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Studies on carbon monoxide and dioxygen binding to cytochrome P-450cam Rajapakse, Nimal 1984

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STUDIES ON CARBON MONOXIDE AND DIOXYGEN BINDING TO CYTOCHROME P-450cam. By NIMAL RAJAPAKSE B.Sc, University of Sri Lanka, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA December 1984 © Nimal Rajapakse,1984 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. N. RAJAPAKSE Department of Chemistry  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 2nd January 1985. DE-6 (3/81) ii ABSTRACT Interest has remained very intense during the last two decades on the heme-containing monooxygenase system, cytochrome P-450. The P-450 hemoproteins are widely distributed in nature and engage in oxygenation of a wide variety of substrates according to the reaction, R-H + 02 + 2H++ 2e~ > R-OH + H20 where R-H represents an unactivated carbon-hydrogen bond. Investigations on binding of small gas molecules such as CO and 02 to the P-450 enzymes are important not only in understanding various aspects of monooxygenation but also in developing protein-free model systems that can mimic the catalytic properties of P-450. This thesis describes gas binding studies carried out on cytochrome P-450cam. A procedure is given for growing the bacterium Pseudomonas putida strain 786 from which soluble, camphor hydroxylating P-450 enzyme is isolated and purified. The binding of CO to the stoichiometrically reduced substrate-free enzyme at different temperatures was studied using a standard spectrophotometric procedure. From these experimental data, the thermodynamic parameters AH0 and AS0 were calculated for the reaction, (P-450)Fe(II) + CO ^ ^ (P-450)Fe(11)-C0 . Attempts to determine such thermodynamic parameters for the binding of dioxygen to the substrate-bound P-450 enzyme were not successful. iii On comparison of the determined thermodynamic parameters for the substrate-free system with the literature values for substrate-bound enzyme, hemoglobin, myoglobin and P-450 model systems, it is concluded that the substrate molecule was bonded in the immediate vicinity of the active-site thereby lowering the CO affinity to the substrate-bound system. iv TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viLIST OF FIGURES viii LIST OF ABBREVIATIONS X ACKNOWLEDGEMENTS xii CHAPTER I INTRODUCTION 1 References 6 CHAPTER II LITERATURE REVIEW 7 11.1 Brief History of Cytochrome P-450 . 8 11.2 Nature and Cytochrome P-450 15 11.3 Structural Considerations 17 11.4 The Mechanism of Catalysis 20 11.4.1 Types of Oxidation Reactions 20 11.4.2 The Catalytic Cycle 24 Binding of Substrate 24 First Reduction 25 Binding of Dioxygen 26 Second Reduction 7 Splitting of Oxygen-Oxygen Bond ... 27 Oxidation of Substrate 28 Dissociation of Product 29 11.5 Electronic Spectroscopy 31 11.6 Interaction of Cytochrome P-450 with Carbon Monoxide and Dioxygen 34 References 43 V CHAPTER III EXPERIMENTAL PROCEDURES 49 111.1 Growth of Bacteria 50 III. 1.1 General Information 50 III.1.2 Media 52 III.1.2.1 Minimal Agar 5111. 1.2. 2 L-Broth 4 III. 1 .2.3 500 mL Shake Flask 5III.1.2.4 14 L Fermenter 6 III. 1.3 Growth Procedure 57 111.2 Isolation and Purification of Cytochrome P-450 63 111.2.1 General Information 63 111.2.2 Materials 6111.2.3 Buffer Solutions 64 111.2.4 Column Chromatography 68 111.2.5 Isolation of Cytochrome P-450 69 Cell-Free Extract 6111.2.5.2 Separation of Cytochrome P-450 .... 69 Ammonium Sulphate Fractionation ... 70 111.2.6 Gel Filtration Chromatography 72 Purification of Cytochrome P-450 .. 72 Removal of Camphor Substrate 72 111.2.7 Spectral Analysis 74 111.3 Determination of Equilibrium Constant at Different Temperatures for the Reaction of Carbon Monoxide with Substrate-Free Cytochrome P-450 ... 77 vi 111.3.1 General Information 77 111.3.2 Procedure 7III.4 Determination of Equilibrium Constant for the Reaction of Dioxygen with Substrate-Bound Cytochrome P-450 .. 80 III. 4.1 General Information 80 III .4.2 Procedure 8References 3 CHAPTER IV RESULTS AND DISCUSSION 84 IV. 1 Growth of Pseudomonas putida Strain 786 85 IV.2 Properties of Cytochrome P-450cam . 87 IV.2 Substrate-Bound Cytochrome P-450cam 87 IV.2.2 Substrate-Free Cytochrome P-450cam 91 IV.2.3 Purity and Stability of Enzyme Preparations 91 IV.3 Determination of Equilibrium Constant at Different Temperatures for the Reaction of Carbon Monoxide with Substrate-Free Cytochrome P-450cam 97 IV.3.1 Acquisition and Treatment of Data . 97 IV.4 Comparison of Measured AH0 and AS0 Values to Those of Other Hemoproteins and Model Systems .... 106 References 111 Appendices 113 vii LIST OF TABLES Table Page 11.1 Kinetic Data for CO Binding to Cytochrome P-450 and Related Systems 37 11.2 Equilibrium Data for CO binding to Cytochrome P-450 and Related Systems 38 11.3 Equilibrium and Kinetic Data for Reversible Dioxygen Binding to Substrate-Bound Cytochrome P-450 and Mb 42 111.1 Volumes of Buffer Solutions Required for a Typical Isolation and Purification of Cytochrome P-450 66 111.2 Quantities of Reagents Required for a Typical Isolation and Purification of Cytochrome P-450 67 IV.1 Absorbance Data for Substrate-Bound Cytochrome P-450cam 89 IV.2 Absorbance Data for Substrate-Free Cytochrome P-450cam 9 3 IV.3 The Equilibrium Constant Values for CO Binding to Substrate-Free Cytochrome P-450cam 102 IV.4 K^ , AH0 and AS0 Values for CO Binding to Some Hemoproteins and Models of Cytochrome P-450 1 05 IV.5 Half-lives of Autoxidation Reaction of Substrate-Bound Cytochrome P-450cam 110 viii LIST OF FIGURES  Figure Page I. 1 The Structure of the Heme Unit 3 II. 1 The First Published Record of the Carbon Monoxide Difference Spectrum of Cytochrome P-450 8 11.2 The Carbon Monoxide Difference Spectra of Cytochrome P-450 and P-420 9 11.3 Electron Transfer Reactions in Bacterial System Leading to Camphor Hydroxylation. ... 12 11.4 The Substrate Induced Spectral Changes for Cytochrome P-450cam 13 11.5 Benzopyrene and its Carcinogenic Metabolite 7,8-Diol-9, 1 0-epoxide 16 11.6 The Catalytic Cycle of Cytochrome P-450. ... 23 11.7 Hypothetical Scheme for the Binding of Substrates to Cytochrome P450 25 11.8 'Proposed Mechanism for P-450 Mediated Hydroxylat ion Reaction 29 11.9 Heme Environments of Myoglobin, Hemoglobin and Peroxidase 35 11.10 Valence Bond Structures of the CO-Complex of Cytochrome P-450 39 111.1 OD660 and pH Profiles for the Bacterial Growth in the First Set of Shake-Flasks .... 58 111.2 OD660 and pH Profiles for the Bacterial Growth in the Second Set of Shake-Flasks ... 60 ix 111.3 OD660 and pH Profiles for the Bacterial Growth in the 14 L Fermenters 61 111.4 The Cell-tonometer 75 111.5 The 10 cm Path-length Cell (Slush Bath) 81 IV.1 The Absorption Spectra of Various States of Substrate-Bound Cytochrome P-450cam 88 IV.2 The Absorption Spectra of Various States of Substrate-Free Cytochrome P-450cam 92 IV.3 The Absorption Spectra of Oxidized States of Substrate-Free and Substrate-Bound Cytochrome P-450cam 94 IV.4 The Absorption Spectra of P-420 Species Formed in Substrate-Free Cytochrome P-450cam Solution 96 IV. 5 Spectral Changes Observed for the P1/2 Experiment no. 5 98 IV.6 A Typical Hill Log/Log Plot 100 IV.7 The van't Hoff Plot 103 X LIST OF ABBREVIATIONS The following abbreviations and common names are used in this thesis along with the standard abbreviations used commonly in chemical literature. A A0 Ace Buffer P Buffer P-100 Buffer T Buffer T-50 Buffer T-100 Buffer T-600 BME o cam or camphor DNase 1 DTT EXAFS ENDOR EPR ESR AH0 Absorbance Observed absorbance at the beginning of an experiment Observed absorbance at the end of an experiment 50 mM phosphate buffer, pH 7.4 50 mM phosphate buffer, pH 7.4, 100 mM KC1 50 mM Tris buffer, pH 7.4, 50 mM Tris buffer, pH 7.4, 50 mM KC1 50 mM Tris buffer, pH 7.4, 100 mM KC1 50 mM Tris buffer, pH 7.4, 600 mM KC1 B-mercaptoethanol Degrees Centigrade D-(+)-camphor Deoxyribonuclease 1 Dithiothreitol Extended X-ray absorption fine-structure spectroscopy Electron nuclear double resonance Electron paramagnetic resonance Electron spin resonance Standard enthalpy change xi Hb Hemoglobin max Maximum Mb Myoglobin NADH Reduced nicotinamide adenine dinucleotide NADPH Reduced nicotinamide adenine dinucleotide phosphate NMR Nuclear magnetic resonance P-450 Cytochrome P-450 P-450cam Camphor hydroxylating P-450 P-450LM Liver-microsomal P-450 RNase A Ribonuclease A RR Resonance Raman SDS Sodium dodecyl sulphate Ago Standard entropy change xii ACKNOWLEDGEMENTS I wish to express my gratitude to Professors D.H.Dolphin and B.R.James for their guidance and encouragement through out the course of this work. My thanks are also due to Messrs. Simon Albon, Gary Hewitt and Lalith Talagala for their help. Finally, I wish to express my deepest appreciation to my family for their love and understanding. CHAPTER 2 I. Introduction The hemoproteins which mediate transportation, storage and activation of dioxygen are vital for most living organisms. For example, in mammals, dioxygen is transported by hemoglobin, stored by myoglobin and reduced to water in the terminal step of respiration by cytochrome c oxidase. Also, decomposition and further activation of hydrogen peroxide formed in biological systems are undertaken by catalase and peroxidase while a number of other hemoproteins function as mono- and dioxygenases. Amongst all these hemoproteins, cytochrome P-450 (P-450) is considered to be one of the most unusual and intriguing. The intense level of research conducted during the last 25 years since its discovery1-5 reflects, in part, the importance of cytochrome P-450 as a drug-metabolizing enzyme, its central role in chemical carcinogenesis, and its broad substrate specificity. The P-450 molecule consists of a single protein chain with a molecular weight of approximately 45,000 , joined to an iron protoporphyrin moiety (Figure 1.1) via a thiolate (-S~ ) ligand derived from a cysteine residue. The P-450 hemoproteins generally catalyze the splitting of molecular dioxygen and insertion of one oxygen atom into an unactivated C-H bond to form an alcohol, the other atom of oxygen being converted to a molecule of water (Reaction 1.1). RH + 02 + 2H++ 2e" > ROH + H20 (1.1) CH, H ca CH2COOH CH2C00H The Structure of the Heme Unit. 4 Therefore, cytochrome P-450's are collectively known as monooxygenases, which are a class of enzymes that incorporate one atom of oxygen from molecular oxygen into a substrate. The ability of P-450 to carry out this hydroxylation reaction under physiological conditions with high stereo- and regiospecificity is in fact remarkable, when compared to the forcing reaction conditions and the number of different products that result, if the same hydroxylation reaction were to be carried out in the laboratory using non enzymatic, chemical reagents.6 Throughout the brief history of cytochrome P-450, numerous attempts to solubilize and purify the membrane-bound enzyme that is present in mammalian systems resulted in the isolation of an 'inactive' form of enzyme.7 However, the discovery of soluble P-450 in the bacterial strain Peudomonas put ida grown on D-( + )-camphor led to the isolation of pure enzyme. Therefore, in the present study, soluble camphor monooxygenase system, P-450cam, has been used as a biological model for mammalian P-450. Reactions of organic compounds with dioxygen in the ground state are restricted by spin conservation.8 This may be overcome by formation of metal-dioxygen complexes which in turn react with the organic substrate. However, the same 5 metal complexes also react directly with alkyl hydroperoxides to generate chain-initiating radicals.8 Since alkyl hydroperoxides are ubiquitous in most hydrocarbon mixtures, the metal-hydroperoxide interactions tend to mask any reaction via 'oxygen activation'. Therefore it is of immense importance to develop catalytic systems that are capable of promoting selective oxidations, preferably using 02 under mild reaction conditions. The study of cytochrome P-450 is thus of great importance not only in widening our knowledge of this remarkable biological system but also in developing protein-free catalytic systems that would mimic the 02 activation capability of these enzymes; such systems bear an unlimited industrial potential.9 In addition to major unanswered questions about the mechanism of P-450 catalytic function, questions concerning the interactions of the substrates with the active-site of the enzyme remain equally challenging. In the present study, the attention was focused to gain some insight into active site-substrate interactions by studying the CO and 02 binding to P-450cam. The thesis begins with an extensive literature survey in Chapter II, that introduces various aspects of monooxygenation mediated by P-450. Chapter III describes the details of experimental procedures. In Chapter IV, the results of experiments are discussed in comparison to other known hemoproteins and model systems, and some conclusions are drawn. 6 REFERENCES 1. D.Y.Cooper,0.Rosenthal,R.Snyder,C.Wilner,Eds.,Cytochrome P-450 and b5 Structure, Function and Interaction, Plenum, New York, 1974. 2. D.W.Cooper,H.A.Salhanick,Eds., Multienzyme Systems in Endochrinology: Progress in Purification and Methods of Investigation, New York Acad. Sci., New York, 1973. 3. T.E.King,H.S.Mason,M.Morrison,Eds.,Oxidases and Related Redox Systems, University Park Press, Baltimore, Maryland, 1973. 4. R.W.Estabrook,J.R.Gillette, K.C.Leibman,Eds.,Microsomes and Drug Oxidations, Williams and Wilkins, Baltimore, Maryland, 1972. 5. M.J.Coon,R.E.White,Metal Ion Activation of Dioxygen,(Ed. T.G.Spiro), Wiley Interscience, New York, 1980, pp73-123. 6. J.H.Groves,W.Kroper,T.E.Nemo, R.S.Myers, J.Mol.Catal., 7, 169 (1980). 7. B.W.Griffin,J.A.Peterson,R.W.Estabrook, The Porphyrins, (Ed.D.Dolphin), Academic Press, New York, VIII, 1979, p.335. 8. R.A.Sheldon,J.K.Kochi,Metal Catalyzed Oxidations of Organic Compounds, Academic Press, New York, 1981, Ch.8. 9. B.R.James,The Porphyrins, (Ed.D.Dolphin), Academic Press, New York; V, 1979, p.205. CHAPTER II LITERATURE REVIEW 8 II.1 Brief History of Cytochrome P-450 The presence in rat liver microsomes of a pigment with a peculiar carbon monoxide binding spectrum was first noted by Williams in 19551 while studying the oxidation-reduction kinetics of microsome-bound cytochrome b5. This novel observation was not published until 1958, at which time Klingenberg1 first reported the carbon monoxide-difference spectrum of this microsomal pigment (Figure II.1). 450 *-i 1 1 1 1 20 10 — 0 T -10 -20 600 400 440 480 520 560 Wavelength (nro) Figure II.1 The first published record of the carbon mono xide-difference spectrum of cytochrome P-450. (from ref.1). Although the binding of carbon monoxide strongly suggested the presence of a heavy metal ion in the chromophore of the pigment, the electronic absorption spectrum showed no resemblance to any known coloured metalloprotein including hemoproteins. In 1962, Omura and Sato2 presented conclusive spectral evidence for the hemoprotein nature of the pigment and confirmed that the pigment was a new b-type cytochrome. They also proposed a 9 'tentative' name "P-450" which meant "a pigment which absorbs at 450 nm". The presence of a- and |3- bands, in addition to intense Soret bands in the carbon monoxide-difference spectrum of the reduced pigment (Figure II.2), clearly revealed the hemoprotein nature of the new pigment. It also 0.4 0.3 02 S 0.1 | 0.0 < -0.1 -0.2 400 450 500 550 600 650 Wavelength (nm) Figure II.2 The carbon monoxide-difference spectra of cytochrome P-450 (A) and P-420 (B).(from ref.l). was observed that treatment of the pigment with detergents converted it quantitatively into a spectrally distinct solubilized form, with a prominent peak at 420 nm in the CO-difference spectrum. Since the establishment of the hemoprotein nature of "P-450", the attention of many biochemists who were engaged in the study of physicochemical properties of hemoproteins became focused on this new b-type cytochrome. Utilizing the photochemical action spectrum technique, in 1963, Estabrook et al.3 demonstrated for the first time the participation 10 of cytochrome P-450 in a steroid C-21-hydroxylation reaction catalyzed by the adrenal cortex microsomes (Reaction II.1). The photochemical action spectrum for the light reversal of the CO-inhibited active monooxygenase enzyme present in these microsomes showed an intense absorption peak at 450 nm that was identical to the reduced CO-difference spectrum of the liver microsomes. In addition, Estabrook et al." were able to demonstrate the participation of cytochrome P-450 in many other mixed function oxidase reactions including hydroxylations and oxidative dealkylation of drugs catalyzed by liver microsomes, confirming the physiological function of cytochrome P-450 to be monooxygenation. As the nature and function of cytochrome P-450 was elucidated, it was also shown that this enzyme could be induced by treating animals with various drugs.5'6 Although cytochrome P-450 was initially thought to be 11 present only in microsomes of animal tissue,7 in a matter of a five-year period from the original discovery in microsomes, it was shown to be present in almost all forms of life.8 In fact, cytochrome P-450 is distributed in nature from primitive bacteria to highly developed mammals, and participates in diversified metabolic reactions as the oxygen activating component of monooxygenase systems. In 1967, Appleby9 presented the first convincing evidence for the presence of P-450 in a bacterial species. The cytochrome P-450 found in the bacterium Rhizobium  japonicum was not bound to membranous cell particles and hence could be partially purified by conventional procedures.9 Shortly afterwards the camphor hydroxylating cytochrome P-450 was isolated from bacterium Pseudomonas  putida 10 and purified to homogeniety,11 and crystallized.12 This source of the enzyme has provided the best material for studying the chemical and physicochemical properties- of this class of enzymes.8 Since the establishment of the physiological function, the next challenge was to determine the mechanism of electron supply to cytochrome P-450. The difficulty in solubilizing the liver microsomal P-450 system, which was very tightly associated with the membranes, caused researchers to look for a more soluble form of P-450. The components of the 1 1 -/3-hydroxylase system of adrenal cortex mitochondria were finally separated by ionic treatment13-1" into a P-450-containing membrane fragment and a soluble 12 NADH-P-450 reductase system, which was later separated by chromatography into an iron-sulfur protein and a flavoprotein. All of these components were required to reconstitute the 1 1 -/^-hydroxylase activity of the system, and the role of iron-sulfur protein was found to be the transferring of electrons from NADPH-linked flavoprotein to cytochrome P-450. In 1968, constituents of the camphor hydroxylating system were separated and identified as an iron-sulfur protein, a flavoprotein and cytochrome P-450 (Figure II .3) . 15 NAD+ NADH +H+ REDUCTASE CFAD>red REDUCTASE (FAD) PUTIDAREDOXIN (Fe3+) PUTIDAREDOXIN (Fe2+) P-450cam(SH) (Fe2+) P-450cam(SH) (Fe3+) 02 SOH +H20 Figure II.3 Electron transfer reactions in bacterial system leading to camphor (SH) hydroxylation. A major breakthrough towards the understanding of the reaction cycle of cytochrome P-450 occurred when Narasimhulu et al.16, in 1965, observed the "substrate binding spectrum" of the C-21-hydroxylase system of adrenal cortex microsomes. When the substrate, 17-hydroxyprogesterone, was added to a suspension of adrenal cortex microsomes, the Soret peak was observed to shift to 13 a shorter wavelength, and the spectrum returned to the original position upon the addition of NADPH. The substrate-induced spectral change for the soluble cytochrome P-450 from Pseudomonas putida was observed17 also and the association constant for camphor binding was calculated to be (0.47±0. 07) 1 06W1 (Figure II.4). 18 0 5 s B *! 01 0 350 400 450 900 550 600 650 7C0 WAVELENGTH <i>o0 Figure II.4 The substrate-induced spectral changes for cytochrome P-450cam. Final camphor concentrati ons (WM) were, 0 ( ) ; 2 ( 0 ) ; and 6 ( 0 ) ; and 20.5 ( A ),(fromref. 18). The association reaction between P-450 and camphor changes the iron(III) from a low to high spin form as detected from EPR experiments,19 and this in turn results in a significant change in the redox potential of the heme iron.20 Thus, the first step in the reaction of this remarkable biocatalyst system became well understood. The microsomal fractions were shown to react with a wide variety of substrates using the photochemical action spectrum technique,3 this being unusual behavior for a single enzyme species. Also, it was observed that the 14 cytochrome P-450 from microsomes of adrenal cortex and liver had essentially the same CO-difference spectrum, although the substrates were chemically very different.21 However, the explanation of this observation was delayed until later separation and purification of microsomal cytochrome P-450. In 1975, it was shown that rabbit liver microsomal P-450 (P-450LM) purified to electrophoretic homogeniety contained as many as six forms of P-450LMs,22-25 designated according to decreasing mobility in SDS-gel electrophoresis as P-450LM,, P-450LM2 and so on.26 Some of these multiple forms (isozymes) have been characterized27 and found to contain different C- and N-terminal residues and other significant structural di f ferences. 15 11.2 Nature and Cytochrome P-450 All human beings and most other animals on earth are chronically exposed to numerous non-nutrient dietary chemicals, to drugs used in treatment or prevention of disease, and to an ever increasing number of man-made environmental pollutants. In order to sustain life in this potentially hazardous surroundings, nature has played a remarkable role in designing and activating relatively efficient ways of detoxification and excretion of these 'deadly' xenobiotics. Since its discovery in 1958, many forms of cytochrome P-450 have been shown to occur in almost all forms of life, functioning as a highly versatile oxygenating catalyst in diversified biochemical reactions. Cytochrome P-450 is known to be present in yeasts,28 fungi,9 plants,29 bacteria,30 - 31 insects,32 fish, birds, reptiles, amphibians,33 • 3 * and mammals.35 In mammals, cytochrome P-450s are found in microsomes of the liver, kidney, small-intestine, lung, adrenal cortex, skin, testis, placenta and several other tissues,8 and are capable of metabolising drugs, food additives, anaesthetics, petroleum products, pesticides and carcinogens.27 In this context, it is conceivable that the basic function of P-450 monooxygenase system is to convert the lypophylic xenobiotic compounds to more polar metabolites which in turn can be excreted from the body. However, the situation is by no means simple, since a number of cases have been reported in which xenobiotics are 16 converted to more hazardous metabolites.36'37 For example, halogenated benzenes are converted to their arene oxides, which bind to cellular macromolecules causing cell damage, and polycyclic hydrocarbons are converted to true carcinogens (see Figure II.5).38 Hence, it appears that Figure II.5 Benzopyrene (A) and its carcinogenic metabolite 7,8-diol-9,10-epoxide (B). nature has failed to a certain extent in designing these enzyme systems by allowing a wider substrate specificity and thus causing the biotransformation of harmless chemicals to potential carcinogens. The wide distribution of cytochrome P-450-containing monooxygenase systems from mammals to bacteria represents an interesting example of how this enzyme system evolved when the surroundings were changed. All components of the bacterial monooxygenase system exist in a 'primitive' soluble form and the more developed mitochondrial system consists of a soluble reductase component and a membrane-bound P-450; in the microsomes, all components are tightly membrane-bound and hence have acquired enough hydrophobicity to become incorporated into biomembranes. 17 11.3 Structural Considerat ions It is of immense importance to elucidate the structure of the cytochrome P-450 enzymes in order to fully understand the mechanisms of oxygenation and dioxygen reduction so that the biological reactions can be mimicked. In this discussion of the structural aspects of cytochrome P-450, attention will be focused mainly on the bacterial P-450cam enzyme, since it is the most studied and most understood. The heme prosthetic group in P-450 is bound to the polypeptide chain via an acid labile, non covalent linkage and hence the system is classified as a b-type cytochrome.39 The cytochrome P-450 is composed of a single polypeptide chain and a single molecule of protoporphyrinatoiron(111) per molecule of protein.39'"0 The immediate environment of the heme group attracted the attention of many investigators in late 1960's because of the abnormal spectral characteristics observed in substrate binding and CO-complexation. Since then, enough direct and compelling evidence has been gathered to indicate the presence of a sulfur atom in the first coordination sphere of the heme-iron, and the iron sulfur distance has been shown to be consistent with thiolate (RS~) ligation in all four states of the enzyme."1 In 1968, Miyake and Galor"2 were able to show using EPR experiments that the substrate-free ferric enzyme is predominantly in low spin (S=l/2) form. This observation was similarly found 18 in alkyl mercaptide complexes of ferric hemoglobin or ferric myoglobin.*3'** The model studies of Chang and Dolphin"5 and others *6 • "7 were able to mimic the spectrum of carbonylated P-450 using simple iron(II)-porphyrins having mercaptide ion and CO ligation. Also the models showed a high energy, low intensity Soret band around 360 nm in the CO-complex which was not observed in the case of the enzyme CO-complex, due to the use of excess sodium dithionite reductant which obscured the absorptions below 380 nm. However, stoichiometrically reduced enzyme, when complexed with CO, gave the second Soret peak at 365 nm. The occurrence of split Soret bands, or "hyper spectra", are characteristic of iron porphyrins coordinated by ligands which can donate high electron density.*8 The measurement of changes in the proton concentration during the binding of CO,02 and exchange of 02 by CO, indicated that the Fe-S linkage remained intact during the react ions.49 Recent direct evidence from Resonance Raman (RR) spectroscopy50 and extended X-ray absorption fine structure (EXAFS)*1 spectroscopy has confirmed thiolate ligation to heme-iron. As in the case of other low spin ferric hemoproteins, the substrate-free cytochrome P-450 is believed to have a sixth ligand coordinated. A nitrogen or oxygen based residue has been proposed as the best candidate.50 Although the problem still remains after extensive investigations, a water ligand has been implicated from the NMR relaxation 19 rate of water protons51 and electron nuclear double resonance (ENDOR)5 2 studies. The amino acid sequence of cytochrome P-450cam consists of 412 amino acid residues wi^th a calculated molecular weight of 46,820 including heme. Of these residues, 210 are hydrophobic, 98 are polar neutral, and 104 are ionic (54 acidic and 50 basic) amino acids.53 There are eight cysteine residues with four in the NH2-terminal half and four in the COOH-terminal half, and all of them are situated in the interior of the polypeptide chain.53 The above data predict that the conformation of the P-450cam molecule consists of 46% a-helix, 16% 0- pleted sheet, 21 /3-turns and 38% random coil structure.53 Based on the nature and the number of amino acid residues present, it is conceivable that the prosthetic group of the enzyme is surrounded mainly by hydrophobic, non-aromatic amino acids. The hydrophobic pocket thus formed would have a cavity large enough for substrate binding and would also prohibit any amino acid group being close to the site of formation of the extremely powerful oxidizing agent. The three dimensional shape of P-450 molecule, which is under investigation in the laboratory of I.C.Gunsalus, has been described as a "donut with a tail".5" 20 11.4 The Mechanism of Catalysis 11. 4 .1 Types of Oxidation Reactions Although the major interest lies in cytochrome P-450 enzymes that activate rather inert C-H bonds, the diversity of substrates and the variety of transformations that the enzyme can execute deserves a brief discussion. The mechanistic cycle will be described with particular reference to C-H hydroxylating systems, although with minor modifications it can be applied to other types of reactions as well. Guengerich and MacDonald55 classify the oxidative reactions of cytochrome P-450 into six categories. (1). Carbon hydroxylation: The formation of an alcohol at a methyl, methylene or methine position. Eg. (2). Heteroatom release; The oxidative cleavage of the heteroatomic portion of a molecule. The molecule is hydroxylated adjacent to the heteroatom and the geminal hydroxy heteroatom intermediate thus formed loses the heteroatom to form a carbonyl compound. Eg. CH3CH2CH2CH2Br > CH3CH2CH2CHBr(OH) > CH3CH2CH2CHO 21 (3) . Heteroatom oxyqenat ion: Conversion of a heteroatom- containing substrate to its corresponding heteroatom oxide. Eg. Me3N > Me3N=0 (4) . Epoxidation: Formation of an oxirane derivative from olefins and aromatic compounds. Eg. (5) . Oxidative group transfer: 1,2-Carbon shift of a group with concomitant incorporation of oxygen as a carbonyl at C-1 position. Eg. C12C=CHC1 > CCI3CHO (6) . Olefinic suicide destruction; Inactivation of the heme of cytochrome P-450 by an enzyme product or an enzyme intermediate. The common feature in all of these reactions is that one atom of oxygen, from the reductive scission of dioxygen, is being inserted into the substrate while the other is reduced to a water molecule. After consideration 22 of the observed stoichiometry, the results of labelled-oxygen experiments,the regioselectivity of the hydroxylation, and the number of electrons required from the reductase , a scheme has been constructed to illustrate the various steps of the oxygenase reaction cycle (Figure II.6).56 Figure 11.6 The P-450 catalytic cycle. 24 II.4.2 The Catalytic Cycle As in any enzymatic catalysis, the first step in the reaction cycle is the formation of an enzyme-substrate complex. This is followed by reduction of iron(III) to iron(II) which allows for dioxygen binding. A second reduction, that induces the splitting of dioxygen, forms the "active oxygen complex" and a molecule of water. Then the 'active oxygen' is inserted into a carbon-hydrogen bond, forming the alcohol product, which in turn is released as the enzyme is returned to the original ferric state. II.4.2.1 Binding of Substrate The associated change in the conformation due to the binding of the substrate to the enzyme causes the spin state of the iron(III) to shift from low spin (S=l/2), to high spin,(S=5/2).19 This change has been observed by EPR spectroscopy19 and by the measurement of the magnetic susceptibility.18 The binding process proceeds at a high rate with a second order rate constant of 3. 7x 106M"1 s"1 at 8°.57 The equilibrium constant in the presence of potassium ions has been estimated to be 4.7x105M ~1 .18 The main bonding forces are believed to be hydrophobic in nature, although there may be some dipole interactions between the substrate and the enzyme. This type of bonding is largely responsible for the observed increase in entropy on transfer of the lipophilic substrate from the aqueous phase into the hydrophobic environment of the active center.58 25 Although the precise location of the substrate is still unknown, inhibition experiments show the bonding site of camphor to be in the immediate vicinity of the oxygen bonding site.59 The process of substrate binding could proceed as indicated in Figure II.7. The most significant change upon substrate binding is a shift in redox potential of the heme iron. The substrate-free cytochrome (low spin) has a reduction potential (E°) of -270 mV while that of substrate-bound enzyme (high spin) is -170 mV.20 Since the E° of putidaredoxin is -240 mV, the spin change in P-450 facilitates reduction by putidaredoxin, thereby enabling the successive binding of dioxygen. First Reduction The next step in the reaction cycle is the transfer of an electron to the cytochrome. Despite the considerable effort devoted to study the nature of this reaction, some questions still remain unanswered. Stopped-flow spectrophotometric studies60 indicate this reduction to be Figure II.7 Hypothetical scheme for the binding of substrates to cytochrome P-450. (from ref. 58). 26 first order biphasic, but no rationale for this behavior has been presented. The rate constants calculated60 indicate this step is not rate-limiting. Although the exact mechanism of electron channeling from putidaredoxin to the iron(lll) center is not known, the putidaredoxin is thought to bind to a highly positively charged region of the P-450 protein, between the 340 and 412 positions from the NH2 terminal end. II.4.2.3 Binding of Dioxygen The association reaction of the reduced substrate-bound enzyme with dioxygen, to form the ferrous-dioxygen complex, has been well established, owing to the fact that the complex is fairly stable at low temperatures.27,61 The formation of oxy-cytochrome P-450cam, as determined from the stopped-flow technique, is first-order with respect t.o both dioxygen and the reduced enzyme, the second-order rate constant being 7.7x10s M"1 s'1 at 4° and pH 7.4.62 The dioxygen complex thus formed slowly decays even at low temperatures to give the oxidized enzyme. This process, known as the "autoxidation", and the structure and stability of dioxygen adduct, will be discussed in detail in Section II.6. The substrate-free enzyme-dioxygen complex undergoes the autoxidation reaction 102 times faster than the substrate-bound enzyme-dioxygen complex.30 This indicates that the oxy-cytochrome P-450cam formed in the presence of camphor is not a simple adduct of dioxygen but a ternary complex of dioxygen,enzyme and camphor. 27 Second Reduction The ability to by-pass the second, third and fourth steps (Figure II.6) of the enzymatic cycle by adding hydrogen peroxide to oxidized enzyme, and still obtain the hydroxylated substrate without any external electron supply, suggests that the nature of the two-electron reduced enzyme-oxo complex to be Fe(III)-02~2 in nature.63 In fact, the model studies indicate that the structure of this species may be high-spin ferric ji2-peroxide.6 * - 6 5 Unfortunately, the model Fe(III)-peroxo complexes, produced either synthetically 64 or electrochemically,65 do not exibit the oxidizing power of the enzyme system possibly because of the absence of thiolate ligand.65 The rate of the second reduction agrees well with observed turn-over number for camphor hydroxylation, and hence appears to be the rate determining step in the catalytic cycle.66,67 11. 4 . 2. 5 S'plitting of the Oxygen-Oxygen Bond The step, where the bond between the two oxygen atoms is cleaved heterolytically, is least understood of the entire mechanistic scheme and has been the subject of much speculation. The conversion of the iron-peroxide complex to the "active oxygen" intermediate involves protonation, followed by heterolytic cleavage of the 0-0 bond with the production of a molecule of water. The remaining oxygen atom stays coordinated to the heme-iron. The overall charge on this two-atom unit, neglecting the contributions of the 28 porphyrin and thiolate, becomes +3, leading to designations such as [Fe(lII )-0]3+, or [ Fe (IV)-0-]3+, or [ Fe (V) -02" ]3+. Evidence has been gathered to demonstrate the occurrence of this "oxenoid" type intermediate by using ferric porphyrins with single oxygen atom donors like iodosylbenzene ,C6H5I-0 68 and peracids 27>69 to effect hydroxylations similar to that of the native P-450,NADPH,02 system. It has been suggested that the presence of thiolate ligand may facilitate the splitting of the peroxide moiety by weakening it through charge repulsion by an electron-rich iron atom.70 But no definitive explanation has been given to the fact that thiolate is not a required ligand for catalase and peroxidase, which undergo similar peroxy-iron intermediates in their reaction cycles. Oxidation of Substrate Since cytochrome P-450 is the only hemoprotein capable of hydroxylating an alkane at an unactivated C-H bond, the electrophi-lic iron-oxenoid species and its reaction with substrate are especially interesting. If the cleavage of a C-H bond is a necessary step in the hydroxylation reaction, then a substrate free-radical would be the lowest energy intermediate that could exist in the strongly hydrophobic active site; any ionic species would be energetically unfavourable. Indeed, isotope effects, and regio- and stereo-selectivity of these reactions, all point towards such a mechanism.27-71'72 Two elegant examples, that account for the existence of an 29 uncharged substrate intermediate have been discussed by Groves et al.73 and Dolphin et al.74 A plausible mechanism of formation and hydroxylation of the substrate radical is outlined in Figure II.8.71 >R:cr,H < >VZH •9: ROH + Fe(lll)P F«(lW)P Figure II.8 Proposed mechanism for the formation and hydroxylation of substrate (RH) radical. The abstraction of a hydrogen atom from substrate RH by the active iron-oxene complex forms an Fe(IV) hydroxide species and a substrate free-radical, R*; this in turn accepts the iron-bound hydroxy radical to form R-OH and regenerates Fe(lII) enzyme ready for the next reaction cycle. Dissociation of Product The hydroxylated product apparently dissociates before a new cycle begins, since diols are not observed in alkane hydroxylations. The hydroxyl group of the product alcohol may be coordinated to iron, but this would make the reduction of the iron(III) center more difficult and also, even if the iron is reduced, would prohibit 02 binding. • « Fe(llf )p+* A 30 Thermodynamically, the binding of a polar molecule will be less favourable and another substrate molecule that is in abundance in the surroundings will be preferred; the high spin Fe(111)-substrate complex formed yields the way for another reaction cycle. 31 11.5 Electronic Spectroscopy Electronic absorption spectroscopy has been used extensively to study the cytochrome P-450 system in relation to molecular structure, geometry, electronic configuration and oxidation state. Other spectroscopic techniques such as electron paramagnetic resonance (EPR), Mossbauer, circular dichroism (CD), magnetic circular dichroism (MCD), nuclear magnetic resonance (NMR) and resonance Raman (RR) have been used to confirm unambiguously the results obtained from electronic spectroscopy. Also, heavy reliance has been placed on comparisons with other known hemoproteins and low molecular weight iron-porphyrin complexes. The P-450 hemoproteins from all known sources are remarkably similar in electronic absorption spectral properties.27 The substrate-free ,ferric ("resting") state of the enzyme is typical of low spin ferric hemeproteins, with a Soret band at 416 nm and well defined a and /3 bands. Upon substrate binding, the hexacoordinate low spin form changes to pentacoordinate high spin form with a shift of the Soret band to 391 nm and disappearance of distinct bands in 500-600 nm region; however, a new low energy,low intensity band at 643 nm is formed. On reducing the substrate-free species in the absence of potential ligands such as CO or 02, the Soret band moves to 408 nm and the a and 0 bands collapse forming a new peak at 542 nm. However, the most striking feature is observed when the reduced 32 enzyme is exposed to a CO atmosphere when a sharp, intense peak appears at 446 nm, an unusually high wavelength for a hemoprotein. The low energy band of the CO complex is observed at 540 nm with about the same intensity as the peak of the reduced species at 542 nm. The spectrum of the dioxygen complex is comprised of a Soret band at 418 nm and a low energy band at 552 nm. The small separation between Soret peaks of the reduced (408 nm) and 02-complex (418 nm) created difficulties in studying the dioxygen binding as a function of gas pressure in efforts to determine the equilibrium constant (see Chapter IV). Normal porphyrin spectra show three sets of bands classified according to orbital symmetry considerations. The bands appear in the visible, Soret and near UV regions."8 These peaks are due to the well characterized 7r-7r* transitions. But some porphyrin-metal complexes show "hyper" spectra, with strong extra bands in the 300-800 nm region, a' common spectral pattern exibited by a number of hyper porphyrins bearing two Soret bands (split Soret), one in the 380 nm region and the other in the 440-480 nm region. The CO and 02 complexes of reduced P-450 species also exibit this feature. As demonstrated by the model studies"5-"7 the presence of thiolate in an axial coordination position is required to mimic the P-450 spectrum, including the split Soret bands. Hansen et al."B using iterative extended Huckel (IEH) calculations were able to show that the split Soret bands arise from the 33 strong interaction of charge transfer transitions (from thiolate p- orbitals to porphyrin 7r*-orbitals) with the porphyrin TT-VT* transitions. 34 11.6 Interaction of Cytochrome P-450 with Carbon Monoxide  and Dioxygen The observation that the addition of carbon monoxide to sodium dithionite-treated liver microsomes resulted in arv unusual Soret peak led to the discovery of cytochrome P-450 (see Section II. 1) . Also, the biological function of this monooxygenase enzyme was determined by a study of inhibition of C-21-hydroxylase reaction of adrenal cortex microsomes via photochemical action spectrum of the CO complex.3 Since these findings, many studies on CO and 02 binding to reduced, native and reconstituted P-450 enzyme and to its model compounds have been reported. 6 1 -7 5 - 8 0 The information gathered from such studies will be discussed and compared to the other heme-containing biomolecules such as hemoglobin (Hb), and myoglobin (Mb), which have been thoroughly studied and are well understood. Although Hb and Mb contain the same prosthetic group as P-450, protohemin, they differ in many ways in their structure and properties. The difference in axial ligation (see Figure II.9) must play an important role.81 It is interesting to note that Hb and Mb do not undergo a change in the oxidation state of the prosthetic groups in any stage of their normal biological functions. In the case of P-450 and Mb, where only one heme unit per molecule interacts, the reaction of CO with the reduced heme unit is represented by reaction II.2, Figure II.9 Heme environments of myoglobin (A), hemoglobin (B), and peroxidase (C). (from ref. 81). 36 Fe(II)+CO ^ ^ Fe(II)-CO, Kco=1on/'bf f <JI«2) where Fe(II) is the reduced heme unit, K co is the equilibrium constant, and kQn and kDff are association and dissociation rate constants, respectively. The ligand association reaction is an over all second order process, while the dissociation reaction is first order. 8 2.8 3 The kinetic and equilibrium data for CO binding to P-450 and related systems are given in Tables II.1 and II.2, respectively. 37 Table II.1 Kinetic Data for CO Binding to Cytochrome P-450 and Related Systems. System Method PH T°C kon (M-'s-1 ) koff (s-1 ) Ref . P-450cam SF 7.4 4 5. 1x10s 2.3 77 (cam.-free) FP 7.4 5 2.7x10s 1 .2 82 1 5 4.7x106 25 8.4x106 P-450cam SF 7.4 4 3.8x10" 77 (cam.-bound) SF 7.0 20 3.6x10s 2.8 84 SF 7.0 24 2.2x10s 1 .7 75 FP 7.0 5 8x1 0" 82 15 1.3x105 25 2.3x105 FP 7.3 12 3.5x10" 85 Microsomal FP 7.4 4 3.4x105 0.068 13 P-450 SF 7.5 4 4.5x10s 0.63 88 Horse Mb FP 7.0 20 5x1 05 0.02 83 FP 7.0 25 3.8x10s 86 P-450 Model FP 23 1 . 1x10s 18 87 SF=stopped-flow, FP=flash photolysis 38 Table II.2 Equilibrium Data for CO Binding to Cytochrome P-450 and Related Systems. System PH T°C KC0(M-1) AH0 (kcal /mol) As0 (cal /mol .deg) Ref. P-450cam 7.4 4 2.2x106 77 (cam.-free) 7.0 24 1 .7X105 75 P-450cam 7.4 4 2.6x105 77 (cam.-bound) 7.0 12 2.6x10s -12 -17 75 7.0 24 1 . 3x105 75 P-450 Model 23 1.1x10" -16 -35 87 Horse Mb 7.0 20 2.9x107 -12.6 -7.4 83 39 The CO and 02 complexes of P-450, Hb and Mb are diamagnetic.89 The metal-carbon bond-order derived from the relationship with the C-0 stretching frequency indicates that carbon monoxide is involved in both a and u—type molecular orbitals of the complex.83 The sp-hybridized lone pair of the carbon atom is involved in 0=C—> Fe type x bonding, while the two pit orbitals overlap with two dir orbitals of iron. The Pauling valence bond structures of the CO complex are given in Figure 11.10. N N N N . \/ -\/ Protein Fe«—CsQ: Protein Fe=C = 0 N N N N I H Figure 11.10 Pauling valence bond structures of carbon monoxide complex of cytochrome P-450. Although CO is thought to bind in a linear fashion to transition metals , it can be attached at an angle in a heme protein to minimize the steric hindrance . In the chironomous hemoglobin CO complex the Fe-C-0 angle is 145±150.83 This observation provides a basis for the explanation of the lower affinity of substrate-bound P-450 towards CO, compared to the substrate-free system (see Table II.2). The camphor molecule bound in the vicinity of the heme moiety77 is considered not only to restrict the access of the CO molecule to the heme unit, but also to have a destabilizing effect on the heme-CO complex once it is formed. However, at 24°, the equilibrium constant value for the substrate-free system, reported by Dolphin et al75, 40 approaches that for the substrate-bound system. In order to understand better the effect of the substrate on CO binding to P-450, the necessity of more studies has been mentioned.87'90 More detailed interpretation of the substrate effect on CO binding can be provided by evaluation of the enthalpy and entropy contributions to the binding constant values at different temperatures. The binding of CO to substrate-bound P-450 is about 200 times weaker than that for Mb (see Table II.2). This difference in affinity towards ligand binding probably reflects the effect of the proximal ligand, imidazole in Mb, and thiolate in P-450. The thiolate anion coordinated to heme may produce an electron-rich iron atom, thereby lowering the affinity for the sixth ligand.91 Since imidazole is a weaker a-donor than thiolate, this could account for the difference in binding constants. The thermodynamic parameters, calculated from the .limited data available 75 for the substrate-bound system (Table II.2), indicate that the AH0 value is comparable in magnitude to that of Mb (-12 kcal/mol for P-450; -12.6 kcal/mol for Mb). Binding of a gas molecule to a metal centre would be unfavourable in entropy terms due to the loss of translational and rotational motion of the gas molecule. But the entropy of binding of CO to P-450 is more unfavourable (AS=-17 cal/mol.deg.) than for Mb; (AS°=-7.4 cal/mol.deg.). Such differences in entropy contributions have been rationalized, based on cis- and trans- effects 41 and steric effects due to the presence of protein as well as the substrate.75-77 The AH0 term for the protein free model system (-16 kcal/mol) is more favourable, but the entropy contribution is not so (-35 cal/mol.deg.). The kinetic and equilibrium data available to date for dioxygen binding to cytochrome P-450 are not as extensive as those for CO binding to P-450, or 02 binding to Mb. The principal reason for this is the instability of the P-450-02 complex at ambient temperatures; in the case of substrate-bound enzyme at room temperature and pH 7.0 the autoxidation (Reaction II.3) occurs with a half-life < 1 minute,92 while the rate of autoxidation of the dioxygen complex of the substrate-free enzyme is at least 100 times faster.75 P-450Fe(lI)-02 + H+ > P-450Fe(III) + H02 (II.3) The binding of dioxygen to substrate bound P-450 is at least 6 times weaker than to Mb (Table II.3). This results mainly from a much slower on-rate in the P-450 system; this could result from a geometrically restricted coordination site due to the presence of the substrate,75 although the electronic effects due to the sixth ligand may also play a role. 42 Table II.3 Equilibrium and Kinetic Data for Reversible 02 Binding to Substrate-Bound Cytochrome P-45075 and Mb.93 System P 1/2 (mmHg) Binding Constant (M-1) ^on (M-1s-M koff (S-1 ) P-450 Horse Mb 2.5 (0°C) •52(20°C) 2.2x105 (0°C) 1.3x10s (20°C) 7.7x10s (4°C) 1 .4x 1 07 (20°C) 3.5 (4°C) 10. (20°C) 43 REFERENCES 1. M.Klingenberg, Arch. Biochem. Biophys . , 7J5 , 376(1958). 2. T.Omura, R.Sato, J.Biol.Chem, 237, 1375(1962). 3. R.W.Estabrook, D.Y.Cooper, O.Rosenthal, Biochem.Z., 338 741(1963). 4. D.Y.Cooper, S.S.Levin, S.Narasimhulu, O.Rosenthal, R.W.Estabrook, Science, 147, 400(1965). 5. L.Ernster, S.Orrenius, Fed.Proc, 24, 1190(1965). 6. S.Orrenius, J.L.E.Erricson, L.Ernster, J.Cell Biol., 25, 627(1963). 7. 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J.A.Peterson, B.W.Griffin, Arch.Biochem.Biophys., 151, 427(1972) . 78. C.Bonfils, K.K.Anderson, P.Morrel, P.Debey, J.Mol. Catal., 7, 299(1980). 79. L.Eisenstein, P.Debey, P.Dousou, Biochem, Biophys.Res. Commun., 77, 1377(1977). 80. J.D.Lipscomb, S.G.Sligar, M.J.Namtvedt, I.C.Gunsalus, J.Biol .Chem. , 25J_, 1116( 1976). 81. R.J.P.Williams, G.R.Moore, R.E.White, Biological Aspects of Inorganic Chemistry, (Ed.A.W.Addison, W.R.Cullen, D.Dolphin, B.R.James), Wiley, New York, 1 977. 82. Ref.20, p.559. 83. E.Antonini, M.Brunori, Hemoglobin and Myoglobin in Their Reactions with Ligands, North Holland-American Elsevier, 1971. 84. B.W.Griffin, J.A.Peterson, R.W.Estabrook, The Porphyrins, (Ed. D.Dolphin), VII, Academic Press, New York, 1979, p.333. 85. C.Bonfils, J.L.Saldana, P.Debey, P.Maurel, C.Balney, P.Dousou, Biochimie, 6±, , 681(1979). 86. B.B.Hasinof f, Biochemistry, J_3, 3111 ( 1 974). 87. C.K.Chang, D.Dolphin, Proc.Natl.Acad.Sci., U.S.A., 73, 3338(1976) 88. P.Debey, G.Hui Bon Hoa, P.Dousou, FEBS Lett., 32, 227(1973) 89. L.Pauling, C.D.Corryell, Proc.Natl.Acad.Sci., U.S.A., 22, 159(1936). 90. S.Albon, M.Sc.Thesis, Dept. Chemistry, U.B.C., (I983)pp.74, 79. 91. D.Dolphin, B.R.James, C.H.Welborn, Ref. 25, p.183. 92. S.G.Sligar, J.D.Lipscomb, P.G.Debruner, I.C.Gunsalus, Biochem. Biophys .Res .Commun. , 6J_, 290( 1974). 48 B.R.James, The Porphyrins, (Ed. D.Dolphin), Academic Press, New York, V, 1978, p.205. CHAPTER III EXPERIMENTAL PROCEDURES 50 111,1 Growth of Bacteria  111.1 . 1 General Information The bacteria Pseudomonas putida strain PpG 786 (ATTC 29607) derived from strain PpG 1 (ATTC 17453) by Gunsalus and Wagner1 was used throughout these studies to produce cytochrome P-450. The parent strain PpG 1 was originally isolated from soil by enrichment on D-(+)-camphor. The strain was kept alive by bi-weekly transfer to minimal agar plates containing D-(+)-camphor as carbon and energy source. The four-stage growth procedure for the mass production of bacteria as described by Gunsalus and Wagner1 was used with several minor modifications. This growth procedure is designed to produce maximum amounts of cytochrome P-450 and other monooxygenase components at the expense of camphor and liquid media and has a relatively short period of growth with a generation time of about three hour's. The strain PpG 786 has the ability to release much of the hydroxylase proteins by freeze-thaw autolysis to circumvent more tedious cell breakage techniques. A single freeze-thaw cycle typically liberates about one half of the cytochrome P-450 content as soluble enzyme in the cell-free extract, based on the total amount of enzyme in the whole cell suspension as determined from the ferrous carbon monoxide difference spectrum. The four stage growth procedure, 1. Minimal agar stage 51 2. L-Broth stage 3. 500 mL Shake flask stage 4. 14 L Fermenter stage typically yielded about 500 g of bacteria based on the wet weight in about 96 hours of elapsed time. All inoculations except for 14 L fermenters were carried out in a laminar flow clean-air station to minimize the possibility of contamination. Three agar plates were inoculated each time and the wire loop used to transfer the bacteria between plates was heated until red hot before each use, in the flame of a Bunsen burner. The L-broth and the shake flasks were inoculated and incubated in a platform shaker thermostated to 30°. Three 14 L fermenters (Labroferm; New Brunswick Scientific, N.J, U.S.A) were used in the final stage of growth. The growth medium was made up to 12 L and stirred at 300 rpm with 15 L/min rate of aeration. The temperature was maintained at 30°. The rate of growth of bacteria was determined turbidometrically by measuring the optical density at 660 nm using a Bausch and Lomb spectrometer model Spectronic 20. During the growth of bacteria in 500 mL shake flask and 14 L fermenter stages, the fluctuation of pH was monitored using a Fisher Accumet model 210 pH-meter equipped with a glass pH-electrode. When the optical density of the medium reached a maximum value, the bacteria were collected using a continuous flow centrifuge (Carl Padberg, Schnell-Zentrifuge, Model LE) at 37, 000 rpm. The bacteria were then stored under dry-ice 52 temperature in a Dewar in 500 g batches. Ill . 1 .2 Media Media for all four stages were prepared according to Gunsalus and Wagner1 using reagent grade chemicals and freshly distilled water. All solutions were sterilized at 121° under pressurized steam using an autoclave (American Sterilizer Co. Model AS-DTT616GE). Solutions of volumes less than 200 mL required 20 min autoclaving while 14 L fermenters required 45 min to ensure complete sterilization. The sterilized solutions were used as early as possible to avoid re-contamination. 111 . 1 . 2.1 Minimal Agar In order to store the bacteria alive, minimal agar plates were prepared using the following ingredients. K2HPOa 3.5-g KH2PO« 1.0 g NaCl 0.06 g MgSO«.7H20 0.05 g (NHa)2SO„ 0.5 g D-( + )-camphor 0.68 g Bacto agar 7.5 g Water 540 mL The salts, camphor and agar were dissolved in water, sterilized, cooled, combined and poured into disposable petri dishes obtained from Canlab. The minimal agar dishes 53 were stored at 5°, three of which were used every two weeks to inoculate with the bacteria. 54 III.1.2.2 L-Broth At the initial stage of large scale growth of bacteria, a very rich medium called L-broth2 was used to facilitate the rapid multiplication of the organism. The following ingredients were mixed, poured into five 50 mL Erlenmeyer flasks and sterilized. Bactotriptone 1.0 g Yeast extract 0.5 g NaCl 0.5 g Glucose 0.1 g Water 1 00 mL This solution was brought to pH 7.0 by adding 1M NaOH dropwise before the sterilization. Ill. 1 .2.3. 500 mL Shake Flask At this stage of growth a phosphate-ammonium (PA) buffer solution was used to maintain the desired pH level. Essential mineral salts were supplied from a hundred-fold strong stock solution (100-X salts) and a carbon and energy source was also added to the medium. In order to maximize the production of cytochrome P-450 content, the 500 mL shake flask stage was performed twice , first with glutamic acid as the carbon and energy source and secondly with camphor as the only carbon and energy source. In each case 1 L of medium was prepared using the following ingredients and divided into five 500 mL Erlenmeyer flasks and 55 sterilized. PA buffer 1 L 100-X salts 16 mL Glutamate 2.9 g or Camphor 2.5 g PA buffer and 100-X salts were prepared by mixing following ingredients. PA buffer: K2HPO, 10.7 g KH2PO« 3.1 g NH«C1 4.0 g Water 1 L 100-X salts: MgSO,.7H20 19.5 g MnSO,.H20 5.0 g FeSO«.7H20 5.0 g CaCl2.2H20 0.3 g L-Ascorbic acid 1.0 g Water 1 L 56 III. 1.2.4 14 L Fermenter The final stage of growth was carried out in a somewhat similar medium to the 500 mL shake flask stage, but it was essential to keep the medium always saturated with camphor during the whole period of growth. The following chemicals were added in each fermenter. K2HPOfl 128.2 g KH2P0„ 36.3 g NH,C1 46.8 g 100-X salts 120 mL Bacto yeast extract 3.0 g Antifoam 1 .0 mL Camphor in DMF (3M) 30 mL Water 1 1 .6 L 57 111.1.3 Growth Procedure Four of the five Erlenmeyer flasks, each containing 20 mL of L-broth medium, were inoculated with the bacteria and shaken at 30° for 10 hours. The fifth flask served as a reference. At the end of the 10 hour period it was noted that the flasks that were inoculated turned turbid, while the reference flask remained clear. On swirling the inoculated flasks, clouds of bacteria could be seen. The third stage of growth was started by inoculating each of the first set of four 500 mL shake flasks with 5 mL of L-broth culture. The fifth flask served as a reference. Each of these flasks contained 200 mL of basal PA buffer , 3.2 mL of 100-X salts and 10 mM glutamate as the carbon and energy source. All five flasks were shaken at 30° until the optical density at 660 nm (OD660) reached 0.3. At this time the induction of camphor 5-monooxygenase system was initiated by the addition of stock camphor solution (3M in DMF) to a final concentration of 5 mM. The OD66o and the pH of one of the inoculated flasks and the reference flask were taken every hour in order to monitor the growth and the metabolism of camphor. Addition of camphor was continued at OD660 0.7, 1.0 and once every three hours thereupon until the growth reaches its late-logrithmic phase (see Figure III.l). At this time the second set .of shake flasks with camphor as the only carbon and energy source was inoculated with 10 mL each of this solution. For this inoculation the culture medium was used only from the 58 59 three shake flasks that were left undisturbed while shaking at 30°. The flask that was used to withdraw aliquots for pH and optical density measurements was never used as an inoculum, in order to avoid any possibility of contamination. The flasks were heated around the neck before opening, by rotating on the flame of a Bunsen burner whenever they were opened either for addition of camphor or for withdrawing aliquots. The needle of the 1 mL disposable syringe that was used to measure the camphor solution was heated to red hot before use. Pre-sterilized 2 mL Pasture pipettes were used to withdraw aliquots from the flasks. The initial rate of growth in the presence of camphor as the only carbon and energy source was smaller compared to that in the presence of glutamate (see Figure III.2), but the optical density reached similar values after about 24 hours. When the growth reached its late-logarithmic phase (about 14 hours), the contents of the three undisturbed flasks were used to inoculate 14 L fermenters. The bacteria were allowed to grow under continuous agitation and aeration (see Section III. 1.1) until the OD6 6 0 reached 0.6 (see Figure III.3), at which time the feeding with camphor was started. A solution of camphor in DMF (3M) as well as solid camphor was used at a rate of 15 mL/h or 6.7 g/h, respectively. The use of a solution of camphor was preferred towards late stages of growth since the solid camphor tended to clog the continuous flow centrifuge. However , the use of solid camphor helped to reduce the T i i 1 1 1 1 1 1 1 1 1 r inoculum for the 14 L fermenters Time (h) Figure III. 2 O.IXggn and pH profiles for the bacterial growth in the second set of shake-flasks. 61 to in to to r-' to tn to to to' I I i 1 r i 1 1 1 r readjusted to 7.4 ' ' I L J L to l_ o O — CO 3rd harvest J I I L J I I L 0.0 16.0 32.0 48.0 64.0 80.0 96.0 Time Ch) Figure III.3 O.UggQ and pH profiles for the bacterial growth in the 14 L fermenters. 112.0 62 amount of DMF added to the media. It is likely that an excess of DMF may interfere witlr the growth. The centrifugation was carried out when the OD660 reached about 1.2 (about 24 hours) and about 150 g of bacteria had collected. The growth was continued until the optical density again reached 1.2 (about 24 hours from last harvest), at which time the centrifugation was carried out again. The pH of the medium drops below 6.0 at this time and enough potassium hydrogen phosphate (K2HPOi,) was added to the medium to bring the pH value to 7.4. Another 120 mL of 100-X salts was added also in order to replenish the medium. The final centrifugation was carried out again when the optical density reached 1.2. The total wet weight of the bacteria collected was 450-500 g. The total growth procedure takes about 5.5 days. The paste of bacteria was stored under dry-ice (-78°) for at least 10 days before being used to isolate cytochrome P-450. 63 III .2 Isolation and Purification of Cytochrome P-450 111.2.1 General Informat ion The freeze-thaw autolized cells of bacteria Pseudomonas putida strain 786 (see Section III.1.3) were used to isolate cytochrome P-450. The method of Gunsalus and Wagner1 was used with some minor modifications. Basically, the isolation procedure involves removal of cell debris by centrifugation of stirred cell suspension in a buffer solution, and separating cytochrome P-450 from other monooxygenase components by a two-step anion exchange column chromatography procedure. The purification of isolated crude cytochrome P-450 was done by a two-step gel filtration column chromatography procedure. Once isolated and purified, P-450 samples can be stored frozen in liquid nitrogen (-196°) or can be kept as a liquid in the refrigerator (5°). The camphor substrate was removed by another two-step gel filtration chromatographic procedure. 111.2.2 Materials The chemicals and sources were as follows. Diethylaminomethyl cellulose (DE 52) from Whatman Chemical Co.; Sephadex G-10 and G-100 from Pharmacia Fine Chemicals; Enzyme grade ammonium sulphate from Schwarz/Mann Chemical Co.; 0-mercaptoethanol (0ME), deoxyribonuclease 1 (DNase) isolated from bovine pancrease, ribonuclease-A (RNase) isolated from bovine pancrease, dithiothreitol (DTT) and 64 t.r is-(hydroxymethyl )aminomethane (Trizma Base) from Sigma Chemical Co.; potassium chloride from American Scientific Chemical Co.. Ill .2.3. Buffer Solutions All buffer solutions were prepared from glass-distilled water, deaerated by repeated aspiration followed by flushing with argon. The argon was passed through a column of copper catalyst pellets (BASF Chemical Co.) heated to 50° to remove trace dioxygen, followed by drying through phosphorus pentoxide and Indicating Drierite (Hammond Drierite Co.). The Tris.CI buffer solutions were prepared from a 1.0M stock Trizma Base (see Section III.2.2.) solution by diluting to 50 mM with distilled water followed by addition of 6M HCl to obtain a pH of 7.4. Four buffer solutions (4 L of each) were prepared with different ionic strengths by adding 0, 50, 100 and 600 mM KC1 to 50 mM Tris.CI buffer. For simplified nomenclature these buffers are specified as T, T-50, T-100, T-600, respectively. Prior to use, all the buffer solutions were made 10 mM with 0ME . The phosphate buffer solutions were prepared by mixing appropriate volumes of 1 M K2HPOi, and 1 M KH2PO« stock solutions to obtain a pH value of 7.4, when diluted to a 50 mM solution. The 50 mM phosphate buffer solution (P-buffer) was made 100 mM with KC1 to produce buffer P-100. Solid camphor was added to appropriate buffers that were stirred for about 6 hours to obtain a saturated (8 mM) 65 solution. The approximate volumes of buffer solutions used in a typical preparation of cytochrome P-450 from about 400 g of bacteria are listed in Table 111. 1 . The quantities of reagents used are listed in Table III.2. 66 Table III.1 Volumes of Buffer Solutions Required for a Typical Isolation and Purification of Cytochrome P-450. Step Buffer Volume Cell-Free Extract T 0.6 L Anion Exchange Chromatography (DE52) (a) Equilibration of column A T 4.0 L (b) Equilibration of column B T-50 4.0 L (c) Separation of protein components T-1 00 4.0 L T-600 4.0 L Gel- Filtration (Sephadex G-100) (a) Column A T-1 00 1 .0 L - (1OOmM DTT) (b) Column B P-1 00 1 .0 L (8 mM Cam.) 67 Table III.2 Quantities of Reagents Required for a Typical Isolation and Purification of Cytochrome P-450. Reagent Quantity Trizma base, 1M 800 mL KH2P04, 1M 20 mL K2HP04, 1M 35 mL KC1 250 g (NH4)2 S04 100 g NH40H, 15M 2 mL B ME 1 0 mL DTT 0.2 g DNase 1 .0 mg RNase 1 .0 mg 68 111.2. 4 Column Chromatography Since the success of isolation and purification of cytochrome P-450 very much depend on the precision exercised in the preparation of chromatographic resins and operation of the chromatography columns, the manufacturer's recommendations were followed with special care. The columns for DE-52 anion exchange chromatography were manufactured by Bio-Rad Laboratories (Model Bio-Rex) and the columns for Sephadex gel filtration column chromatography were constructed in the mechanical shop of the Chemistry Department, U.B.C. The ends of the DE-52 columns were fitted with porous polyethylene and 100 mesh nylon inserts. The ends of Sephadex columns contained only 100 mesh nylon inserts. Both DE-52 columns were equilibrated in the descending flow mode using a peristaltic pump (Buchler Instruments) to achieve constant flow rates. The Sephadex columns were equilibrated under gravity feed. The enzyme samples were collected with a LKB Broma, 7000 Ultrarac fraction collector, and the enzyme samples were concentrated using an Amicon ultrafiltration apparatus fitted with a YM-10 Diaflow ultrafiltration membrane (10, 000 molecular weight cut-off). 69 111.2.5. Isolation of Cytochrome P-450  III .2.5.1. Cell-Free Extract About 400 g of bacteria, which had been frozen for at least two weeks in dry-ice, were thawed at room temperature and made into a creamy paste with 320 mL of T-buffer. This suspension was then stirred in the cold room (5°) for 8 hours at which time 2 mg each of DNase 1 and RNase A were added, and the stirring continued overnight (ca. 12 h) at 5°. Another 120 mL of T-buffer were then added and the mixture centrifuged at 10,000 rpm for 15 minutes in a Sorvall RC-2 refrigerated centrifuge equipped with a GSA rotor. This removed most of the cell debris from the enzyme extract. The resultant cloudy liquid was again centrifuged on a SS-34 rotor at 15,000 rpm for 30 minutes to obtain a clear pink coloured solution. This cell-free extract was kept on ice at all times under an argon atmosphere. Ill .2.5.2.' Separation of Cytochrome P-450 Separation of cytochrome P-450 from the other monooxygenase components was carried out at 25°, unless otherwise specified, using a two-step anion exchange column chromatographic procedure. The cell-free extract derived from about 400 g of bacteria was applied in the descending mode at a flow rate of 180 mL/h to a DE-52 anion exchange column (150 g, 5x15 cm) previously equilibrated with buffer T (see Table II 1.1). This column was then washed with buffer T-100 for 0.5h and the effluent discarded. 70 Subsequent effluent from this column was then applied in the ascending mode to a second DE-52 anion exchange column (300 g, 5x30 cm) equilibrated with buffer T-50. The elution with buffer T-100 continued until the three enzyme bands were well separated. At this time the yellow putidaredoxin reductase band was running in front of the pink cytochrome P-450 band which was followed by the brown putidaredoxin band. At this point a linear salt gradient from T-100 to T-600 was started in order to further separate yellow and pink bands from the brown band. The first DE-52 column was disconnected when the yellow and pink bands entered the second column, and the elution of second column was continued. The yellow band which eluted first was discarded and the pink cytochrome P-450 band was passed through a 6 m coil of small diameter tubing immersed in ice prior to collection at 5°. The total time required for running the two columns was about 14 hours. Ammoniurn Sulphate Fractionation The crude enzyme solution from the second DE-52 column was made 1mM with D-(+)-camphor and fractionated with ammonium sulphate (enzyme grade) by adding 200 g/L (36% saturation) over a period of 30 minutes. The mixture was kept on ice at all times under an argon atmosphere with gentle stirring. To maintain a constant pH value, 1 mL of concentrated NH„OH was slowly added to the mixture for each 30 g of (NHjJzSO,, added. The mixture was stirred for 71 another 30 minutes and centrifuged at 10, 000 rpm for 10 minutes; the resulting white apo-cytochrome P-450 precipitate was discarded. The supernatant was precipitated by adding 140 g/L of (NH„)2SO» (60% saturation) using the same procedure. The precipitate was collected by centrifugation and the yellow supernatant discarded. The precipitate was resuspended in a minimum volume of buffer p-100 containing 8 mM camphor. This crude enzyme solution was stored under argon at 5° in a Schlenk tube until purified by gel filtration chromatography. 72 III.2.6. Gel-Filtration Chromatography Purification of Cytochrome P-450 The crude enzyme samples were made 50 mM with DTT and incubated for 30 minutes at room temperature prior to purification by two Sephadex G-100 (particle size 40-120 micron) columns equilibrated with T-100 (10 mM DTT) and P-100 (8 mM camphor) buffers, respectively. All enzyme samples were concentrated to 1-2 mL (ca. 0.5 mM) aliquots by ultra-filtration before applying to the columns. The enzyme solution from 400 g of bacteria was divided into two portions and purified separately in order to avoid overloading the columns. Both columns were equilibrated and run at 5° under gravity flow with a head-pressure less than 200 mm. The fractions from the first column were selected by inspecting the colour density so that about 60-70% of the pink enzyme elute was included; after being concentrated to about 2 mL those fractions were then applied t-o the second column. The middle fractions of this column contained the most pure enzyme samples which were spectroscopically analyzed. The usual yield calculated from a molecular weight of 45,000 Daltons amounted to about 135 mg of protein (ca. 3 mL of 1 mM solution) isolated from 400 g of bacteria. Removal of Camphor Substrate The camphor substrate was removed by another two-step gel-filtration column chromatographic procedure using Sephadex G-10 at 5°. Pure cytochrome P-450 solutions were 73 made 50 mM with DTT and incubated at room temperature for 30 minutes. Camphor was then removed by gel-filtration on a Sephadex G-10 column (1.Ox 20 cm) equilibrated with T-100 buffer containing 1OmM DTT. Only about 1.0 mL of enzyme solution was used for each run and the head-pressure was adjusted so that the column ran over a 24 hour period. The effluent enzyme solution was then chromatographed on a second Sephadex G-10 column equilibrated with buffer P-100 in order to effect a buffer exchange and to remove the excess thiol reagent. During the removal of camphor, special care was taken to avoid the glassware and 'tubing which came in contact with camphor, as this sometimes led to the isolation of partly substrate-bound enzyme. The enzyme samples prepared in this manner demonstrated no apparent residual bound camphor, as indicated by the absence of an optical absorption shoulder at 391 nm in the presence of 50 mM potassium ion (see Section III.2.7.). 74 111.2.7 . Spectral Analysis The purity of cytochrome P-450 samples can be estimated conveniently by correlating the extinction coefficient of the electronic absorption maximum of the heme protein with the electrophoretic mobility in sodium dodecyl sulphate (SDS) gels.1 The analytical SDS-tube gel electrophoresis by the integration of densiometric recordings from an automatic gel scanner using the method of Weber and Osborn,3 combined with the optical absorption spectral data of the heme protein, indicate that crystalline cytochrome P-450 (greater than 99% homogeneous by the SDS-tube gel electrophoresis) is characterized by an optical absorption ratio of the peak at 391 nm to the peak at 280 nm of 1.63. 1 Purified but non crystallized samples greater than 95% homogeneous have an absorption ratio, A3 91:A2 so or 1.45. Electronic absorption spectra were recorded from 250-700 nm in a Cary spectrophotometer model 17D at room temperature, a special cell tonometer (Figure III.4) designed to handle enzyme solutions anaerobically being used. The cell tonometer consisted of a clear rectangular quartz cell (1x1x5 cm)attached to a sidearm reservoir (10 mL) through glass tubing (20 cm) containing a silicon rubber septum fitting. A high- vacuum O-ring joint (Kontes Glass Co.) with a B-14 sidearm socket was utilized for attachment to the vacuum-gas handling line. The total volume of the cell tonometer was 60 mL. 1 cm quartz cell gure III.4 The cell-tonometer. 76 Previously degassed. P-100 buffer (4.8 mL) was transferred to the sidearm reservoir and repeatedly flushed with argon and evacuated three times. Stock enzyme solution (ca.0.2 mL) was added to the reservoir, mixed by swirling and then transferred to the cell cavity by tilting the tonometer. The optical absorption spectrum of the ferric state of the enzyme was then recorded. Stoichiometric reduction of the enzyme to the ferrous state was carried out by adding small amounts of a sodium dithionite solution (ca. 4 mg/mL in degassed buffer P-100) through the rubber septum using a syringe, until a small absorption peak appeared at 314 nm due to excess dithionite. Any excess sodium dithionite was removed by adding small amounts of dioxygen via a gas-tight syringe; the optical spectrum of reduced enzyme was then recorded. The carbon monoxide complex of the reduced enzyme was conveniently formed by gently bubbling the gas through the solution for 30 seconds at room temperature. 77 111.3 Determinat ion of the Equilibrium Constant for the  Reaction of Carbon Monoxide with Substrate-free Cytochrome  P-450 at Pifferent Temperatures 111 . 3 . 1 General Informat ion The equilibrium constant for the reaction of carbon monoxide with reduced substrate-free cytochrome P-450 at a given temperature can be measured conveniently by observing the electronic absorption spectral changes as a function of gas pressure. The procedure for measuring the spectral changes as a function of gas pressure and determining the equilibrium constant via Hill log/log plots has been described previously." • 5 In order to allow for the addition of small partial pressures of CO gas (ca.0.8 mm Hg), a vacuum-gas handling apparatus was constructed with an incorporated di-n-butylphthalate (DBT) manometer instead of the usual mercury manometer.. Partial pressures of CO in mm DBT were converted to mm Hg for data analysis using the formula, mm DBT 7.73X10~2 = mm Hg, derived using density data. 111 . 3.2 Procedure All buffer solutions were freeze-thaw degassed and argon-saturated before use. About 4.8 mL of camphor-free P-100 buffer was added to the optical cell tonometer and the system evacuated and refilled with argon three times. The cell was then transferred to the thermostated cell compartment of the Cary 17D and the base-line spectrum 78 recorded from 250-700 nm. For thermostating , two Haake model FK constant temperature baths were utilized, one connected to the special cell compartment of the spectrophotometer and the other at the CO-gas handling line. Before the begining of each experiment, the constant temperature baths were brought to the required temperature (±0.2°) and allowed to equilibrate at the temperature for at least 30 minutes . The cell tonometer was again attached to the argon line and about 0.2 mL of stock substrate-free enzyme solution added anerobically with gentle swirling. At all times, care was taken not to form bubbles in the enzyme solution as this can lead to denaturation of 'the protein. Then the cell tonometer was again placed in the thermostated cell compartment and the spectrum of oxidized substrate-free enzyme recorded. The stoichiometric reduction of the enzyme was then carried out as described in Section III.2.7 and the spectrum recorded. The cell tonometer ' was then taken to the the CO-gas handling line and thermostated to the desired temperature by immersion in a Dewar full of water containing a spiral copper coil attached to the water circulation port of the constant temperature bath. Two thermometers were used to measure the temperature (±0.2°), one at the CO-line and the other at the spectrophotometer.The cell tonometer was then slowly evacuated to remove the argon atmosphere and the system allowed to equilibrate ( >5 minutes) before reading the vapour pressure of the enzyme solution. This procedure was 79 repeated three times. The value of vapour pressure obtained was converted to mm Hg and checked with published data6 for accuracy, in order to make sure that there were no leaks in the apparatus. Then a small amount of CO gas was carefully admitted to the system and allowed to equilibrate (3 minutes). The pressure was recorded and the cell tonometer was closed, removed from the gas handling line, slowly swirled and taken to the spectrophotometer and allowed to equilibrate for another 3 minutes prior to the spectrum of partially carbonylated enzyme being recorded. Once the spectrum was recorded, the cell was taken back to the CO-line and the pressure was readjusted using the same procedure as above. At least five different CO-pressures were added and the spectra recorded in each case. The final spectrum.was recorded under 1 atmosphere of CO. This general procedure was followed at four different temperatures in order to investigate the temperature dependence of the equilibrium constant. 80 111.4 Determination of Equi1ibr ium Constant for the  Reaction of Dioxygen with Substrate-bound cytochrome  P450cam 111.4.1 General Informat ion The equilibrium constant for the reaction of dioxygen with substrate-bound P-450 can be estimated by an experimental procedure similar to that used for the determination of CO binding constant, discussed in the previous section. In order to equilibrate the system at sub-zero temperatures a slush bath (Figure III.5) was used.Therefore, the use of constant temperature baths as well as the special cell compartment was not necessary. The buffer solution used was 50% diethyleneglycol in P-buffer. 111 .4 .2 Procedure The cell-cavity of the slush bath was filled to 2/3 of its volume ( 30 mL ) with the buffer solution and the base line spectrum was recorded in the Cary spctrophotometer as described _ in Section III.3.2. Stock substrate-bound enzyme solution was then added to a final concentration of about 2 1CT6 M and the spectrum recorded. Then, the stoichiometric reduction was carried out as given in Section III.3.2. The cell was then cooled to the desired temperature (± 1°) using the suitable slush7 from the list below. Slush Temperature Benzonitrile/liquid N2 -13° Carbon tetrachloride/liquid N2 -23° o-Xylene/liquid N2 -2981 Serum cap To vacuum line Dewar flask Constant temperature bath Optical cell Quartz windows Figure in.5 10 cm path-length optical cell (Slush Bath). 82 The temperature was maintained constant by the addition of small amounts of liquid N2 periodically. Small partial pressures of 02 were then added using a low pressure gas handling line as described in Section III.3.2, and the corresponding spectra were recorded. Attempts were made to record the spectrum of the fully formed oxygenated species under 1 atmosphere of dioxygen (see Section IV.4). 83 REFERENCES 1. I.C.Gunsalus,G.C.Wagner,Methods Enzymol.,52,166(1978). 2. E.S.Lenox, Virology, J_, 190(1955). 3. K.Weber, M.Osborn, J. Biol. Chem., 244, 4406(1969). 4. D.Dolphin, B.R.James, H.C.Welborn, Biochem. Biophys. Res. Commun., 88, 415(1979). 5. D.V.Stynes,B.R.James,J.Am.Chem.Soc.,96,2733(1974). 6. Handbook of Chemistry and Physics, 41st Edn, Chemical Rubber Publishing Co., Cleveland(1959-60)p.2326. 7. R.E.Randeau, J. Chem. Eng. Data, 11 , 124( 1966). CHAPTER IV RESULTS AND DISCUSSION 85 IV.1 Growth.of Pseudomonas putida Strain 786 The monooxygenase enzyme system present in the bacterium Pseudomonas putida has provided a definitive biological model for extensive biochemical and physical studies relevant to the cytochrome P-450 mediated hydroxylation reactions occurring in mammalian systems.1 In the present study, the Pseudomonas putida strain 786 was used to isolate the camphor hydroxylating monooxygenase component, cytochrome P-450cam. The strain 786 possesses several advantages over the parent strain PpG 1 which was originally isolated from soil by enrichment on D-(+)-camphor.1 Among these advantages are the shorter generation time, resistance to lysis by bacteriophage, and the ability to release the hydroxylase proteins by freeze-thaw autolysis, thereby providing easy access to the P-450 enzyme. In order for the bacteria to retain the ability to metabolize camphor, and hence to produce the P-450 enzyme, it was necessary to maintain the strain in minimal agar plates containing camphor as the only carbon source. Failure to do so results in the bacterium losing the ability to multiply in camphor saturated media, with subsequent long lag-phases in the growth curves.2 It was observed that the pH trends of the culture media depend on the basic carbon and energy source provided for the growth. In the case of L-broth and 500 mL shake flask stages, where glucose and glutamic acid were used, 86 respectively, the pH of the media increased, while the growth on camphor resulted in a decrease of pH. The pH decrease during camphor metabolism is known to be due to the accumulation of acidic metabolites,3" while no clear explanation has been given for the increase in pH during the metabolism of the other organic substrates. The basic difficulty encountered in the growth of the bacteria is the 'foaming out', despite the use of antifoaming agents, at the later part of the 14 L fermenter stage. The foam caused the exit air filters to clog and the air supply to the culture medium was thus cut off by the build-up of high pressure in the fermenters. This process, which usually happened during overnight growth, resulted in the death of bacterial cells; the use of an external container, connected to the fermenters via rubber tubing (with air filters removed), in order to collect the foam (which contained pink bacterial cells) served as an alternativ-e, but this also resulted in the death of a considerable fraction of the cells due to lack of aeration and agitation. The duration of the growth procedure was found to be at least twice as long as that given in the literature procedure,1 although all the instructions were followed closely. As a consequence of the lengthy growth period, the amount of enzyme isolated was generally smaller than that reported.1 87 IV.2 Properties of Cytochrome P-450cam  IV.2.1 Substrate-Bound Cytochrome P~450cam The absorption spectra of the various states of pure, substrate-bound cytochrome P-450cam (Sections II.2.5 and II.2.6.1) are given in Figure IV.1. The wavelengths of the absorption maxima (Amax), as well as the corresponding millimolar extinction coefficients (e mM) are presented together with literature data in Table IV.1. The oxidized, substrate-bound enzyme absorbs at 280 nm (protein peak), 391 nm (Soret peak), and 510, 542, and 643 nm (visible peaks). The calculated EmM values agree reasonably well with the published data, except for the absorption at 280 nm, which is due to aromatic groups in the protein chain. The intensity of the 280 nm peak varied slightly (± 5%), in different enzyme preparations, due to the presence of small amounts of apo-cytochrome P-450. On reducing the oxidized state with stoichiometric amounts of sodium dithionite (^max 314 nm), the colour of enzyme solution changes from light brown to light pink with generation of absorption peaks at 408 and 544 nm; the protein absorption remains unchanged as expected. The CO-complex of the reduced enzyme, formed by bubbling CO through the enzyme solution for a few seconds, has a golden yellow colour with absorptions at 365 and 445 nm (near uv and low energy bands of a split Soret) and 550 nm. The discrepancy in values for the 365 nm peak (Table IV.1) is due to the interference of excess dithionite in the 1.0 0) o c (0 M O 5 oxidized reduced reduced-CO Dithionite absorption oo 300 400 —I 500 600 Wavelength (nm)700 oo oo Figure IV.1 Various states of pure, substrate-bound cytochrome P-450cam. 89 Table IV.1 Absorbance Data for Substrate-Bound Cytochrome P-450cam. State (a) Expt. Prep. (b) Lit . Prep. (c) Lit . Prep. X max emM X max emM X max emM Oxidized 280 81.2 - - 280 63.3 391 1 02 391 101 391 102 510 12.2 510 - 510 13.0 542 10.9 540 - 540 11.2 643 4.2 645 - 645 5.4 Reduced 408 80.7 408 83 408 86.5 544 14.5 542 15.2 542 16.0 Reduced-CO 365 51.1 - - 364 60.8 445 1 20 446 1 19 446 120 550 13.8 550 13.0 550 14 (a) Determined in P-100 buffer, pH 7.4, 8 mM camphor at 23+2°; e mM calculated assuming emM=120 for reduced-CO species at 445 nm. (b) From ref. 5 (c) From ref. 1 90 literature preparation; and the smaller e value obtained in the present work is considered more accurate. 91 IV.2.2 Substrate-Free Cytochrome P~450cam The absorption spectra of the various states of pure, substrate-free cytochrome P-450cam (Section II.2.6.2) are shown in Figure IV.2; the xmax and emM values for the experimental preparation are given with the literature data in Table IV.2. Examination of the spectral characteristics of various states of the substrate-bound and substrate-free enzyme (Tables IV.1 and IV.2) reveals that both forms have very similar absorption spectra in the reduced and reduced-CO states. However, the oxidized states of the forms have remarkably different spectral properties (Figure IV.3). The binding of camphor to substrate-free enzyme is strong and occurs rapidly (Section II.4.2.1). Because of the similiarities in spectra of reduced states (and reduced-CO states) of substrate-free and substrate-bound enzymes, it was essential to make sure that no trace camphor was present i-n the buffers or glassware used in the studies of substrate-free enzyme. IV.2.3 Purity and Stability of the Enzyme Preperations The isolated and purified (Sections II.2.5 and substrate-bound enzyme samples were at least 80% homogeneous,1 having an absorption ratio A391:A2B0= 1.14. The absorption ratio for some of the literature preparations was 1.00 5, 1.13 2, 1.37 6, 1.45 (crystalline sample)1. All substrate-free enzyme samples were of the same Dithionite absorption Figure IV.2 Various states of pure, substrate-free cytochrome P-450cam. 93 Table IV.2 Absorbance Data for Substrate-Free Cytochrome P-450cam. State (a) Expt Prep. (b) Lit Prep. (c) Lit. Prep. X max emM X max emM X max emM Oxidized 278 107 - - 280 68.3 360 41.0 - - 360 36.7 416 117.9 418 1 04 417 1 15 535 9.7 535 10.3 535 11.6 568 9.3 570 10.4 569 11.9 Reduced 408 78.9 408 69.0 408 76.7 540 12.5 540 13.5 540 15.1 Reduced-CO 365 445 120 447 104 447 120 550 12.5 550 12. 550 14.3 (a) Determined in buffer P-100, pH 7.4, no camphor added, at 23±2°; emM calculated assuming emM=120 for reduced-CO species at 445 nm. (b) From ref. 5 (c) From ref. 1 95 order of purity as the substrate-bound enzyme, from which they were prepared. But, storage at 5° under argon for more than 48 hours caused the formation of detectable amounts of P-420 species (denatured P-450) as seen in the absorption spectrum (Figure IV.4). Therefore, substrate-free enzyme samples for the equilibrium ligand binding studies were prepared on a daily basis. The substrate-bound enzyme, once purified, could be stored at 5° for over 6 months without the formation of any P-420 species. The substrate-bound enzyme could be stored indefinitely at liquid nitrogen temperature without detectable loss of purity. Figure IV.4 P-420 species formed in substrate-free cytochrome P-450cam soluti 97 IV.3 Determination of the Equilibrium Constant at Pifferent  Temperatures for the Binding of Carbon Monoxide by  Substrate-Free Cytochrome P-450cam  IV.3.1 Acquisition and Treatment of Pata Equilibrium constant values for the binding of CO with substrate-free cytochrome P-450 were estimated from the experimental data obtained as given in Section III.3. At each temperature, at least three different determinations were carried out to give a mean value for the equilibrium constants. The spectral changes observed for experiment no.5 (Table IV.3) are given in Figure IV.5. At a fixed enzyme concentration (7-8 10 " 6 M), the CO pressure was varied from 0.4-760 mm Hg, resulting in a decrease of the 408 nm peak (reduced) and an increase in 445 nm peak (CO-complexed). Isosbestic points were observed at 373, 430, 466 and 583 nm. Raw data from all the experiments are given in Appendix (i), along with the processed data. The degree of formation of the CO-complex of the substrate-free cytochrome P-450cam (Reaction II.2) can be determined using the familiar Hill log/log plot which yeilds the Pw2 and hence K value. J CO The ratio of the Fe(II)-CO to the decarbonylated complex present at a given CO pressure (Pco^ can De expressed as (A-A0 )/(Aao-A), where, A = absorbance of Fe(Il)-CO at a known PCQ AQ = absorbance of Fed I) enzyme when Pco=0 Figure IV.5 Spectral changes observed for the P. /0 experiment no. 5. 99 A^ absorbance of fully formed Fe(II)-CO Clearly, A-A0 a. [Fe(II)-CO] , and A^-A oc[Fe(II ) ] . Since, =[Fe(II)-CO]/[Fe(II)][CO], the following relationships are derived. Log KCQ=Log (A-A0)/(AOD-A) - Log [CO] Log (A-A0)/(Aoo-A)=Log Pco+ Log K' where, K'= x.K and x= the solubility of CO in water expressed in M/(mm Hg) at the reaction temperature. Therefore, the plot of log (A-A0)/(A^-A) vs. log PCO should give a straight line with a theoretical gradient equal to unity for the simple equilibrium; and when A-A0 =Aoo_A or, log (A-A 0)/( A^-A) =0 , the corresponding PQ-, value would be the P,y2. The KCO(M -1 ) values can be calculated using the equation, Kco=1/X.P1/2 The log (A-A0)/(Aro-A) values were calculated from the corresponding absorbance values at 445 nm. P^-, values were converted from mm DBT to mm Hg as described in Section III.3.1. The data were plotted as in Figure IV.6 using the computer program "ace:graph" available from the computer facility at U.B.C. The gradients of all the plots were in the range 0.90-1.15; in certain instances , removal of an experimental data point (considered to contain a large error), applying the condition that at least four other data points remained, resulted in a much more satisfactory 100 101 correlation of data, according to least-squares linear regression analysis. The plots obtained for all P,^ experiments are given in Appendix (ii). Each P1/2 value calculated from these plots w recorded and the mean value calculated (Table IV.3). The P,^ values were converted to equilibrium constant (Kco,M_1) values using published data7 for the solubility of CO in water at various temperatures. An important aspect of the study of reactions of hemoproteins with ligands is the determination of the thermodynamic parameters AH0 and AS0. In the case of biological systems, the determination of such parameters has usually been via van't Hoff plots,8 although highly sensitive microcalorimetric techniques are also available.9 The van't Hoff plot, (log KCQvs. 1/T ; Figure IV.7) was obtained using the computer program mentioned above, and the thermodynamic parameters were calculated. The thermodynamic relationship, Log K = -(AH°/2.303R).1/T + AS°/2.303R , allows for a plot of log K vs. 1/T to give &H° from the gradient and AS0 from the y-intercept. Good linearity is observed in these plots owing to the fact that the measurements are done in a narrow temperature range8 (usually 0°-40°) because of the instability of proteins at higher temperatures. The AH0 value for the CO binding to reduced substrate-free cytochrome P-450cam was estimated to be -18±1 kcal/mol 102 Table IV.3 The Equilibrium Constant Values for CO Binding to Substrate-Free Cytochrome P-450cam. T(°C) # Pimm Hg) 1/2 Mean P^ KCQ (M* ) Mean Log KCO l/TxlCT3 4.0 1 0.372 1 .55X106 2 0.423 0.398 1.37X106 1.46x10s 6.1644 3.608 3 0.398 1 .45x1 06 12.0 4 1 .38 5.07x105 5 1 .32 1 .40 5.30X105 5.00x10s 5.6990 3.507 6 1 .57 4.63x10s 18.0 7 2.63 2.95x10s 8 2.69 2.78 2.88x10s 2.80x10s 5.4472 3.434 9 3.02 2.57x10s 24.0 10 4.68 1.57x10s 1 1 4.47 4.59 1.65x10s 1.60x10s 5.2041 3.365 1 2 4.62 1.59x10s 103 10 3.36 3.4 3.44 3.4B 3.52 3.56 3.6 3.64 1/T*10 Figure IV.7 The van't Hoff plot. 104 The AS0 value was -37±4 cal/mol.deg. (standard state of 1 M). Table IV.4 summarizes published thermodynamic data for the CO binding to substrate-bound cytochrome P-450cam, horse Mb, sheep Hb (per mole of heme), and to some model compounds. Given also are the values determined in this study for substrate-free cytochrome P450cam. 105 Table IV.4 Kco, AH0 and AS0 Values for CO Binding to Some Hemoproteins and Models of Cytochrome P-450. System Kco,25° AH0 AS0 Ref . (M-1 ) (kcal/mol) (cal/mol.deg.) P-450cam 1.3x105 -12 -17 1 1 (sub.-bound) Horse Mb 2.5x107 -12.6 -7.4 8 Sheep Hb - -16 - 8 Human Hb - -17.7 - 9 Model* (a) 1.3x10' -16 -35 1 0 (b) 2.8x103 -19 -48 1 0 P-450cam 1.3x105 -18±1 -37±4 this (sub.-free) work * Fe(II)Pp(IX)DME-BuS complex (a) in a polar medium (N,N-dimethylacetamide) (b) in a non-polar medium (toluene) 106 IV.4 Comparison of Measured AH° and AS0 Values to those  of Other Hemoproteins and Model Systems Structural analyses of carbonylated hemoproteins reveal that the CO unit is bent and/or tilted from the perpendicular to the porphyrin plane, due to interactions with the distal residues.12 However, in the protein-free model systems, as expected, the Fe-C-0 unit is linear and normal to porphyrin plane.13 It has been proposed 1 *"17 that in hemoproteins, the CO affinities are lowered significantly due to this 'distal-side steric effect' which causes the Fe-C-0 unit to be bent. Results from the present study show that the binding of CO to substrate-free cytochrome P-450cam is at least 5 kcal/mol more favourable in terms of AH0 compared to the published value11 for the substrate-bound system (Table IV.4). The reverse trend observed in terms of AS0 is considered to be due mainly to differences in conformational changes in the protein chain.18 As proposed by Lumry and Rajender,18 there exist a specific linear relationship between the entropy change and enthalpy change within a variety of protein reactions termed 'enthalpy-entropy compensation phenomena'. It has been demonstrated that the 'compensation effect' produces parallel enthalpy and entropy changes so that the net change in the free-energy of the system under investigation is relatively minor. Although this principle is not rigorously est lished, the feeling is that expansions and 107 contractions of the protein result in changes in the 'free volume' of water, which contributes to the entropy change of the system. For example, the effect of varying ethanol concentrations on the reversible thermal unfolding transition of ribonuclease A at various temperatures is to produce parallel AH0 and AS0 changes that give rise to the same A(AH0)/A(AS0) value at each temperature. Also similar compensation effects have been noted in reactions of hemoglobin and myoglobin with ligands such as F~, N3, CN~, SCN-.18 The compensation hypothesis appears to apply to the results of the present study; the large negative entropy contribution observed in the case of CO binding to substrate-free P-450cam compensates for the large enthalpy change observed, the overall process resulting in no change in the AG0 of the reaction, compared to the substrate-bound system. Qualitatively, the observed less-favourable AH0 for the binding of CO to substrate-bound enzyme, compared to the substrate-free system, may be due mainly to the presence of the substrate molecule sitting close to the active-site, perhaps causing the CO molecule to bind in a more angular fashion. In fact, such a hypothesis indirectly supports the observations by Peterson et al.19 in their inhibition experiments where they conclude that the bonding site of the substrate must lie in the immediate vicinity of the heme iron. 108 The relatively larger CO binding constant to myoglobin (Table IV.4) may result from the weaker 6"-donor property of the fifth ligand, imidazole, compared to the thiolate ligand in P-450. The AH0 term for the CO binding to sheep Hb is more favourable than that for substrate-bound P-450cam or Mb, and indeed it is similar to the AH° determined for the substrate-free P-450cam. However, the entropy term for the sheep Hb system is not available for comparison. The relatively small entropy change in the Mb system may reflect that only minor conformational changes occur in the protein chain upon CO binding compared to the substrate-bound P-450 system; this results in a much higher binding constant. The binding of CO to the model system (Table IV.4) is equally favourable in terms of AH0 as the binding of CO to substrate-free P-450cam. Therefore, it is conceivable that the Fe-C-0 unit is linear (or almost so) in the substrate-free P-450-CO complex. There are no thermodynamic data available on 02 binding to P-450 system. In the present work, a large number of attempts were made to determine the P^ values, at temperatures ranging from 0° to -30°, for the substrate-bound system using the procedure given in Section III.4. None of these experiments were successful because of several difficulties that were not resolved. At temperatures near 0°, the enzyme-02 complex was formed, but autoxidized (according to reaction II.3, to give the Fe(III) enzyme) during the time of the experiment. The only 109 reported P,/2 value for 02 binding to P-450 enzyme at 0° was determined using the same conventional cell tonometer method used in the present study to determine the CO binding constants. It should be noted that the same report gives the half life of autoxidation at 0° as 75 minutes.11 However, the published 11.20,21,22 rates of autoxidation of the substrate-bound P-450cam-dioxygen complex (Table IV.5) are rather inconsistent; the rates determined in the present work were higher than most of the reported values [Table IV.5; Raw Data in Appendix (iii)]. At lower temperatures (-10° tO -30°) the autoxidation reaction was indeed prevented, but the co-solvent system used (ethyleneglycol/water) became more viscous, thus causing small bubbles to form when the slush-bath was shaken to mix the added gas. These bubbles floated to the surface over a long period of time (ca. 1 h), and a single bubble rising to the surface during the recording of a spectrum altered absorption considerably such that the isosbestic points were not observed. After many unsuccessful attempts it was decided that the current approach was not suitable for determination of the P1/2 value (and AH0, AS0 values) for the binding of dioxygen to substrate-bound cytochrome P-450cam. 110 Table IV.5 Half-lives of Autoxidation Reaction of Dioxygen Complex of Substrate-Bound Cytochrome P-450cam. pH T (°C) Half-life (min.) Ref . 7.0 0.0 75 1 1 7.4 4.0 45 20 7.4 4.0 1 15 21 7.4 2.0 21 22 7.4 4.0 18 22 7.4 10.0 9.2 22 7.4 0.0 20 this work 7.4 10.0 6.7 this work Unfortunately, no equilibrium or kinetic data are available on P-450 model systems that do not use a vast excess of a 'non-innocent' ligand.23 Mercaptan-tai1 porphyrins, prepared recently by Collman et al.23 by covalently attaching a thiol group to the porphyrin so that the thiol can serve as a built-in axial ligand, appear to be good candidates for studies on CO vs. 02 binding for comparison with P-450 systems in the context of steric considerations. Ill REFERENCES 1. I .C.Gunsalus, G.C.Wagner, Methods Enzymol., 5_2, 166 (1978). 2. S.Albon, M.Sc Thesis, Dept. of Chemistry, U.B.C., (1983) p.198. 3. H.E.Conrad, R.Dubus, I.C.Gunsalus, Biochem. Biophys. Res. Commun., 6, 293,(1961). 4. J.Hedegard, I.C.Gunsalus, J. Biol. Chem., 240, 4038 (1965). 5. J.A.Peterson, Arch. Biochem. Biophys., 144, 678(1971). 6. K.Dus, M.Katagiri, C.A.Yu, D.L.Erbs, I.C.Gunsalus, Biochem. Biophys. Res. Commun., 40, 1423(1970). 7. A Dictionary of Chemical Solubilities-Inorganic, (Ed. A.M.Comey, D.A.Hahn) 1921 p.158. 8. E.Antonini, M.Brunori, Hemoglobin and Myoglobin in their Reactions with Ligands, (North Holland) 1971. 9. H.T.Gaud, B.G.Barisas, S.J.Gill, Biochem. Biophys. Res. Commun., 59, 1389(1974). 10. C.K.Chang,-D.Dolphin, Proc. Natl. Acad. Sci., U.S.A., 73, 3338(1976). 11. D.Dolphin, B.R.James, C.H.Welborn, J. Mol. Catal., 7, 201(1980). 12. a. E.J.Heidner, R.C.Ladner, M.F.Perutz, J. Mol. Biol., 104", 707( 1976). b. J.C.Norvell, A.C.Nunes, B.P.Schoenborn, Science, 190, 568(1975). c. R.Huber, O.Epp, H.Formanek, J. Mol. Biol., 102, 349(1970). d. E.A.Padlen, W.E.Love, J. Biol. Chem., 249, 4067(1975). 13. S.Peng, J.A.Ibers, J. Am. Chem. Soc, 98, 8032( 1976). 14. J.P.Collman, J.I.Brauman, K.M.Doxsie, Proc. Natl. Acad. Sci., U.S.A., 76, 6035(1979). 15. a. J.P.Collman, J.I.Brauman, T.R.Halbert, K.S.Suslick, Proc. Natl. Acad. Sci., 73, 3333(1976). b. J.P.Collman, J.I.Brauman, B.L.Iverson, J.L.Sesler, R.M.Morris, Q.H.Gibson, J. Am. Chem. Soc, 105(10), 3052(1983). 112 16. P.W Tucker, S.E.Phillips, M.F.Perutz, R.Houtchens, W.H.Caughey, Proc. Natl. Acad. Sci., U.S.A., 75, 1076(1978). 17. W.J.Wallace, J.A.Volpe, J.C.Maxwell, W.S.Caughey, S.Charache, Biochem. Biophys. Res. Commun., 68, 1379(1976) . 18. R.Lumry, S.Rajender, Biopolymers, 9(10), 1125(1970). 19. J.A.Peterson, V.Ullrich, A.Hilderbrandt, Arch. Biochem. Biophys., J_45, 531(1971). 20. R.W.Estaboook, J.Baron, J.A.Peterson, Y.Ishimura, Biochem. J., 121, 3(1971). 21. J.A.Peterson, Y.Ishimura, B.W.Griffin, Arch. Biochem. Biophys., J_49, 197(1972). 22. L.Einstein, P.Debey, P.Dousou, Biochem. Biophys. Res. Commun., 7_7, 1377 ( 1 977). 23. J.P.Collman, S.E.Groh, Hemoglobin and Oxygen Binding, (Ed. Chien Ho), Elsevier Biochemical, New York, 1982 p.37. 113 APPENDICES Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4 ± Q±2° C Vapour Pressure of Water: 79 mm DBT Experiment No. 1 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) P rco (mm DBT) A445nm 1 - 76 - 0.177 2 5 80 4 0.327 3 10 89 13 0.416 4 20 97 21 0.443 5 40 111 35 0.467 6 100 183 107 0.492 Buffer: P-100 PH: 7.4 Processed Data Pco (mm Hg) A-A 0 A.-A A-A 0 A— -A Log A_Ao A.-A L°8 P™ CO 0.309 0.150 0.180 0.833 -0.07918 -0.5098 1.01 0.239 0.091 2.626 0.4194 -0.002120 1.62 0.266 0.064 4.156 0.6187 0.2104 2.71 0.290 0.040 7.250 0.8603 0.4323 8.27 0.315 0.015 21.00 1.322 0.9176 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4f0.2°C Vapour Pressure of Water:79 Experiment No. 2 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) P rco (mm DBT) A445nm 1 - 76 - 0.205 2 5 80 4 0.385 3 10 85 9 0.475 4 15 91 15 0.490 5 25 98 22 0.548 6 - - 0.063 Buffer: P_100 PH: 7.4 Processed Data Pco (mm Hg) A-A 0 A.-A A-A 0 A<« -A Log A_Ao A..-A Log P CO 0.309 0.189 0.245 0.735 -0,1339 -.5100 0,696 0.270 8.155 1.742 0.2410 -0.1600 1.16 0,285 -.140 2.036 0.3087 0.0643 (?) 1.70 0,343 0.082 4,183 0.6215 0.2306 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4+0.2°C Buffer: P-100 Vapour Pressure of Water: 79 mm DBT pH: 7.4 Experiment No. 3 Raw Data Processed Data Species P CO wanted (mm DBT) ptotal (mm DBT) P rco (mm DBT) A445nm 1 - 74 - 0.150 2 5 79 5 0.350 3 20 95 21 0.485 4 40 lit 0.520 5 - - 0.560 Pco (mm Hg) A-A o A-A QD A-A o A- -A Log A-Ao Log P CO 0.387 0.200 0.210 0.9524 -0.0212 -0.4129 1.62 0.335 0.0750 4.466 0.6499 0.2104 3.48 0.370 0.0400 9.250 0.9661 0.5414 Appendix (1) contM. Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 12+0.2°C Buffer: P-100 Vapour Pressure of Water: 135 mm DBT pH: 7.4 Experiment No.4 Raw Data Processed Data Species P CO wanted (mm DBT) ptotal (mm DBT) p RCO (mm DBT) A445nm Pco (mm Hg) A-A o A.-A A-A o A<» -A Log A_Ao A.-A Log P CO 1 - 135 - 0.195 2 5 140 5 0.285 0.387 0.090 0.355 0.254 -0.5960 -0.4129 3 10 164 29 0.480 2.24 0.285 0.160 1.78 0.2507 0.3506 4 30 215 80 0.565 6.18 0.370 0.075 4.93 0.6931 0.7913 5 50 405 270 0.610 20.9 0.415 0.030 13.8 1.141 1.320 6 oo - - 0.640 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 12+ Q 2oc Buffer: p_l00 Vapour Pressure of Water: 135 mmDET 7.4 Experiment No. 5 Raw Data Processed Data Species P CO wanted (mm DBT) ptotal (mm DBT) P rco (mm DBT) A445nm Pco (mm tt^) A-A 0 A.-A A-A 0 Ao -A Log A_Ao A.-A Log P CO 1 - 134 - 0.222 2 5 138 4 0.260 ? 0.310 0.038 0.600 0.0633 -1.198 -0.5100? 3 10 141 7 0.397 0.541 0.175 0.463 0.378 -0.4225 -0.2668 4 20 163 29 0.610 2.24 0.388 0.250 1.15 0.1909 0.3506 5 50 196 62 0.727 4.79 0.505 0.133 3.79 0.5794 0.6806 6 150 290 156 0.787 12.1 0.565 0.073 7.74 0.8887 1.081 7 OO - - 0.860 00 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 6 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) p rco (mm DBT) A445nm 1 _ 135 - 0.190 2 10 153 18 0.372 3 20 163 28 0.430 4 40 188 53 0.478 5 60 246 111 0.530 6 CO - - 0.582 Buffer: pH: Processed Data pco (mm Hg) A-A o A-A GD A-A o A— -A Log A Ao A^-A Log P CO 1.39 0.182 0.210 0,867 -0.0621 0.1435 2.16 0.240 0.152 1.58 0.198^ 0.3353 4.10 0.288 0.104 2.77 0.4424 0.6125 8.58 0.340 0.052 6.54 0.8155 0.9335 Appendix (i) (cont'd) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 18± 0.2°C Buffer: P-100 Vapour Pressure of Water: 200 mm DBT pH: 7.4 Experiment No. 7 Raw Data Processed Data Species P CO wanted (mm DBT) ptotal (mm DBT) p (mm DBT) A445nm (mm Hg) A-A o A.-A A-A o A<» -A Log A_Ao A.-A Log P CO 1 0 192 - 0.220 2 10 200 8 0.325 0.618 0.105 0.445 0.236 -0.6270 -0.2087 3 20 219 27 0.470 2.09 0.250 0.300 0.833 -0.0792 0.3195 4 40 268 76 0.595 5.88 0.375 0.175 2.14 0.3310 0.7690 5 80 313 121 0.635 9.35 0.415 0.135 3.07 0.4877 0.9710 6 120 371 179 0.685 13.8 0.465 0.085 5.47 0.7380 1.141 7 oO - - 0.770 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 8 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) p rco (mm DBT) A445nm 1 0 203 - 0.165 2 10 212 9 0.252 3 20 313 110 0.482 4 40 403 200 0.528 5 80 429 226 0.565 ? 6 oo - - 0.585 Buffer: pH: Processed Data Pco (mm Hg) A-A o A.-A A-A o A- -A Log A_Ao A„-A Log P CO 0.696 0.0870 0.333 0.261 -0.5829 -0.1576 8.50 0.317 0.103 3.08 0.4882 0.9295 15.5 0.363 0.057 6.37 0.8040 1.189 17.5 0.400 0.020 20.0 1.301 1.242 Appendix (1) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 9 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) p rco (mm DBT) A445nm 1 0 205 - 0.225 2 10 216 11 0.310 3 20 221 16 0.348 4 40 239 34 0.400 5 CO - - 0.605 Buffer: pH: Processed Data Pco (mm Hg) A-A 0 A.-A A-A o A- -A Log A_Ao A.-A Log P CO 0.850 0.085 0.295 0.288 -0.5404 -0.07040 1.24 0.123 0.257 0.479 -0.3200 0.09230 2.63 0.175 0.205 0.854 -0.06872 0.4197 Appendix (i) (cont'd) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 24±0.2° C Buffer: P_ioo Vapour Pressure of Water: 290 mm DBT pH: 7.4 Experiment No.IQ Raw Data Processed Data Species P CO wanted (mm DBT) ptotal (mm DBT) p rco (mm DBT) A445nm Pco (mm Hg) A-A o A-A CD A-A o A«j -A Log A_Ao A^-A Log P CO 1 0 250 - 0.290 2 15 266 16 0.326 1.23 0.036 0.500 0.0720 -1.1427 -0.1118 3 25 275 25 0.390 1.91 o.ieo 0.436 0.229 -0.6395 0.2810 4 40 286 36 0.490 2.82 0.200 0.336 0.595 -0.2253 0.4502 5 65 318 68 0.600 5.25 0.310 0.226 1.37 0.1373 0.7201 6 100 360 110 0.652 8.51 0.362 0.174 2.08 0.3182 0.9299 7 - - 0.826 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 12 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) p 'co (mm DBT) A445nm 1 0 263 - 0.190 2 10 355 102 0.415 3 30 454 191 0.455 4 80 533 270 0.480 5 - 0.540 Buffer: pH: Processed Data Pco (mm Hg) A-A o A.-A A-A o AA -A Log A-Ao A»-A Log P CO 7.88 0.225 0.125 1.80 0.2553 0.8968 14.81 0.265 0.085 3.12 0.4938 1.170 20.9 0.290 0.060 4.83 0.4842 1.320 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 11 Raw Data Species P CO wanted (mm DBT) ptotal (mm DBT) p cco (mm DBT) A445nm 1 0 272 - 0.220 2 5 277 5 0.260 3 10 281 9 0.293 4 20 290 18 0.340 5 50 356 84 0.543 6 80 387 115 0.590 7 - - 0.770 Buffer: PH: Processed Data Pco (mm Hg) A-A o A-A OD A-A o Aaa -A Log A-Ao Log P CO 0.387 0.040 0.510 0.0784 -1.1055 -0.4129 0.696 0.073 0.477 0.153 -0.8152 -0.1577 1.39 0.120 0.430 0.279 -0.5543 0.1435 6.49 0.323 0.227 1.42 0.1532 0.8125 8.89 0.370 0.180 2.06 0.3129 0.9489 Appendix (ii) Appendix (ii) (cont'd) -r~n—1—1—r -,—T 1 1 1 r—i " r -0 2 0? OS • • • 1 1 1 L 10 u i' 22 -0.3 -01 i.< log Pco r 6 Experiment No. 4_ — ~~ The Hill log/log plots obtained for the CO-binding to substrate-free cytochrome P-450cam at 12°C. Appendix (ii) (cont'd) The Hill log/log plots obtained for the CO-binding to substrate-free cytochrome P-450cam at 18°C. ro oo Appendix (ii) (cont'd) i«« Experiment No. 10 1 — The Hill log/log plots obtained for the CO-binding to substrate-free cytochrome P-450cam at 24°C. Appendix (iii) Determination of rate of autoxidation of the dioxygen compl of reduced, substrate-bound cytochrome P-450cam. Raw Data Expt. No. : 1 Temperature : 0 ± 0.2 °C Buffer : P-100 A =1.68 A Aoo-A -In (Aoc-A) Time (min.) 1.28 0.396 0.9263 1.0 1.45 0.230 1.4697 15 1.50 0.176 1.7323 20 1.55 0.130 2.0402 30 1.58 0.100 2.3025 36 1.62 0.0560 2.8824 55 Expt. No. : 2 Temperature : 0 ± 0.2°C Buffer : P-100 ^=1.09 A A^-A -In(A^-A) Time (Min; 0.790 0.306 1.1841 1.0 0.856 0.240 1.4271 7.0 0.920 0.176 1.7372 16 0.996 0.100 2.30 6 30 1.05 0.0460 3.0790 54 Expt. NO. : 3 Temperature : 10 + 0.2°C Buffer : P-100 A~=1.63 A Aoo-A -In (Aoo-A) Time (Min.) 1.38 0.254 1.3704 1.0 1.48 0.146 1.9241 5.0 1.53 0.100 2.3026 9.0 1.57 0.0600 2.8134 14 1.61 0.0200 3.9120 24 


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