nitrogen, the only heat leakage surface is the t wall and that seems to be neglegible. Again, temperature is Fored by an imbedded thermocouple and regulated by gas flow. This system, despite its awkward appearance, produces signal equal to tter than the original Dewar arrangement. The temperature is ly controlled in the range of -192 C to -182 C (liquid N2 - -l96 h warmer temperatures possible with some effort. Since this is more difficult to set up, it is not normally used for routine Figure 45 EPR Tube r/ Spinner N. Go: m \p 1 / Frozon ”\- Sample Lid ‘- N' l Coll ‘ ,1 action 1 Optic: TW‘ g I § - \Bock Scattering Mirror ‘ F (Prisms m coming at? .... Boom Figure 2.3 Liquid nitrogen temperature backscattering Dewar system. work with the I obtain Raman 8] intermediates. The basis 0 rig is a SYStem use of EPR tube: Problem. The wi< spectrometer is run) and curved e sample giving er with the acquisi which has 3 f°°m still poor r613tj was considerably minimized by the down to a width 0 the sample to reci more of it would 1 shown in Figure 2. Using a clear liqu transmitted relati buch smaller than l Instl’lmnent to achil baseline with this Pathlength calibrat Centered. This r1 8 46 >rk with the more stable compounds, but it has added the capability to >tain Raman specta of highly unstable compounds and enzyme itermediates. The basis of the low temperature optical absorption spectroscopy g is a system like the original Raman Dewar arrangement. However, the a of EPR tubes for absorption spectra presented a slightly different >blem. The width of the light beam in the average absorption :ctrometer is '2 to 4 mm. The narrow inside diameter of the tubes ('3 and curved edges allowed light from the spectrometer to bypass the ple giving erroneous absorption values. The situation was improved h the acquisition of a Perkin-Elmer Lambda 5 spectrophotometer, ch has a focused beam width of ~1 mm, however light throughput was 11 poor relative to the light bypassing the sample. The situation considerably worse with frozen samples. These problems were mized by the use of a cylindrical lens to focus the incident light to a width of ~0.2 mm at the sample. Another lens was added after sample to recollimate the light diverging from the sample so that of it would reach the detector. A drawing of this Dewar system is in Figure 2.4. Inset in the top is a schematic of the light path. ; a clear liquid as a blank, approximately 15% of the light is .mitted relative to the unhindered reference beam. Although this is smaller than hoped, there is still enough dynamic range in the ument to achieve good spectra even with concentrated samples. The ine with this rig is flat over most of the visible range and the ength calibrates to 3 mm, indicating that the beam is narrow and ‘ed. This rig has been an essential part of my research. Positive Light Path Aim). \— (Yokogo “Sure 2 .4 EPR Tube Pro-Focus Lens I F Air Jets Post-Focus Lens (10 hoop Donor from fogging) ‘ Thermocouple Figure 2.4 Low temperature Dewar system for optical absorption spectroscopy. identificatior more 6°“fident temperature SY spectrum can b‘ decorllp°5iti°n : c. ANAEROBIC Many 0f the sensitive. sinc the basic requii anaerobic/vacuul it offered a hié anaerobic train This design is b (personal commun highly reduced m4 selectable rough The anaerobic pal oxygen scrubbing trap or solvent s pressure releases anaerobic line is alternately evacue used with this can tint and able to manifold, ‘— ‘ 48 dentification of sample species by their absorption spectra allows ore confident interpretation of their Raman spectra. With this low emperature system, the absorption spectrum taken after a Raman pectrum can be compared with the one taken before as a test for sample composition in the laser beam. ANAEROBIC TECHNIQUES Many of the compounds I have worked with have been oxygen and water nsitive. Since the normal atmosphere contains large amounts of both, e basic requirement for Such work is a well made glove box or a good erobic/vacuum train. The latter was chosen for my experiments since offered a higher degree of anaerobicity at a lower cost. The serobic train I designed for this purpoSe is shown in Figure 2.5. .s design is based on one designed and used by Dr. John Ellis rsonal communication) at the University of Minnesota for work with hly reduced metal species. The vacuum part consists of alternately ectable rough or oil diffusion pumps with two cold traps in series. anaerobic part consists of an Ar gas source, BASF (R3-ll catalyst) :en scrubbing column (generously donated by Dr. James Dye), moisture or solvent saturation bubbler, Ar gas reservoirs, and mercury pool sure releases. The connecting unit between the vacuum line and the robic line is the Schlenk manifold which allows an apparatus to be 'nately evacuated or pressurized with Ar gas. The specific vessel with this can take on any variety of forms but it must be vacuum and able to attach to the ground glass joint on the Schlenk old. '1 l 5: Us ‘1‘)“ I, In yr 1;: “v : i i yell \ i. 5 (IE Figu 49 ’7'" 15] in i ' , ' ‘1 e5 0 Figure 2.5 Anaerobic/vacuum system. Dillon“ Pump J A Vent Examples I flasks are set manifold and if over the septu the flask is u‘ removed to alh attached high \ removed from th reattached late an EPR tube, wh suentific) higl allows modular l exPeriment. 0the glass “W for e exPeUSiVe. The 0 SolutiOn from thl fl -..., _,, 50 Examples of some of my more versatile and frequently used anaerobic Flasks are seen in Figure 2.6. The upper flask attaches directly to the Ianifold and features only a septum port. The plate of glass is clamped ver the septum to assure a complete seal during the vacuum cycles. If he flask is under a positive pressure of Ar gas, this plate can be emoved to allow syringe access to the flask. The lower flask has an :tached high vacuum stopcock. When this is closed, the cell can be moved from the manifold without risk of air contamination and attached later as needed. This flask also features a syringe port and EPR tube, which are connected to the main body with FLOTITE (Pope dentific) high vacuum heat shrink tubing. This is useful because it lows modular pieces to be assembled quickly for a specific >eriment. Otherwise, specific cells would have to be made by the 155 shop for each experiment, which would be both time consuming and ensive. The other advantage is that it allows for easy "seal-offs". ution from the main body of the flask can be poured into the EPR 2 arm. When the bridging part of the FLOTITE is heated, it will .apse on itself. If it is crimped while warm, the tube will be .tly sealed and can be cut off the main body without loss of robicity. Unlike a traditional glass seal off, this is destructive. The FLOTITE can be easily removed later and the EPR reused. This procedure was especially useful for the preparation man samples, since the EPR tube cannot be spun unless it is ed from the main body of the flask. :en a flask is first attached to the manifold, the sample within e purged of oxygen by "freeze, pump, thaw" cycles. As the name Figure 2.6 51 Glass Plate Anaerobic glassware . implies. the completely fr! Pumped 0‘“ unt nitrogen is re Ar gas Simply evaporation as gases ‘10 be re! or other volat1 depends 0n the the sensitiVl-t)’ cYCles are Perf' Although hi! septum ports, Ca syringe needles under a positive septum will call“ the second end is above atmosphere) anaerobically. If the higher pressm transfer these so] ‘10 complex anaerob 52 - implies, the cell is immersed in liquid nitrogen until the sample is completely frozen. While still immersed in the liquid N2, the flask is pumped out under vacuum for ~5 to 30 minutes. Finally, the liquid . nitrogen is removed and the cell is filled with Ar gas as it thaws. The Ar gas simply provides an inert atmosphere which minimizes solvent evaporation as the solution thaws. This allows for oxygen and other gases to be removed from the sample without losing much of the solvent or other volatile components of the sample. The number of cycles used depends on the sample size, the surface area of the frozen sample and the sensitivity of the final product to oxygen. Ordinarily 5 to 12 cycles are performed. Although high quality gas tight syringes can be used with the septum ports, cannulae are generally more useful. These are long :yringe needles with points on both ends. If a flask is maintained nder a positive Ar gas pressure, insertion of the cannula through the eptum will cause Ar gas to flow out preventing air from flowing in. If he second end is inserted in a flask of lower pressure (but still Jove atmosphere), Ar gas will flow from one to the other, still iaerobically. If the cannula point is pushed below the fluid level in 3 higher pressure flask, liquid will be transferred. The ability to ansfer these solutions anaerobically is the final tool necessary to complex anaerobic chemistry as will be described in later chapters. p. ELEC'FROG One powerf electrochemist the technique ‘ oxidase, is me] reagents commor the ring SUbSti undesirable Sin porphyrin which Previous “duct: the careful tit: results and wast system since the there is n0 fad electrodes. Sinm the literature 56 designed my 0W! e Current flows bet wire acts as a pS separated by glas: but to prevent 1a: solutions are free then poured in uni reduction potentia determined by perft W of current flc low only during an 53 D. ELECTROCHEMISTRY One powerful synthetic tool often overlooked by chemists is electrochemistry. For some of the reactions I performed, it was clearly the technique of choice. Heme g, the prosthetic heme in cytochrome oxidase, is more difficult to study than most hemes because the reagents commonly used to reduce Fe+3 to Fe+2 can also easily reduce the ring substituent formyl group (see Figure 1.3). This is very undesirable since it is the r-conjugation of this group into the Jorphyrin which gives heme a some of its unique characteristics. ’revious reduction schemes (Vansteelandt-Frentrup et al. 1981) required he careful titration of reductant, which often produced unpredictable esults and wasted heme g. Electrochemistry is ideal for this heme ystem since the reduction potential can be specifically adjusted and mere is no facile mechanism for formyl reduction on simple platinum Lectrodes. Since none of the electrochemistry cell designs reported in m literature seemed to meet my specific needs (Kadish 1983), I signed my own electrochemistry cell. This cell is seen in Figure 2.7. rrent flows between the working and counter electrodes while a silver re acts as a pseudo reference electrode. These compartments are Jarated by glass frits to allow electronic contact and ion transport : to prevent large amounts of mixing. The sample and electrolyte .utions are freeze, pump, thawed in their individual side arms and n poured in unison into their electrochemistry compartments. The uction potential of heme a relative to the pseudo-reference is ermined by performing cyclic voltammetry (Figure 2.8 a), i.e., a : of current flow versus electrode potential. Since current will 'only during an oxidation or reduction process, we can determine Figul'e 2H 54 To Anaerobic Train Electrical Contact: Freeze, Pump, Thou Arms Silver Wire Pseudo-reference Electrode (reference compartment) Working Electrode (sample compamnenl ) BEGINS IN THIS POSITION Figure 2.7 Electrochemistry cell for small volume anaerobic work. ) b mozsthetic porphyrin in this enzyme has been postulated to be a chlorin porphyrin with a reduced pyrrole ring) by Sibbert and Hurst (1984) i Babcock et al. (1985), it was not known whether this anomalously v Fe+3 ~CN vibration was due to porphyrin effects or protein effects. Ice no results have been reported in which the effects of the :phyrin structure on the binding of CN' were characterized, we iertook such a study. In this chapter, the Raman spectra of cyanide mplexes of iron deuteroporphyrin (deuteroheme), iron deuterochlorin, l heme a are compared with previously reported Raman spectra of >tein and model species and cyano-horseradish peroxidase. The Lteroporphyrin system was chosen because of its availability, its mical stability, and the fact that its Soret optical absorption peak in good resonance with our 406.7 nm laser line. MATERIALS AND METHODS l-methylimidazole (Aldrich) was vacuum distilled over calcium ride and stored over molecular sieves. All other materials were used .out further purification. Fe(III)deuteroporphyrin dimethyl ester Fe(III)deuterochlorin were generously supplied by Dr. C. K. Chang. dimethyl ester of Fe(III)deuterochlorin was prepared by refluxing :hlorin with trifluoroacetic anhydride under nitrogen in a Ilene chloride/methanol mixture (Wang et al., 1958). Bis-cyanide exes of the porphyrins and chlorin were prepared as follows. ximately 0.004 g of solid KCN (Fisher) were dissolved in 1 ml of a red detergent solution (0.1 M phosphate, pH 8.4, 0.2% W/V Brij 35 1)) to produce a final pH of 10.7. The iron porphyrin or chlorin ssolved in methylene chloride'and vortexed with the cyanide solution unde transfer to t bis-cyanide c mono-l-methyl bis-cyanide c< the above solL in optical abs Horseradish pe PhOSphate buff. addition of a 1 sallples were 131 lSN, Cambridge OPtical abs. SUV/Visible Sp, air referenCe. l Ramalog scanning Photomultiplier operatic“ is ach built interfaCe. 406.7 llm excl-tat: Contained in a SI for all gems. 60 olution under a stream of argon. As the methylene chloride evaporates, ransfer to the aqueous solution results. The formation of the is-cyanide complex was confirmed by optical absorption spectra. The ono-l—methylimidazole, mono-cyanide complexes were prepared from the is-cyanide complexes by the addition of 20 pl of l-methylimidazole to he above solution. This was again verified by characteristic changes n optical absorption and resonance Raman spectra (see below). orseradish peroxidase (Sigma type IV) was dissolved in a 20 mM hosphate buffer at pH 7.5. The CN‘ complex was produced by the idition of a few crystals of KCN. The final pH was 7.8. C15N labeled amples were produced by identical methods in which KClSN was used (99% SN, Cambridge Isotope Laboratories). Optical absorption spectra were obtained with a Perkin—Elmer Lambda UV/visible spectrophotometer by using a 0.5 cm quartz cuvette with r reference. Resonance Raman spectra were obtained with a Spex 1401 nalog scanning double monochromator; a cooled RCA 031034 atomultiplier tube was the detector. Data collection and instrument mration is achieved from 3 DEC LSI-ll/2 computer through a house 1t interface. All spectra were recorded by using 20 mW of power at .7 nm excitation (Spectra—Physics model 164 Kr+ ion). Samples were tained in a spinning cell; a 90 degree scattering geometry was used. Raman spectrometer was calibrated to the 1004 cm‘1 line of toluene all scans. C. RESULTS The struc shown in Figu except that t protons. The increase the .I any acid/base distinguished alteration res degeneracy. It differences in these perturbaz bis-imidaZole’ are Shown in F1 SPectra of the : relativg, to tho: also one less pa and the peak bet sistems retain t Qbands (bands t. between the 1513-, distinguishes the the red of 600 mm The low frequ‘ Porthrin Species System, a band occ 61 RESULTS The structures of the iron deuteroporphyrin and deuterochlorin are hown in Figure 3.1. The deuteroporphyrin is similar to protoporphyrin (cept that the vinyl groups at positions 2 and 4 are replaced by rotons. The dimethyl ester (of the proprionic acid groups) is used to mcrease the solubility of this species in organic solvents and block y acid/base chemistry of the group. The deuterochlorin is stinguished by the reduced pyrrole ring at positions 3 and 4. This teration results in reduced ring conjugation and a loss of the X, Y generacy. It is expected that if the CN' binding is sensitive to Fferences in porphyrin structure, we should see differences due to se perturbations. The optical absorption spectra of the bis-cyanide, -imidazole, and mixed ligand compounds of the porphyrin and chlorin shown in Figures 3.2 and 3.3, respectively. The peaks in the :tra of the bis-cyanide complexes are considerably red shifted ttive to those of the bis-imidazole species. In each case there is . one less peak in the 500 to 700 nm range with bis-cyanide ligation the peak between 330 and 340 nm is much stronger. The mixed ligand ems retain the shape of the bis-cyanide complexes but the Soret and ads (bands to the red of the Soret) are intermediate in location Ben the bis-cyanide and bis-imidazole cases. The feature which most Lnguishes the chlorin from the porphyrin is the additional band to 'ed of 600 nm. he low frequency region resonance Raman spectra of the three Vrin species are presented in Figure 3.4. In the mixed ligand n, a band occurs at 451 cm'1 which is not present in either of the Figure 3 2 3 a) H CH3 1 H3C H CH CH 7 i 2 l 2 6 CH2 CH2 l I COOCH3 COOCH3 FeIII Deuferoporphyrin DME 2 3 b) H H CH3 1 “sc 2 4 CH2 CH2 7 CH2 CH2 5 l l COOH COOH FeIII Deuterochlorin Figure 3.1 The structures of iron deuteroporphyrin DME and iron deuterochlorin. Figure 3. moz.:WZM._Z_ Zroduced results identical to the unesterified species. No other .sotope sensitive modes are observed in these samples. The absorption pectrum of native and ON bound horseradishperoxidase (HRP) are shown n Figure 3.8. Although the spectrum of the CN bound species is red hifted relative to the deuteroporphyrin mixed ligand system, the basic stern is very similar. Figure 3.9 shows the low frequency Raman )ectra of the native and CN‘HRP. Again, looking at the expanded low tequency region (Figure 3.10) we see a peak at 457 cm'1 which shifts .452 cm‘1 upon isotope substitution. We assign this peak to the Fe-CN) stretching frequency. DISCUSSION The results from the mixed ligand systems are consistent with the :viously reported results for models (Tanaka et a1. 1984) and protein :cies (Kerr et al., 1984; Yu et al., 1984). They are quite different m the myeloperoxidase results, however. The presence of the isotope Figure 3.5 ISO >k~WZUhZ~ ZL._WZU#Z~ 68 CN’,NMI _ 0) Fem Deuieroporphyrl” DME c‘SN b) Fe”l Deuterochiofln cl‘N‘ " {clsN’ Au=4087 nm 550 ' -1 50 RAMAN SHIFT (cm ) Figure 3.6 Raman spectra of NMI, CN' NMI/CN' ligated FeIII deuteroporphyrin and deuterochlorin with isotope labeled CN'. )\ :406.l ex RAMAN INTENSITY Figure 3.; 69 A =4067 nm BX M M C‘SN— «l b) C‘4N- Fen] Deuierochlodn zoo - RAMAN SHIFT (cm“) 600 Figure 3.7 The Raman spectra of bis-CN' FeIII deuteroporphyrin and deuterochlorin with isotope labeled CN'. Figure 3. 300 moz.—._WZU._.Z. Z<2e asymmetric (lack planar symmetry) and would be expected to have a [aman allowed v(Fe-CN) stretching frequency. The broadness of the Soret and in the optical spectra of the bis~cyanide samples suggested that he sample was probably not homogeneous. The possibility that the bserved signal was due to impurities was ruled out by additional erriments. Increasing the CN' concentration decreased the broadness 1d further red shifted the Soret as expected for the bis-cyanide >ecies. The Raman spectrum of the sample higher in CN‘ concentration rd a peak of greater intensity at 458 cm'l. This would seem to rule t the possibility that this peak is associated with a ve-coordinated sample or the H20 complex. However, the increase in ' concentration is associated with an increase in pH which may favor a hydroxy complex. This possibility was rendered unlikely by :ration to lower pH to produce a sample which also had a more logeneous optical spectrum and a Stronger 458 cm‘1 band. This observation bis-cyanide presence of , presence of 1 the loss of p pyrrole ring properties of 02' ligand bi in which the withdrawing al l’(Fe-ligand) : 11Sand heme a detergent emu] 2 in heme é, a (Positions 6 a which WEakens be a P°rPhyrin hemes (Kerr at to 453 cm‘l) 0t Porphyrin eff“ The frequen consistent with of PreViOUS wot] -1 cm ) is higher This obsematim S teric COnStrair 75 observation also indicates that the 458 cm’1 band is due to the bis-cyanide sample. The optical inhomogeneity is most likely due to the presence of some residual u-oxo dimer which is broken up in the presence of high CN' concentrations or upon lowering the pH. Other than the loss of planar symmetry, it is clear that the reduction of a pyrrole ring to produce a chlorin has little effect on the binding properties of CN'. This is consistent with results from other 02 and 02' ligand binding studies (Tsubaki et al. 1980; Kean and Babcock 1986) in which the heme peripheral substituents were varied in their electron withdrawing ability with little or no resultant change in the u(Fe-ligand) frequency. The slightly lower frequency of the mixed ligand heme é sample (447 cm‘l) may result from a difference in detergent emulsification. The long hydrophobic "tail“ on ring position 2 in heme a, across from the hydrophilic proprionic acid groups (positions 6 and 7), may result in a different solubilization geometry vhich weakens the CN' binding. Alternately, this frequency lowering may re a porphyrin substituent effect as has been observed for CO ligated .emes (Kerr et al., 1983). The near constant u(Fe-CN) frequency (~451 o 453 cm‘l) observed for mixed ligand models and protein species makes orphyrin effects seem less likely. The frequency of the v(Fe-CN) stretching frequency of the CN-HRP is >nsistent with the results of our model compounds and with the results 3 previous work. It is interesting to note that the frequency (457 F1) is higher than that in both models and most other heme proteins. is observation is indicative of a slightly stronger bond or less eric constraint in the HRP species. Yoshikawa et al. (1985) have implicated e histidine re effect is ex sufficient t configuratio frequency in speculate as strict steric a large decre 76 implicated a hydrogen bonding interaction between a protonated histidine residue and the CN' nitrogen atom in HRP. Although this effect is expected to be weak in the Fe(III) protein, it may be sufficient to stabilize the CN‘ in a more strongly binding configuration. It is predicted that the anomalously low u(Fe-CN) frequency in myeloperoxidase is due to protein effects. We can only speculate as to the nature of these but it would seen probable that strict steric constraints would have to be involved to account for such a large decrease in frequency. l \ A. INTRODUC syntheCic the underStanl heme Containix interiJretaCior have been synt structure and/ references Wit function 0f he] ProtiC or 3P”? specific steri< been sucCeSSfUI henoglobins, Wa Babcock, 1981; sophisticated “1 rigid structure: models would id‘ (octaethylporph) naturally occurr Unfortunately, t to the floppines CHAPTER 4 CHARACTERIZATION OF CYTOCHROME g3 MODEL COMPOUNDS A. INTRODUCTION Synthetic hemes (iron porphyrins) have played an important role in the understanding of the chemistry and ligand binding properties of heme containing proteins. Not only have they been important in the interpretation of spectroscopic data of heme proteins, but heme models have been synthesized which mimic heme enzymes in some aspects of their structure and/or function (see Lever and Gray eds., 1983, and references within). Some of the variables important in the specific function of heme proteins are the following: specific axial ligation, protic or aprotic environment, proximity of other metal centers, and specific steric constraint. Axial ligation and general environment have been successfully reproduced in simple solution studies (i.e., hemoglobins, Walters et al., 1980; cytochrome oxidase, Callahan and Babcock, 1981; cytochrome P-450, Sakurai and Yoshimura. 1985) but more sophisticated modeling requires covalent attachment of the porphyrin to rigid structures which can mimic very specific protein effects. These models would ideally utilize fi-substituted porphyrins (octaethylporphyrin (OEP) types), since they are the type found in all naturally occurring heme proteins (Smith and Williams, 1970). Unfortunately, the mOdels in this class often suffer from disorder due t0 the floppiness of the side chains used for the covalent attachment 77 (Young and ‘ types) have 197821; Colln 1985; Schaei ease of synt their substi difficulty 0 and Chang, 1% the four pher SPECtrum (Gou Raman spectru the physiolog Structural am Spectroscopic the TPP mOdels Mint and Chan phenfl groups , pheny1 substitl of the 03p type compmmise for rigidity of the the naturally 0 important to“ : characteristics reported. We hat SpecieS to deter Enema, more cl 78 (Young and Chang, 1985). Meso-substituted tetraphenylporphyrins (TPP types) have found extensive use for model construction (Burke et al., 1978a; Collman et al., 1983; Kerr et al., 1983; Schappacher et al., 1985; Schaeffer et al., 1986; and Bruice et al., 1986) because of their ease of synthesis, chemical stability and the increased rigidity of their substituent groups. The problem with making TPP models is the difficulty of selectively derivatizing the phenyl substituents (Young and Chang, 1985). From a spectroscopic point of view, the presence of the four phenyl substituents produces changes in the optical absorption spectrum (Gouterman, 1978) and dramatic alterations in the resonance Raman spectrum (Burke et al., 1978; Chottard et al., 1981) relative to the physiological (OEP type) hemes, making it difficult to infer structural analogies in heme proteins just from comparison of spectroscopic data. A heme type that may represent an improvement over the TPP models is the meso-diphenylporphyrin (Gunter et al., 1981; Young and Chang, 1985). Models with different derivitization of the two phenyl groups can be easily prepared and separated, and with only two phenyl substituents, the optical absorption spectra are more like those of the OEP type hemes. These meso-diphenyl derivatives represent a good compromise for heme protein models because they exhibit the structural rigidity of the TPP types but have optical characteristics similar to the naturally occurring hemes. Although Raman spectroscopy is an important tool for the study of heme proteins, the Raman spectral characteristics of these meso-diphenylporphyrins have not yet been reported. We have undertaken the Raman characterization of these species to determine if the Raman spectra, like the optical absorption spectra, more closely resemble the OEP type hemes than do the TPP types. Since species have Chang, 1985) different ax spectra of F The appl. particular it the oxygen re To model this must be able the heme and between the t alw 1978). A (Berry 8t al. were ”59d, hax overall failec In addition, a unambiguously “Produced the centers in oxi 9ta1,,1980; l meso“liphenylpl sYnthesiZed 0t} oxidase, which resonance Ramar Interpretation : V 79 types. Since the optical absorption spectra of only a few ligation species have been previously reported (Gunter et al., 1981; Young and Chang, 1985), the first part of this study establishes the effects of different axial ligation on both the optical and resonance Raman spectra of Fe+3 meso-diphenyl porphyrins. The application of meso-diphenylporphyrin derivatives that is of particular interest to us is their use in the synthesis of models of the oxygen reduction site of cytochrome oxidase (Gunter et al., 1981). To model this center (in the resting oxidase) accurately, these models must be able to chelate a copper ion in close proximity to the iron of the heme and display strong anti-ferromagnetic exchange coupling between the two metal centers through a bridging ligand (Tweedle et al., 1978). Although previous models, in which either TPP type hemes (Berry et al., 1980) or meso-diphenyl type hemes (Gunter et al., 1981) were used, have succeeded in this copper binding, the models have overall failed to mimic the magnetic coupling properties of the enzyme. In addition, actual ligand binding states of these models could not be unambiguously assigned. Some iron and copper containing species have reproduced the strong magnetic coupling that occurs between the metal centers in oxidase, but none of them contains the heme structure (Okawa et al., 1980; Morgenstern-Badarau and Hickman, 1985). Using the meso-diphenylporphyrin structure as a basis, K00 and Chang (1987) have synthesized other models of the oxygen reduction center of cytochrome oxidase, which we have characterized by using optical absorption, resonance Raman, and EPR spectroscopies as structural tools. Interpretation of these results was-made possible by the previous characterize second Part characterist suitability spectroscoPi B. MATERIA Methyl hYdride and 1 distilled 0‘” further Purl"f Asaad salehi The copper"1i by WOW Seo hemes, as prej chloride and 1 ligation was 1' with aqueous F M, pH 8.4). to chloride or di was produced by DMSO solution ‘ characteristic toluene/tetrah) methylene ch10! mixture of to It produced by the —_'——'"'I 80 characterization of the simpler meso—diphenylporphyrin species. In the second part of this chapter, I will discuss the structural characteristics of these cytochrome oxidase model species and their suitability as oxidase models, as established by the observed spectroscopic results. B. MATERIALS AND METHODS l-methylimidazole (Aldrich) was vacuum distilled over calcium hydride and stored over molecular sieves. Methylene chloride was distilled over calcium hydride. All other materials were used without further purification. Meso-diphenyletioporphyrins were synthesized by Asaad Salehi and Myoung Seo Koo as described in Young and Chang (1985). The copper-ligating cytochrome oxidase model compounds were synthesized by Myoung Seo K00 (K00, 1986; K00 and Chang, 1987). These various hemes, as prepared, generally consisted of a mixture of five-coordinate chloride and hydroxide ligated species. Homogeneous hydroxide ion ligation was induced by shaking the heme solution (methylene chloride) with aqueous KOH ('1 M) followed by washing with phosphate buffer (.1 M, pH 8.4), total solvent evaporation, and re-solution in methylene chloride or dimethyl sulfoxide (DMSO). Homogeneous chloride ligation was produced by bubbling HCl vapor through the methylene chloride or DMSO solution until the absorption spectrum remained constant and characteristic of chloride ligated hemes. Samples in toluene/tetrahydrofuran (THF) were prepared by evaporation of the methylene chloride from the desired sample and resolution in a 50:50 mixture of toluene/THF. Bis-N-methylimidazole ligated heme samples were produced by the addition of excess N-methylimidazole to the methylene chloride sol spectrum we: Optical 5 UV/visible air referenc. regulating De 1401 Ramalog photomultipli achieved from All spectra w excitation (S in a Spinning Collected by , Spectrometer ‘ scans. EPR Spa new) by Usir a Bruker ER 2C and high Spin observed EPR 3 solutions of c respectiVely. ‘. hepes butter 21: equatim“ s-(n IS the magnetic 81 chloride solution until no further changes in the optical absorption spectrum were observed. Optical absorption spectra were obtained with a Perkin-Elmer Lambda 5 UV/visible spectrophotometer by using a 0.5 cm quartz cuvette with air reference, or in a 4 mm O.D. EPR tube suspended in a temperature regulating Dewar. Resonance Raman spectra were obtained with a Spex 1401 Ramalog scanning double monochromator and a cooled RCA 31034C photomultiplier tube. Data collection and instrument operation were achieved from a DEC LSI-ll/Z computer through a house built interface. All spectra were recorded by using 20 mW of power at 406.7 nm excitation (Spectra—Physics model 164 Kr ion). Samples were contained in a spinning EPR tube (at room temperature) and the signal was collected by utilizing a ~170 degree scattering geometry. The Raman spectrometer was calibrated to the 1004 cm-1 line of toluene for all scans. EPR spectra were recorded at '10 K (or specific temperature as noted) by using an Oxford Instruments ESR 9 liquid helium cryostat and a Bruker ER 200D X-Band spectrometer. Concentrations of ligated copper and high spin hemes were estimated by double integration of the observed EPR signal and comparison with the signals from standard solutions of copper (II) EDTA (1 mM) and metmyoglobin fluoride (.1 mM) respectively. Both of these standards were prepared in 50 mm aqueous hepes butter at pH 7.4 The "g" values were calculated by using the equation: g=(714.47*u)/H, where v is the frequency in gigahertz and H is the magnetic field in gauss. C. MESO-D The stri Figure 4.1a buty1(benzan porphine), c blocked/bloc groups preve‘ (under alkal: toluamido, (; imidazole/blc one simple bl Species are d macrocyclesy . different 9X0! in"fingered ; in this group of the phenyl simplify the d single Capital imidaZOle/bloc} ligands are 1m “Elite 4 lb . In Figure 4 and six-coOrdin are both five~c, consistent Wi th 82 C. MESO-DIPHENYLPORPHYRIN RESULTS The structures of the diphenylporphyrin derivatives are shown in Figure 4.1a. The top species (Trans-5,15-bis [o—(p-tert- butyl(benzamido)) phenyl]-2,8,12,l8 tetraethyl-3,7,l3,l7 tetramethyl porphine), called Trans bis(p—tert-butyl(benzamido))DPE or more simply blocked/blocked, represents the simplest structure. These blocking groups prevent aggregation of the heme and formation of p-oxo-dimers (under alkaline conditions). The second structure (Trans[(N-imidazolyl) toluamido, (p-tert—butyl)benzamido]DPE), referred to as imidazole/blocked, has one intramolecular ligating imidazole group and one simple blocking group. The schematic diagrams for these different species are depicted to the right of each one. With these two basic macrocycles, a variety of heme species can be made by the addition of different exogenous ligands to the system. The species that were investigated are shown in Figure 4.lb in short hand notation. Included in this group is a macrocycle with an imidazole group attached to both of the phenyl substituents to yield a imidazole/imidazole structure. To simplify the discussion, each of parent macrocycles has been assigned a single capital letter designation: blocked/blocked, A; imidazole/blocked, B; and imidazole/imidazole, C. Additional axial ligands are indicated in parentheses after the letter as is shown in Figure 4.lb. In Figure 4.2, optical absorption spectra are shown for the five- and six-coordinate species of macrocycle A. Species A(Cl') and A(0H') are both five-coordinate high-spin species and these spectra are consistent with those previously published (Young and Chang, 1985). Ammo, FiSure 4. 83 ij = Z o .7 U W C?” C? C2" [:7 J O A(Cl') A(0H-) A(Cl-,DMSO) MOH-DMSO) Cb Ci“ Ca C: 51527 I} A(NMI)2 B(CI-) B(OH') C Figure 4.1 Structures of FeIII meso - diphenylporphyr in species. 84 MG?) \ \ \ \\ g _ _ 0H Fe 9 2 ’5‘ .4 MOH'DMSO) A(CI'.DMSO) \ C555 5&7 MW“), WAVELENGTH (nm) Figure 4.2 Optical absorption spectra of FeIII meso-diphenylporphyrin species. Species A(C The differe‘ the similar: five-coordii ligand to fc spectrum in A(NMI)2. The Visible band spectrum of ( that the cove its ligation In Figure SPeCies are c, Six'cool‘dinatg has a Spectrum visible region deSCribed as S band nearly id are quite diff. A(OHVDMSO) is new bands appe; similarity of t six-coordinate Probably reflec species Owing t. species. These ( —i——" "7 85 Species A(Cl',DMSO) and A(OH',DMSO) are both six—coordinate high~spin. The differences between the spectra of A(Cl',DMSO) and A(OH',DMSO) and the similarities of the spectra to those of their respective five—coordinate analogues, suggests that DMSO is not a strong enough ligand to form A(DMSO)2 when Cl' and OH' are present. The final spectrum in Figure 4.2 is that of the bis-(N—methylimidazole) species, A(NMI)2. The narrow red shifted Soret band (412 nm) and the single weak visible band (534 nm) identify this species as low-spin. The absorption spectrum of C (not shown) is identical to that of A(NMI)2, indicating that the covalently attached ligand is not significantly weakened in its ligation strength relative to free solution NMI. In Figure 4.3, the optical spectra of macrocycle B six-coordinate species are compared with the corresponding spectra of the - six-coordinate DMSO ligated species (A(Cl',DMSO), A(OH',DMSO)). B(Cl') has a spectrum similar to that of A(Cl',DMSO) in both the Soret and visible regions, indicating that B(Cl') is probably accurately described as six-coordinate high-spin. B(OH‘) has a Soret absorption band nearly identical to that of A(OH‘,DMSO) but the visible regions are quite different. The strong band-at '575 nm in the spectrum of A(OH',DMSO) is almost completely absent in the spectrum of B(OH') and new bands appear at ”492 and ~620 nm in the B(OH‘) spectrum. The similarity of the Soret regions suggests that these species are both six-coordinate high-spin. The differences in the visible region probably reflect different charge transfer absorptions in the two species owing to the different ligands trans to the OH' in the two species. These differences will be discussed in more detail below. C?" \ mow) Figure 4 86 A(CI'DMSO) x5 0 a N O ( Cl F o g N ; ‘D 8(Cl‘) ‘ I: In K on “ t7 " ISA A(OH 'DMSO) ( OH in N mow) WAVELENGTH (nm) Figure 4.3 Comparison of the optical absorption spectra of six-coordinate high-spin FeI I meso~diphenylporphyrin species. The big macrocycle those which the heme spa presented ir of frequency arrows. With species are spectra. Th1; Vibrational r Verifies the Previously un figures are s COusecutiVely rePresents a l fiSures. AsSiE Similarity to élssieffluents ar could not b e a Wis. The impj The Raman 5 absorption Spec confirmS the pr 0f the inlidazoh 3(01‘) and 3(0H‘ those of the DME — 7 87 The high frequency resonance Raman spectra of the complexes of macrocycle A are shown in Figure 4.4. The peaks marked with arrows are those which are sensitive to or characteristic of the axial ligation of the heme species. The low frequency region, for these same species, is presented in Figure 4.5. Again, peaks which show significant variation of frequency or intensity with change of ligation are marked with arrows. With the exception of A(Cl',DMSO) and A(OH‘,DMSO), all the species are easily distinguished from each other by their Raman spectra. This spin or ligation state sensitivity of the normal vibrational modes allows us to make initial mode assignments and it verifies the potential of Raman spectroscopy to identify species of previously undetermined ligation. The peaks observed in these two figures are summarized in Table 4.1 with the peaks being numbered consecutively from high to low frequency. An asterisk (*) in the table represents a peak which is marked with an arrow in either of the figures. Assignments are made for some of these peaks based on their similarity to peaks observed with OEP or TPP type hemes and these assignments are also included in Table 4.1. Most of the observed peaks could not be assigned by direct comparison with these other porphyrin types. The implications of this will be discussed below. The Raman spectrum of the macrocycle C heme, like the optical absorption spectrum, was identical to that of species A(NMI)2. This confirms the previous observation that there is no detectable weakening of the imidazole ligand in the strapped species. The Raman spectra of B(Cl') and B(OH‘) (see next section) were also essentially identical to those of the DMSO ligated analogs A(Cl',DMSO) and A(OH‘,DMSO), despite The big macrocycle . those which the heme spa presented ir of frequency arrows. With species are spectra. Thi; vibrational r. Verifies the Previously un fiSures are s consecutively rellresents a 1 figures. ASSig Similarity to assignments ar °°uld not be a tl'Pes. The impj The Rainan s abs°rption Spec 87 The high frequency resonance Raman spectra of the complexes of macrocycle A are shown in Figure 4.4. The peaks marked with arrows are those which are sensitive to or characteristic of the axial ligation of the heme species. The low frequency region, for these same species, is presented in Figure 4.5. Again, peaks which show significant variation of frequency or intensity with change of ligation are marked with arrows. With the exception of A(Cl',DMSO) and A(OH‘,DMSO), all the species are easily distinguished from each other by their Raman spectra. This spin or ligation state sensitivity of the normal vibrational modes allows us to make initial mode assignments and it verifies the potential of Raman spectroscopy to identify species of previously undetermined ligation. The peaks observed in these two figures are summarized in Table 4.1 with the peaks being numbered consecutively from high to low frequency. An asterisk (*) in the table represents a peak which is marked with an arrow in either of the figures. Assignments are made for some of these peaks based on their similarity to peaks observed with OEP or TPP type hemes and these assignments are also included in Table 4.1. Most of the observed peaks could not be assigned by direct comparison with these other porphyrin types. The implications of this will be discussed below. The Raman spectrum of the macrocycle C heme, like the optical absorption spectrum, was identical to that of species A(NMI)2. This Confirms the previous observation that there is no detectable weakening 0f the imidazole ligand in the strapped species. The Raman spectra of B(Cl-) and B(OH') (see next section) were also essentially identical to those of the DMso ligated analogs A(Cl',DMSO) and A(OH',DMSO). despite Ce .7 A(Cl') A(OH') 88 1353 [\r We 1 1143 '31 1224 'G——— 1257 1269 1373 1408 1470 1228 1435 A(OH') I A(NMT), 12'00’ 'RAwAN' SHIFT (cm") 1700 Figure 4.4 High frequency Raman spectra of_FeIII mesa-diphenylporphyrin species. 89 “347* -1134 3U 243 330 l Amm q 4 l J J 251 43:7 Afiflf) m a! 0 ( CI F0 9 oS~ ? i Aunumusoo I ?. ( OH I ' Ft A«Jflilflflfl 861 AGGWD; v r _r I V ‘ V 150 ' emu SHTFT (cm'l) “50 Figure 4.5 Low frequency Raman spectra of Fe111 mesa-diphenylporphyrin species. Table 4.1: . Peak 1e (C. 1 161 *2 16: 3 161 *4 15: *5 155 6 151 *7 14s 8 147 *9 , 10 14c 11 137 12 135 13 . *14 126 15 125 *16 122 17 120 18 116 *19 114 20 113. 21 109. 22 106. 23 101 24 _ 25 961 26 911 *27 85: 1:28 82. *29 802 *30 751 31 731 1132 . 33 671 *3“ 631 *35 60: 36 551 37 49: 38 451 *39 39: 4o , "(Fe'ch 34; 1141 . [‘2 287 *43 90 Table 4.1: Raman Peaks of FeIII Meso-Diphenylporphyrins. (Cl‘, (OH', compo- equiv Peak # (Cl') (OH') DMSO) DMSO) (NMI)2 sition mode 1 1640 1640 1640 - - substituent ? *2 1622 1618 1626 - 1627 CaCm? V10 3 1608 1606 1605 1603 1605 phenyl A *4 1577 1579 1572 1570 1583 Cbe V2 1571 *5 1557 1555 — - - ? ? 6 1511 1511 '- 1507 - ? ? *7 1493 1492 1484 1486 1505 CaCm V3 8 1470 1472 1472 1471 1467 ? ? *9 - 1435 - - 1438? ? ? 10 1408 1410 1407 1406 1411 ? ? 11 1372 1374 1372 1372 1375 ? ? 12 1353 1354 1352 1352 1356 CaCN V3 13 - 1313 1316 1310 1308 ? ? *14 1269 1269 1275 1275 1274 CaCN u13 15 1257 1260 1260 1258 1257 Cm-Phenyl? *16 1224 1228 1231 1229 - (phenyl-H) C 17 1209 1209 1211 1213 1211 Cm-phenyl ? 18 1169 1180 1189 1181 1181 ? ? *19 1143 - 1146 1145 1147 ? ? 20 1134 1135 1137 1135 1135 ? ? 21 1094 1088 1095 1097 1093 ? ? 22 1064 1061 1061 1058 1065 ? ? 23 1011 1004 1004 1007 1010 Ca-Cm V6 24 - 948 - - 1000 phenyl F 25 960 961 958 958 - ? ? 26 910 922 913 - — ? ? *27 855 855 855 857 861 ? ? *28 827 827 829 827 - ? ? *29 801 800 802 802 802 ? ? *30 758 759 - - - ? ? 31 738 742 743 742 741 ? ?. *32 - 724 722 722 722 9 ? 33 674 674 ? ? 671 9 ? *34 638 638 637 639 640 phenyl G *35 605 1 604 604 607 607 ? ? 36 558 550 545 550 548 ? ? 37 493 493 497 496 498 ? 7 38 456 457 462 463 460 ? ? *39 395 394 391 390 393 (porphyrin) ug? 40 ? 372 373 374 377 ? ? v(Fe-C1) 347 *41 - 330 325 323 325 ? ? 42 287 290 287 287 291 (porphyrin) ? *43 243 251 246 244 248 (porphyrin) ? the fact th. absorption 1 clearly dis1 and A(OH')) verifies the high-spin ir absorption a spin and lig D. CYTOCHR The meso 0f Compounds oxidase. Thi: maCrOCYCle t1 held in close (trans-5.[(p_ (2‘(2~pyridy1 4.6a along m- t° by the she designation 1: imidazOle gro by the letter studied and t Cl‘ and 0H‘ e species which expected to f. addition of OJ 91 the fact that there were some differences in the corresponding optical absorption spectra. The Raman peak positions (see Table 4.1) are also clearly distinct from those of the five-coordinate high-Spin (A(Cl‘) and A(OH')) and six-coordinate low-spin (A(NMI)2) Species. This verifies that these species of macrocycle B are indeed six-coordinate high-spin in nature. These results establish the utility of optical absorption and resonance Raman spectroscopy for the characterization of spin and ligation states of meso-diphenylhemes. D. CYTOCHROME OXIDASE MODEL COMPOUND RESULTS The meso-diphenylporphyrin can be used as a basis for the synthesis of compounds which model the oxygen reduction site of cytochrome oxidase. This is accomplished by the addition of a copper chelating macrocycle to one of the phenyl groups, so that a copper ion can be held in close proximity to the heme iron. This structure (trans-5-[(p-tert-butyl(benzamido)]-15-[o-(6-(N,N-bis (2-(2-pyridy1)ethyl)amido)methyl nicotinamido]DPE) is shown in Figure 4.6a along with its shorthand diagram. For simplicity, it is referred to by the shorthand name blocked/chelate and it is given the letter designation D. A companion species, imidazole/chelate, has a ligating imidazole group attached to the other phenyl group and it is designated by the letter E. Various ligated species of these two macrocycles were studied and these are drawn schematically in Figure 4.6b. These include Cl' and OH‘ exogenous ligands with and without chelated copper. For species which have a chelated copper, a u-oxo bridge (Fe-O-Cu) is expected to form between the two metal centers (see below) upon addition of OH“ and these species will be referred to as oxygen a] d. .. ./ \ b] D(Cl') D(OH ‘) D(Cu,Cl‘) CU C‘u Cl 9 e 8 Fe Fe NL: NJ NL: N1] E(C|‘) E(OH‘) E(CU,CI') E(CU,O‘3) Figure 4.6 Structures of mesa-diphenylporphyrin cytochrome oxidase model compounds. bridged. Po the exogeno ambiguous a In Figu: changing one ligand. It : D(Cu,Cl'), i nearly ident high-spin sp (and oxygen 4.8. Note, h Progressivel; and then the Series of Si: and E(Cu,C1‘j absorption p1 ligand Streng four Species oxygen bridge the Previous these differe these, the pe and intensity Six'coordinat. : I 93 bridged. For several of the five-coordinated species, the position of the exogenous ligand (i.e. above or below the heme plane) is possibly ambiguous and these possibilities will be discussed below. In Figure 4.7, we examine the effects on the absorption spectrum of changing one of the phenyl derivatives while maintaining a Cl‘ axial ligand. It is evident that the three species (A(Cl‘), D(Cl'), and D(Cu,Cl'), including one with ligated copper (D(Cu,Cl‘)), produce nearly identical spectra which are characteristic of five-coordinate high-spin species. A similar phenomenon is observed with OH' ligated (and oxygen bridged) species (A(OH'), D(OH'), and D(Cu,0'2)) in Figure 4.8. Note, however, with these samples that the band at '570 nm becomes progressively broader and weaker upon addition of the chelating group and then the bound copper. In Figure 4.9 the spectra are shown for a series of six-coordinate Cl' bound species (A(Cl',DMSO), B(Cl'), E(Cl') and E(Cu,Cl‘)). Again there is little difference between the optical absorption properties of these species, and thus, despite the stronger ligand strength of the attached imidazole versus the solution DMSO, all four species appear to be high-spin. The final series, OH' ligated (or oxygen bridged) six-soordinate species, is shown in Figure 4.10. Unlike the previous series, there is significant variation in the spectra of these different species. Although the Soret peak is similar in all of these, the peaks to the red of the Soret vary considerably in position and intensity. We suggest that these species are still all six-coordinate high-spin; the variation in the optical spectra will be discussed below. Cs; Met) 5. Fe— 0(Cl‘) C‘s. FQ‘ men Figure 4 94 'co ('3 a: co co ( CI F7 A(Cl‘) a: a n V V ‘9 511 K] 544 o«nuui WAVELENGTH (nm) Figure 4.7 Comparison of the optical absorption spectra of five-coordinate, Cl' ligated FeII mesa-diphenylporphyrin species. “8111‘s 1 x5 9 o v to a N a Is to v to n ( OH Fe AKHO N '0 ca 0 w v E“ Fe D(OH) 570 WAVELENGTH (nm) Figure 4.8 Comparison of the optical absorption spectra of five-coordinate, OH' ligated/ 0'2 bridged FeIII mesa-diphenylporphyrin species. Ce .§- Mama) EM) E(CuCI-) F1Eur. ( CI Fe O K7 0| 510 505 628 ,§_ A(CI‘DMSO) ( C I F a V N‘ : N n 8(Cl‘) \ ‘° E C I F e o N : c, co ,— a EU? “’ Co a K C l N F 97 3 g N E(Cu.Cl‘) WAVELENGTH (nm ) Figure 4.9 Comparison of the optical absorption spectra of six-coordinate, Cl’ ligated FeIII mesa-diphenylporphyrin species. t OI- Fe- ,§— «on-m I Fe~ L/ E(Cuo-z) Figure 97 406 x5 575 492 620 m E" m m [X qqxo'g gqo d \L I >>n / WAVELENGTH (nm) Figure 4.10 Comparison of the optical absorption spectra of six-coordinate, OH' ligated/ O' bridged Fe II mesa-diphenylporphyrin species. In Fig demonstrat optical ab and six-co. This same 1 shown). The effects on 0H" ligated respective A(OH',DMSO) species wit] OH. (or 0‘2: Figures 4.1: intensity ar 0H. ligatior for the Rama DESpite meso‘diphenyj tl’PeS, both 1 these cl’tochx confidently e Species. The indiCate Vari are not detec six'co°rdinate res ' ting form C IIIIIIIIIII_________________________—__fi_‘fT‘m' 98 In Figures 4.11 through 4.14, the high frequency Raman results demonstrate the same pattern that was observed in the corresponding optical absorption spectrum; all the oxidase model species, both five— and six-coordinate, have spectra characteristic of high spin species. This same trend is also reflected in the low frequency Raman data (not shown). The presence of the chelating group and bound copper have small effects on the optical absorption spectra of those species which are OH' ligated or oxygen bridged, but no distinctions are observed in the respective Raman spectra. Although the Raman spectra of A(Cl‘,DMSO) and A(OH',DMSO) could not be clearly distinguished, the six-coordinate species with a ligating imidazole do show a distinction between Cl‘ and OH” (or 0'2) ligation. Examination of the peaks marked with arrows in (peak 7 from Table 4.1), shows a gain of Figures 4.13 and 4.14 intensity and a shift to higher frequency on going from 61' ligation to OH' ligation. The low frequency region (not shown) is nearly identical for the Raman spectra of all the six-coordinate species. Despite the fact that the optical and Raman spectra of these meso-diphenylporphyrins seem to be distinct from both the OFF and TPP types, both techniques have been valuable to the characterization of these cytochrome oxidase model compounds. We have been able to confidently establish the spin and ligation state of these various species. The small changes in the optical absorption spectra may indicate variation in charge transfer transitions, yet these effects are not detected in the respective Raman spectra. The fact that the six-coordinate species are high-spin is encouraging because in the resting form of cytochrome oxidase, the heme is six-coordinate A(Cl') 1200 RAMAN SHIFT (cm") 1700 Figure 4.11 Comparison of the high frequency Raman spectra of five-coordinate, Cl' ligated FeIII mesa-diphenylporphyrin species. I u 8 cl F 100 1200 RAMAN SHIFT (cm“) 1700 Figure 4.12 Comparison of the high frequency Raman spectra of five-coordinate, OH‘ ligated/ O‘2 bridged FeIII meso-diphenylporphyrin species. 101 1351 E(Cu.Cl') 1200 RAMAN SHIFT (cm’1) 17°° Figure 4.13 Comparison of the high frequency Raman spectra of six-coordinate, Cl' ligated FeIII meso-diphenylporphyrin species. r u g .1. F . fl ,-..'_4- ,_.2_ . .‘___A~ W -——~‘ ~4‘A": A—h -- 102 ~1352 ~1570 EKMNYW L 1260' jRANANVSHIFTTcrfi“) .1700 Figure 4.14 Comparison of the high frequency Raman spectra of six-coordinate, OH' ligated/ 0'2 bridged FeIII mesa-diphenylporphyrin species. [0. Fe- ’5‘ A(OH'N K 0b F.- La B(OH-} 5.. F0- E(0H') La E(Cu.°-: Figure 4. -1352 ~1570 { OH Fe O -a— A(OH'DMSO) 4 ~113 4 1253 1372 1406 * ~1486 15 .——— -1229 ~1603 ~1275 l 1470 8(OH') E(Cu.0" ) 1 12’00 RAHAN SHIFT (cm-1) 170° Figure 4.14 Comparison of the high frequency Raman 2 spectra of six-coordinate, OH' ligated/ O‘ bridged FeIII mesa-diphenylporphyrin species. high-spin. OptiI however, cannot between metal CI coupling similaI EPR to study the The EPR spec demonstrated by prominent featur smaller signal a compounds (A(Cl‘ fluoride as demo signal is an ind Signals are over a glass forming (Figure 4.15c). With Chelated co attributed mostl The Concentr. calculated by dox rhombic signals 1 metmyoglobin flm intensities for t and D(Cu_c1-)) a: favorably with tb SpeCtra (‘ . 25 mM) 103 high-spin. Optical absorption and resonance Raman spectroscopy, however, cannot establish whether there is magnetic exchange coupling between metal centers. To determine if our models exhibit exchange coupling similar to that observed with cytochrome oxidase, we have used EPR to study these model compounds. The EPR spectrum of a typical high spin heme (see discussion) is demonstrated by that of metmyoglobin fluoride (Figure 4.15a). The prominent features are the large signal with a value of g=~6 and a much smaller signal at g=~2. The EPR specra of the five-coordinate Cl' bound compounds (A(Cl'), D(Cl')) are similar to that of the metmyoglobin fluoride as demonstrated in Figure 4.15(b-d). The splitting of the g=6 signal is an indication of some rhombicity (see discussion) but the signals are overall narrow and fairly homogeneous. Samples prepared in a glass forming solvent (toluene/THF) exhibited the narrowest lines (Figure 4.15c). The spectrum of the five-coordinate Cl' ligated species with chelated copper (D(Cu,Cl‘)) exhibits a strong g=2 signal which is ttributed mostly to a signal from the copper (Figure 4.15e). The concentrations of the EPR detectable high-spin hemes were alculated by double integration of the g=6 signals (or the split hombic signals near g=6). These values, standardized against etmyoglobin fluoride, are reported in Table 4.2. The integrated ntensities for the five-coordinate Cl‘ bound species (A(Cl'), D(Cl'), nd D(Cu,Cl')) are consistently high (above .150 mM) and compare vorably with the concentrations estimated from the optical absorption ectra (-.25 mM). The EPR spectra of the five coordinate OH' ligated bF' nmufiOK b) ,3“? AW) dTM/THF Fe 104 8 CHZCl2 J o. a)mdeF' 10K 3, 4.3 Mm? c) Tol/THF . ' ‘JHfi | Q~ INC?) ) Cu ( CI' F. l ‘9 I “2 Is N a “CROW " Figure 4.15 EPR spectra of Met Mb F' and five-coordinate, Cl' ligated FeIII meso-diphenyl porphyrin species. Table 4.2: Comp. Cono EPR 1 Species U m :> 3222:“ Table 4. 3: Slope Frequr Peak K (Sl< l u 2 304.3 3 u § 214.2 6 121.2 7 357.1 8 - 9 mm 10 89.3 105 Table 4.2: Comparison of High-Spin FeIII Meso-Diphenylporphyrin Concentrations as Determined by Optical Absorption and EPR Spectroscopy. Conc mM Conc mM EPR Species Solvent (Optical) (EPR) Temp K A(Cl ) CH2012 25 195 10 A(Cl ) Tol/THF 25 180 10 D(Cl’) CHZCIZ 25 255 11 D(Cu,C1 ) CH2C12 25 186 11 E(Cl') CH2C12 25 144 11 E(Cu C1 ) CH2612 25 099 12 A(OH') CH2C12 25 086 11 A(OH’) Tol/THF 25 104 11 D(OH ) CH2C12 25 115 11 D(Cu 0'2) CH2C12 25 031 11 E(OH‘) . CH2012 .25 .051 11 E(Cu,0'2) CH2C12 .25 .023 11 Cable 4.3: Slope and Intercept Values for the Core Size Dependent High Frequency Raman Peaks of FeIII Meso-Diphenylporphyrins. ’eak K (Slope) A(Intercept) Composition Mode 1 - — ? 7 2 304.3 7 33 CaCm? V107 3 - - phenyl 4 214.2 9.37 Cbe V2 5 - - ? ? 6 121.2 14.47 Cbe? ? 7 357.1 6.20 CaCm V3 8 - - 2 2 130.4 13.0 Cbe? ? 89.3 17.79 ? ? ble 4.4: Comparison of the Vibrations of Three Different Porphyrin Types. FeIIIOEP FeIIIDPP FeIIITPP ode # (NMI)2 (NMI)2 (NMI)2 Cbe V2 1599 1583 1568 CaCm V3 1505 1505 1545? CaN uh 1375 1356 1370 species (A(OH-) (extending to h broad 8'2 Sigma solvent resulte some Of this ca: unusual and wil: five-Coordinate bridged specieS} the iron and col: the C1‘1bound SP integrated C°nce ligated Compound the decrease in fact that the Sii its broadness. H< (D(Cu,0'2)) is 5‘ (Table 4.2). Whi‘ The EPR spect has a more rhombi species and a rat the six-coordinat displays a simila intensity of the 1 corr ‘ esponding f iVI °°pper (E(Cu,C1 )‘ Species. The EPR 106 species (A(OH‘), D(OH')) are characterized by very broad g=6 bands (extending to higher g values than the other samples), and very large, broad g-2 signals (Figure 4.16). Again, the use of toluene/THF as a solvent resulted in a more resolved line shape (Figure 4 16b). Although some of this can be attributed to rhombic distortion, these spectra are unusual and will be discussed further below. The spectrum of the five-coordinate species with chelated copper (D(Cu,0'2)) (an oxygen bridged species) has a prominent copper signal (Figure 4.16), but both the iron and copper signals are weak in comparison to the spectrum of the Cl' bound species with chelated copper (Figure 4.15). The integrated concentrations of high-spin heme are lower for these OH' ligated compounds than for the Cl- ligated species (Table 4.2). Part of the decrease in intensity (see discussion) may be attributed to the fact that the signal extended beyond the integration window owing to its broadness. However, the value for this oxygen bridged compound :D(Cu,0‘2)) is still much lower than that of the OH" ligated species :Table 4.2), which also exhibit a broad signal. The EPR spectrum of the six-coordinate Cl' bound species (E(Cl')) as a more rhombic g-6 signal than the corresponding five coordinate ecies and a rather large g=2 signal (Figure 4.17a). The spectrum of e six-coordinate Cl' species with chelated copper (E(Cu,Cl')) 'splays a similar g—6 signal and a g-2 copper signal. The integrated tensity of the high-spin heme for (E(Cl‘)) is less than for the rresponding five-coordinate compound, while the sample with chelated >pper (E(Cu,Cl_)) exhibits the smallest value of all the Cl' ligated :ecies. The EPR spectra of the six-coordinate OH" ligated species are Cain... A(OH‘) b) Tol/THF W\ Figure 4.1I 107 b) Tol/THF Figure 4.16 EPR spectra of five-coordinate, OH' ligated/ 0'2 bridged FeIII meso-diphenylporphyrin species. 3) :H C! Fe KG“) 7 EXCKG’) c) Cw Fe E(0H') d) Clo ( .0 Fe “W0 Figure 4. 1 108 Figure 4.17 EPR spectra of six-coordinate FeIII mew-diphenylporphyrin species. unusual. The 5 displays a bro features (Figu indicate the p? spectrum of th‘ displays a ver) which does not values of the i are low. These To obtain a high-spin heme, (five-coordinate to insure inclus concentration of the bulk solutio at least .25 mM. equilibria, the 1‘ function of temps temperature depen by Curie law and (see Figure 4.18) co“Sistent with a high-spin hemes (1 Signal and the ab: lines in the spect t . his temper-attire I 109 unusual. The spectrum of the imidazole/chelate species (E(OH')) displays a broad multifeatured g=6 signal and several other noisy features (Figure 4.17c). The appearance of a broad signal at g=~2.6 may indicate the presence of a low-spin component in the sample. The spectrum of the corresponding species with chelated copper (E(Cu,O‘2)) displays a very small g=6 signal but a comparatively large g=2 signal, which does not appear to be a simple copper signal (Figure 4.17d). The values of the integrations of the g=6 signals for both these species are low. These spectra will be further discussed below. To obtain a more accurate indication of the loss of detectable high-spin heme, a careful study was done with compound (D(Cu,0'2)) (five-coordinate oxygen bridged). By using a large integration window to insure inclusion of all the high-spin (g='6) signal, the effective concentration of detectable high-spin iron integrated to ~.OS mM while the bulk solution concentration (from optical absorption spectra) was at least .25 mM. To test for temperature dependent spin state equilibria, the integrated value of the g=6 signal was determined as a function of temperature over the range of ~4 K to ~20 K. The observed temperature dependence of this signal roughly followed that predicted by Curie law and paralleled that of the metmyoglobin fluoride standard (see Figure 4.18). The observed deviations from linearity are consistent with a zero-field splitting of -10 cm‘1 which is typical of high-spin hemes (Palmer, 1985). The temperature dependence of the g=6 Signal and the absence of low-spin (g—3) and intermediate-spin (g=4 75) lines in the spectra indicate that no spin state equilibria exist over this temperature range. Integration of the g=2 signal, which is 25606. I Enigma: m0 a... 110 .pasoaaou Hopes moonwxo oeoHSoouxo pompaun N.o mustapuooo-o>wu o5» one -m p: out you ABNA oHusov H\H m3muo> huumcoucm acumHm no uqu < wH.q oufiwwh b: 3 . e n. ma. out n a. T1 u _ _ h \. <\\\\ \ \ \\°\ 4 \ \ \ \ \ \ \ \\ o \ \ \ \\ \\ a \\ \ .fi \\ \\ o \\ \ \ \ \\ \ \\ \ \\ \ \ \ \.\ \ - \\ \ \d \ \ o A7965 \\ \\ 28m”- { m6 I x o \ so \ \ \ bx - I \ . 2.58 -uasaoe no I O .. easv P9331591!“ annaleu f O N 1 O P) expected t° or concentration ‘ copper signal 6 a nearby metal E_ DISCUSSION Mesa-diphen between those ° through 4~3I th‘ axial ligand WE the bis-imida201 presence of an a of Cl' ligation characteristic 0 does not seem to which has a trans in the charge tré imidazole ligand character. This 5 region of B(0H') (Makinen and Chur, Six-coordinate hi; position). With or Symmetry of the me Similar drop in sy breakdown of X and lMinds. This does n 111 expected to originate predominantly from the copper, yielded a concentration of ”.07 mM (against a copper (II) EDTA standard). This copper signal exhibited fast relaxation behavior which is indicative of a nearby metal center (Goodman and Leigh, 1985). E. DISCUSSION Meso-diphenylporphyrins exhibit optical absorption properties between those of the OEF type and TPP types. As is seen in Figures 4.2 through 4.3, the optical absorption bands are also quite sensitive to axial ligand type and coordination number, and with the exception of the bis-imidazole ligated species, all appear to be high-spin. The presence of an absorption band between ~630 and ~645 nm is diagnostic 3f Cl‘ ligation while a strong band at "570 to ~580 nm is :haracteristic of the OH' ligation, although this latter generalization ioes not seem to apply well to the six-coordinate OH‘ ligated species hich has a trans imidazole ligand (B(OH‘)). This may reflect a change n the charge transfer absorption bands owing to the addition of the midazole ligand (Asher and Schuster, 1979) and less iron out of plane aracter. This seems reasonable since the peak pattern in the visible egion of B(OH‘) bears a strong resemblance to that of metmyoglobin iakinen and Churg, 1983) which contains imidazole ligated Lx-coordinate high-spin heme (with H20 ligated to the other axial Isition). With only two phenyl porphyrin substituents, the effective mmetry of the metalloporphyrin is reduced from -D4h to ~D2h- A milar drop in symmetry for free base porphyrins results in a eakdown of X and Y axis degeneracy. and a splitting of the visible nds. This does not occur for the meso-diphenylporphyrins because the phenyl substitI Yaxes (througl on both axes. S (Gouterman, 196 axes and split substituents do The Raman both OEP and TP POrphyrin ring . the normal coorr Abe and Kitagaw; limitation, asSj with extensive c 1978; Stein et a make some assigr al_, 1975; and C Smng et al., 19 p”Wynn Core S straight line Wi °°mP°Sitiom The °f different met with iron Porphy- and Gouterman, 1! through 10 are p: G°utermam 1983) The core SiZe (CE 112 phenyl substituents, unlike the free base hydrogens, are off the X and Y axes (through the pyrrole nitrogens) and exert an equal perturbation on both axes. Since the optical transition dipoles are along these axes (Gouterman, 1961), free base perturbations remove the degeneracy of the axes and split the observed absorption peaks, but the two phenyl substituents do not. The Raman spectra of the meso-diphenyl hemes were distinct from both OEP and TPP type hemes. The presence of the phenyl groups on the porphyrin ring seriously perturbs the vibrational properties such that the normal coordinate assignments (mode composition and frequency) of Abe and Kitagawa (1978) are not directly applicable. Despite this limitation, assignments for the vibrations of OEP type hemes, along with extensive characterization of the TPP type hemes (Burke et al., 1978; Stein et al., 1984; and Blom et al., 1986), should allow us to ake some assignments. It has been demonstrated for OEP (Spaulding et 1., 1975; and Choi et al., 1982) and TPP (Huong and Pommier, 1977; tong et al., 1980) type hemes that a plot of frequency versus 1, produces a orphyrin core size, for porphyrin modes over 1450 cm‘ traight line with a slope and intercept characteristic of mode omposition. The porphyrin core size can be varied either by insertion f different metals (Spaulding et al., 1975, and references within), or ith iron porphyrins, by variation of spin and ligation state (Scheidt nd Gouterman, 1983). In Figure 4.19, the frequencies of peaks 1 rough 10 are plotted versus the estimated core size (Scheidt and uterman, 1983) for the ferric meso-diphenylporphyrins of Figure 4.4. e core size (center to pyrrole nitrogen distance) increases for the F1SUre 113 p e a k 6 -Coordinate 5 -Coordinate 6 -Coordinate # Low-Spin High-Spin High-Spin 1650< 1.989 A 2,012 A 2.045 A I ' A Ania 2 I I Y ‘Y I l 1500-p- 1550-"- 1’11 cm Isoo-- I l 1 l l I I I I I, g l a b T' I I | I I I 1450'”- ' l l 9 L ' | l P \- I l I l I I l 10 e h . In I. .I . T . 1406 1 Ti *1 1,97 199 2.01 2.03 2.05 (CI-NM Figure 4.19 The core size dependence of the high frequency Raman vibrations of FeII mesa-diphenylporphyrin species. I I series six-(10‘ and intercept the estimated ana10g°us OEP those for both type POrPhyrin mode ComPOSiti( both the OEP ar Despite the modes of the fe exhibit overall Peak 4 (see Tab] spin state (and frequency range probably has a V hemes. Peak 7 is intensity with V6 frequency range ( (Table 4.3) all i hemes. Peak 12 sh Oertling, unpubli: the spin state ant frequency region w dependence and thi These three modes sIlecies of OEP me‘ 114 series six-coordinate low-spin to six-coordinate high-spin. The slope and intercept values from this plot are listed in Table 4.3, along with the estimated dominant mode component and the assignment of the analogous OEP mode. The slope values for this plot are much lower than those for both the OEP (Choi et al., 1982) and TPP (Stong et al., 1980) type porphyrins (and the intercepts, higher) which suggests that the mode compositions may be altered or significantly perturbed relative to both the OEP and TPP types. Despite the unusual core size dependences of the high frequency modes of the ferric meso-diphenylporphyrins, three of these modes exhibit overall behavior similar to modes in their OEP counterparts. Peak 4 (see Table 4.1) undergoes changes in frequency with changes in spin state (and ligation) in the range of 1570 to 1583 cm'l. Its frequency range and core size dependence (Table 4.3) indicate that it probably has a vibrational composition comparable to V2 in OEP type hemes. Peak 7 is also is most notable for the dramatic changes in intensity with variation in the axial ligand. This behavior, its frequency range (1483 to 1505 cm'l), and the core size dependence (Table 4.3) all indicate a close correspondence to V3 in the OEP type hemes. Peak 12 shifts to ~1345 cm'l upon reduction of the heme (W. A. Oertling, unpublished results) but exhibits only a small dependence on the spin state and ligand type. This is the only peak in the high frequency region which exibits any significant oxidation state dependence and this property makes it analogous to V4 of the OEP types. These three modes are listed in Table 4.4 for the bis-NMI ferric Species of OEP, meso-diphenylporphyrin, and TPP species. No consistent trends are rev for the three perturbatim‘s different mode of these vibrat For hemes W vibrations With and A2g (AP), w or anomalously I 99). Of these, 1 enhanced with S< Upon addition of lowered to D2h' Ag (D211) and Blg being polarized polarized) (McCla enhanced by Sorel totally symmetric depolarized (32g) Our results indie me$0~diphenylporp the TPP types. In for the three por] we Would expect t< frequencies of OE} There-“re, it appe 115 trends are revealed by comparison of the frequencies across the series for the three different modes. This may result from the different perturbations of u2, V3, and V4 by the phenyl substituents owing to the different mode composition types (Cb-Cb, Ca-Cm, and Ca-N respectively) of these vibrations. For hemes with D4h symmetry, resonance Raman spectroscopy enhances vibrations with the following characters: Alg (P), 32g (DP), Blg (DP), and AZg (AP), with the predicted polarizations (polarized, depolarized, or anomalously polarized) as indicated in parentheses (Spiro, 1983, Pg 99). Of these, the totally symmetric Alg modes are most strongly enhanced with Soret excitation and will normally dominate the spectrum. Upon addition of meso diphenyl substitution, the symmetry will be lowered to D2h‘ Alg and B2g Vibrations (D4h) will now both transform as Ag (DZh) and Blg and A2g as Blg (Wilson et al., 1955), with Ag modes being polarized and Blg modes modes being depolarized (or anomalously polarized)(McClain, 1971). The net result is that more modes should be enhanced by Soret excitation in D2h symmetry since more of them are now :otally symmetriC‘in nature; some of the modes that were previously iepolarized (32g) in D4h should be polarized (Ag) under D2h symmetry. >ur results indicate approximately the same number of modes for the eso~diphenylporphyrins as for the OEP types, but many more than for he TPP types. In addition, there are very few direct mode correlations or the three porphyrin types. If only symmetry effects were involved, a would expect to see a closer correlation between the vibrational :equencies of OEP types and those of the meso-diphenylporphyrins. erefore, it appears that, in addition to symmetry effects, the specific effe‘ determination mesa-diphenyl] for these varfi more of the mc Although t porphyrin ring been able to a size or oxidat other vibratim for ferric mes< state sensitivi Altthh We hax be used to inte °°mPOUnds. Thus 4'5 (asterisk i ferric meso-dip1 substituent modr TPP types (Burkr the Phenyl subs: above , The °Ptical show a Strong re mes°‘diphenylpor Phenyl Sub Stitue‘ 116 specific effects of phenyl substitution are also involved in the determination of the specific vibrational patterns observed for the meso—diphenylporphyrins. Excitation profiles and depolarization ratios for these various meso-diphenylporphyrin may be necessary to assign more of the modes and establish the actual extent of symmetry lowering. Although the interaction of the phenyl substituents with the porphyrin ring makes vibrational mode assignments difficult, we have been able to assign three of the high frequency modes through the core size or oxidation state dependence of their frequencies. Many of the other vibrations observed in Figures 4.4 and 4.5 (listed in Table 4.1) for ferric meso-diphenlyporphyrins also display ligation and/or spin state sensitivity through variations of intensity and/or frequency. Although we have not been able to assign most of them, they can still be used to interpret the Raman spectra of previously uncharacterized compounds. Thus, the peaks marked with arrows in the Figures 4.4 and 4.5 (asterisk in Table 4.1) serve as spin and ligation standards for ferric meso-diphenylporphyrins. We have also assigned several phenyl substituent modes (see Table 4.1) on the basis of those observed in the TPP types (Burke et al., 1974). This further affirms the interaction of the phenyl substituents with the porphyrin macrocycle as suggested above. The optical absorption spectra of the cytochrome oxidase models show a strong resemblance to those of the simpler meso-diphenylporphyrin species. The presence of the chelating pyridine phenyl substituent, as well as the bound copper produced very little perturbation < species (Figux general red sh group and then visible region modeling cytoc oxidase is six seems to be on: in close proxir bound or oxyger particularly an that the result he explained as Characteristic This idea is St‘ (1936) with "ba: covalently Strap of this band Wit six.coordinate A sixth ligand, in OH'. Addition of Plane or out of 1 and decrease of : bands at 492 and hYdrogen bend to 117 perturbation of the optical spectra of the five-coordinate Cl' bound species (Figure 4.7). The six-coordinate Cl' bound species show a general red shift of the Soret peak upon addition of the chelating group and then bound copper (Figure 4.9) but no major changes in the visible region. These results are encouraging from the perspective of modeling cytochrome oxidase. The heme of cytochrome g3 in cytochrome oxidase is six-coordinate and high—spin in the resting state and there seems to be only a small perturbation from the presence of the copper in close proximity (Blair et al., 1982). The optical spectra of the OH‘ bound or oxygen bridged species exhibit more varied behavior, particularly among the six—coordinate species (Figure 4.10). We beleive that the results from these OH' ligated and oxygen bridged species can be explained as follows. The presence of the 578 nm band is characteristic of displacement of the iron towards the ligating OH'. This idea is strongly supported by the results of Schaeffer et al. (1986) with "basket-handle" porphyrins, in which alkoxo groups are covalently strapped across iron axial positions. The strong intensity of this band with the five-coordinate A(OH') species and the six-coordinate A(OH’,DMSO), which has the weakly coordinating DMSO sixth ligand, indicate displacement of the iron toward the ligating OH'. Addition of the imidazole ligand (B(OH‘)) pulls the iron back into plane or out of plane toward the imidazole, resulting in a blue shift and decrease of intensity of the ~578 nm band and the appearance of bands at 492 and 620 nm. The chelating pyridine groups (E(OH')) may hydrogen bond to the OH' axial ligand, thereby increasing its ligating strength and stabilizing a structure with the iron out of plane toward the OH'. This results in the reappearance of a peak at ~570 nm. The addition of c and induces t species is ap constraints, r decrease in i1 five-coordinat consistent wit heme plane. Th chelating pyri Complex may ha‘ out of plane t] the ligation st Some of the brc D(CU,0H‘) may l: OPPOSite to the no steric const Peak at ‘570 mm The“: resul of CytOChrome 0: an abs°rption he states) which is copper in the Cy that the absorpt to the °°mParab1 this 655 nm band 620 mm bands of —7————”‘ 118 addition of copper to the chelating group removes this hydrogen bond and induces the formation of the p-oxo species. The iron in the p-oxo species is apparently back in plane, probably owing to simple steric constraints, as evidenced by the appearance of the 620 nm peak. The decrease in intensity and wavelength of the ~578 nm band in the five—coordinate series A(OH‘), D(OH'), D(Cu,OH') (Figure 4.8) is consistent with increased steric constraint pushing the iron into the heme plane. This is not inconsistent with hydrogen bonding to the chelating pyridines. The optimum geometry for this hydrogen bonded complex may have the iron out of plane toward the OH‘ but perhaps less out of plane than for the unconstrained species (A(OH'), even though the ligation strength of the hydrogen bonded complex may be stronger. Some of the broadness of the visible bands of the species D(OH') and D(Cu,OH‘) may be accounted for by OH' ligating to the axial position ' opposite to the chelating group. In this case the OH" would encounter no steric constraints and would display a peak at 578 nm along with peak at ~S70 nm (or less) from the constrained species. These results have interesting implications for the interpretation of cytochrome oxidase absorption spectra. Cytochrome oxidase exhibits an absorption band at ~655 nm in the resting enzyme (and some ligated states) which is associated with ligand coupling between the iron and copper in the cytochrome a3 site (Beinert et al., 1976). If we consider that the absorption spectra of heme a species are red shifted relative to the comparable meso-diphenyl hemes (FeIII Heme a Cl', ~ 670 nm), this 655 nm band in cytochrome oxidase could correspond to the ~630 or ~620 nm bands of the respective C1‘ or OH' bound six-coordinate meso-diphenyl different pos: One possibilit in resting oxi result in the that 0‘2, or a enzyme with th bridging, or p1 loss of the 65.‘ Despite the meso-diphenyl h of Cl‘ or OH' 1 35 was discusse mes°'diPhenylpo structures of t] spectra of the I high‘sPin (Figui Raman spectral c “3 and 4.14). a broadness of p sample inh°m0gen This may reSult ( hacrocy C19- decOI m a low Spin cm —7—‘_" 119 meso-diphenyl hemes. If this analogy is correct, it suggests two different possibilities for the bridging ligand in cytochrome oxidase. One possibility is that a halide ligand bridges the two metal centers in resting oxidase, and displacement by other exogenous ligands could result in the loss of the 655 nm band. The other possibility suggests that 0‘2, or a similar ligand, bridges the metal centers in the resting enzyme with the iron in plane. Exogenous ligands which disrupt this bridging, or pull the iron out of plane, could likewise result in the loss of the 655 nm band. Despite the inability to assign most of the Raman lines of the meso-diphenyl heme species, the Raman spectra are consistent in terms of Cl‘ or 0H' ligation, five- or six-coordinate, and high- or low-spin, as was discussed above. As such, the Raman spectra of the simpler meso-diphenylporphyrins allow for straightforward analysis of the structures of the more complex cytochrome oxidase models. The Raman spectra of the D species indicate that they are all five-coordinate high-spin (Figures 4.11 and 4.12). The species made from E have the Raman spectral characteristics of six-coordinate high-spin (Figures 4.13 and 4.14). The spectra of the species E(OH') and E(Cu,OH') exhibit a broadness of peaks 4 ("1573 cm'l) and 7 (~1490) which may indicate sample inhomogeneity, such as a small population of low-spin species. This may result from extraneous pyridine or imidazole ligands (macrocycle decomposition products) or a small percentage of the sample in a low spin configuration. Optical a establish whe iron and cow whether there a reasonable n established; 1 to other techn spectroscopic bridging in th‘ six-coordinate indicate that 1 Well as some cc E(Cu,c1‘) (Koo, model Compounds between the met The EPR der ligand Symhletry lineshape comm! with g Perpendn Symmetry is Clea Perturbaticms Wh distortion) caus from the mo non spectra of the f which display a 1 indicates that tr 120 Optical absorption or Raman spectroscopy cannot definitively establish whether the bound C1‘ or OH' (0'2) is bridging between the iron and copper in those species with chelated copper and if so, whether there is magnetic coupling between the two metal centers. To be a reasonable model for resting oxidase, both of these aspects must be established; ligand bridging and magnetic coupling. We therefore turn to other techniques which may give us this information. IR spectroscopic studies (Koo, 1986) suggest that oxygen (0'2) is probably bridging in the five-coordinate model D(Cu,0‘2) and possibly with the six-coordinate model E(Cu,0'2) also. Magnetic susceptibility studies indicate that there may also be magnetic coupling in these species, as well as some coupling with the six-coordinate Cl' ligated species E(Cu,Cl') (K00, 1986). We have utilized EPR spectroscopy on some of the model compounds to verify the presence and extent of magnetic coupling between the metal centers of the copper chelating species. The EPR derivative line shapes expected for hemes of different ligand symmetry and spin state are discussed by Palmer (1985). The lineshape common to the high spin ferric hemes is the axial symmetry with g perpendicular (gX—gy) at '6 and g parallel (g2) at ‘2. Axial symmetry is clearly demonstrated with the Mb F' (Figure 4.15a) sample. Perturbations which remove the equivalency of the X and Y axes (rhombic distortion) cause a splitting of the g=6 signal into individual signals from the two non-equivalent axes. This phenomenon is seen in the spectra of the five-coordinate Cl' bound species (Figure 4.15b-e), which display a small splitting of the g=6 signal. Palmer (1985) indicates that this behavior arises most often from changes in the type or orientatio meso-diphenyl some extent i aromatic meso compounds (Fi, (Figure 4.17a This is obser signal and a ( resulting line different axie distortion. Ir relative to th Premature to r SOlvent instea for both Sampl Since the THF line shape is there is also . solvent SyStem c”Pounds are I the "glass" pr: CHzcl2 (which ( that similar u, bound TPP tYpe low-spin Chara: hYdrogen bondir 121 or orientation of axial ligands. In the case of these meso-diphenylporphyrins, since this rhombic distortion is present to some extent in all observed species, asymmetry induced by the tw0 aromatic meso substituents may also occur. With the OH' ligated compounds (Figure 4.16) and some of the six-coordinated compounds (Figure 4.17a,d), we see a much greater distortion of this pattern. This is observed as a broadening and loss of intensity of the g=6 signal and a dramatic increase in the intensity of the g=2 signal. The resulting line shape most closely resembles a composite of the two different axial symmetries, or perhaps a more severe rhombic distortion. In this case, the specific orientation of the axial ligand, relative to the meso substituents, may be responsible. It is also premature to rule out aggregation effects. The use of a toluene/THF solvent instead of CH2C12 in tw0 cases, resulted in sharper line shapes for both samples and less distortion in the spectrum of the 'OH sample. Since the THF weakly ligates, it is difficult to say if the sharper line shape is due to the "glass" quality of the solvent, or whether there is also a ligation effect. It is also difficult to say which solvent system would be more prone to aggregation. Although these compounds are less soluble in Toluene/THF overall, the THF ligation and the "glass" properties upon cooling may cause less aggregation than CH2012 (which crystallizes upon freezing). It is interesting to note that similar unusual line shapes have been reported for OH' and R0' bound TPP type hemes (Schaeffer et al., 1986). The appearance of some low-spin character in the spectrum of E(OH') may confirm that there is hydrogen bonding to the pyridine chelating group and increased ligation strength towa: rule out ligat I Although it spin iron (III study, the EPR chelated speci signals is ind ligand bridgin errors in the 4.2, these err low integratio Six-coordinate comParison wit] magnetic coupl; Careful study ( (”(Cuv0‘2n. wt inteStation val values. For D(( Value of ‘05 mm this is taken 1 much leSS for s determined cone (D(Cu,0~2)) our centers which a r91aXat10n of t‘ he exchange cou- 122 strength toward the iron. However, the signal is not strong enough to rule out ligation by extraneous pyridine or imidazole. Although we have focused primarily on the identification of high spin iron (III) through the detection of the g=6 signal in this EPR study, the EPR detection of the copper II (g=2) signal for copper chelated species is also of importance. Absence or attenuation of these signals is indicative of coupling between metal centers by means of ligand bridging or aggregation effects. Although there may be large errors in the absolute values of the integrations reported in Table 4.2, these errors should be systematic. Considering this, the unusually low integration values for both the five— (D(Cu,0'2)) and six-coordinate (E(Cu,0'2)) copper chelated oxygen bridged compounds, in comparison with their copperless counterparts, is strong evidence for magnetic coupling between the metal centers in these compounds. The careful study of the five-coordinated oxygen bridged species (D(Cu,0'2)), which uses a larger integration window, confirms that the integration values in Table 4.2 are probably lower than the true values. For D(Cu,0'2), the value in Table 4.2 is -38% lower than the value of .05 mm obtained with the larger integration window. Even if this is taken into account, the EPR detectable high-spin heme is still much less for several species than the corresponding optically determined concentration. These results suggest that for species (D(Cu,0'2)) our sample contains ~20% non-bridged or non-coupled metal centers which are still in close enough proximity to enable fast relaxation of the copper copper center._The other ~80% is presumed to be exchange coupled to yield an even'spin species which is undetectable under convent susceptibilit discussed bro non-coupled h (opposite sid‘ chelating grox would not nece and would onl) Although no at are confident bridged specie bridged C°mpou and additional Based on t1 Raman, and EPR meso-diphenyl 5 Pr°Perties of t The model that be the °Xygen b Six‘°°°rdinate magnetic Coupl i‘ —__—'-__V” 123 under conventional EPR conditions. This analysis is consistent with the susceptibility results and also correlates with the previously discussed broadness in the optical absorption spectra. This ~20% non-coupled heme may be accounted for by backside OH" ligated heme (opposite side to the chelating group) or by heme in which the Cu or chelating group are absent. Small quantities of these different species would not necessarily be distinguished in the optical or Raman spectra and would only contribute to the observed broadness or inhomogeneity. Although no other species have been this carefully characterized, we are confident that coupling also occurs for the six-coordinate oxygen bridged species. A similar coupling may occur in the six-coordinate Cl' bridged compound, but the evidence for this species is not as strong and additional studies are required. — Based on the combined results of optical absorption, resonance Raman, and EPR spectroscopies, we conclude that some of these meso—diphenyl substituted hemes mimic the geometry and spectroscopic properties of the oxygen reduction site in resting cytochrome oxidase. The model that most accurately represents the structure in oxidase may be the oxygen bridged six-coordinate species E(Cu,0'2) in that it is six-coordinate high-spin, like oxidase, and it displays apparent 3+ magnetic coupling between Fe and Cu2+ through a bridging ligand. CHAT A. iNTRODUC'. Heme prote processes. Hen their intrinsi for a wide var probably the m Adar, 1978, an and storage, 1;. CytOChrome oxiw the thermodynar reduction of 0) P450, members to metaboliZe w (Griffin et al. biochemiCal oxi their diSPl‘Opor Although’ mOSt j center) CytOChr. been identified Tsubaki et al. variatio“, in tr CHAPTER 5 CHARACTERIZATION OF "OXY" AND "OX0" HEMES AS MODELS OF HEME PROTEIN REACTION INTERMEDIATES A. INTRODUCTION Heme proteins perform prominent roles in many biochemical processes. Hemes are especially important in aerobic organisms, where their intrinsic affinity and reactivity toward oxygen has been utilized for a wide variety of specialized functions. Hemoglobin and myoglobin, probably the most extensively studied of all the heme proteins (see Adar, 1978, and references within), are utilized for oxygen transport and storage, respectively, in mammals and some other higher organisms. Cytochrome oxidase terminates the electron transport chain and provides the thermodynamic drive for aerobic respiration through the catalytic reduction of oxygen to water (Wikstrom et al., l981). Cytochromes P-450, members of the group of heme containing oxygenases, utilize 02 to metabolize various compounds through specific oxygen insertion (Griffin et al., 1979). Peroxidases use peroxides as specific biochemical oxidants, whereas catalases remove peroxides by catalizing their disproportionation to H20 and 02 (Hewson and Hager, l979). Although, most heme proteins utilize protoheme as the prosthetic center, cytochrome oxidase uses heme g and additional heme types have been identified in various other proteins (see Adar, 1978, pg. l80; Tsubaki et al., 1980). The specific biochemical purposes of heme Variation, in these various enzyme systems, is not well understood. 124 The centr mechanisms by the intermedi structural far Although the .‘ have been obte Love, 1974; T2 studies have g stable interme conditions or less direct te expected to be may not be pos enzyme samples extremely Valu “Ch 35 solven 1975), axial 1; restrictiOns (1 it a1., 1981; 1. Wing (MiSpe] with model Stud used to interpr complicated pro TWO Species ll ferrous OXy" a' pH . (e ‘02) is ex, 125 The central goals of heme protein research are to elucidate the mechanisms by which these reactions occur, as well as the structures of the intermediate species, and to determine the heme and protein structural factors which determine the specificity of a given reaction. Although the structures of some of the above mentioned heme proteins have been obtained with X-ray crystallographic techniques (Padlan and Love, 1974; Takano, 1977; Fermi, I975; Poulos et al., 1980), these studies have generally been limited to resting enzyme states or highly stable intermediate species. Structures of these systems under turnover conditions or in less stable states have so far been probed only by less direct techniques. Since a variety of environmental factors are expected to be involved in the specific chemistry of a given enzyme, it may not be possible to separate these effects in experiments with enzyme samples. For this reason, model compound studies have been extremely valuable in elucidating heme reaction mechanisms. Factors such as solvent polarity (Brinigar et al., 1974; Chang and Traylor, 1975), axial ligation (Kerr et al., 1983; Collman et al., l983), steric restrictions (Yu et al., 1983), heme peripheral substitution (Traylor et al., 1981; Kerr et al., 1983; Kean et al., 1987), and hydrogen bonding (Mispelter et al., 1983), can often be varied independently with model studies. The results of these various studies may then be used to interpret results obtained from experiments with the more complicated protein species. TWO species of great interest in heme chemistry research are the "ferrous oxy" and "ferryl oxo“ species. The ferrous oxy species (Fell-02) is exemplified by hemoglobin and myoglobin, which bind 02 reversibly wk intermediate Chance, 1986, (Babcock et a been identifi (Champion at . ferrous perox: III), which me 1983). Althoug oxy species of reactive. Unde crucial to the Ferryl oxo Cycle of cytoc the °XYgen don. intermediates , Jones, 1984). 1 hemes can also Irvine, 1952; I Peisach, 1981). enzymes and the expécted that h and chemical re 3P8cies, Compar Protein SPecies 126 reversibly when the heme is in a ferrous (Fe+2) state. The initial intermediate in the reaction of cytochrome oxidase (see Naqui and Chance, 1986, and references therein) is a ferrous oxy compound (Babcock et al., 1984, 1985) and a ferrous oxy species has recently been identified as a reaction intermediate of cytochrome P-450 (Champion at al., 1986). Dioxygen binding can also be easily induced in ferrous peroxidase samples to produce a ferrous oxy species (compound III), which may be involved in plant growth regulation (Smith et al., 1983). Although the ferrous oxy complexes of the globins are stable, oxy species of other proteins and most free solution hemes are very reactive. Understanding the factors which control this reactivity is crucial to the understanding of heme chemistry. Ferryl oxo species (FeIV=O) have been postulated in the catalytic cycle of cytochrome g oxidase (Wikstrom, 1981; Blair et al., 1985), as the oxygen donating species in cytochrome P-450 (Groves, 1985), and as intermediates in the reactions of catalases and peroxidases (Frew and Jones, 1984). Addition of peroxide to the normally unreactive globin hemes can also induce the formation of ferryl species (George and Irvine, 1952; Dalzial and O’Brien, 1954; Aviram et al., 1978; Uyeda and Peisach, 1981). Given the diverse chemistry catalyzed by these various enzymes and the lack of reactivity of the globin species, it is expected that heme pocket modulation strongly influences the structure and chemical reactivity of these oxy (Fell-02) and oxo (FeIV=O) species. Comparison of the properties of these species for each given protein species and between different protein systems should help characterize their diverse ' Resonance study of ferr< recently been several ferryl species (Terne 1986a,b; Sitte and five - and al., 1984; Pro: a1" 1987)y th‘ 5'6). This is ; PerturbatiOns j also vary in pr and referenCes smaller range ( in the Optical Species (see Ta SPECtra are sen these Changes 5' this paper, We 7 SiX'COOrdinate ; contain l‘methyj Protoheme and he octaethylheme F 127 characterize these different heme pockets and provide insights into their diverse reactivities. Resonance Raman spectroscopy has been applied extensively to the study of ferrous oxy species (both protein and model compounds) and has recently been used for the identification and characterization of several ferryl oxo species. In a comparison of ferryl peroxidase species (Terner et al., 1985; Hashimoto et al., 1984; Hashimoto et al., l986a,b; Sitter et al., 1986), ferryl myoglobin (Sitter et al., 1985a), and five- and six-coordinate ferryl oxo heme model compounds (Bajdor et al., 1984; Proniewicz et al., 1986; Schappacher et al., 1986; Kean et al., 1987), the frequency of u(FeIV=O) varies by ~85 cm’l (see Table 5.6). This is indicative of a high sensitivity of this bond to perturbations in the local environment. Observed values of u(FeII-02) also vary in protein species and heme model compounds (see Spiro, 1983 and references within; Van Wart and Zimmer, 1985) but over a much smaller range (~10 cm'l). In addition, there are distinct differences in the optical spectra of various ferrous oxy and ferryl oxo protein species (see Table 5.5 and cited references). Optical absorption spectra are sensitive to subtle changes in the heme environment and these changes should also correlate with the observed Raman results. In this paper, we present optical absorption and resonance Raman data for six-coordinate ferrous oxy and ferryl oxo heme model compounds which contain 1-methy1imidazole in the trans axial position. Octaethylheme, protoheme and heme a were used for the ferrous oxy models, and octaethylheme, protoheme, and tetraphenylheme were used for the ferryl oxo models. . reactivity 01 B. MATERIAI Methylene to use. Tolue 1972). Toluen molecular sie‘ purification. anhydrous cop; over calcium r Chemical Inc.) were stored in PUrification. Without furche pr°t°13°rphyrin (Fel‘PP), and i] by W. Anthony ( from bovine has at 31-. 1976), complex“, (NMI Chang (1974) an proceflures are . M) were prepar: .lOO-fold eXCesg line glaSSware’ pressure of argc 128 oxo models. These results provide insight into the structure and reactivity of the heme proteins mentioned above. B. MATERIALS AND METHODS Methylene chloride was dried by reflux over calcium hydride prior to use. Toluene was distilled from benzophenone ketyl (Gordon and Ford 1972). Toluene-d8 (Cambridge Isotope Laboratories) was dried over molecular sieves (4 and 5 angstrom) and used without further purification. Dimethylformamide (DMF) was vacuum distilled over anhydrous copper sulfate. l-methylimidazole (NMI) was vacuum distilled over calcium hydride. Sodium dithionite (a generous gift from Virginia Chemical Inc.) and tetrabutylammonium borohydride (TBAB, from Aldrich) were stored in vacuum dessicators and used without further purification. Iron protoporphyrin IX (Sigma, bovine hemin) was used without further purification for preparation of the oxy models. Iron protoporphyrin IX dimethylester (FePPIXDME), iron tetraphenyporphyrin (FeTPP), and iron octaethylporphyrin (FeOEP) were generously provided by W. Anthony Oertling (Michigan State University). Heme g was isolated from bovine heart cytochrome g oxidase as previously described (Babcock et al., 1976). The procedures for the preparation of the oxy heme complexes, (NMI)FeII-02, were based on those reported in Brinigar and Chang (1974) and Babcock and Chang (1979). The details of the procedures are as follows. Solutions of the various hemes ('50 to 250 pM) were prepared in methylene chloride or DMF which contained an ~lOO-fold excess of NMI. These were then purged of oxygen, in Schlenk line glassware, by using 5 to 10 freeze-pump-thaw cycles. A positive pressure of argon gas was maintained over the sample during the thaw stages. Fen] addition of a reduced by ti reductions we Oxygen bindin samples (-45 monitored opt displacement < illumination ( hemes, (NMI)Fe et a1. (1983) carried out in box. Although for FePPIXDME Protoheme Was I I”“1310 label; achieved by the respective gym obtained with a temperature Opt built °Ptica1 D were “Rained temperature by j with a Spex 140: detector) by usj model 164 Kr+ it in EPR tubes, We 129 stages. FeIIIOEP in methylene chloride was reduced to FeIIOEP by the addition of a slight excess of solid TBAB. The hemes in DMF were reduced by titration with a degassed aqueous dithionite solution. These reductions were monitored by using optical absorption spectroscopy. Oxygen binding was achieved by the addition of oxygen to the cooled samples (—45 to -70 C) with a gas tight syringe. This binding was monitored optically (see below) and shown to be reversible by displacement of the 02 with CO followed by degassing under strong illumination (Brinigar and Chang, 1974). Preparation of the ferryl oxo hemes, (NMI)FeIV=O, was performed according to the procedure of La Mar et al. (1983) with the exception that the anaerobic steps were all carried out in Schlenk line glassware instead of an anaerobic glove box. Although it was not previously reported, the procedure worked well for FePPIXDME in addition to FeOEP and FeTPP. Use of the non-esterified protoheme was prohibited by its negligible solubility in toluene. Isotopic labeling of both the ferrous oxy and ferryl oxo compounds was achieved by the use of 1802 (98% Cambridge Isotope Laboratories) in the respective synthetic procedures. All optical absorption spectra were obtained with a Perkin-Elmer Lambda 5 UV/Visible spectrophotometer. Low temperature optical absorption spectra were obtained by using a house built optical Dewar mounted in the Lambda 5 (Kean, 1987). The samples Were contained in EPR tubes which were cooled to the desired temperature by flowing cold nitrOgen gas. Raman spectra were obtained with a Spex lhOl scanning monochromator (with an RCA 31034C PMT detector) by using 15 mW incident power at 406.7 nm (Spectra-Physics model 16A Kr+ ion) in a backscattering geometry. The samples, contained in EPR tubes, were spun continuously in a dewar while the desired temperature sloping back in the figurr C. RESULTS The optic OEP are show reported for 1974). The ab samples (not (Brinigar and The peak posi1 reported resul from the absor °XY Species f1 Shift of the fi band blue shif bis-NMI Specie obserVed for a Warn of the W are Shown 3 shifts to 547 ( Peak is assigne shift of 26 Cm‘ simple harmOnic .l _ cm in the res 5.2d, respectiv —f——————77 ' 130 temperature was maintained by flowing cold nitrogen gas. A linear sloping background was subtracted from some Raman spectra, as indicated in the figure legends, but no smoothing was done. C. RESULTS The optical absorption spectra of ferrous bis-NMI and ferrous oxy OEP are shown in Figure 5.1. As expected, this is similar to that reported for an iron mesoporphyrin derivative (Brinigar and Chang, 1974). The absorption spectra of the ferrous oxy protoheme and heme a samples (not shown) are in agreement with previously reported spectra (Brinigar and Chang, 1974, and Babcock and Chang, 1979, respectively). The peak positions are listed in Table 5.1 along with some previously reported results for ferrous oxy species of other porphyrins. As seen from the absorption spectra in Figure 5.1, the formation of the ferrous oxy species from the ferrous bis-NMI species is accompanied by a red shift of the fl and a hands and a change in their intensities. The Soret band blue shifts to a value close to that of the corresponding ferric bis-NMI species (see Table 5.1). This same characteristic behavior is observed for all of the porphyrins used in these experiments. Raman spectra of the ferrous oxy complexes of FeOEP, FePPIX, and heme a in DMF are shown in Figure 5.2. For ferrous oxy FeOEP the peak at 573 cm'1 shifts to 547 cm'1 upon substitution of 160 by 18O; consequently this peak is assigned to the Fell-02 stretching vibration. The frequency shift of 26 cm'1 is in agreement with the value predicted by using a simple harmonic oscillator model. Peaks observed at 573 cm'1 and ~576 Cm‘l in the respective FePPIX and heme a samples (Figures 5.2c and 5.2d, respectively) also demonstrate a 26 cm‘1 downshift upon 180 m020 FeIVPPIXDMEmM FelvTPPmmno Fe TmTP545 CH2c12 RT this work FeIIOEP(NMI)02 404 530=563 CH2C12 —so this work FeIIOEP(NMI)02 ~404 531=564 DMF -45 this work FeIIPPIX(NMI)02 415 540=574 DMF —45 this work and Brinigar & Chang (1974) FeIIHeme a(NMI)02 426 579<595 DMF —45 this work and Babcock & Chang (1979) FeIITPP(PYR)02 - 547>583 cuzc12 -80 Anderson et al. (1973) FeIITPivPP(NMI)02 - 548>>580 BZ RT Collman et al. (1973) FeIITPivPP(THF)02 419 538 THF -70 Schappacher et al. (1985) FeIVOEP(NMI)O ' 406 535,546=573 TOL -90 this work and Kean et al. (1987) FeIVPPIXDME(NMI)O 416 543,555=584 TOL -90 this work and Kean et al. (1987) FeIVTPP(NMI)O 427 SSS,563>597 TOL -90 this work FeIVTmTP(NMI)O 420 ~544,‘556>59o TOL -9o Chin et al. (1980) FeIVTPivPP(THF)O 419 550 THF -70 Schappacher et . al. (1985) FeIVTPiVPP(NMI)O 426 sso>>59o THF -70 Schappacher et al. (1985) FeIV(ETIO)(NMI)O — “535,550<572 TOL -80 La Mar et al. (1983) Abbreviations: OEP, octaethylporphyrin; PPIX, protoporphyrin IX; TPP, tetraphenylporphyrin; TPivPP, "picket fence" porphyrin (Tetra- (O-pivaloylphenyl) porphyrin); TmTP, tetramesitylporphyrin; ETIO, etioporphyrin; NMI, l-methyl imidazole; PYR, pyridine; THF, tetrahydrofuran; DMF, dimethylformamide; TOL, toluene; BZ, benzene <,>, or = sign reflects relative intensities of the absorption bands. OI, CH, h, p. 04-01 u,c-n,C z \ C 042-0 lat-+1, 01; 94, vs 0") OCIAETHYLHEME If 0) 1 tn, 1 CH CH) "It CH .911 ”'c , CH, f"! 2", EH’ f": “’0“ coon 'O'ONemg °\ ’0-01 0‘3 NJ: .04! 0:: H N c ’ 5“? EN; F") f”! (OOH “OH hem. ! Figur. 133 N #- - 120°C cu, CH3 xex = 406.7nm (in, é“: ch‘ch CHECH, chdfic th:—CH, 9= 9* CH) CH! OCTAETHYLHEME l6 0) 02 l8 — b) 02 cm, a m, H3c cn-cn, H,c CH, $“2 9» cu, c‘Hz l COOH COOH cu, HJC -cHz o=c (H H s 54H: 9*: CH, H 4oooob '7obooo RAMAN SHIFT (tm") Figure 5.2 Identification of V(FeII-02) for ferrous oxy octaethylheme, protoheme, and heme a. substitution experiment w decompositio low temperat1 spectra. It a region which and heme g 55 fluorescent p of these latt absorption ba excitation li done in methy and we have if frequency reg 0f the corresI frequency regj AlthoUgh the 5 the Peak posit biS'NMI Specie with those of The optica and TPP Specie; Shift in the $1 the increasing SUbStituehts_ ,1 With thOSe of c 134 substitution (not shown) and are likewise assigned to u(FeII—02). This experiment was complicated by the formation of fluorescent heme decomposition products in DMF. The fluorescence is more pronounced at low temperature and obscured the high frequency region of the Raman spectra. It also produced a sloping background in the low frequency region which is subtracted from the data in Figure 5.2. The protoheme and heme a samples were more susceptible to the formation of this fluorescent product. In addition, there is less resonance enhancement of these latter two species owing to the wavelenghts of their Soret absorption bands, which are considerably to the red of the 406.7 nm excitation line. Because of these problems, the remaining oxy work was done in methylene chloride, in which the heme decomposition is minimal, and we have focused on the FeOEP complex. A portion of the low frequency region is displayed in Figure 5.3 along with the same region of the corresponding FeIII bis-NMI species. Figure 5.4 shows the high frequency regions in the -127 C Raman spectra of these two samples. Although the spectrum for the ferrous oxy species is overall weaker, the peak positions are nearly identical with those of the ferric bis-NMI species. The peak positions are summarized in Table 5.2 along with those of a variety of other FeOEP species. The optical absorption spectra of (NMI)FeIV-O for the OEP, PPIXDME, and TPP species are displayed in Figure 5.5. There is a progressive red shift in the spectra in going from OEP to PPIXDME to TPP which reflects the increasing electron withdrawing ability of the respective ring substituents. The peak positions are also tabulated in Table 5.1 along with those of other ferryl oxo model species which have been previously >tmzuez_ z<§ Table 5.5 Ferrous Ox normal: FeHPPIX(N MbOz Hb02 oxy HRP (M oxy IN 2,3 LiP cmpIII red-shifter BPO cmpIII LPO cmpII] IPO cmpII] CPO cmpIIl cyt‘s P-45C cyt P-450 cyt P-450 Ferryl Oxo normal: FEIVPPIXDME Mb1V=0 HbIV=o Leg Hb Ivao HPP II L1? 11 CCP II red-shifted, BPO II LPO 11 IPO II CPO II BMC II Hb, human he BP07 bromope CPO, chIOrop Cyt P'450 In C, bacteri ’ 0r 7 r. 149 Table 5.5: Optical Absorption Peaks of Protoheme Containing Ferrous Oxy and Ferryl Oxo Species. Ferrous Oxy series: normal: FeIIPPIX(NMI)02 415 540=574 This work and Brinigar and Chang (1974) Mb02 418 542<58O Makinen and Churg (1983) HbO 415 541<577 Antonini and Brunori (1971) oxy HRP (MPIII) 418 541>574 Wittenberg et a1. (1967) oxy IN 2,3,D 415 541<576 Sono (1986) LiP cmpIII 419 543>578 Renganathan and Gold (1986) red-shifted: BPO cmpIII 424 552>588 Manthey and Hagar (1985) LPO cmpIII 428 551=590 Kimara and Yamazaki (1979) IPO cmpIII 430 553>59O Kimara and Yamazaki (1979) CPO cmpIII 432 555>586 Nakajima et a1. (1985) cyt's P—450: cyt P-450 cam 418 555 Peterson et a1. (1972) cyt P-450 1m 418 555 Oprian et a1. (1983) Ferryl Oxo Series: normal: ' FeIVPPIXDME(NMI)O 416 542,555-584 This work MbIV=o 420 550‘580 George and Irvine (1952) HbIV=0 . 418 545>575 Dalzial and O'Brian (1954) Leg HbIV—o 418 545>575 Aviram et al. (1978) HPP II 418 527-553 Blumberg et a1. (1968) LiP II 420 525<556 Renganathan and Gold (1986) CCP II 419 529<56l Yonetani (1965) red-shifted: BPO II 433 534>564 Manthey and Hager (1985) LPO II 433 537>568 Kimura and Yamazaki (1979) IPO II 436 538>565 Kimura and Yamazaki (1979) CPO II 438 542>571 Nakajima et a1. (1975) BMC II 428 530<568 Theorell and Ehrenberg (1952) abbreviations: IN 2,3,D, indoleamine 2,3-dioxygenase; Mb, sperm whale myoglobin; Hb, human hemoglobin; Lip, lignin peroxide; LPO, lacto peroxidase; BPO, bromopenoxidase; IPO, intestine (hog) peroxidase; CPO, chloroperoxidase; Cyt P-450 cam, cytochrome P-450 camphor; Cyt P-450 1m, cytochrome P-450 liver microsomal BMC, bacterial catalase; 11, compound II. <, >, or - reflect relative intensities of the absorption bands. .577 nm. l their simi general re region fro and they a group is t hemes but ( compare the oxo proteir spectra sim and to the more red sh the globins 5-5) have Sc bands are b1 "red'Shifted ralative to (Soret red 8] 0bsemacions differences 1 manifestecl ir spectra of th differenCe fr (redox State Perphyfiny am globins and t1 the "red Shift 150 ~577 nm. We shall designate these as “normal" heme proteins because of their similarity to the simple model. The next group represents a general red shift relative to the normal hemes, with Soret peaks in the region from 424 to 432 nm, fl-bands at ~553 nm and a-bands at ~588 nm and they are therefore designated as "red shifted" hemes. The final group is the cytochromes P-450 which have a Soret band like the normal hemes but exhibit only a single band in the fi/a region at 555 nm. If we compare these observations with the data for the corresponding ferryl oxo protein species, we see that only the ferryl globin species have spectra similar to the ferryl oxo PPIXDME model (also in this table) and to the ferrous oxy compounds. The ferryl oxo model, however, has more red shifted 5- and a-bands and a blue-shifted Soret relative to the globins. The ferryl oxo species of the "normal" peroxidases (Table 5.5) have Soret bands similar to the ferryl model but the fi- and a- bands are blue shifted relative to the model. The ferryl species of the "red-shifted" peroxidases still exhibit a red shifted optical spectrum relative to the "normal" peroxidases but demonstrate mixed behavior (Soret red shift, fl/a blue shift) relative to the model. The above observations suggest the following: 1) there are environmental differences between the globins and the peroxidases which are manifested in the ferryl oxo but not the ferrous oxy species, 2) the spectra of the "red shifted" peroxidases display a fairly constant difference from the normal peroxidases (~10 nm) which suggests an iron (redox state and/or axial ligand) independent perturbation to the porphyrin, and 3) the cytochromes P-450 are distinct from both the globins and the peroxidases. Bacterial catalase seems to fall in with the "red shifted" peroxidases but its behavior is also distinct, with a larger se than the of the in references perturbati macrocycle are not st as other In nature of 1 after the ( Upon co Table 5.2 a Six-coordin. [DOSE of the electron der Similar in t On the iron the Spectrum 0X0 Species aPPfiars to C, the fOur Coo; impurity Comm any Fen SPEC oxygen eVen a is that this slx‘COOrdinatl —7—_—____,,, ' 151 larger separation of the 6- and a-bands and a Soret at lower wavelength than the "red shifted" peroxidases. Due to the interaction and mixing of the iron orbitals and the porphyrin orbitals (Adar, 1978, and references within), the observed optical spectra are sensitive to small perturbations of both the iron environment and to the porphyrin macrocycle. For this reason, interpretation of changes in heme spectra are not straightforward and they do not usually follow the same trends as other metalloporphyrins. Further discussion as to the possible nature of these different heme environments will be reserved until after the discussion of the Raman results. Upon comparison of the Raman peaks for the various FeOEP species in Table 5.2 a strong homology is seen for the FeIII bis-NMI, ferrous oxy, six-coordinate ferryl oxo, and five-coordinate ferryl oxo species in most of the characteristic marker bands. This implies that the net electron density on the metal, as experienced by the porphyrin, is very similar in these four species even though the formal oxidation states on the iron vary in the range of ~+2 to ~+4. It is also evident in both the spectrum and listed peak positions that the six-coordinate ferryl oxo species is not homogeneous. This is clear in the V3 region which appears to contain contributions from both p-oxo (or peroxo) dimer and the four coordinate FeII species. The p-oxo dimer contamination is an impurity common to the synthetic procedure but it is unexpected that any FeII species should remain, since it reacts very quickly with oxygen even at these low temperatures. The most reasonable explanation is that this is the immediate product of the photoreduction of the six—coordinate ferryl species. Photoreduction has been proposed as the mechanism (Stillman protoheme strong re. and 174 see l-"eIII to 1 species ir negligible the Raman demonstrat oxy and fe: occurrence Place of t} Perturbatic light of th the differe p°rPhyrin c. The frec Table 5.6 fc Protein samp Show only mi trend in ”(R mloglobin sat compmmds’ We sensitivity t I ”(Fe V‘O) for —7—fih’77 ' 152 mechanism for light induced decay of heme protein ferryl species (Stillman et al., 1975). Similar results are demonstrated for the protoheme species (Table 5.3). The ferryl oxo model species bears a strong resemblance to the ferryl oxo protein species. Although V2, V3, and V4 seem to exhibit a general increase in frequency in the series FeIII to ferrous oxy to ferryl oxo, differences between individual species in these latter two groupings are fairly subtle and may be negligible within the variability of sample preparations. Comparison of the Raman data of various FeTPP type model compound (Table 5.4) again demonstrates the spectral similarities of the ferric bis-NMI, ferrous oxy and ferryl oxo species, with the interesting addition that even the occurrence of a trans thiolate ligand (cytochrome P-450 model), in place of the NMI (Chottard et al., 1984), produces only small perturbations in the porphyrin modes. This trend is interesting in light of the large variations of the Fe-ligand (axial) vibrations in the different systems and it indicates only a small perturbation in the porphyrin core size. The frequencies of these iron—ligand vibrations are summarized in Table 5.6 for various ferrous oxy and ferryl oxo model compound and protein samples. The frequencies for V>E mm L9€l E: Seen} 173 Table 6.1: Distinguishing Characteristics and Raman Peaks of Frozen Cytochrome Oxidase Species. Distinguishing Characteristics Resting Photo- Pulsed Cmpd 580 nm 580 nm Re- reduced C +CO oxidized Optical 424 424 428 428 (nm) 655 606 607 537,580 EPR g=5 rhombic a3 CUB y site LS FeII Soret Excitation Raman Peaks (cm'l) (frozen samples) Assignment Resting Photo- Pulsed Cmpd 580 nm 580 nm Re- reduced C +CO oxidized u(C=O) a3 1675 1672 1677 1675 1676 1676 1676 1661 le(LS) + 1647 1640 1648 1650 1647 1650 1648 v(C=O..H 1645 1644 1647 u(C=C)aI 1622 ? 1625 1628 1628 ulO(HS) a3 1612 1610 1616 1613 1613 1613 u(C=O)aII(LS) 1610 ”1083II u2(LS) 1589 1585 1595 1594 1595 1594 1594 u2(HS) a3 1575 1572 1573 1577 ?1581 1570 ? 1562 1551 1565 1562 1561 V11 ?1511 1523 ?1520 ?1519 ?1517 u3(LS) 1504 1504 1506 1505 1505 1508 u3(HS) a3 1482 1481 1488 1484 1482 V28 1472 1471 1472 1478 1479 1473 1474 ? 1439 1440 1437 1436 1440 1442 1438 ? 1395 1394 1397 1395 1395 1400 1399 V4 1372 1370 1375 1374 1377 1375 1374 1360 1364 ? 1335 1327 1338 1338 1337 1335 1335 ? 1303 1302 1310 1309 1309 1311 1308 7 1289 1284 1284 1291 1291 1291 1292 7 1246 1250 1249 1253 1253 1252 ? 1225 1228 1225 1225 1230 1228 Assignments are based on those of Choi et a1. (1986) according to the scheme of Abe et a1. (1978). a = cytochrome a heme, a3 = cytochrome a3 heme Note: Rows without labels are a continuation o (1983) and Babcock indicate 2 peaks with the same assignment (i.e., inhomogeneous samples). f the one above and a3 and a or 174 Table 6.2: Raman Peaks of Heme a Model Compounds.a ! -------- FeII -------- z 1 ------------- FeIII -------------- 1 . 6 Coord 5 Coord 6 Coord 6 Coord 5 Coord' ASN Low Spin High Spin Low Spin High Spin High Spin u(C=O) 1642 1660 1670 1672 1576 v(C=O H) 1633 1640 1656 - 1656 u(C=C) 1622 - - - - v10 - 1607 1642 1615 1632 u37 ~1605 1565? - - - u2 1587 1578 1590 1572 1581 v19 1583 - - - - 1138(1) 1563 - - - 1540 V38(2) - - - - 1.520 V11 1.511. - - " ‘ U3 1493 1473 1506 1482 1492 V28 1468 1455 1474 - - V29/20 1.391 1394 ' ' ‘ V4 1360 1357 1374 1373 1374 6(-CH2) 1329 1333 - - - v21 1307 1314 1312 - - aFrom Babcock (1986) 175 In Figure 6.2, the high frequency Raman spectra of non-photoreduced resting oxidase at O C (liquid) (6.2a) and -120 C (frozen) (6.2b) are displayed. The liquid spectrum is consistent with previously reported studies (Babcock et al., 1981; Woodruff et al., 1982; and Copeland et al. 1985). Peaks from both heme centers are observed but several of the peaks from the cytochrome a3 center (high-spin) are prominent. This is due to better resonance of the 406 7 nm exiting line with the the absorption band of the high-spin cytochrome a3 site than that of the low-spin heme in the cytochrome a site. The frozen sample retains many of these features but is overall poorer in quality owing to the higher background fluorescense at the lower temperature and the weaker Raman scattering from frozen aqueous solutions (relative to the analogous liquid samples). There are also some changes evident in the frozen sample. The band centered at 1648 cm'l, which is a composite of several modes (Babcock 1986), is more symmetric and narrower than in the liquid sample, and the 1675 cm'1 peak, assigned to VCO of the heme a ring formyl group of cytochrome a3, is increased in intensity. The increase of intensity at 1589 cm'1 indicates increased scattering from a low—spin species. There is also a small increase of intensity at 1610, 1628, and ‘1511 cm'l. The nature of these changes will be discussed below. Despite the increased experimental difficulty, most of the details observed in the liquid spectrum are also resolved in the spectrum of the frozen sample and the peak positions are the same in the two spectra (except as noted above). For the frozen sample (2b), the peak positions and assignments are listed in Table 6.1. In resting displa} studies al. 195 peaks f due to absorpt low-spi of thes backgro scatter liquid sample, modes (1 sample, formyl g of inter low-spin 1628, an below, D details SPECtmm the tWo the peak 175 In Figure 6.2, the high frequency Raman spectra of non-photoreduced resting oxidase at 0 C (liquid) (6.2a) and —120 C (frozen) (6.2b) are displayed. The liquid spectrum is consistent with previously reported studies (Babcock et al., 1981; Woodruff et al., 1982; and Copeland et al. 1985). Peaks from both heme centers are observed but several of the peaks from the cytochrome a3 center (high-spin) are prominent. This is due to better resonance of the 406.7 nm exiting line with the the absorption band of the high-spin cytochrome a3 site than that of the low-spin heme in the cytochrome a site. The frozen sample retains many of these features but is overall poorer in quality owing to the higher background fluorescense at the lower temperature and the weaker Raman scattering from frozen aqueous solutions (relative to the analogous liquid samples). There are also some changes evident in the frozen sample. The band centered at 1648 cm'l, which is a composite of several modes (Babcock 1986), is more symmetric and narrower than in the liquid sample, and the 1675 cm'1 peak, assigned to vCO of the heme a ring formyl group of cytochrome a3, is increased in intensity. The increase of intensity at 1589 cm'1 indicates increased scattering from a low-spin species. There is also a small increase of intensity at 1610, 1628, and ~1511 cm'l. The nature of these changes will be discussed below. Despite the increased experimental difficulty, most of the details observed in the liquid spectrum are also resolved in the spectrum of the frozen sample and the peak positions are the same in the two spectra (except as noted above). For the frozen sample (2b), the peak positions and assignments are listed in Table 6.1. 1372 Aex=406.7 nm 3 mW 4 Sec/pt Liquid 0°C {5% , Frozen ‘ N -120°C b) g 1200 RAMAN SHIFT (cm-1) 1700 Figure 6.2 High frequency Raman spectra of resting cytochrome oxidase (liquid and frozen). 177 In Figure 6.3, the high frequency Raman data are shown for the species with 580 and 537 nm peaks in the absorption spectrum (580 nm species). Spectra 3a and 3b were obtained with liquid samples and spectrum 3c with a frozen sample. Spectrum 3a was obtained with a previously unirradiated sample. Spectrum 3b was obtained on the same sample after '1 1/2 hours of laser irradiation. The initial scan (Figure 6.3a) shows a clear dominance of contributions by low-spin species. This is exemplified by the increased intensity at 1643 and 1589 cm‘1 and the decreased intensity at -1572 cm'1 which suggests a low spin heme in the cytochrome a3 site. The 1505 cm'1 peak is more intense than in the resting enzyme and V4 (~1374 cm'l) is 2 cm'1 higher in frequency than in the resting enzyme. The laser irradiation greatly accelerates the decay of these samples which are ordinarily stable for several hours at O C. In addition, there is no evidence of photoreduction. By comparison with spectrum 3b, it is apparent that this sample decays with time into what appears to be the resting enzyme. In spectrum 3c (the frozen sample) there appears to be a complete conversion of the cytochrome a3 heme to low spin and the V4 frequency is 4 cm'1 higher than in the resting enzyme. Again, there is no evidence for photoreduction. In contrast to the liquid sample (3a), the frozen sample can be irradiated for a full day with no detectable change in the Raman spectrum. This indicates a dramatic increase in stability of this sample at the low temperature (-120 C). This observation, along with the more homogeneous appearance of the frozen sample spectrum, suggests that the spectrum observed for the liquid sample (3a) already displays substantial decay of the 580 species. 178 580 nm Species Aex=406.7 nm 1374 3 mW 4 Sec/pt Liquid 0°C Fresh 1.5 hr. in Laser Frozen c) 4 g g. -120 c ‘I'IfI‘l‘l‘l‘l'III‘l - o 1200 RAMAN SHIFT (cm 1) ‘7 0 Figure 6.3 High frequency Raman spectra of 580 nm species of cytochrome oxidase (liquid and frozen). 179 Since the 580 nm species accounts for only about 60 % of the frozen sample, the percentage in the liquid sample must be significantly less. Our Raman spectra of liquid samples of Compound C (not shown) displayed a strong resemblance to the spectrum of resting oxidase, but optical spectra taken after the scans showed significant sample degradation to a pulsed or resting species. This suggested fairly rapid decay of the liquid compound C samples in the laser beam, such as that which was observed for the 580 nm species. The compound C samples were therefore scanned at low temperature (frozen) to determine if they were more stable under these conditions. These spectra are displayed in Figure 6.4. Figure 6.4a shows the spectrum of compound C produced by the addition of 02 to the mixed valence CO bound (MVCO) oxidase. The spectrum of compound C, produced by the addition of a stoichiometric amount of peroxide to the pulsed enzyme, is displayed in Figure 6.4b. Both of these samples should be at the level of peroxide bound fully oxidized enzyme. For comparison, the spectrum of pulsed enzyme is shown in Figure 6.4c. The Raman scattering from the pulsed and compound C species was extremely weak overall. In addition, the peaks characteristic of high-spin heme species were reduced in intensity or virtually absent but there were only small increases in the intensity of the peaks which are characteristic of low-spin heme species. Compound C samples produced by two different methods produced similar but not identical results. The pulsed plus peroxide sample (Figure 6.4b) seems to be more homogeneously low-spin (low intensity at 1577 cm'l) and it displays broader features at ~1510 and 1675 cm’1 than the MVCO plus 02 sample (4a). Both of these samples had Raman spectra 180 x0x=4oa7 nm Frozen ~120°C 3 mW 1544 25 Sec/pt Compound C (MV + 00) 3) Compound C (Pulsed + peroxide) 6) c) Pulsed I I ' 1 ' 1 ' I I ! r—fi . x 1 r . , 1200 1700 RAMAN SHIFT (cm-1) Figure 6.4 High frequency Raman spectra of frozen compound C cytochrome oxidase (MVCO + 02 and pulsed plus peroxide) and pulsed cytochrome oxidase. 181 similar to that of the pulsed enzyme. However, the spectrum of the pulsed enzyme was noticeably weaker overall and it is distinguished by a broader band at ~1645 cm-1 and the absence of the 1628 cm'1 peak. All three of these spectra exhibit V4 at ~1374 cm‘l. Photoreduction does not appear to be appreciable in any of these three spectra. Raman spectra of two other species of interest were obtained in the frozen state and these results are shown in Figures 6.5 and 6.6. The first of these is produced by chemical oxidation (PPD) of the enzyme during turnover conditions and it is distinguished by the detectability of CuB in the EPR spectrum (Witt et al., 1986). The Raman spectrum of this sample (Figure 6.5) is dominated by contributions from low‘spin species (increased intensity at 1508, 1594, and 1648 cm‘l) indicating that the cytochrome a3 heme is probably low-spin in this sample. Overall it is a more strongly Raman scattering sample than the pulsed and compound C samples but not as strong as the 580 nm sample. The final sample is produced by the reaction of the 580 nm species with CO, which produces 002 and a ligated (02 or CO) ferrous heme species (Blair et a1. 1985, Witt et al. 1986). This Raman spectrum (Figure 6.6) is almost indistinguishable from the spectrum of the starting material (the 580 nm species). However, in Figure 6.6, the spectrum displays increased intensity at '1473 and "1484 curl, and V4 is at 1375 cm'1 in contrast to 1377 cm‘1 for the 580 nm species. Resonance Raman spectra were attempted for frozen samples (-120 C) of the resting, 580 nm species, compound C and pulsed samples in the low frequency region. The compound C and pulsed samples (not shown) 182 .wmwuHXO waouzocuzo :puufipflxoou= :oNoum mo aduuooam Sufism mucuskuw swam m.w ouzwwm cot ATEQV Kim z<2>E m r 979l CaCN—.1 choun. a: Sin} 183 .omdpfixo waoucoouho mo mofiuonm an own couomuu 00 mo azuuowam Sufism xozwpvoum 5w“: o.o oudwwm 8: 9-83 5.:m z>E N O ONT :39“. E: 33”} IIIIIIIIIIIIIT_____________________________mZ‘_——I‘7fl7~_F__u___________________'—_—'w"777 184 displayed weak scattering and little detail. Spectra for the resting and 580 nm species exhibited stronger scattering and these results are shown in Figures 6.7, 6.8 and 6.9. Since the 580 nm species has been proposed to be a ferryl oxo species (FeIV=O), we examined samples made with 16O and 18O in an attempt to identify the (FeIV=O) stretching frequency via an isotope shift. The spectra of both isotopes obtained over the low and middle frequency ranges are shown in Figures 6.7 and 6.8. In Figure 6.7, we observe that ”8 for the 580 nm species (347 cm'l) is decreased in intensity and increased in frequency relative to the resting enzyme (341 cm'l). In addition, the peak at 376 cm‘1 (resting) is esentially absent for the 580 nm species and the 412 cm‘1 peak is decreased in intensity with a slight increase of intensity at 422 cm'l. The spectrum of the 180 labeled 580 nm species (7c) is not identical to that at the 160 labeled sample (7b) but none of the differences are reproducible. Overall, the spectrum of the 160 sample most clearly displays the spectral characteristics that are reproducible and unique to the 580 nm species. The peak at ~491 cm'1 and the broad band centered at ~415 cm"1 are mostly due to scattering from the quartz EPR tube. In Figure 6.8, the spectra of 16O and 180 labeled 580 nm species are compared with each other and the spectrum of resting enzyme. Of the small differences observed between the two isotope species, the only one that is reproducible is the slightly higher intensity at 749 cm‘l. If this were the v(FeIV=l8O) frequency, the corresponding v(FeIV=l6O) frequency would be expected at ~785 cm'l (~36 cm'l higher). However the intensity at ”785 cm'l, for the 160 sample is not reproducibly higher and we must concede that we cannot yet assign a u(FeIV=O) peak. The most dramatic differences between the 185 .ommuon uaounoou%o Ao H van ow v as 0mm cam MGaUmuu cououw we uuuoo m Enema M _ 9-53 Kim 25.5. o... b.—.—.nLP.—. CON — p p . Ow. . 990 £69 Z 9 w Z ononvouu 304 n.o wuswwm r 1.3 i 4 Am 689 03 z} wmzomam E: own u a 8:8: m u mmflwm g G untomw mm >>E m ObNT :39”. EC 33”} h. 186 .ouppwxo quunoouko Aoma use emav a: cam paw mnaumou conouw mo wuuoonm cuaum mucopvouu oudeoauoucH w.w uudmfim 9-23 rim is: _ _-_- .—.—u—._._-—. 0mm 03 .3 Om: mofioam E: owm museum .303 mm 3:. e 6.0m? 586; e: 23”} . m 187 .wmmpwxo waousoouxo om.H new 0 My a: own new magnum Cououw mo wuuoo m :mEmm ususkum ouwwpoauoucfi yuan: o6 ouswwm 9-23 :__:m 3:1...08 a... Olll Slit BELL onoam E: owm mczwom 1631 6911 2511 Pitt 338 mm as m ooowwl co~ouu E: 33“} IIIIIIIIIIIIIIIIIIIII_____________________________7_________—__‘ ‘ "7"”7 ’7 “ '““"' "” 188 Spectrum of the resting enzyme (8a) and those of the 580 nm species (8b,c) is a loss of intensity at 765 and 795 cm'1 for the 580 nm species and an increase of intensity at ~749 cm'l. In Figure 6.9, we see an increase of intensity at 1138 and 1233 cm'1 for the 580 nm species (relative to the resting), as well as a decrease of intensity at ~1170 cm‘1 and a downshifting of the 1132 cm‘1 peak to 1125 cm‘l. The peaks observed in Figures 6.7 through 6.9 are summarized in Table 6.3. D, DISCUSSION The characteristic signs of photoreduction, as demonstrated in Figure 6.1, are the increase of intensity of the peaks at 1360, 1587, ”1520, 1610, and 1621 cm'1 and a loss of intensity of the -l370, and 1676 cm'1 peaks and the ~1646 cm‘1 band. The most dramatic of these changes is the intensity increase at 1610 and 1621 cm'1 and the loss of intensity at ~1646 cm'l, and the onset of these characteristics were used as a criterion for the occurrence of photoreduction. Comparison of Figure 6.1a with Figure 6.1b demonstrates extensive photoreduction in both the frozen and liquid samples at high laser power (~25 mW). This is similar to results previously reported by Bocian et al. (1979) under similar conditions. Since the effects of photoreduction on the sample integrity of these various samples is not known, the laser power was lowered on all further samples (~3 mW) to minimize or eliminate this photoreduction. The similarities of the Raman spectra of liquid (0 C) and frozen (—120 C) resting oxidase, Figures 6.2a and 6 2b, assure us that freezing the solution does not cause any major perturbation of the heme environments in resting oxidase. Except for the 1550 to 1660 cm'1 189 Table 6.3: Assignment of the Mid— and Low-Frequency Raman Peaks of Resting and 580 nm Species of Cytochrome Oxidase. Assignment Resting 580 nm Species v13(a3) 1232 1232 ? 1189 1188 V30 or V43 1174 1170 V6 + V8 1138 1138 V22 1132 1125 1232(a3) 795 792 ? 765 ”16(a3) 756 :755 U47(a3) 738 _741 ? 713 716 ? 700 ~703 v7(a3) 1683 -684 ? 650 648 ?(a3) 420 422 *6(Cb-Ca—Cfl)(a3) 412 - 20135) or 7(Cb-S) 376 - ”8(33) ~341 347 ? 296 - ? - -258 ? 226 227 * Ca, C are carbons on the vinyl substituent assignment scheme based on that of Abe and Kitagawa (1978). fi—r 7* g, {any 190 region, there are no significant changes in peak positions in the frozen sample relative to the liquid sample. The increased intensity at 1589 cm'1 for the frozen sample would best be explained by the presence of low—spin heme species in the cytochrome a3 site. Since no exogenous strong field ligands are present in solution, these low-spin species would most likely be accounted for by accumulation of low‘spin intermediate species during slow photoreductive turnover. Since this additional low-spin component is not detected by low temperature EPR or with magnetic susceptibility (Tweedle et al., 1978), it is unlikely that it represents a low temperature induced protein conformation or a thermal spin state equilibrium. , The spectrum of the 580 species exhibits strong Raman scattering and the peak positions and intensities are consistent only with a ’ low-spin cytochrome g3 center. The higher frequency of V4 and the stronger V3 at 1506 cm‘1 than low-spin ferric hemes is characteristic of a ferryl oxo species (FeIV=O) of a physiological type heme (Campbell et al., 1980; Terner & Reed, 1984; Hashimoto et al., 1986; Proniewicz et al., 1986; & Kean et al., 1987). Hydroxide ligation is capable of producing low-spin heme species (Beetlestone & George 1964, Yonetani et al. 1971); however, formation of this species would not be favorable under the experimental conditions used (pH 7.4). In addition, the position and intensity of V3 (1506 cm'l) for the Raman spectrum of the 580 nm species are not consistent with that observed for hydroxy metmyoglobin (1479 cm'l, Ozaki et al., 1976) and the EPR properties are not consistent with a FeIII-OH species as discussed by Blair et al. (1985). Simple peroxide ligation cannot be ruled out since its ligation —7——'——" ' 191 characteristics are not well known, but it seems unlikely that this would be a strong enough ligand to induce this low spin conformation. The only other reasonable possibility, considering the lack of exogenous ligands in the system, is that the species is a ferrous oxy compound (Fell-02). The observed high frequency modes are consistent with those of other ferrous oxy heme proteins (see Van Wart & Zimmer, 1985 for example). They are also similar to high frequency modes (1374, 1585, and 1650 cm‘l) reported by Babcock et a1. (1984) for a transient "oxy" species observed during the reaction of oxygen with reduced oxidase. Although an oxy species cannot be positively ruled out on the Raman data alone, the peroxide chemistry of hemes in peroxidase systems (Terner et al., 1985; Hashimoto et al., 1984), globin species (Sitter et al., 1985a), and model compounds (Bruice et al., 1986) are consistent in favoring the formation of a ferryl species when peroxide is present. Our results, like those discussed in Witt et al. (1986), are consistent with the assignment of the 580 nm species as an oxo ferryl heme. This same assignment has been proposed by Wikstrom (1981) as the result of studies with mitochondria poised in a highly oxidized state . Attempts at conclusive identification of the 580 nm species as a ferryl oxo heme, through observation of the (Fer=O) stretching frequency, were not successful since no oxygen isotope sensitive modes could be assigned (see Figure 6.8). This in itself is not significant since several factors could account for the absence of the this peak or the absence of an isotope shift. The mode may be weak like the V(FeIV=O) peak of cytochrome c peroxidase compound ES which was 192 recently reported by Hashimoto et al. (1986a). Considering the poor signal to noise ratio in Figure 6.8 and the large number of heme modes in this region of the spectrum, the V(FeIV=O) could easily be obscured. Oertling and Babcock (1987) have determined the excitation profile for the V(FeIV=O) peak in horseradish peroxidase compound II with Soret region excitation and have established that the intensity of the V(FeIV=O) exhibits a strong dependence on the excitation wavelength. If 406.7 nm is not near the maximum of the excitation profile for ferryl heme a, then the V(FeIV=O) intensity may be weak and easily obscured. Excitation at wavelengths closer to the Soret maximum (428 nm) than 406.7 nm may be needed to observe this mode. It is possible that the 180 labeled ferryl sample (580 nm species) undergoes a hydrogen bond mediated exchange with the predominantly 16O of water. This behavior has been demonstrated with horseradish peroxidase compound II at neutral pH by Hashimoto et al. (1986b). It is also noted that the intensity of the V(Fe1v=0) peak decreases in intensity upon hydrogen bonding. The 580 nm species is unstable at liquid temperatures and appears to revert to a resting or pulsed species under laser illumination at 0 C (Figure 6.3a,b). This is in contrast to the the results for the frozen sample which indicate that at a temperature of -120 C and with low laser power, no significant decomposition occurs in the course of a full day of laser irradiation. Like the spectrum in Figure 6.3a, previously reported spectra of species that may be the same as the 580 nm species (Woodruff et al., 1982, resting + H202; and Copeland et al., 1985, "420 nm" + H202) may also demonstrate significant decomposition —7——* “W ' 193 before it is identifiable by optical absorption measurements on the bulk sample. We believe that the use of low temperature (-120 C) frozen samples may allow more reliable Raman characterization of unstable species than previously used liquid samples techniques. In the discussion below, we will also compare our Raman spectra of frozen samples of pulsed and compound C species with those reported for liquid samples by the above mentioned authors. It is hoped that this will help bring a consensus to the field as to the identities of these different species and whether any of the previously reported samples suffer from any major decomposition or heterogeneity. The pulsed form of oxidase (Soret 420 nm, 424 in our samples) was reported as distinct from resting oxidase and peroxide bound species by Kumar et al. (1984). The Raman spectrum of pulsed cytochrome oxidase has been previously shown, for liquid samples, (Copeland et al. 1985) to resemble that of resting oxidase. These results are contrasted by results from Woodruff et a1. (1982) in which the spectrum of pulsed oxidase was quite different from the spectrum of the resting species. This difference was primarily manifested in an overall weakness of the spectrum and a distinct loss of intensity at ~1573 nm (high-spin a3). Our Raman spectrum of the frozen pulsed enzyme (Figure 6.4c) displays the same trends observed in the results of Woodruff et al. (1982) There is evidence for only a small high-spin contribution (weak 1575 cm'1 peak) and the overall Raman scattering intensity is weak for this spectrum in comparison with those of the resting and 580 nm species. Since resonance Raman scattering is strongly dependent on the optical absorption spectrum, we examine the differences in optical absorption 194 properties between resting and pulsed. Three types of Soret optical absorption changes could account for this reduced scattering intensity: 1) broadening; 2) a decrease of the absorption intensity; or 3) red shifting (further off resonance). The Soret band is not-any broader for the pulsed enzyme than the resting enzyme so the first possibility can be excluded. Although the Soret maximum is at approximately the same wavelength as that of the resting enzyme ("424 nm), the Soret band of pulsed enzyme is decreased in intensity and slightly skewed to the red relative to the resting enzyme. This decrease in the intensity of the Soret absorption and slight red shift could account for a decreased Raman scattering intensity and may explain the observed behavior although other factors may be involved too. Since changes in absorption properties often accompany changes in axial ligation (with oxidation state unchanged), the difference in Raman scattering (at low temperature) between the pulsed and resting forms of the enzyme is probably indicative of a difference in heme axial ligation (in the cytochrome a3 center) for the respective enzyme samples. A difference in ligation could account for the difference in reactivity and ligand binding rate observed by Brunori et al. (1979) for the pulsed enzyme relative to the resting enzyme. This idea is further supported by EXAFS studies of both the resting and pulsed (Chance et al. 1983) enzyme species. Woodruff et a1. (1982) have interpreted the results of their Raman and magnetic circular dichroism (MCD) studies for the pulsed enzyme as indicative of magnetic coupling between an intermediate spin ferric heme, in the cytochrome g3 site, and the CuB center. Intermediate spin ferric heme models have been observed (Scheidt & Gouterman, 1983) in both five- and six-coordinated species with weak 195 axial ligation. The unusually weak iron-imidazole (proximal) bond in cytochrome a3 ( V(FeII-His), 214 cm'l), as determined by Raman spectroscopy in the five-coordinate ferrous state (Van Steelandt—Frentrup et al., 1981; & Ogura et al., 1983), may be conducive to the formation of an intermediate spin species in the ferric state. Compound C is postulated to be a species with peroxide bound to the heme of cytochrome a3, possibly bridging to CuB (Chance & Leigh 1977). This species has been produced by two different methods, addition of 02 to the half reduced (mixed valence) enzyme and direct binding of peroxide to the fully oxidized enzyme, which produce samples with nearly identical optical absorption spectra. In our Raman studies, however, we observed slight differences in the Raman spectra (“1510 and ~1570 cm'l regions) of the compounds C produced by different different methods, MVCO + 02 (Figure 6.4a) and pulsed + peroxide (Figure 6.4b). This suggests that they are two distinct species or that both samples contain the same compound C species but that they are heterogeneous and contain different impurities. We favor the latter explanation since the differences between these two samples are small and is not necessarily significant. Since these two samples are made by different methods, it would not be surprising for them to contain different contaminant species. Scattering is so weak from the compound C samples that small amounts of other species could readily alter the observed Raman spectra. The similarity of the spectra of the compound C species (4a,b) with that of the pulsed enzyme samples (4c) was also a source of concern. The two most likely interpretations of this similarity are: l) 196 one of the samples rapidly photo-reacts into the other one on a time scale that is short in comparison to the data collection time, or 2) the two species share very similar heme structures. Because of the limited region of laser exposure on these frozen samples, it has not been possible to check the samples effectively by optical absorption spectroscopy after Raman data collection for a transition from one to another. The first possibility does not seem likely since the spectra of these frozen samples do not change significantly over a full day of scanning. This indicates a lack of significant turnover in these samples, which would suggest that they are distinct species. In addition, if the compound C sample is simply a peroxy bridged species as postulated, a one electron reduction of compound C should produce the ferryl species (580 nm), which seems resistant to photoreduction and exhibits a very strong Raman spectrum. It seems unlikely that compound C would photoreduce to the pulsed form without accumulation of detectable amounts of the ferryl species. This conclusion is consistent with the results of both Woodruff et al. (1982) and Copeland et a1. (1985), who reported similarities between the Raman spectra of their pulsed and compound C (oxygenated) samples, yet identified them as different species (note that oxygenated oxidase should be the same species as our compound C which was produced by the addition of stoichiometric amounts of peroxide to the pulsed enzyme). The spectrum of oxygenated oxidase, reported by Babcock et a1. (1981), also resembles the spectra of the compound C species reported by these other researchers, but it seems to exhibit a greater contribution from low spin hemes. 197 If we accept that compound C is distinct from the pulsed enzyme, the similarity of the low temperature Raman spectra of the compound C samples to those of the pulsed enzyme is surprising considering the difference in optical absorption spectra. The red shifted Soret band of the compound C samples (428 nm) relative to the resting and pulsed samples (424 nm) indicates a greater contribution from low-spin species. This would be expected to produce a Raman spectrum with stronger low-spin characteristics. This does occur to a limited extent but the effect is small in comparison to that observed for the 580 nm species which also has a Soret maximum at 428 nm. This observed behavior may indicate a mixture of high- and low-spin species or intermediate- and low-spin species in the cytochrome a3 site of the compound C with both species only weakly scattering. Babcock et al. (1981) discuss the Raman, MCD, and EPR results of the oxygenated oxidase (compound C in our studies) as possibly due to the magnetic coupling of a low-spin heme to the CuB+2 in cytochrome a3. Woodruff et a1. (1982) conclude that compound C is also an intermediate-spin species (like the pulsed enzyme). The discussion of Copeland et a1. (1985) asserts that the spectra of pulsed enzyme and compound C (oxygenated?) are similar and may reflect inhomogeneity or the presence of intermediate-spin ferric heme. They emphasize however that increased photoreduction of the pulsed sample indicates a protein conformation different from that of the compound C. Our final conclusion is that the pulsed and compound C species are different. It is likely that none of the spectra published to date for pulsed enzyme or compound C (including our own) represent scattering from a homogeneous sample and that sample-to-sample variations represent Raman signal from more 198 strongly scattering heme impurity species. If pulsed cytochrome oxidase is a non-ligand bound species, and compound C is the corresponding peroxide bound species, the peroxide apparently has little effect on the Raman properties of the heme and may reflect weak or disordered binding. The Raman spectrum of the reoxidized enzyme (Figure 6.5) is dominated by contributions from low-spin species. This spectrum is similar to the spectrum of the 580 nm species, but it is not as strongly scattering and V4 is lowar (1374 cm'l). The sample is also distinguished from the 580 nm species, by its EPR properties, as noted above, and it has been postulated to be a ferrous oxy species (Fell-02) based on the EPR studies (Blair et al., 1985). The Raman results are consistent with a ferrous oxy species although the ferryl oxo or ferric hydroxy species should not be ruled out from the Raman data alone. Due to the method of preparation, the reoxidized cytochrome oxidase sample is not likely to be homogeneous and it may contain a variety of intermediate species. Fortunately, it is unlikely that any pulsed or compound C type species would be distinguished due to their weak Raman scattering. The observed Raman spectrum would therefore reflect the presence of at least moderate amounts of ferrous oxy or ferryl oxo species in this sample. Further identification cannot be made with the data available. The reaction of the 580 nm species with CO is expected to yield an 02 or CO ligated ferrous heme species (Witt et al., 1986). The observed Raman spectrum of this sample (Figure 6.6) demonstrates that the heme 199 of the cytochrome g3 site is predominantly low—spin which would seem to be consistent with either ligated species (02 or C0). However, experiments with a mixed valence CO bound sample (not shown) demonstrated that the CO undergoes extensive photolysis even when the laser power is lowered to '2 mW. This was evident in the Raman spectrum by an overall weaker scattering and a shift of V2 to 1578 cm‘l, which is characteristic of ferrous five-coordinate high-spin heme a. By analogy to the mixed valence CO bound species, a ferrous CO species would most likely photolyze and it would not account for the observed spectrum. We therefore conclude that the Raman spectrum of the C0 reacted 580 nm species is most likely due to scattering from residual unreacted 580 nm species and/or scattering from an oxy ferrous species. In conclusion, the study of cytochrome oxidase intermediates and related species has been hindered by a lack of common nomenclature and by confusion over the ligation and redox states of the metal centers in these different species. In an attempt to help clarify this situation, we have obtained low temperature resonance Raman spectra of what we believe to be six distinct cytochrome oxidase species: resting, pulsed, compound C, 580 nm, reoxidized, and 580 nm plus CO. These results suggest that the structures of the cytochrome a3 sites of pulsed oxidase and resting oxidase are different and that the spectral differences are best observed at low temperature. The low temperature Raman spectrum of compound C strongly resembles that of the pulsed sample. This may reflect a coincidental similarity in heme geometry and spin state in the two different species. The latter three species exhibit spectra characteristic of a low-spin heme in the cytochrome g3 200 site. The 580 nm form is assigned to a ferryl oxo Species owing to the conditions of its formation and its reactivity with CO. The latter tw0 samples are probably not homogeneous and the spectra may be dominated by contributions from ferrous oxy and ferryl oxo species. CHAPTER 7 CONCLUSIONS AND FUTURE WORK A. CONCLUSIONS The goals of my research, as outlined in my second year research proposal, centered on the use of resonance Raman spectroscopy to elucidate the catalytic pathway and intermediate structures in the reductiOn of oxygen by cytochrome oxidase. The initial technical phase of this research has been completed in full. This included: 1) building hardware and writing software for the interfacing of our computer to our Raman spectrometer; 2) construction of optical Dewars and additional equipment to enable collection of low temperature optical absorption and Raman spectra; and 3) development of specific anaerobic procedures and equipment. Attempts to produce the low temperature trapped intermediates and collect Raman spectra (work in collaboration with Dr. Patricia Moroney) were not successful owing to the difficulty of sample preparation, inherent sample inhomogeneity, sample fluorescence and inexperience with the handling of and data collection from low temperature samples. At this point we realized that much more preliminary experimental work would be necessary before we could hope to do the experiments with these trapped intermediates. The direction of my project shifted from the strict enzyme work to one involving the use of model compounds. Synthetic procedures were available in the literature for a number of heme species which had structural characteristics similar to those proposed for the oxidase 201 202 intermediates. Only a few of these had been characterized by Raman spectoscopy. The Raman spectral studies of ferryl and oxy heme species (Chapter 5) was conducted to fill gaps in the existing literature. Our goal was to improve our understanding of the chemistry and biochemistry of these species and to be able to predict the spectroscopic properties that these species would demonstrate if they occurred in cytochrome oxidase. In parallel with this, W. Anthony Oertling was characterizing reaction intermediates of a variety of other heme enzymes which may have analogies with the chemistry of oxidase. The studies of cyanide binding to hemes in solution (Chapter 3) were initiated to try to understand the unusual Raman results from cyanide bound samples of one of these proteins. Other studies were initiated even more closely related to oxidase. We entered into a collaboration with Dr. C. K. Chang which involved the characterization of a synthetic porphyrin species designed to model the oxygen reduction site of cytochrome oxidase (Chapter 4). My studies using resonance Raman, optical absorption, and EPR spectroscopies were complemented by the magnetic susceptibility and IR studies of M. S. Koo, the student who synthesized these compounds. These studies provided insights into the interactions of copper and iron (heme) atoms in close proximity and the effects of axial ligand variation on the spectroscopic properties. A final series of experiments, in collaboration with the research group of S. 1. Chan (California Institute of Technology), utilized low temperature Raman spectroscopy to investigate a number of chemically induced "pseudo" intermediates of cytochrome oxidase (Chapter 6). These experiments provided experience 203 with data collection from low temperature samples, yet they were more stable, more homogeneous, and simpler to prepare than the cold trapped species. In this study, we characterized what appear to be a ferryl oxo, ferrous oxy and several ferric species of cytochrome oxidase. These experiments have reaffirmed that the chemistry of oxidase is complicated and that Raman spectroscopy will not easily provide unambiguous information about the structures of the cold trapped intermediates without good low frequency data. However, we have demonstrated that we can obtain meaningful results even under these extreme experimental limitations and with fairly inhomogeneous samples. With the technology and expertise we developed while doing these experiments, and the results we obtained, additional work with trapped intermediated shoud be possible and informative. In the course of the above research, we have learned a great deal about the structure and chemistry of hemes. We have determined that the peripheral porphyrin substituents have little or no affect on the bond strengths of the axial ligands as monitored by their Fe-ligand vibrational frequencies. The chemistry of species such as the ferrous oxy (Fell-02), ferryl oxo (FeIV=O) and ferric cyanide (FeIII-CN') seem to be controlled by out-of—plane effects such as trans ligand strength, steric constraints, and hydrogen bonding. This raises further questions as to the purpose of the unusual heme a ring substituents for the functioning of cytochrome oxidase and the purpose of different iron porphyrin variations in other proteins. In contrast to the Raman spectra, the optical absorption spectra of hemes are easily perturbed by a variety of effects. These include ligation, oxidation state, and 204 planarity of the iron relative to the heme ring as well as environmental perturbation to the porphyrin macrocycle. Although optical absorption spectroscopy does not give specific structural information, these results reaffirm its importance in the characterization of heme protein species. It has become obvious, in the course of our research, that the interaction of peroxides with cytochrome oxidase is not well understood and past interpretations may have been oversimplified. Further studies will be required to clarify the situation. B. FUTURE WORK Low temperature experiments with trapped intermediates should be pursued further. The results from the pseudo-intermediate studies cast doubt as to whether the trapped intermediate studies will yield unambiguous results. However, if samples can be produced which are of more homogeneous composition than those used in our study, it should be possible to identify the intermediate through the assignment of Fe-ligand vibrations in the low frequency region of the spectrum. The alternative, to the cold trapped intermediate studies, is the use of time resolved resonance Raman techniques which can characterize intermediate species at liquid temperatures. Preliminary work has already been done (Babcock et al., 1984, 1985) and more experiments are planned. One problem, that has been a continual hindrance to the use of Raman spectroscopy for the study of cytochrome oxidase, is sample fluorescence and photoreduction. The fluorescence seems to be caused by residual flavins of other fluorescent biomolecules that are accidentally isolated along with the cytochrome oxidase (Adar and 205 Yonetani, 1978). The amount of fluorescence varies dramatically with preparation technique and even from one prep to another with the same technique. This fact gives hope that a preparation scheme could be developed that would minimize this fluorescence. An alternate approach to the problem is the use of fluorescence rejection Raman techniques. These are based on the fact that Raman scattering is fast (10'12 sec) compared to fluorescence (~10'9 to 10'6 sec). By using‘picosecond pulsed lasers and fast detector gating, data collection can be gated off before significant fluorescence occurs (Van Duyne et al., 1974). Alternately, the intensity of a continuous wave laser can be modulated at a high frequency (~megahertz range) and coupled to the detector through a lock in amplifier. Raman scattered photons will be in phase with the amplitude modulation; photons from fluorescence will fall out of phase and can be rejected (Van Hoek and Visser, 1985). Photoreduction is a problem that may be directly linked to fluorescence in cytochrome oxidase but the exact relationship is not clear. The magnitude of photoreduction has been observed to be dependent on enzyme conformation (Copeland et al., 1985), although it is not known whether the reducing equivalent arises from a protein residue from exogenous impurities. This phenomenon should be studied more thoroughly to establish the source of this reducing equivalent and whether photoreduction can be minimized through modification of the isolation procedure. Although the interaction of hemes with peroxides is fundamental to the chemistry of peroxidase and catalase enzymes, the characterization of this chemistry in solution studies have relied almost exclusively on 206 kinetic studies, with little direct structural information about the reaction pathways (see Traylor et al., 1984; and Bruice et al., 1986; and references within). The time resolved Raman techniques utilized by Oertling and Babcock (1985) for the characterization of horseradish peroxidase intermediates, may be ideal for characterization of heme-peroxide solution chemistry. The advantage of these solution studies over the use of only protein species, is that axial ligation, solvent polarity, hydrogen bonding interactions, and steric constraints can be varied independantly to determine their effect on the chemistry. Additional studies with heme a models may be useful in further identification of cytochrome oxidase structure and intermediates. Although the chemistry may not be altered by variation of the porphyrin ring substituents (see above), the unusual substituents of heme a produce unique optical absorption and resonance Raman properties which make it difficult to model oxidase with other hemes. Heme a species that may be useful in cytochrome oxidase model studies include: ferryl oxo, ferric (NMI,H20), and ferric (NMI,OH') as well as additional studies with the ferrous oxy species. Heme a is difficult to work with because the formyl substituent (ring position 7) is easily reduced by reagents commonly used to reduce the iron. In addition, like all physiological hemes, heme a readily aggregates or forms u-oxo dimers under some solution conditions. Heme reduction, necessary for the synthesis of ferryl oxo and ferrous oxy species, may be done more reliably by using the electrochemical techniques which I haVe described in Chapter 2. Formation of ferric (NMI,H20) and ferric (NMI,OH') may require the use of attached imidazole ligands and/or anchoring of the heme to a polymer substrate (to prevent aggregation). A procedure for 207 the attachment of imidazole ligands to the related protoporphyrin species has been described by Brinigar and Chang (1974), and a procedure for attachment to a polymer suport has been described by Tsuchida et al. (1982). Both procedures utilize the heme propionic acid substituents as a linking point and should work for heme a as well as for protoheme. Additional species that may be of interest are heme a with attached imidazole in one axial position and sulfur ligands (either free in solution or also attached) in the other axial position. A bridging sulfur ligand (between heme a and Cu) has been proposed for cytochrome a3 in the resting state of the enzyme (see Naqui and Chance, 1986, and references within). Sulfur ligated models may give some insight into the expected properties of such a structure. The studies we have performed on the meso-diphenylporphyrin model compounds may have only begun to utilize their potential as models of the oxygen reduction site of cytochrome oxidase. In the studies discussed in Chapter 4, we examined the properties of these compounds as models for the resting form of cytochrome oxidase, which contained metal centers in their fully oxidized (Fe+3, Cu+2) forms. What yet remains is the study of these model species with reduced metal centers (Fe+2 and/or Cu+1) and reactive ligands (02' and H202). Since there are only two metal centers (as opposed to four in oxidase), experiments should be easier to perform and the results should be easier to interpret. Through the use of electrochemistry (in non aqueous solvents), it should be possible to reduce one of the metal centers of the copper chelated models selectively; the iron would probably be more easily reduced. It is expected, based on cofacial mixed metal porphyrin ————'*Wewwwe—~u .- . 208 models, that with only iron reduced, the sample will form a stable oxygen bound species (Ward et al., 1981). With the iron and copper reduced, reduction of the oxygen to the peroxide level may occur. Further reduction steps may be possible, in a controlled fashion, by reductive titration or by electrochemistry at room temperature or low temperature. These processes should be detectable by Raman spectroscopy as well as by other physical techniques. The copper chelating group of the meso-diphenylporphyrin models is likely to mimic cytochrome oxidase better than the copper porphyrin of the cofacial models, and the optical spectra or the mesa-diphenylporphyrin models are not complicated by the presence of the copper chelating porpyrin. Facilities are already available in the chemistry department for all these experiments. As discussed above (Chapter 4), additional work will be necessary in order to assign the remaining vibrational modes of the meso-diphenylporphyrins. These studies should initially include depolarization ratios and excitation profiles which will both aid in peak assignment and help establish the extent of symmetry reduction relative to D4h symmetry. If this is not sufficient to make the assignments, studies with isotope substitution (2H, 15N, and 13C) may be required. Although we are relatively confident of the structures of these model compounds, additional verification would help establish the credibility of our results. Ideally, this could be done with X-ray crystallography, but EXAFS may suffice if crystals cannot be obtained. If these meso—diphenylporphyrin models can be emulsified in aqueous detergents or derivatized to increase their solubility in water, 209 peroxide binding and reaction studies should be attempted. Soluble hemes have already been demonstrated to exhibit peroxidase and catalase activity when peroxide is added to the aqueous solution (Bruice et al., 1986). It would be informative, in terms of cytochrome oxidase chemistry, to determine if the presence of a nearby chelated copper ion modifies this peroxide chemistry or selectively favors one of the possible processes. Finally, attempts should be made to bind bridging sulfur ligands between the iron and copper of these models, to determine if these species will also demonstrate antiferromagnetic exchange coupling in a six-coordinate high-spin state as was determined for the oxygen bridged species discussed in Chapter 4. APPENDIX APPENDIX 1 COMMERCIAL COMPONENTS OF THE RAMAN SPECTROSCOPY SYSTEM Spectrometer Scanning double monochrometer w/1200 groove/nm gratings blazed at 500 nm (Spex Ramalog 1401) Sample illuminator (Spex 1419A) Photomultiplier tube (PMT) (RCA 31034C) High voltage power supply and photometer unit (Spex Ramalog 4) PMT chiller and associated power supply (Products for Research) Chart recorder (Linear 1200) Computer Enclosure (Netcom HV-1123-ll-736) Diskette drive (Data Systems DSD 480120) Terminal (Zenith HE-WH19) and graphics retrofit board (Northwest Digital Systems Graphics-Plus) Plotter (Houston DMF-2) Real Time Clock (Data Translation DT2769) Quad Serial Board (DEC DLVll-J) LSI 11/2 CPU (DEC KDll-HA) Floating point and expanded instruction set (DEC KEVll) 64KByte memory (Christlin 01—1103) Photon counting board (custom made with an AM 9513 counting chip) Lasers Krypton ion laser, with high-field magnet (Spectra Physics Model 164-ll) with red (647.1/676.4 nm), U.V. (350.7/356.4 nm), and special blue (406.7/413.1 nm) optics Argon ion laser (Spectra Physics Model 165) With all lines optics and tuning prism Power supply (Spectra Physics Model 265) . Dye laser and dye circulator (Spectra Phy51 375, 376N, respectively) Helium-cadmium laser and power supp Powermeter (Scientech 362) cs Models 1y (Liconix 4240) 210 LIST OF REFERENCES LIST OF REFERENCES Abe, M., Kitagawa, T., and Kyogoku, Y. (1978) J. Chem. Phys. 62, 4526-4534. Adar, F. (1978) in "The Porphyrins", Vol. 2, Dolphin, D. (ed.). Academic Press: New York, pp. 167-209. Adar, F. and Yonetani, T. (1978) Biochem. Biophys. Acta 592, 180-86. Anderson, D. L., Weschler, C. J., and Basdo, F. (1974) J. Am. Chem. Soc. 96, 5599-5600. Andersson, K. K., Lipscomb, J. D., Valentine, M., Munck, E., and Hooper, A. B. (1986) J. Biol. Chem. 261, 1126-1138. Andersson, L. A., Loehr, T. M., Chang, C. K., and Mauk, A. G. (1985) J. Amer. Chem. Soc. 107, 182-191. Antonini, E. and Brunori, M. 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