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Studies on the succinate oxidase system of E. coli Kim, In-Cheol 1971

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STUDIES ON THE SUCCINATE OXIDASE SYSTEM E. COLI by IN-CHEOL KIM B.S.P., Seoul National University, 1960 M. Sc., Seoul National University, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biochemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Bri t ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree tha permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Biochemistry The University of Bri t ish Columbia Vancouver 8, Canada ABSTRACT The succinate oxidase system of E. coli has been studied from three main viewpoints: (a) the preparation and properties of succinate dehydrogenase (SDH), (b) the function of nonheme iron, and (c) the se-quence of the components of the respiratory chain. Three different preparations containing SDH activity were isol-ated from this organism. These were the particulate fraction,soluble respiratory complex, and soluble SDH. The partial purification and characterization of these enzymes or complexes was performed. The particulate fraction consisted of membrane fragments which contained the whole respiratory chain and which oxidized succinate and NADH. The soluble respiratory complex contained both SDH and cyto-chrome bi. The molecula* weight was 1.6 x 106. The soluble SDH did not contain cytochrome bi and had a molecular weight of 100,000. One of the characteristic properties of SDH of the particulate fraction and the soluble respiratory complex was activation. If the enzyme was prepared in phosphate buffer both succinate oxidase and SDH activities could be activated by heating at 38° in the presence of succinate. The enzyme was stabilized by succinate in the absence of heating. Activation of succinate oxidase seemed to be mainly due to the activation of SDH. A second activation phenomenon which was independent of heat treatment was also observed. When the enzyme was prepared in Tris i i buffer with succinate the activated enzyme was formed at 0°. Heating did not further increase its activity. Activation by heat was irreversible. The heat-activated enzyme deactivated to a form which could not be reactivated. The heat-independent activated enzyme was more stable. The two activation phenomena thus seemed to be different. In contrast, the soluble SDH did not show the activation phenomenon nor was it stabilized by sub-strate. A mechanism for the activation of SDH is proposed. The nature, properties, role and location of nonheme iron in the particulate fraction of E. coli was investigated. The level of non-heme ferrous or ferric iron in the particulate fraction was determined spectrophotometrically using o-phenanthroline or Tiron. Analysis of iron by both chemical and spectrophotometric methods showed that only 45% of the total iron reacted with o-phenanthroline ("o-phenanthroline-reacting iron"). Heme iron constituted 5% of the total iron. The rest of total iron was not exposed by treating the particulate fraction with detergents or urea. The nature of the remainder of the total iron (50%) is unknown. Half of the o-phenanthroline-reacting iron reacted directly with o-phenanthroline ("directly-reacting iron"), but the other half only reacted after addition of dithionite ("dithionite-reducible iron"). Directly-reacting iron appeared to be ferric iron which was lo-cated in the hydrophobic region of the particulate fraction. This ferric iron could be reduced by sulfhydryl groups of the protein, > The dithionite-reducible iron was probably located at the surface of the particulate fraction and could not be reduced by sulfhydryl groups. Part of the dithionite-reducible iron was reduced by NADH or succinate. This substrate-reducible iron, probably less than 10% of the total iron, was located in the cytochrome bx region of the respir-atory chain. It was not associated with SDH. The effect of ultraviolet irradiation, inhibitors and extraction of ubiquinone on the activities of SDH and succinate oxidase was exam-ined. From these experiments, and those outlined above, a scheme for the sequence of the succinate oxidase chain of E. coli is proposed. iv TABLE OF CONTENTS Page ABSTRACT -TABLE OF CONTENTS iv LIST OF TABLES x LIST OF FIGURES x i i ABBREVIATIONS USED xvi ACKNOWLEDGEMENTS ... xvii INTRODUCTION 1 MITOCHONDRIAL RESPIRATORY CHAIN 1 BACTERIAL RESPIRATORY CHAIN 5 COMPOSITION OF THE RESPIRATORY CHAIN 7 Cytochromes . 7 Cytochrome b and c 8 Cytochrome oxidases 122 Cytochromes of E. coli. 15 Lipids and quinones 16 Nonheme iron proteins 20 Dehydrogenases 21 NADH dehydrogenase 22 Succinate dehydrogenase 23 MATERIALS AND METHODS 28 (i) Chemicals and solvents 28 (ii) Reagents and buffers 29 V Page (i i i ) Enzyme assay procedures «• 29 (is) A. Succinate oxidase activity 30 B. Catalase 30 C. SDH (or succinate PMS-DCIP reductase) 31 D. DCIP reductase 32 E. Ferricyanide reductase 32 (iv) Determination of protein? 32 (v) Determination of phosphate 33 (vi) Determination of molecular weight 33 (vii) Irradiation with near-ultraviolet light- 33 (viii) Column chromatography 34 A. Adsorption chromatography on calcium phosphate gel v> 34 B. Gel filtration 34 (ix) Measurement of cytochromes and flavoprotein 35 A. Reduced minus oxidized difference spectrum...... 35 B. Carbon monoxide difference spectrum 35 (x) Determination of cytochrome b^, cytochrome o, and flavin 36 (xi) Quantitative assay of ubiquinone in the particulate fraction 36 (xii) Qualitative analysis of quinone from whole cell...* 37 (xiii) Determination of the extinction coefficient f errous-o-phenanthrolinate 38 (xiv) Determination of the extinction coefficient 58 of ferrous-o-phenanthrolinate in a turbid solution and in the presence of dithionite. 38 (xv) Determination of total iron 39 vi Page (xvi) Determination of electron paramagnetic resonance spectrum 40 (xvii) Growth of cells.... 41 (xviii) Preparation of cell-free extract 41 (xix) Fractionation of cell-free extract by ultra-centrifugation and preparation of particulate fraction 42 (xx) Isoelectric precipitation of cell-free extract 42 (xxi) Ammonium sulfate fractionation of cell-free extract... 43 (xxii) Preparation of purified particulate fraction obtained by ammonium sulfate fractionation from cell-free extract 4 43 (xxiii) Preparation of soluble respiratory complex from cell-free extract 44 (xxiv) Preparation of acetone powder of E. coli. f 45 (xxv) Extraction of soluble SDH from the acetone powder 45 (xxvi) Purification of acetone powder extract by chromatography on calcium phosphate 46 (xxvii) Ammonium sulfate fractionation of acetone powder extract 46 (xxviii) Purification of acetone powder extract by chromatography on a column of a Sepharose 4B 47 (xxix) Purification of acetone powder extract by chromatography on a column of Sephadex G-200 47 (xxx) Determination of Km for succinate 48 (xxxi) Determination of Ki for malonate. 48 (xxxii) Activation of SDH v 48 PART I. STUDIES ON SUCCINATE DEHYDROGENASE (SDH) 49 RESULTS AND DISCUSSION 49 v i i Page Cl) PREPARATION OF SDH 49 1. Localization of SDH 49 2. Liberation of SDH by sonication 51 3. Isoelectric precipitation of the cell-free extract 51 4. Purification of the particulate fraction 5 from the cell-free extract 54 5. Disintegration of the membrane and purification of the cleaved respiratory chain by detergent 54 6. ;! Extraction and purification of soluble SDH from acetone powder 61 7. Ammonium sulfate fractionation of acetone powder extract and its purification by gel filtration 65 8. The role of cyt bx in SDH. 70 (2) PROPERTIES OF SDH v. 71 1. Determination of SDH activity. 71 2. Kinetics of SDH 72 3. Activation of SDH v 85 4. Lability of SDH y 99 5. Effect of PCMB on activation of SDH 107 6. Mechanism of activation of SDH....? 107 PART II. THE ROLE OF NHI IN E.CQLI 114 RESULTS 114 1. Spectrophotometry determination of ferrous iron using o-phenanthroline 114 (i) Absorption spectrum of ferrous-o-phenanthrolinate 115 v i i i Page (ii) Determination of the extinction coefficient of ferrous-o-phenanthrolinate 116 (iii ) Determination of the extinction coefficient of ferrous-o-phenanthrolinate in a transparent solution in the presence of TCA 119 (iv) Determination of bound ferrous iron in the particulate fraction 121 (v) Determination of the extinction coefficient of ferrous-o-phenanthrolinate in a turbid solution in the presence of dithionite 124 2. Spectrophotometric determination of ferric iron using Tiron 127 (i) Reaction of Tiron with ferric iron at pH 7.7 130 (ii) Reaction of Tiron with ferric iron at pH 9.2.... 134 (iii ) Determination of the extinction coefficient of ferric Tiron complex in a turbid particulate fraction 137 3. Measurement of NHI bound to the particulate fraction 143 (i) Total iron v 143 (ii) o-Phenanthroline-reacting iron 143 (ii i ) Tiron-reacting iron...- 147 4. Nature of iron in the particulate fraction 147 5. Binding of iron by the particulate fraction 160 6. SySolubilization of o-phenanthroline-reacting iron 163 7. Effect of succinate on iron in the particulate fraction.... 163 DISCUSSION.... 174 PART III. COMPOSITION OF TIE RESPIRATORY CHAIN IN THE PARTICULATE FRACTION 182 RESULTS 182 ix Page (1) The role of flavoprotein 182 (2) The role of UQ 188 (3) The role of cytochrome o i 196 (4) The role of NHI (see Part II) 196 (5) Effect on the succinate oxidase system of detergents, ultraviolet irradiation and inhibitors 196 (a) Effect of detergents or denaturing agent 199 (b) mm-irradiation 199 (c) Effect of inhibitors on SDH and succinate oxidase activities 207 DISCUSSION i . s - . * . . 210 BIBLIOGRAPHY 215 X LIST OF TABLES Table Page I. The principal cytochromes of certain bacteria 9 II Properties of crystalline bacterial cytochromes 10 III Properties of cytochrome oxidases 13 IV The quinones of mitochondrial and bacterial systems 18 V Properties of NADH dehydrogenases and oxidases solubilized from bacterial membranes.;- 24 VI Localization of NADH dehydrogenase and SDH 50 VII Liberation of SDH as a function of son ication time 52 VIII Recovery of SDH activity in the isoelectric precipitate 53 IX Distribution of SDH and succinate oxidase activities in fraction precipitated by ammonium sulfate from the cell-free extract. 55 X Extraction of soluble SDH from acetone powder with phosphate buffer, pH 7.5, in presence or absence of succinate ? 63 XI Distribution of SDH activity in fractions from ammonium sulfate fractionation of acetone powder extract 66 XII Solubilized or purified SDH from various sources 83 XIII Km and Ki for different preparations of SDH from E. coli. 84 XIV Activation of purified particulate fraction 94 XV Effect of Triton X-100 on SDH activity 97 XVI Lability of SDH activity 106 XVII Relationship between o-phenanthroline-reacting iron and total iron 146 x i T a b l e Page XVIII Liberation of soluble iron compounds from particulate fraction by substrate 164 XIX Concentration of respiratory components in the particulate fraction 185 XX Reducibility of electron transport components of unwashed particulate fraction 186 XXI Effect of flavin on SDH activity of proteins obtained from cell-free extract by isoelectric precipitation -. 189 XXII Effect of detergents and urea on succinate-linked reduction of flavin and cytochrome b^ 202 XXIII Effect of irradiation with near-ultraviolet light on SDH of cell-free extract 203 XXIV Effect of irradiation with ultraviolet light on succinate oxidase activity of combined particulate and supernatant fractions 204 XXV Effect of ultraviolet irradiation on the reduction of cytochrome b^  by substrate in particulate fraction 206 XXVI Effect of inhibitors on SDH and succinate oxidase activities 208 Mi LIST OF FIGURES Figure Page 1. A general scheme for the respiratory chain 2 2. Mitochondrial respiratory chain by Chance 2 3. The arrangement of the four complexes (I, II, III, IV) of the respiratory chain by Green 4 4. Typical respiratory chain of aerobic bacteria 4 5. The general respiratory chain of facultative anaerobes under anaerobic conditions 4 6. Separation of AS-particulate fraction on Sepharose 4B 57 7. Purification of soluble respiratory complex on a column of Sepharose 4B 59 8. Rechromatography of the soluble respiratory complex on a column of Sepharose 4B 60 9. Purification of acetone powder extract on a column of calcium phosphate 64 10. Purification of acetone powder extract by chrom-atography on a column of Sepharose 4B 67 11. Purification of acetone powder extract by chrom-atography on a column of Sephadex G-100 69 12. Determination of Km for succinate with a whole cell suspension 74 13. Determination of Km and Ki with cell-free extract 76 14. Determination of Km and Ki in the presence of KC1 with the cell-free extract 78 15. Determination of Km and Ki with the soluble respiratory complex 80 16. Determination of Km and Ki in the presence of KC1 with the soluble respiratory complex 82 x i i i Figure Page 17. Time course of DCIP reduction by SDH 87 18a. Conditions for activation of SDH activity 89 18b. Effect of flavin on SDH activity 89 19A. Determination of the optimal incubation time for the activation of SDH and succinate oxidase activities at 38° ,. 92 19B. Spontaneous inactivation of activated SDH 92 20. Irreversibility of activation of SDH 96 21. Time course of DCIP reduction by SDH solubilized from acetone powder 98 22. Effect of buffer composition on ageing of SDH activity of the cell-free extract 101 23. Effect of oxygen on ageing of SDH activity 102 24. Activation of SDH under different conditions 105 25. Effect of PCMB on SDH activity of cell-free extract 109 26. Proposed mechanism for activation of SDH 113 27. Absorption spectrum of ferrous-o-phenanthrolinate 117 28. Determination of the extinction coefficient of ferrous-o-phenanthrolinate 118 29. Determination of minimum concentration of o-phenanthroline required to react with 4 uM ferrous iron solution 120 30. The absorption spectrum of ferrous-o-phenanthrolinate in the particulate fraction 123 31. Determination of the extinction coefficient of ferrous-o-phenanthrolinate in the presence of the particulate fraction 126 32. Determination of the extinction coefficient of ferrous-o-phenanthrolinate in the presence of the particulate fraction and dithionite 129 xiv Figure Page 33. Conditions for the reaction of Tiron with ferric iron (part 1) 133 34. Conditions for the reaction of Tiron with ferric iron (part 2) 136 35. Conditions for the reaction of Tiron with ferric iron (part 3) 138 36. The absorption spectrum of the ferric-Tiron complex 140 37. Determination of ferrous and ferric iron levels in the particulate fraction 142 38. Effect of PCMB on the reaction of o-phenanthroline with the particulate fraction 145 39. Effect of Triton X-100 on the reaction of NHI of the particulate fraction with o-phenanthroline 152 40. Effect of urea on the reaction of NHI of the particulate fraction with o-phenanthroline 154 41. Liberation of SDH, cytochrome bi, protein, directly-reacting and dithionite-reducible iron from the particulate fraction by Triton X-100 156 42. Recovery of SDH, cytochrome bi, protein and NHI after treating the particulate fraction with Triton X-100 159 43. Binding of Fe+^to particulate fraction in the absence and presence of dithionite 162 44. Reduction of NHI by substrate 167 45. Effect of HQNO, succinate and dithionite on the reaction of particulate fraction with o-phenanthroline 169 46. Effect of HQNO and succinate on reduction of cyt bi and reaction of o-phenanthroline with the particulate fraction 172 47. Reduced minus oxidized difference spectrum of particulate fraction 184 48. Spectra of fractions obtained during separation of quinones from E. coli cells 194 XV Figure Page 49. The absorption spectrum of ubiquinone 194 50. Carbon monoxide difference spectrum of particulate fraction 198 51. Effect of detergents on cytochrome bx reduction by succinate 201 52. The NADH and lactate oxidase systems of E. coli as proposed by Cox et al. 211 53. The respiratory chain of E. coli as proposed by Hendler et al. 211 54. Proposed sequence of the succinate oxidase chain of E. coli 212 xvi ABBREVIATIONS USED Amytal Sodium amylobarbitone CCCP Carbonyl cyanide m-chlorophenylhydrazine Cyt Cytochrome DHBG 2,3-Dihydroxy benzoyl glycine DHBS 2,3-Dihydroxy benzoyl serine DCIP 2,6-D ic hioropheno1indopheno1 Dithionite Hydro sulfite Dicumarol 3,31-Methylene-bis-(4-hydroxycoumarin) Dipyridyl 2,2,-Bipyridine DNP 2,4-D in it ropheno1 EPR Electron paramagnetic resonance EDTA Ethylenediamine tetraacetate Fd Flavoprotein of respiratory chain-linked NADH dehydrogenase Fs Flavoprotein of respiratory chain-linked succinate dehydrogenase HQNO 2-Heptyl-4-hydroxy-qu inoldne-N-oxide Isooctane 2,2,4-Tr imethylpentane Menadione 2-Methyl-1,4-naphthoqu inone MK Menaquinone NHI Nonheme iron PCMB p-Chloiromercuribenzoate PCMS p-Chloromercuriphenyl sulfonate PMS Phenazine methosulfate Pi Phosphate; SDH Succinate dehydrogenase TCA Trichloroacetic acid Tiron 4,5-Dihydroxy-m-benzenedisulfonic acid Tris Tris-(hydroxymethyl)-aminomethane TTFA Thenoyltr ifluoroacetone UQ Ubiquinone uv Ultraviolet Ve Elution volume Vo Void volume xvii ACKNOWLEDGEMENTS I wish to express my deepest thanks to my supervisor, Dr. P.D. Bragg, for his guidance, advice and encouragement throughout my thesis work, and for his constructive crtiticism in the preparation of this manuscript. I also wish to thank Dr. S.H. Zbarsky and Dr. W.J. Polglase for the use of equipment, and Dr. G.F. Herring and Mr. R. Tapping of the Department of Chemistry for the determination of electron paramagnetic spectra. I would like to extend general thanks to my laboratory colleagues for discussions and suggestions. I thank my wife, Hiroko, for help in typing and arranging this manuscript. This research was supported by grants to Dr. P.D. Bragg from the Medical Research Council of Canada. 1 INTRODUCTION The respiratory chain is a highly organized, multienzyme complex which transfers pairs of electrons from electron donors, such as NADH and succinate, to electron acceptors. Molecular oxygen is the usual terminal electron acceptor for aerobic cells but sulfate, nitrate, and other substrates may function this way in anaerobic systems. During the oxidation of substrate the released energy may be trapped as ATP by the process known as oxidative phosphorylation. The respiratory chain is located in the inner membrane of the mitochondrion and in the cytoplasmic membrane of bacteria. A general scheme for the respiratory chain of either aerobic or anaerobic cells is shown in Fig. 1 (214). MITOCHONDRIAL RESPIRATORY CHAIN Since Keilin's work on cytochromes (181), the respiratory chain of the mammalian mitochondrion has been studied extensively by many re-searchers. Chance and his collaborators proposed that the mitochondrial electron transport chain is composed of a set of continuous monomeric respiratory proteins arranged in a particular sequence, as shown in Fig. 2 (63, 64, 65, 68, 69). Later Green proposed that the mitochondrial respiratory chain is not composed of monomeric protein, but is a multiprotein, phospholipid-containing enzyme complex (120, 122, 124, 125, 127, 129). It has been possible to divide the electron transport system into four discrete complexes (I, II, III, IV) with two mobile components (UQ, cyt c). In S u b s t r a t e s — > C i > C 2 > > > n ( A D P + P i ) O t h e r s C ; i n t e r m e d i a t e e l e c t r o n t r a n s p o r t c a r r i e r s n F i g . 1. A g e n e r a l scheme f o r t h e r e s p i r a t o r y c h a i n NADH —> Fd-i -> F d 2 — > [Cyfc b-UQ] /N ( A D P + P i ) ATP S u c c i n a t e ->Cyt C j ' C > C y t a«a 1 r 1 (ADP+Pi) ATP ( A D P + P i ) ATP -> Fs F i g . 2. M i t o c h - o n d r i a l r e s p i r a t o r y c h a i n by C h a n c e ( 6 8 ) . 3 addition, the reconstitution experiments of Hatefi have shown that the four complexes can be spontaneously reassembled to form the complete respiratory chain. Each of these complexes contains three or more catalytic proteins arranged in a stepwise order of oxidoreduction po-tential, and they cannot be further resolved into their component pro-teins without destroying their activity. Complex I, which contains UQ, NHI, lipids and flavoprotein, catalyzes the oxidation of NADH by UQ(140, 142); complex II (flavoprotein, cyt b, NHI and lipids) the oxidation of succinate by UQ (140); complex III (cyt b and c, NHI, UQ and lipids) the oxidation of reduced UQ by cyt c (140, 141); complex IV (cytochrome oxidase, copper and lipids) the oxidation of reduced cyt c by molecular oxygen (100, 130, 137). In addition, other proteins (129, 219, 269, 270, 296, 297, 298) are present. The site where succinic and NADH dehydrogenases converge is not clear yet. This may be a pool of cyt b, UQ, nonheme iron or other factors where various inhibitors bind to block the transferring of electrons to cyt c x. The free energy liberated by the series of oxidoreductions is conserved by a subsequent synthesis of ATP. Three ATP are synthesized during NADH oxidation, while two ATP are formed during succinate oxi-dation. In complex II, no ATP is formed, because the redox potential of succinate-fumarate system is nearly the same as that of the reduced UQ-oxidized UQ. The arrangement and order of the complexes of the respiratory chain according to Green is represented in Fig. 3 (128, 122). (ADP+Pi) ATP (ADP+Pi) (ADP+Pi) ATP J ± _ NADH Succinate >Cyt c II III IV Fig. 3. The arrangement of the four complexes (I,II,III,IV) of the respiratory chain by Green (128). Substrates > Dehydrogenases (NADH, succinate, lactate, malate, etc.) Fig. 4. Typical respiratory chain of aerobic bacteria. Quinones > Cyt (b or c) > Cyt oxidase (a and/or o type) Substrates — ^ Dehydrogenases > Quinones > Cytochromes > Nitrate reductase Nitrite reductase Sulfate reductase NO, -> NP2"1 -> SO, Thiosulfate reductase—> SO,' 2 3 etc. Fig. 5. The general respiratory chain of facultative anaerobes under anaerobic conditions. 5 BACTERIAL RESPIRATORY CHAIN Most bacteria have a respiratory chain which is similar to that of mammalian mitochondria except that bacterial systems are more di-verse in their composition and are tightly bound to the cytoplasmic membrane (301). The components and mechanism of the bacterial respir-atory chain are not as well known as the mammalian system. The main reasons for this may be: 1. Physiological differences between bacteria, e.g. obligate anaerobes, facultative anaerobes, and obligate aerobes, result in different compositions and structures of the respiratory chain. In the obligate aerobes, energy is obtained by the complete oxidation of the substrates via pyridine nucleotides, flavoproteins and cytochromes, with oxygen serving as the final electron acceptor. In the obligate anaerobes, the u l t i -mate electron acceptor is not oxygen but other substrates. The actual pathway in facultative anaerobes varies with the environmental conditions of growth, that is, the electron acceptor may be either oxygen or other substrates, e.g. nitrate, sulfate, and others (99, 159, 262, 354, 355, 326). 2. For a single species of organism, the slight change in growth conditions, medium or environmental conditions, alters the respiratory chain. (91, 108, 235, 236, .327, 337). The cytochromes of yeast are qualitatively different between aerobically and anaerobically grown yeast (301). The content 6 of cyt a 2 in E. coli is greater when the bacteria are grown at lower oxygen tension (236), and this cytochrome is absent when aerobically-grown cells are harvested in the logarithmic growth phase (301). Anaerobically-grown E. coli contains cyt c. 3. It is difficult to investigate the individual components of the bacterial respiratory chain because they are tightly bound to particulate structures. In cell-free extracts of bacteria having oxidative activity the activity is associated with broken cytoplasmic membrane fractions which differ only in size and shape, as shown by electron microscopy (1, 307). There is no structure comparable to the mitochondrion. A general scheme of a typical respiratory chain of aerobes is represented in Fig. 4 (110). As shown in Fig. 4, electrons can enter the respiratory chain through various dehydrogenases which are firmly bound to the whole en-zyme complex of the respiratory chain. The bacterial respiratory chain differs from the mammalian mitochondrial system, where electrons enter the main chain primarily through two dehydrogenases, i.e. SDH and NADH dehydrogenase. The prosthetic groups of bacterial dehydrogenases are flavin nucleotides. Therefore, electrons transferred to flavoprotein are, in turn, donated to the cytochrome system. The reduced cyto-chrome is reoxidized by cytochrome oxidase with the reduction of mole-cular oxygen to water. The cytochromes and terminal oxidases are characterized by great diversity (299, 300). The respiratory chain of facultative anaerobes (E. coli, P. pentosaceum) is very similar to that of aerobes except in the terminal 7 electron acceptor. Under anaerobic conditions, various terminal electron acceptors such as inorganic oxidants can be used for res-piration. Under aerobic conditions, oxygen is also used, so that the electron transport system may then be very similar to the strictly aerobic bacteria. The general respiratory chain of facultative an-aerobes grown under anaerobic conditions is represented in Fig. 5. Obligate anaerobic bacteria lack cytochromes and quinones, and use soluble flavin enzymes for electron transport in a substrate to substrate chain (110, 299). COMPOSITION OF THE RESPIRATORY CHAIN Cytochromes Keilin, who rediscovered cytochromes in 1925 (181), recognized the existence of three different types, named cyt a, b, and c. Since then, many kinds of cytochromes have been identified and also isolated. Yeast and mammalian systems contain at least three kinds of cytochromes in the mitochondrial membrane, Cyt b. occurs in the membrane of endo-plasmic reticulum (308, 309). Bacteria have cyt a, b, c and o types which are bound to membrane structures. The exact composition varies according to the species and also depending on the growth conditions for a given species. Some bacteria do not have any cytochrome (299), for example, obligatory anaerobes having no oxidative metabolism (Clostridium, butyric acid bacteria). Identification of cytochromes is based on their absorption maxima in the oxidized and reduced forms. The absorption maxima of their 8 hemes with cyanide and pyridine (cyanide- and pyridine-ferrohemochro-mogens) are also used. Four major cytochromes are classified. Those cytochromes in which the heme is loosely bound, cyts b, a and a 2, have protoheme,heme:a and iron-chlorin prosthetic groups, respectively (324). The function of cytochrome is to transfer electrons between flavoprotein and oxygen in the aerobes (or other electron acceptors in anaerobes). Cytochromes are also involved in oxidative phosphorylation during the electron transfer through the respiratory chain. The val-ance of iron is changed during oxidation and reduction. The principal cytochrome components of bacteria, and those which have been crystallized, are shown in Tables I and II, respectively. Cytochromes b and c Cytochromes of b-type are the major constituents of the respir-atory chain of aerobic and facultative anaerobes (301). They are firmly bound in the cell membrane, and so it is very difficult to characterize them. Detergents, bile salts, lipolytic or proteolytic enzymes, sonic oscillation or other methods have been utilized to liberate cyt b from the membraneous structures. Cyt b contains protoheme bound in non-covalent bonds. The heme can be removed from the apoprotein by treating with acidified aqueous acetone. If the a-peak of the alkaline pyridine ferrohemochromogen spectrum is at 562 to 565 nm, the cytochrome is named cyt b. If the peak is at 557 to 560 nm the cytochrome is cyt bj (180). These cyto-chromes do not react with CO or cyanide. 9 TABLE I The principal cytochromes of certain bacteria Cytochromes Species Reference a l ° b558 c552 c554 A. suboxydans 58.59, 167,302 a l a2 o bj A. vinelandii 58,59,172,173,328 ai a 2 o A. aerogenes* 58,59,328 o bj A.. aerogenes** 58,59,328 a a3 b c l B. subtilis 59,180,302 a a3 0 bj^  B. megaterium 41,203 a a3 b564 c552 C. diphtheriae 253.281,282 a l a2 o b^  E. c o l i * 58,59,182 o bi E. coli** 58,59,182 a l a 2 o bi CS53 H. parainfluenzae 344,345,346 a a3 0 b C C l M. phlei 10,11,180 a l a2 o bi P. vulgaris* 58,59 o bi P. vulgaris** 58,59 a a3 b C C l Mammalian mitochondria 64,68,118,128 * ** in the stationary phase of growth in the log phase of growth TABLE II Properties of crystalline bacterial cytochromes Absorption : Ln re-Cytochrome duced form (nm) Redox po- M.W. Source Reference 3' tential (V) a Y b 562.5 532.5 429 -0.34 28,000 Beef heart 112,113,248,283 bi 557.5 527.5 425 -0.34 66,000 E. coli 84, 102 bi 557.5 527 425 -0.21 . - R. rubrum 96, 180 b2 557 528 423.5 0.12 186,000 L-LUH of yeast 7,8,234,358 b 5 557 526 421 0.02 13,000 Microsome 179, 308,309 b558 558 - 437 -0.34 48,000 A. suboxydans 167, 168 b562 562 531.5 427 0.113 12,000 E. coli 164,165 c 550 520 416 0.26 12,400 Horse heart 226,227 Cl 554 524 418 0.22 53,000 Beef heart 53,272,284 c 2 550 521 415 0.3.65... . 12,000 R. rubrum 151,152 552 522 418 -0.205 12,000 D. vulgaris 151,152 ch 551 522 416 0.30 11,200 A. vinelandii 240, 329, 330 c 5 551 526 420 0.32 11,600 A. vinelandii 240, 329,330 c550 550 521 414 - - E. coli 103, 104 c550 550 520 415 0.25 12,000 B. megaterium 335 c551 551 520 416 0.286 9,000 P. flucreseens 4 c551 551 521 416 0.25 8,100 P. aeroginosa 3, 4, 149, 150 C552 552 523 420 -0.20 "" 11,000 E. coli 21, 103,104,132,337 C553 553 522 417 - 250,000 A. peroxydans 239 c554 554 525 416 0.225 - P. aeroginosa 149, 150 oxidase 605 517 444 - 100,000 Beef heart 157, 349, 363 * cc« 550 - 426 0.009 26,000 P. denitrificans 311 ** c c;r 550 - 424 -0.01 26,000 R. rubrum 18, 152,320,336 * Cryptocytochrome ** Cytochromoid c or Rhodospirillum heme protein(RHP) 11 Several cytochromes of b-type have been isolated and purified from mammalian mitochrondria and bacterial sources (Table II). Purified dehydrogenases isolated from bacterial respiratory chains often contain tightly bound cytochrome components. For example, cyt b in the SDH complex of C. diphtheriae (253, 254, 255), cyt bt in the formic dehydrogenase-nitrate reductase complex (158, 160, 162, 163) or formic dehydrogenase (218, 353) of E. coli, or SDH of P.  pentosaceum (213), cyt b 2 in lactic dehydrogenase of yeast (7, 8), cyt b562 in glucose dehydrogenase of B. antitratum (135), and cyt C553 in alcohol dehydrogenase (239) or lactic dehydrogenase (167, 168) of Acetobacter. Cyt b is also found in the SDH complex of beef heart mitochrondria (372). The cytochrome in the mammalian SDH complex may be a functionally and structurally essential component of the enzyme complex. Thus, there may be two kinds of cyt b in mammalian mito-chondria. One is reducible and is involved directly in electron trans-port, while the other may be a structural component of succinic UQ re-ductase. The latter does not undergo oxidation and reduction (42,359). Cyt c is defined as having a covalently linked protoheme. The spectrum of the alkaline pyridine ferrohemochromogen has an a-peak at 550 nm. Cyt c occurs universally except in obligate anaerobes such as Clostridium and St reptoc occus. Cytochromes of c-type from various sources have been isolated, crystallized, and their sequences deter-mined (226, 227, 242, 306). No cyt c of the mitochondrial type has been found in bacteria, and bacterial cyt c is generally not oxidized by cytochrome oxidases from other sources (6). 12 The primary structure of bacterial cyt c from several sources has also been determined (cyt c551 from P. aeruginosa (4), cyt c 2 from R. rubrum (95), and cyt c 3 from D. vulgaris and D. gigas (180) ). Cytochrome oxidases (a, a l 4 a 2, a 3, o) The function of cytochrome oxidase is to transfer accepted elec-trons directly to molecular oxygen, or to other terminal electron acceptors (nitrate, sulfate). The terminal cytochrome oxidases contain cyt a, a., a 2, a 3, or o. Some bacteria have only one cytochrome oxi-dase but others may have more than one. For example, a-type and o-type cytochromes are often present in facultative anaerobes (Table I). Cyt a contains non-covalehtly bound heme a which can be removed by acidified acetone from the apoprotein. The alkaline pyridine ferro-hemochrombgen shows an a-peak at 580 nm to 590 nm. Cytochrome oxidases are generally sensitive to CO, cyanide and azide. Carbon monoxide binds to cytochrome oxidase forming a particular absorption band which can be dissociated by light. The CO-treatment is widely used to deter-mine the presence of cytochrome oxidase. It is s t i l l unknown i f bacterial cyt o is a b-type cytochrome. Iwasaki has purified and crystallized cyt b558 from Acetobacter  suboxydans, and this is possibly cyt o (168). Two b-type cytochromes have also been isolated from Vitreoscilla (343) and these have the properties of cyt o. The absorption peaks of cyt oxidases are given in Table III. Cyt a and a 3 have the same absorption peaks in the reduced spectrum but the a-band of cyt a 3 is shifted in the presence of CO while cyt a is not. 13 TABLE III Properties of cytochrome oxidases Absorption maxima in the reduced form Binding with Cytochrome a -region Soret region CO CN" a 600- 605 nm 440--445 nm - -a l 585- 595 nm 435--445 nm + + a 2 (d) 620- 630 nm nm + + a3 605 (*590) nm 445 (*430) nm + + 0 *557 -*567 nm *415-420 nm + -* Absorption peak with CO 14 The cytochrome oxidase from the mammalian mitochondrion contains cyt a, a 2 and Cu (94, 245, 246, 341, 349, 304). It has been suggested that Cu takes part in oxidation and reduction in the respiratory chain (24, 131). No copper has been found in bacterial cyt c oxidases. Sol-uble copper-containing blue proteins which may function in the bacterial respiratory chain have been isolated. One from P. aeruginosa has a molecular weight of 17,000 and has been crystallized (149, 150, 74). Others have been extracted from P. fluorescens (4), denitrifying bac-teria (311), several species of Bordetelle, and other sources (310). These blue proteins contain one atom of copper per molecule. The level of cytochrome oxidase may depend on the oxygen tension during growth and on the growth phase of the bacteria (235, 236, 346, 347). Oxygen deficiency stimulates biosynthesis of cyt a 2 in E. coli,  A. aerogenes and H. parainfluenzae (17, 235, 236, 355, 356). Cyt a 2 is not found in the logarithmic phase of growth but appears in the station-ary phase with E. coli, P. vulgaris, and A. aerogenes. It is not found under anaerobic growth conditions in the presence of nitrate (58, 351). Cyt o predominates in the logarithmic phase of aerobic growth in these organisms. The biosynthesis and level of cyt a x is independent of the aeration rate (235, 236). In anaerobic nitrate respiration, the level of cyt a t is increased, which may indicate that i t is associated with nitrate reduction (347). Oxygen and nitrate compete as electron acceptors in the facultative anaerobes because nitrate reductase is induced by nitrate but repressed by high oxygen levels in E. coli (350, 351) and A. aerugenes (235). 15 Few bacterial cytochrome oxidases have been solubilized. Only that from P. aeroginosa grown anaerobically in the presence of nitrate has been crystallized (154). This cytochrome oxidase contained cyt c551 and cyt a 2. It catalyzed the rapid oxidation of reduced cyt c551 and blue protein by oxygen or by the reduction of nitrite (354, 355, 357). Cytochrome c554 was oxidized more slowly. Another cytochrome oxidase which contains cyt a t and c551 has been partially purified from aerobically grown P. aeruginosa (12, 13). A cytochrome oxidase complex (cyt a-a.-6) has been solubilized by de-tergent from M. phlei. Cytochrome o could be liberated from this com-plex by pancreatic lipase (267, 268). This enzyme also releases cyt o from the cyt a3-o particle derived from B. megaterium (41). The nature of the mechanism of reaction of cyt oxidase with oxy-gen is not completely known (220, 366). Cytochromes of E. coli Depending on growth conditions, E. coli may contain cyt a x, a 2, b x, and o. These are located in the cytoplasmic membrane (158, 163, 182, 299). Cyt bx from aerobically grown E. coli has been crystallized (84, 102, 352). It has been shown to be a component of certain respiratory enzymes: formic dehydrogenase-nitrate reductase complex (162, 163), formic dehydrogenase complex (218, 233), nitrate reductase (160, 319). A soluble cyt b562 (105), which is quite distinct from membrane-bound cyt bt (164), has been prepared from the aerobically grown cells. It has been crystallized (164) and its amino acid sequence reported 16 (166). Cyt t>562 is widely distributed in Enterobacter, but it occurs '«at a lower concentration than cyt bx . Its concentration is not varied by growth conditions (105). In addition to the soluble cyt b562, two soluble cyt c, cyt c550 and c552, are also found in the cytoplasmic fraction of anaerobically 'grown Enterobacteriaceae (103, 104, 105, 132). These cytochromes are also found in E. coli grown anaerobically with nitrate (71, 72, 73). Cyt c550 and c552 have been partially purified (103, 104, 105) but the soluble cyt c552 is located at the cell surface, probably outside the •cytoplasmic membrane (106) and it may be involved in anaerobic nitrite reduction (72, 107). Nitrate and nitrite, particularly with nitrite as inducer, induce the biosynthesis of cyt c552 (107, 350, 351) but oxygen inhibits its formation even in the presence of nitrate or nitrite (107, 350, 351). Cyt c550 has been detected in aerated culture of E. coli also. This cytochrome may be involved in sulfite reduction (21). Lipids and quinones Lipids, especially phospholipids, play an important role in the respiratory chain. Phospholipids are absolutely required.for elec-tron transport because they are required for the formation of mem-branes (125, 126, 127, 128, 143). Phospholipids are also required for the interaction between mobile components (UQ, cyt c) and respiratory chain complexes (126, 143, 333). Several kinds of lipophilic quinones have been found to be assoc-iated with the respiratory chain in animals, plants and bacteria (79, 17 119, 123, 152, 204, 205, 252). There are four major groups of quinones: ubiquinones, plastoquinones, menaquinones and phylloquinones. For ex-perimental purposes menadione is often used because of its good water solubility. Plastoquinone is found in the chloroplast of plants, whereas the predominant quinone of mammalian cells is ubiquinone (UQ) (5, 79, 205). Bacteria may contain UQ (s), menaquinone(s), or both. Most gram-negative bacteria contain UQ (300), while gram-positive bacteria contain menaquinones (215, 300, 29). Certain bacteria, in-cluding E. coli (28, 177, 178), synthesize both UQ and menaquinone. The menaquinone content of some gram-positive bacteria depends on the conditions of cultivation (110). In E. coli, aerobic conditions favor synthesis of UQ, whereas menaquinone is formed under anaerobic con-ditions (261). In E. coli, in addition to the predominant UQ8 (215) and mena-quinone (28, 29, 176), a 2-demethyl menaquinone, a series of UQ's (UQj_8, 82), and various menaquinones have been isolated and ident-ified (56). In M. phlei and some aerobic Micrococci, at least three menaquinone iso-prenylogs are found ( 55, 171). The occurrence of several quinones in bacterial systems is a point of difference to higher organisms. The quinones are found exclusively in the cytoplasmic membrane fraction, with the cytochromes (10, 29, 93, 176, 203, 251, 344). The role of quinone in the bacterial respiratory chain has been extensively studied (Table IV). The possible role of quinone as an intermediate of oxidative phosphorylation of M. phlei was once TABLE IV The quinones of mitochondrial and bacterial systems Organism Quinone System Reference Beef heart mitochondria UQg Succinate oxidase 5.78, 80,89,118,215,216 UQ § mitochon-drial lipid NADH oxidase 97, 316, 317 A. vinelandii NADH oxidase 313 Succinate cyt c reductase s 172,323 B. megaterium MK NADH and malate oxidases fumarate reductase 203 B. subtil is NADH oxidase 93 B. stearothermophilus K i ' Succinate oxidase 92 C. diphtheriae MK9 Succinate oxidase 206,281, 282 E. coli UQg Formate-cyt b^  reductase Succinate oxidase NADH oxidase 161 178 174 MKg NADH oxidase 177,178 H. parainfluenzae 2-Demethyl MK7 Succinate, NADH, lactate and 344 formate-tetrazolium reductases M. phlei MKg(IIH) NADH oxidase 10,11,109 19 suggested (39, 40, 121, 176), but is now considered to be unlikely (79, 156). In E. coli grown under the condition of anaerobic nitrate respi-ration it has been shown that cyt b_ is connected to formic dehydro-genase by UQ8 (160), and UQ8, among other quinones, effectively rest-ored the respiratory activity which had been lost after cold acetone extraction (161). The E. coli strains used by Bragg (31) and the author contained only UQ. There was no loss of the NADH oxidase activity after UQ ex-traction (31), which indicates that UQ is probably involved in a minor pathway or not directly in the main respiratory pathways. However, UQ could be reduced by NADH (31). Kashket and Brodie (178) suggested that menaquinone was specifically required for NADH oxidation, but this re-sult has not been confirmed by other workers (31, 76, 174). Additional evidence that UQ is involved in the oxidation of lactate, ma late, and NADH, was obtained using an E. coli mutant lacking UQ. The quinone appears to be located at two different sites which are not directly in the main respiratory chain. The quinone may be complexed with nonheme iron (76). Bragg and Hou have solubilized a NADH-menadione reductase from a particulate fraction of E. coli (31). This enzyme would also reduce UQ, and it may be the enzyme which reduces this quinone in the intact respiratory system. However, the exact location of quinones and their mode of reac-tion in the bacterial respiratory chain s t i l l remain to be determined. 20 Nonheme iron (NHI) proteins It has been suggested that NHI proteins exist universally among animals, plants and bacteria (27, 133). Ferredoxin, or proteins sim-ilar to ferredoxin (molecular weight less than 20,000) have been iso-lated and well characterized from plants (354) and photosynthetic (356) and nitrogen-fixing (257) bacteria. NHI proteins from Clostridium (48, 237, 238) and Azotobacter (47, 87, 285) take part in electron transport during nitrogen fixation. Spinach ferredoxin is involved in light-dependent electron transport (9, 66, 67), and adrenodoxin from adrenal glands in steroid hydroxylation (247). Beinert and his colleagues, employing electron paramagnetic res-onance (EPR) spectroscopy, observed a characteristic asymetric EPR signal, major component at g = 1.94 and a minor component at g = 2.0, at 77° K in substrate-reduced beef heart mitochondria and submitochon-drial preparations which were rich in NHI (23, 27). The signal was attributed to a specific type of NHI protein (23, 27). The same signal was observed in the presence of substrate in SDH (25, 88, 90, 198, 199), NADH dehydrogenase (23, 25, 26, 273), xanthine oxidase (2), hepatic aldehyde oxidase (2), and in each complex of the respiratory chain (23, 141, 142, 143, 139, 369). The NHI protein from complex III has been isolated after succinylation (269). It was concluded that the NHI of EPR signals g = 1.94 is a func-tional group of these enzymes because the rate of appearance and dis-appearance of the signal is in good agreement with the overall rate of the enzymatic reaction (26). 21 In bacterial systems NHI may be a functional component of certain enzymes as also is the case with some mammalian enzymes. This has been shown in mammalian SDH (14, 15, 198, 288, 372) and NADH dehydrogenase (275, 295, 223, 334); SDH of M. lactilyticus (339, 340) and of P__ pentosaceum (213); NADH dehydrogenase (31, 115, 116), NADH oxidase (35), nitrate reductase (277, 338) with Mo (319) of E. coli; succinic-cyt c reductase of A. vinelandii (323); NADH oxidase of M. tuberculosis (207, 114); and succinic oxidase of M. phlei (208, 209, 210, 211). NHI proteins may be involved in oxidative phosphorylation (40, 45, 176, 250). During the succinic oxidase reconstitution experiments carried out by Yamashita and Racker, it was shown that no other NHI was required than that which was present in SDH (44, 359, 360, 361). Although i t was previously believed that NHI giving an EPR signal at g = 1.94 was involved in energy coupling at site I (219), this has recently been found not to be so (249). The experiments of Hatefi suggest that the NHI of SDH is essential to this enzyme i f i t is to react with particle-bound cytochromes (14, 15). The exact role of NHI s t i l l remains to be determined. Dehydrogenases There are numerous dehydrogenases which are involved in glycolysis, the TCA cycle, photosynthesis and fermentation in mammalian and bacterial cells. Most of them, including the electron trans-ferring flavoprotein, are located in the cytoplasm. The respiratory chain-linked dehydrogenases are firmly bound in the membrane. 22 There are two general types of dehydrogenases: 1. Pyridine nucleotide-dependent dehydrogenases, which require either NAD or NADP as co-factors. These groups of dehydrogenases are in the soluble state in the cytoplasm and can be easily extracted. 2. Flavin-dependent dehydrogenases which contain flavin, FAD or FMN, as the prosthetic group. Most of these dehydrogenases are tightly bound to membrane structures and are not easily solubilized. Bacterial dehydrogenases and mitochondrial dehydrogenases linked to the respiratory chain belong to this group. In the mitochondrial respiratory chain, the only flavoprotein dehydrogenases involved in electron transport are succinate, NADH, glycerophosphate, and choline dehydrogenases, and the electron trans-ferring protein. In bacterial systems some other dehydrogenases are linked to the cytochromes of the respiratory chain, e.g. D-lactic and L-malate dehydrogenases. The function of the respiratory chain linked flavoproteins is to be the primary electron acceptor site from substrates and to transfer the accepted electrons to molecular oxygen through the cytochromes in the aerobic respiratory chain. Succinate and NADH oxidase systems probably converge at the cytochrome b or ubiquinone level in the mammalian as well as in the bacterial respiratory chain. NADH dehydrogenase Various NADH dehydrogenase preparations have been extracted by snake venom phospholipase, by acid-ethanol treatment, by sonication 23 or by other treatments, from electron transport particles from mito-chondria (223, 259). All these preparations contain FMN with tightly bound NHI, but the quantitative composition, physical and chemical properties differ slightly from one another (81, 83, 86, 142, 194, 195, 222, 224, 232, 259, 266, 273, 278, 332). The assay method for mitochondrial NADH dehydrogenase activity is based on the ferricyanide reductase activity. This method is used because the "solubilized" enzyme cannot transfer electrons to cyt b or UQ, which are probably the natural electron acceptors in the par-ticulate respiratory chain. In bacterial systems, either ferricyanide reductase or menadione reductase is measured. With bacterial systems less intensive work has been done on NADH dehydrogenase because of the difficulty in isolating i t . Solubiliz-ation of NADH dehydrogenase as well as of SDH has been obtained by treating membrane particles with detergents. Recently, two different soluble NADH dehydrogenases, having menadione reductase activity, have been partially purified from E. coli particles by Bragg and Hou (33, 34). A short summary of the properties of NADH dehydrogenases and oxidases solubilized from two different bacterial sources is given in Table V. Succinate Dehydrogenase Mitochondrial SDH is, like NADH dehydrogenase, an iron-flavoprote but with FAD covalently bound to the apoenzyme (189, 297). The penta-peptide around FAD as the active site has recently been isolated and its sequence determined (190). TABLE V Properties of NADH dehydrogenases and oxidases solubilized from bacterial membranes GrgaQrganism Enzyme Prosthetic group E. coli Menadione reductase I FMN.FAD 2.5 X IO"4 Menadione ca. 35, 000 Menadione, 31 DCIP E. coli Menadione reductase II FMN,FAD 1.4 X IO"5 Menadione ca. 100, 000 UQ8, DCIP, 34 c menadione E.„ coli NADH dehydrogenase FMN,FAD 0.8 X i o" 5 Ferricyanide MK, DCIP, 115 ferricyanide E. coli NADH cyt c reductase FAD 6 X IO"5 Cyt c Cyt c 38 * R. rubrum NADH cyt c reductase FMN 5 X IO'6 Cyt c 33, 000 Cyt c, UQ 155 R. rubrum NADH cyt c reductase FAD 5 X IO"5 Cyt c 26, 000 Cyt c, UQ 155 R. rubrum NADH dehydrogenase FMN.FAD 5 X 10-6 .Menadione DCIP, menadione, 50 ferricyanide R. rubrum NADH oxidase - 7 X IO"6 - Cyt c 49 E. coli NADH oxidase FAD 5 X IO"5 0 UQ, DCIP, cyt c, 34 0?? Acceptor KmNADH for Km Molecular Electron Reference cletermin-(M) nation weight acceptors 25 SDH has been solubilizecl and purified from only few bacterial sources. In the enzyme from the obligate anaerobe, M. lactilyticus, the flavin moiety is not covalently linked and this dehydrogenase is a fumarate reductase rather than an SDH (339, 340). The facultative anaerobe, P. pentosaceum, has two forms of SDH - a soluble and a particulate form. The latter form has been solubilized. Both forms have been purified and found to be the same (213). Both enzymes con-tain cyt bj in addition to NHI as a functional component. In E. coli, it has been reported that there are two species of particle-bound SDH, The activities of the two enzymes vary independently with growthcon-ditions. Under aerobic growth condition, the SDH which is found acts primarily in the direction of converting succinate to fumarate. The enzyme primarily formed under anaerobic conditions is mainly a fumarate reductase (148). Thus SDH seems adapted to the environmental conditions or phys-iological requirements of the cells. With dehydrogenases from aerobic cells succinate is oxidized more quickly than fumarate is reduced, be-cause the Km for succinate is lower than that for fumarate. Since the reaction of SDH is reversible, the assay method for SDH activity can be based on either the spectrophotometric determinat-ion of 2, 6-dichlorophenolindophenol (DCIP) reduction with phenazine methosulfate (PMS) as a catylyst (191, 196), or by reduction of FMN by fumarate (339, 340). One characteristic property of SDH from mammalian and yeast cells is its activation by substrate or by certain competitive 26 inhibitors (186, 187, 188, 225, 226, 230, 325). It has been proposed that this substrate or inhibitor activation, through the same mechan-ism, involves a reversible conformational change in the dehydrogenase (188). Conversion of the inactive form to the active form can be brought about by several minutes incubation with succinate, malonate, and phosphate, or other compounds which bind to the active site of SDH. On removal of the activators the enzyme is inactivated, but i t may be reactivated by repetition of the activation treatment (202). Thorn has reported the activation of a particulate SDH by heating the enzyme to 38° in the absence of substrate or competitive inhibitors (325). It was later found that the activation was due to the presence of an endogenous activator (230). Activation of SDH from bacterial sources by substrate has not been reported until the present work. NHI is present in SDH. Ziegler suggested that thenoyl t r i -fiuoroacetone (TTFA) inhibits succinic-UQ reductase activity by binding the NHI of SDH (372). Beinert and Sands have interpreted the EPR signal at g = 1.94 given by SDH as being due to iron-sulfur groups in the enzyme (25). According to their EPR titration experi-ments, only 8% of total NHI in SDH responds readily to reduction by succinate (88). It seems, therefore, that not a l l of the NHI in SDH is involved in a catalytic activity of the enzyme. The succinate oxidase system of E. coli has been l i t t l e invest-igated. SDH has not been solubilized from this organism nor its properties examined. This thesis describes the isolation of three different soluble preparations containing SDH activity from E. coli. 27 The partial purification and characterization of these enzymes are described. The heat-substrate activation of SDH, as well as of succinic oxidase activity, is reported for the fi r s t time for a bacterial system, and some experiments to determine the mechanism of activation of SDH are given. In addition, this thesis reports on the role and the location of NHI in the electron transport chain of the membrane, as well as on the site of action of inhibitors and on the sequence of the components of the succinate oxidase chain of E. # coli. 28 MATERIALS AND METHODS (i) Chemicals and solvents All chemicals employed were of reagent-grade purity. Disodium succinate, sodium 2, 6-dichlorophenolindophenolate, and disodium malonate were purchased from Eastman Organic Chemicals; spectroscopic-grade isooctane and benzoquinone, from British Drug Houses, Ltd.; Horse cyt c, HQNO, lithium DL-lactate and PCMS from Sigma Chemical Co.; potassium borohydride from Metal Hydrides Inc.; Triton X-100 and pig thyroglobulin from Mann Research Laboratories; sodium cholate and quinacrine hydrochloride from Nutritional Biochemicals Corp.; Human y-<jlobulin, PCMB, FMN, FAD, PMS, bovine serum albumin, dithiothreitol, NADH, dicumarol, glycylclycin, 2, 2 1-dipyridyl from Calbiochem; acetone, petroleum ether (b.p. 40°-60°), diethyl ether, sodium dith-ionite, trichloroacetic acid, thenoyltrifluoroacetone, glycerol, urea, sodium azide, o-phenanthroline, ferric ammonium sulfate and ferrous ammonium sulfate, from Fisher Scientic Fo.; Sephadex G-25, G-100, and G-200, and Sepharose 4B from Pharmacia; Bio-Gel P-300 from Bio-Rad Laboratories; Tiron from J. T. Baker Chemical Co.; nitrogen gas from Canadian Liquid Air, Ltd.; carbon monoxide from Matheson (Canada); CCCP was a generous gift from Dr. D. C. Heytler, E. I. Du Pont de Nemours and Co. (U.S.A.). (ii) Reagents and buffers o-Phenanthroline was dissolved in 20% alcohol unless otherwise stated. 29 TTFA and dinitrophenol were dissolved in ethanol. Solutions of dicumarol and HQNO were prepared by dropwise addi-tion of 1 N and 0.002 N NaOH, respectively, to a stirred aqueous sus-pension until the substance dissolved. Then the solutions were diluted to the appropriate concentration with water. The concentration of HQNO was checked spectrophotometrically (75). Solutions of succi-nate and malonate were adjusted to pH 7.5 with 0.2 N NaOH. Tris buffer was prepared by titrating the appropriate concen-tration of Tris solution with N HCI to the desired pH. Phosphate buffer, for the range pH 7.0 - 8.0, was prepared by titrating 0.1 M dipotassium phosphate with 0.1 M monopotassium phos-phate to the desired pH. Phosphate-NaOH buffer for the range pH 9.0 - 9.5, used for the ferric iron-Tiron reaction, was prepared by titrating disodium phos-phate solution with N NaOH to the required pH. Phosphate-succinate buffer was prepared by dissolving disodium succinate in dipotassium phosphate solution and adjusting the mixture to pH 7.0 or 7.5 with monopotassium phosphate solution. The final concentrations of phosphate and succinate were 0.01 M and 0.025 M, respectively. (i i i ) Enzyme assay procedure All assays were carried out at room temperature. Changes in optical density were measured in a Cary 15 spectrophotometer. One unit of activity is defined as the oxidation of 1 umole substrate 30 per minute. Specific activity in all cases is defined as the oxidation of 1 umole of substrate (for dehydrogenases) or the consumption of 1 uatom oxygen (for oxidase) per minute per mg enzyme protein. A. Succinate oxidase activity Oxidase activity is measured as uptake of dissolved oxygen by the system at room temperature. The oxygen uptake was determined using an Aminco-Chance vibrating reed platinum electrode inserted in a 5 ml vial containing: 0.2 ml 0.5M disodium succinate, 2.0 ml 0.01M phosphate buffer, pH 7.5, and 0.1 ml M NaCl (and 0.2 ml of inhibitor for the ex-periments concerning the inhibition of oxidases). The final volume was adjusted to 3.5 ml (or to 3.7 ml when inhibitor was used) with the same phosphate buffer. The reaction was initiated by the addition of the appropriate amount of enzyme solution and the oxygen uptake was recorded on a 10 mV Varian model recorder. The experiments were performed at room temper-ature . B. Catalase To a cuvette containing 2.0 ml hydrogen peroxide solution the appropriate amount of enzyme was added. The change in absorbance at 220 nm was measured. The hydrogen peroxide solution was prepared as follows: 0.25 ml of 3% hydrogen peroxide was mixed with 24.75 ml 0.1 M phosphate buffer, pH 7.0. This activity was employed to determine the elution position of catalase during column chromatography and so was not quantitated (22). 31 C. SDH (or succinate PMS-DCIP reductase) The method of King (196) was adopted with the slight modification of omitting serum albumin. Each solution was prepared fresh before use. To the cuvette containing 0.9 ml 0.1M phosphate buffer, pH 7.5, 0.1 ml 0.045M potassium cyanide, 0.1 ml 1.5 mM DCIP, 0.2 ml 0.15 M di-sodium succinate, pH 7.5, the appropriate amount of 9 mM PMS was added. 0.1 ml to 0.2 ml of activated or non-activated (control) enzyme sample was added to start the reaction. The absorbance change at 600 nm was measured. The amount of SDH enzyme to be added was controlled by diluting it so that it gave an absorbance change at 600 nm in this assay of between 0.05 and 0.1 per minute with 0.3 mM PMS. The activity is dependent on the concentration of PMS. Thus i t was necessary to use several concentrations of PMS to determine the maximal velocity (Vmax). The amount of PMS used in this experiment was 0.3, 0.6, 0.9 and 1.2 mM. The final volume in the cuvette was 3.0 ml. To calculate the rate, the absorbance change during the first and second minute (i.e. the initial rate) after the addition of enzyme was recorded. This value was corrected for nonenzymatic ab-sorbance change by separate experiments with each level of PMS from which the enzyme had been omitted. The extinction coefficient of 21 liter npmoles-^cm"1 was used (191). 32 The activity and specific activity of SDH was determined without activation (i.e. "non-activated" or "control"), unless stated as "activated". D. DCIP reductases The procedure is similar to that for SDH except that PMS was omitted. To a cuvette containing 0.2 ml 0.5 M succinate or 0.2 ml 9.0 mM NADH, 0.1 ml 1.5 mM DCIP and 2.0 ml 0.1 M phosphate buffer, pH 7.5, the appropriate amount of enzyme was added. The same wavelength and extinction coefficient as in the SDH assay were used. E. Ferricyanide reductases To a cuvette containing 0.2 ml of 0.5 M succinate or 0.2 ml of 9.0 mM NADH, 0.1 ml of 0.02 M potassium ferricyanide and 0.1 M phosphate buffer, pH 7.5, was added, to give a final volume of 3.0 ml. The appropriate amount of enzyme was added after the rate of the non-enzymatic reduction had been measured. The reduction of ferricyanide was recorded as the change in absorbance at 420 nm. For calculation of enzyme activity an extinction coefficient of 1.0 liter mmoles"lcm_1 (139) was used. (iv) Determination of protein The concentration of protein in the enzyme preparation was det-ermined by the method of Lowry et al. (221) with crystalline bovine serum albumin as a standard. 33 (v) Determination of phosphate Phosphate was assayed according to Sumner's method (312). (vi) Determination of molecular weight The molecular weight of SDH was determined by gel filtration on Sephadex G-200 (378) and Sepharose 4B (379). The elution position of the enzyme was compared with that of appropriate standards. For chromatography on Sepharose 4B the standard proteins were thyroglobulin, catalase, bovine serum albumin and ^-globulin. With Sephadex G-200, catalase, bovine serum albumin, cytochrome c and y-globulin were used. The standards were applied to the same gel column after chromatography of the sample. They were eluted with the same buffer. The elution volume (Ve) of the standard proteins divided by the void volume (Vo, determined with blue dextran) was plotted against the logarithm of the molecular weight of the standard proteins. (vii) Irradiation with near-ultraviolet light The enzyme solution (ca. 3 ml; protein concentration as in legends) was placed in a 50 ml beaker cooled in ice. It was irradiated for varying times while being stirred, using a General Electric Mercury Black Light (H 100-FL 4) which was at a distance of 7 cm from the surface of the enzyme solution. The maximum emission from this lamp is of light at 360 nm. 34 (viii) Column chromatography All chromatography was carried out at 4°. A. Adsorption chromatography on calcium phosphate gel 100 ml of 3% calcium phosphate gel suspension (315) was mixed with 200 ml of 10% cellulose powder suspension (Whatman CF11 Fibrous Cellulose Powder). The mixture was deaerated before f i l l i n g the column as described by Massey (229). After the sample had been app-lied to the column, elution was carried out first with 0.01 M phosphate buffer, pH 7.0, and then with a linear gradient of 0.01 M to 0.2 M phosphate buffer, Ph 7.0. Finally the column was eluted with 1.0 M phosphate buffer, pH 7.0. B. Gel filtration Chromatography on columns of Biogel P-300, Sephadex G-25, and G-100, and Sepharose 4B was by the "descending" method, whereas with Sephadex G-200 the "ascending" method was employed. The suspension of preswelled gel in the appropriate buffer was kept overnight in the cold room (4°) before pouring into the column. The column was equilibrated at least 24 hours with the buffer to be used before the sample was applied. The sample was dissolved in the minimum volume of the appropriate buffer before it was applied to the column. 35 (ix) Measurement of cytochromes and flavoprotein Difference spectra were scanned at room temperature with a Gary 15 spectrophotometer, using quartz cuvettes of 1 cm light path. A. Reduced minus oxidized difference spectrum The base line was scanned with equal volumes of enzyme prepar-ation in both the sample and reference cuvettes. After the addition of reducing agent or substrate to the sample cuvette, the spectrum was scanned from 650 nm to 400 nm (an equal volume of the same buffer was added to the bottom cuvette to correct for any volume change). For the dithionite reduced minus oxidized difference spectrum, a few cry-stals of sodium dithionite were added to the sample. Scanning was repeated until the spectrum was constant. If the base line was tilted, the difference spectrum as scanned was corrected by putting the base line at zero absorbance. B. Carbon monoxide difference spectrum The procedure was similar to that for obtaining the reduced minus oxidized difference spectrum. However, substrate or reducing agent was added to both cuvettes. When cyt bx was completely re-duced, a baseline was scanned as described before. Carbon monoxide gas was then gently bubbled into one cuvette for about 30 seconds and the difference spectrum was scanned as described above. Gassing with carbon monoxide was repeated until no increase in the peak of 419 nm was observed. 36 (x) Determination of cytochrome b x, cytochrome o and flavin For the calculation of cyt bx levels in the enzyme preparations, the extinction coefficient of 16 liter mmoles^cm"1 (84, 163), for the height of a-band at 558 nm above the baseline in the reduced minus oxidized difference spectrum was used. In some experiments where the level of reduction of cyt b-. was followed, the height of the Soret band at 427 nm relative to 410 nm in the reduced minus oxidized diff-erence spectrum was taken as a measure of the cyt bx content. The extinction coefficient for the Soret band was 153 liter mmoles-1cm-1 (163) in the reduced minus oxidized difference spectrum. For cytochrome o, the extinction coefficient of 170 liter mmoles"1cm~1 (16) for the peak (416 nm) to trough (430 nm) height of the carbon monoxide difference spectrum was used. For flavin, the extinction coefficient of 10.3 liter mmoles"1cm"1 (120) for the depth of trough below the baseline of 450-460 nm in the reduced minus oxidized difference spectrum was used. (xi) Quantitative assay of ubiquinone in the particulate fraction Particulate fraction (14.2 mg protein) suspended in 0.01 M phosphate buffer, pH 7.5, was placed in a tube (130 x 19 mm). Methanol-petroleum ether (1:1), 5 ml, was added to the tube and the solution mixed vigorously for two minutes using a Vortex-Genie mixer. To this suspension another 5 ml of the solvent mixture was added and the mixing repeated. The mixture was then centrifuged at 10,000 x g for ten minutes. The upper layer was saved and 5 ml of petroleum 37 ether (b.p. 40° - 60°) was added to the lower aqueous layer in the Thunberg tube. The suspension was mixed and centrifuged as before. The upper solvent layers were combined and evaporated to dryness under • reduced pressure. The residue was dissolved in 3 ml ethanol. The spectrum of the solution was scanned between 400 and 240 nm before and after addition of potassium borohydride. The amount of ubiquinone was determined from the difference between the oxidized and reduced spectra at 275 nm, using an extinction coefficient of 12.25 liter mmoles-'cm-1 (266). (xii) Qualitative analysis of quinones from whole cells This procedure is based on the method described by Bragg and Polglase (32). Two g packed cells were suspended and homogenized in 25 ml of 0.01 M phosphate buffer, pH 7.5, with a Potter-Elvehjem Teflon-glass homogenizer. The homogenate was lyophilized. The lyophilized cells were suspended by homogenization in 20 ml of petroleum ether (b.p. 40° - 60°) and mixed for 30 minutes with a Vortex-Genie mixer. The suspension was then filtered through Whatman fi l t e r paper (No. J ) . The residue was re-extracted with an additional 50 ml of petroleum ether as before and the extract filtered. The combined petroleum ether extracts were evaporated to dryness under reduced pressure in a water bath at 30° - 35°. The residue was dissolved in 2 ml isooctane. This solution was applied to a column (10 x 11 mm) of Magnesol-Celite (5:1) which had been washed with isooctane until free from UV-absorbing material. The column was eluted sequentially with the 38 following solvents: three 5 ml portions of isooctane, two 5 ml portions of diethyl ether:isooctane (1:100), three 5 ml portions of ethanol: isooctane (1:100). The spectrum of each fraction was measured. Those fractions which contained ubiquinone (Xmax = 275 nm) were evaporated to dryness under reduced pressure at 40°-45°. The dried material was dis-solved in 2 ml ethanol and the spectrum scanned from 400 nm to 230 nm. (xiii) Determination of the extinction coefficient of  f errou s-o-phenanthro1inate To a cuvette containing 1 ml of freshly prepared ferrous iron solution (ferrous ammonium sulfate), 25 yl of 0.05 M o-phenanthroline was added. After mixing, the spectrum was measured between 400 and 600 nm versus the same iron solution in the reference cuvette. The absorbance difference at 510 nm relative to 550 nm was recorded. The ferrous-o-phenanthrolinate complex shows an absorption peak at 510-515 nm. For determination of the extinction coefficient of the same complex in the presence of 5-8% trichloroacetic acid, 0.5 ml of 0.05 M o-phenanthroline was added to the 1 ml of ferrous iron solution containing this concentration of trichloroacetic acid. (xiv) Determination of the extinction coefficient of  ferrous-o-phenanthrolinate in a turbid solution  and in the presence of dithionite To 1 ml particulate fraction in 0.01 M phosphate buffer, pH 7.5, was added 25 yl 0.05 M o-phenanthroline. The absorbance difference 39 at 510-515 nm relative to 550 nm was measured. When the absorbance had become stable, various amounts of freshly prepared ferrous ammonium sulfate were added and the absorbance difference at 510 nm relative to 550 nm of ferrous-o-phenanthrolinate was recorded. These values, after correction for the absorbance difference obtained in the absence of added iron (i.e. "directly-reacting iron" of the particulate prepar-ation), were a linear function of the iron concentration added. To measure the same extinction coefficient in the presence of dithionite in the particulate preparation (that is to measure "dithionite-reducible iron") a similar experiment to the above was carried out, ex-cept that a few crystals of sodium dithionite were added to the particulate fraction prior to the addition of o-phenanthroline. To minimize the contribution to the absorbance by the reduced cyt bx in the particulate fraction, the enzyme solution was diluted so that the absorbance of the a-band of cyt b_ at 560 nm was less than 0.01. (xv) Determination of total iron The method of King (200) was employed without modification. The enzyme solution (total protein ca. 35 mg) or an equal volume of phos-phate buffer (for control's) were placed in 100-ml Kjeldahl flasks. Each sample was set up in triplicate. The samples were lyophilized overnight in the Kjeldahl flasks. Concentrated sulfuric acid (2 ml; iron less than 0.2 ppm) was added to each flask. The samples were digested first at a "low" heat setting for 30 minutes and then at a "high" heat setting for one hour until the solution became a trans-parent deep red color. After cooling to room temperature 0.25 ml of 40 30% hydrogen peroxide (Fisher S c i e n t i f i c Co.) was added dropwise. Heating was then continued at the high heat setting. Addition of hydrogen peroxide followed by heating was continued u n t i l the s o l -utions were transparent and colorless. Then 5 ml d i s t i l l e d water were added to each fla s k and heating continued for one hour at the high heat setting. The samples were then transferred to 25 ml v o l -umetric f l a s k s . The Kjeldahl f l a s k s were washed f i v e times with 2.5 ml aliquots of water. The washings were transferred to the volum-e t r i c f l a s k s . To each volumetric f l a s k was added 1 ml 0.25% acqueous o-phenanthroline, 0.5 ml of 1% aqueous hydroquinone, and 2.5 ml of 25% sodium c i t r a t e . The pH of the contents of the fl a s k s were ad-justed to between pH 4 and 5 by addition of concentrated ammonium hydroxide using pH paper (Alkacid, Fisher S c i e n t i f i c Co.). The con-tents of the f l a s k were f i n a l l y diluted to 25 ml with water. After standing at room temperature f o r one hour, the absorbance at 510 nm of the solutions were recorded against water as a blank. The absor-bance, corrected for absorbance in the control, was used for the cal c u l a t i o n of t o t a l iron. The extinction c o e f f i c i e n t used was 11.4 l i t e r mmoles _ 1cm _ 1 (365). (xvi) Electron paramagnetic resonance To an EPR tube, 0.2 ml particulate f r a c t i o n (29.3 mg protein per ml, 0.01 M phosphate pH 7.5) was added without substrate, or after reduction by d i t h i o n i t e or 7.5 mM substrate (succinate or NADH). The X-band spectra were recorded as f i r s t - d e r i v a t i v e spectra at 77° K 41 using a Varian E3 spectrophotometer. The recording conditions were as follows: microwave frequency, 9.15 GHz; modulation level, 3.2 or 5G; modulation frequency, 100 kHz; power level 50 mW; time constant, Is; gain setting, 5 x 106; scan rate, 100 G per minute. (xvii) Growth of cells Escherichia coli var. communis (strain Dm of the collection of the Department of Bacteriology, Provincial Dairy School, St. Hyacinth, Quebec) was grown with vigorous aeration at 37° in 15 1 patches in a "Biogen" (American Sterilizer Company, Brampton, Ontario). The following medium was used: 0.7% KjUPO^ , 0.3% KHjPO^ ,0;02% MgSO^ , 0.1% ( N H i J g S O i , , 0.05% sodium citrate, 0.1% yeast extract (Nutritional Bio-Chemicals Corporation), and 0.5% disodium succinate. The cells were harvested near the end of the logarithmic phase of growth (i.e., when the absorption at 420 nm reached ca. 1.85) and were washed once with 0.9% sodium chloride. The washed cells (80-85 g) were stored at -20°. This strain of E. coli was chosen because it grew well on the above medium. (xviii) Preparation of cell-free extract All the procedures were carried out at 0°-4°. The frozen cells were thawed and suspended in buffer solution by homogenization in a Potter-Elvehjem Teflon-glass homogenizer, to give a 10% (W/V) suspension. The viscous homogenate (25-30 ml) was sonicated at 4° in a Bronwill 20-kc sonic oscillator for the approp-42 riate time, ranging from 5 to 15 minutes. The sonicate was centrifuged at 10,000 x g for 20 minutes. The resulting supernatant fluid ("cell-free extract") was used for further studies. The different sonication times, buffer composition and pH, and amount of cells used, are given in the description of the individual experiment. (xix) Fractionation of cell-free extract by ultra-centrifugation  and preparation of particulate fraction A suspension of 0.2 g packed cells per ml 0.01 M phosphate buffer, pH 7.5, was sonicated for 15 minutes, unless noted otherwise, and the cell-free extract prepared as described before. The extract was cen-trifuged for 2 hours at 175,000 x g ("unwashed particulate fraction" and "supernatant"). The unwashed pellet was resuspended in the original buffer. Unless stated otherwise, the suspension was again centrifuged at 110,000 x g for 90 minutes. The pellet obtained ("particulate fraction") was used for experiments after suspending in the same buffer, to give ca_. 1.5-2.5 mg protein per ml. (xx) Isoelectric precipitation of cell-free extract 0.6 g of packed cells was suspended in 20 ml of 0.01 M tris buffer, pH 7.5, and sonicated.for 5 minutes. The cell-free extract was obtained as described before. By gradual addition of cold 0.1 N HCl, the isoelectric point of pH 4.3 was obtained by observing the separ-ation of sedimenting protein from the transparent fluid. The pre-cipitate was recovered by centrifuging the solution at 10,000 x g for 43 20 minutes. It was resuspended in the same buffer and the activity of SDH determined on aliquots (0.37 mg protein for non-activated c e l l -free extract, 0.074 mg protein for activated cell-free extract, 1.32 mg protein for activated isoelectric precipitate suspension) of the solution (Table XV). (xxi) Ammonium sulfate fractionation of cell-free extract 8.5 g of packed cells were suspended in 60 ml of 0.01 M phosphate buffer, pH 7.0, and sonicated for 15 minutes. The cell-free extract was obtained as described previously. Solid ammonium sulfate was added to the extract to give 0.2 saturation (377). The pH was adjusted to 7.0 by addition of 0.2 N NHi+OH and the solution was stirred for 10 min-utes. This solution was centrifuged at 10,000 x g for 15 minutes. In the same manner as before the supernatant was fractionated with solid ammonium sulfate at 0.5 and 0.65 saturation. The pellets obtained at 0.2 saturation ("0.2F"), 0.2-0.3 saturation ("0.3F"), 0.3-0.5 satura-tion ("0.5F") and 0.5-0.65 saturation ("0.65F") of ammonium sulfate were suspended in 3 ml of phosphate buffer and dialysed against 4 liters of the same buffer overnight at 4°. (xxii) Preparation of purified particulate fraction obtained by  ammonium sulfate fractionation from cell-free extract A suspension of 2.5 g packed cells in 20 ml phosphate-succinate buffer, pH 7.5, were sonicated for 15 minutes. The cell-free extract was obtained as described previously. The extract was fractionated with solid ammonium sulfate. The fraction precipitating between 0.2 44 and 0.6 saturation was collected. The pH was maintained at pH 7.5 during precipitation. The sedimented protein was suspended in the same buffer to give a volume of 5 ml (160 mg protein). This was app-lied to a Sepharose 4B column (41.5 x 2.8 cm) and elution was performed with the same buffer. Fractions from the column were analyzed for SDH activity and cytochrome b1. This purified particulate fraction is arbitrarily termed the "AS—particulate fraction" to differentiate i t from the "particulate fraction" obtained from the cell-free extract by ultracentrifugation as described previously. (xxiii) Preparation of soluble respiratory complex from cell-free  extract A suspension of 8 g packed cells in 100 ml of phosphate-succinate buffer, pH 7.5, was sonicated for 15 minutes. The cell-free extract was obtained as previously described. To this extract (90 ml) solid ammonium sulfate was added to give 0.4 saturation. During the addition of salt the pH was mainl^ ed at 7.5 with 0.2 N NH^H. After stirring for ten minutes the mixture was centrifuged at 10,000 x g for 15 min-utes. The pellet (488 mg protein) was resuspended in the same buffer and 2% sodium cholate solution was added to give a final concentration mg. of 1.5 mg per/protein. The mixture was stirred for 15 minutes at 0° and then brought to 0.3 saturation with solid ammonium sulfate. After adjusting the pH to 7.5 the solution was stirred for a further 30 min-utes. The mixture was centrifuged as above. The supernatant was now brought to 0.6 saturation with ammonium sulfate, the pH being kept at 7.5. After stirring for 15 minutes the solution was again centrifuged 4 5 as before. The sedimented pellet was suspended in the same buffer to give a final volume of 6 ml (66 mg protein). This solution was app-lied to a Sepharose 4B column (43 x 2.8 cm) and elution was carried out with the same buffer. Fractions from the column were analyzed for SDH activity and cyt b x. The fractions containing SDH and cytochrome bx were termed "soluble respiratory complex". (xxiv) Preparation of acetone powder of E. coli E. coli cells were suspended in 10 volumes of 0.01 M phosphate buffer, pH 7.0. To this suspension, ten times its volume of cold acetone (-20°) was added dropwise with constant stirring. The temp-erature of the solution was kept below 4 ° . The precipitate was f i l -tered on a Buchner funnel and dried by suction. The dried precipitate was washed twice by suspending i t in cold acetone and collecting i t by filtration. The precipitate was allowed to dry completely at room temperature, with care being taken to avoid exposure to direct sun-light. It was ground to a fine powder and stored over anhydrous copper sulfate and concentrated sulfuric acid in an evacuated desiccator at room temperature and in the dark. Approximately 2 g acetone powder was obtained from 10 g wet weight of packed cells. The SDH activity of this powder was relatively stable for a month. (xxv) Extraction of soluble SDH from the acetone powder The SDH was extracted from the acetone powder by homogenizing it with the specified buffer for 15 minutes at 0°. Insoluble material ("residue") was removed by centrifugation at 10,000 x g for 25 min-utes. The acetone powder was extracted once only unless stated otherwise. 46 (xxvi) Purification of acetone powder extract by chromatography  on calcium phosphate 500 mg of acetone powder was homogenized in 50 ml of phosphate-succinate buffer, pH 7.0, and centrifuged at 10,000 x g for 30 min-utes. The extract (44 ml; 81 mg protein) was applied to a column of calcium phosphate gel (2x8 cm) which was prepared as described before. The column was eluted with 5 ml of 0.01 M phosphate buffer, pH 7.5, and then with a linear gradient of 0.01 M to 0.2 M phosphate buffer, pH 7.0. Finally 1 M phosphate buffer, ph 7.0, was applied. Fractions from the column were analyzed for SDH and cytochrome b x. (xxvii) Ammonium sulfate fractionation of acetone powder extract 770 mg acetone powder was suspended in 45 ml of phosphate-succinate buffer, pH 7.0. The soluble SDH was obtained as described previously. The extract was brought to 0.5 saturation with solid ammonium sulfate, the pH being kept at 7.0 by addition of 0.2 N ammonium hydroxide. The mixture, after stirring for 10 minutes, was centrifuged at 10,000 x g for 15 minutes and the pellet was kept. In a similar manner the proteins precipitating between 0.5 and 0.7 sat-uration, and 0.7 and 0.9 saturation, were obtained. Each pellet (0.5F, 0.7F, 0.9F) was dissolved in 3 ml phosphate buffer and de-salted by passing the solution through a column (1.8 x 17 cm) of Sephadex G-25 (fine). Elution was performed with the same buffer. SDH activity was determined in the fractions from the column. 47 (xxviii) Purification of acetone powder extract by chromatography  on a column of Sepharose 4B One g of acetone powder was extracted twice with 40 ml of phos-phate-succinate buffer, pH 7.0, as previously described. Solid ammonium sulfate was added to a final concentration of 0.7 saturation, the pH being maintained at 7.0 by addition of 0.2 N ammonium hydroxide. This mixture was centrifuged at 10,000 x g for 15 minutes and the pellet was suspended in the same buffer to a final volume of 5.0 ml. The solution (105 mg protein) was applied to a Sepharose 4B column (18 x 19 cm) and elution was performed with the same buffer, with the addition of 0.1 M NaCl. The fractions from the column were examined for cyt bj and SDH as previously described. (xxix) Purification of acetone powder extract by chromatography  on a column of Sephadex G-200 One g of acetone powder was extracted with 50 ml of phosphate-succinate buffer, pH 7.5. The extract was centrifuged at 10,000 x g for 30 minutes. The supernatant was brought to 0.6 saturation with solid ammonium sulfate and the pH was adjusted to 7.5. The precip-itated protein recovered by centrifuging the liquid at 10,000 x g for 15 minutes was suspended in the same buffer. The solution (7 ml; 69 mg protein ) was applied to a column of Sephadex G-200 (35 x 2.8 cm) and eluted with the same buffer. 48 (xxx) Determination of Km for succinate The SDH activity of the enzyme preparation was determined, with-out activation, as described in Materials and Methods, except that different final concentrations of succinate, ranging from 0.1 to 4.0 mM, were used. Activity is expressed in units using 1.2 mM PMS in the assay. To calculate Km, Lineweaver-Burk curves were plotted. (xxxi) Determination of Ki for malonate The enzyme was incubated with each of two concentrations (0.075 or 0.225 mM) of malonate at 0° for 5 minutes and 0.2 ml of the incu-bated mixture was removed to determine SDH activity. The final concentration of malonate in the cuvette was 0.005 or 0.015 mM. Determination of SDH activity was performed, without activation, as described in Materials and Methods, except that the final succinate concentration ranged from 0.1 mM to 4.0 mM. Activity is expressed in units using 1.2 mM PMS in the assay mixture. Lineweaver-Burk curves were plotted to calculate Ki. (xxxii) Activation of SDH Unless indicated otherwise, an appropriate amount of enzyme solution was diluted with an equal volume of 0.15 M disodium succinate, pH 7.5, and incubated at 37°-38° for 15 minutes. Aliquots were then removed for assay of SDH or succinic oxidase activities. 49 PART I. STUDIES ON SUCCINATE DEHYDROGENASE (SDH) Since few studies on the succinate oxidase system from E. coli have been made (147, 148, 178), this system was studied by the author. The isolation and purification of SDH was attempted, and the proper-ties of SDH have been studied. This is described in this section of this thesis. The role of NHI of the respiratory chain is described separately in Part II of this thesis, and the sequence of the components of the succinate oxidase chain is proposed and described in part III of this thesis. RESULTS AND DISCUSSION (1) PREPARATION OF SDH 1. Localization of dehydrogenases The whole cells have active NADH, lactate and succinate oxidase activities. As shown in Table VI, SDH is localized almost completely in the particulate fraction, while NADH-ferricyanide reductase is found in the supernatant. This indicates that SDH is tightly bound to the membrane frag-ments as has been found by Hirsch et al. (148, 256, 285, 286). About 40% of the succinate oxidase activity of the cell-free extract was recovered in the particulate fraction, which also indicates that the succinate oxidase system is membrane bound. TABLE VI Localization of NADH dehydrogenase and SDH NADH d e h y d r o g e n a s e S D H Fraction Total protein (mg) Electron acceptor Control Control Activated Specific Total Specific Total Specific Total activity activity activity activity sctiv ity activity Cell-free PMS-DCIP 0.063 14.8 0.23 53.3 extract 229 DCIP 0.13 27.9 0.0009 0.19 0.0043 0.98 Ferricyanide 1.73 396.0 0.0041 0.94 0£0I)37> 3.12 PMS-DCIP 0.014 2.3 0.026 4.2 Supernatant 161 DCIP" 0.15 24.0 0.0005 0.09 0.0017 0.27 Ferricyanide 1.82 293.0 0.0036 0.58 0.0071 1.15 Unwashed PMS-DCIP _ _ 0.204 13.0 0.83 53.3 particulate 62.5 DCIP 0.014 " 0.9 0.0021 0.13 0.009 0.56 fraction Ferricyanide 0.13 8.0 0.0096 0.60 0.031 1.92 (Washed) PMS-DCIP _ _ 0.437 12.5 1.21 34.7 particulate 29 DCIP 0.008 0.2 0.0037 0.11 0.015 0.45 fraction Ferricyanide 0.04 1.0 0.0166 0.48 0.050 1.44 The fractionation was carried out as described in Materials and Methods, starting from a suspension of 7 g packed cells in 70 ml 0.01 M phosphate buffer, pH 7.5, which was sonicated for 5 minutes. The enzyme assays and activation procedure are described in Materials and Methods. 51 2. Liberation of SDH by sonication-Since SDH is not easily released from the membrane, sonic oscil l -ation was employed to break the cytoplasmic membrane of E. coli. As shown in Table VII, the longer the period of sonication the more the protein that was released, and accordingly the greater the degree of solubilization of SDH. It was also found that the activities of SDH and succinate oxidase were decreased by sonicating the cells for longer than 15 minutes. Therefore, during this thesis work the time of sonication was not allowed to exceed a total of 15 minutes. Generally, two 7 minute per-iods of sonication were employed. The chamber was cooled to 0° between the periods of sonic oscillation. 3. Isoelectric precipitation of the cell-free extract Experiments were carried out to explore whether isoelectric pre-cipitation of the cell-free extract would permit recovery of SDH activity without its destruction. If this was possible, we hoped to use this as a primary step toward purification of the enzyme. The pH of the isoelectric point was 4.3. Though 90% of protein was re-covered during this procedure the recovery of SDH activity was very low (Table VIII) . Addition of flavin to the isoelectric precipitated protein did not reactivate the inactivated SDH (Table XXI). Thus, the low SDH activity in the isoelectric precipitated protein is not due to the loss of flavin but is due to the denaturation of SDH protein. "Heat 52 TABLE VII Liberation of SDH as a function of sonication time Sonication Total First day Second day time (min) Protein (mg) Control Activated Control Activated 5 74 2.49 11.0 1,21 10.5 (0.0337) (0.149) (0.0169) (0.142) 15 106 3.94 32.1 3.28 27.0 (0.0371) (0.302) (0.0309) (0.254) Two suspensions of 0.6 g packed cells in 20 ml of 0.01 M Tris buffer, pH 7.5, were sonicated for 5 and 10 minutes. The cell-free & extracts were prepared as described in Materials and Methods. The extracts were kept at 0° to assay their activity on the second day. Aliquots (0.74 mg and 0.074 mg protein for non-activated and activated extract, respectively, obtained after 5 minutes sonication; 0.53 mg and 0.07 mg protein for non-activated and activated extract, respect-ively, obtained after 15 minutes sonication) were assayed for SDH as described in Materials and Methods. Specific activity is given in parenthesis. 53 TABLE VIII Recovery of SDH activity in the isoelectric precipitate Preparation Total Protein Specific Activity Recovery (mg)„ Control Activated (%) Cell-free extract 29.6 0.031 0.254 100 Isoelectric precipitate 26.4 Very low 0.056 22 The isoelectric precipitate was prepared from the cell-free extract, as described in Materials and Methods, and suspended in 4 ml of 0.01 M Tris buffer, pH 7.5 (6.6 mg protein per ml). Aliquots (0.074 mg protein or 0.22 mg protein for the cell-free extract and the isoelectric precipitate suspension, respectively) were employed for the determination of SDH activity after activation. Protein recovery is 90 %. 54 activation" in the presence of succinate was without effect. Therefore, it was concluded that the isoelectric precipitation was accompanied by serious damage to the active site and conformation of the SDH protein. 4. Purification of the particulate fraction from the cell-free extract. The cell-free extract was fractionated with ammonium sulfate. As shown in Table IX, the activities of SDH and succinate oxidase were mostly found in the fraction precipitated at 0.2 to 0.3 saturat-ion. Cyt bj was also found to this fraction. The fraction (0.2 to 0.6F) was applied to a column of Sepharose 4B, to separate and purify succinate oxidase or dehydrogenase. As shown in Fig. 6, SDH having succinate oxidase activity as well as cyt bj, was excluded from the column. This indicates that the active material has a very large molecular weight. It may be composed of fragments of the cytoplasmic membrane which was broken down during sonication. This fraction was arbitrarily termed the AS-particulate fraction. It also contained a l l the membrane-bound respiratory carriers. 5. Disintegration of the membrane and purification of the cleaved respiratory chain by detergent The cleavage of the mitochondrial respiratory chain into indiv-idual enzymes or complexes has been successfully performed using sodium cholate (140, 145, 193) or deoxycholate (145). TABLE IX Distribution of SDH and succinate oxidae activities in fractions precipitated by ammonium sulfate from the cell-free extract Total SDH* Succinate oxidase"3 Cyt b x Fraction protein (mg) Specific activity Total activity Spec if ic activity total activity 0.2 F 40 0.279 11.2 0.159 6.4 + 0.3 F 215 0.415 112.0 0.168 36.3 + 0.5 F 340 0.067 22.8 0.020 6.7 - + 0.65 F 319 0 0 0 0 -f tha activity was determined after activation in the presence of 15 mM succinate. b the activity was assayed after activation in the presence of 50 mM succinate 56 Fig. 6. Separation of AS-particulate fraction on Sepharose 4B . The fraction precipitated from the cell-free extract by ammonium sulfate between 0.2 and 0.6 saturation was chromato-graphed on Sepharose 4B as described in Materials and Methods. SDH activity assayed in the presence of 1.2 mM PMS is expressed as absorbance units per ml eluant, where one unit equals an absorbance change at 600 nm of 1.0 per minute;- The enzyme was activated as described before. Cytochrome b^ is expressed as the height of the Soret band in the difference spectrum. X 100 150 200 250 3 0 0 EI ut ion Vol ume, m I Fig. 6. Separation of AS-particulate fraction on Sepharose 4B 58 In bacterial systems there has been l i t t l e work on the successive resolution and reconstitution of the respiratory chain. Detergents have been routinely used to cleave the bacterial membrane system. Sol-ubilization of NADH dehydrogenase from B. megaterium with deoxycholate (98), nitrate reductase from A. aerogenes (274) and NADH dehydrogenase from R. rubrum with Triton X-100 (51), formate-cyt b1 reductase (218), formate dehydrogenase and cyt bj-nitrate reductase (160, 163), and several NADH dehydrogenases (menadione reductases) (31, 34) from E. coli with deoxycholate or sodium dececyl sulfate (33), have been reported. The particulate fraction obtained by ammonium sulfate fractiona-tion from the cell-free extract was treated with an anionic detergent, sodium cholate, in the presence of ammonium sulfate and purified by Sepharose 4B gel filtration. As shown in Fig. 7, SDH was eluted to-gether with cytochrome b_. A molecular weight for this L'soluble respiratory complex" of approximately 1.6 x 106 was obtained by com-parison with:the elution positions of standard proteins. The elutants having SDH activity were pooled and precipitated again with ammonium sulfate. This precipitated material was re-chroma to graphed on the same Sepharose 4B column. As shown in Fig, 8, SDH with cyt b1 was mostly excluded in the void volume of the column in the same way as the particulate fraction (Fig.8). This suggests that the respiratory complex had reaggregated in the presence of ammonium sulfate into macroprotein. It is not known whether the polymerization leads to the formation of the original particulate fraction. 59a Fig. 7. Purification of soluble respiratory complex on a column of Sepharose 4B. The soluble respiratory complex was prepared and chromatographed on Sepharose 4B as described in Materials and Methods. SDH was ass-ayed after activation and is expressed as absorbance units per ml eluant using 1.2 mM PMS in the assay mixture, where one unit equals an absorbance change at 600 nm of 1.0 per minute. The amount of cyt bj is expressed as the height of the Soret band in the difference spectrum, "c" indicates the elution position of catalase. Elution Volume, ml 60a Fig. 8. Rechromatography of the soluble respiratory complex on a column of Sepharose 4B. The fractions having SDH activity from the first separation of the soluble respiratory complex on Sepharose 4B were combined and brought to 0.6 saturation with solid ammonium sulfate. The pH was maintained at 7.5 by addition of 2N ammonium hydroxide. After stirr-ing for 10 minutes at 4°, the solution was centrifuged at 10,000 x g for 15 minutes. The pellet was suspended in the minimum volume of phosphate-succinate buffer, pH 7.5, and both cytochrome bj and SDH activity were measured as in the first separation, a, b, and c indi-cate elution positions of thyroglobin, catalase and bovine serum albumin respectively. Vo, void volume. 100 150 200 250 Elution Volume, ml Fig. 8. Rechromatography of the soluble respiratory complex on a column of Sepharose 4 B . 61 A s i m i l a r soluble r e s p i r a t o r y complex has also been separated from a d i f f e r e n t s t r a i n of Ii. c o l i and extensively studied by B a i l l i c , Itou and Bragg (16). They reported that the soluble r e s p i r a t o r y com-plex was not d i r e c t l y l i berated from the p a r t i c u l a t e f r a c t i o n by det-ergent but was reconstituted from smaller species of proteins (such as SbH and a cyt bj-cyt o complex)which were separately l i b e r a t e d from the membrane by detergent. Both NAuH and succinate oxidase a c t i v i t i e s were recovered by addition of extra ubiquinone although the r e s p i r a t -ory complex s t i l l contained ubiquinone (16). The soluble r e s p i r a t o r y complex may be formed when detergent and ammonium sul f a t e are removed during chromatography. The treatment with cholate d i d not e f f e c t the reduction of f l a v i n and of cyt b x by succinate (Table XXII) which indicates that the action of cholate i s not at the l e v e l of the ind i v i d u a l r e s p i r a t o r y c a r r i e r s . Cholate probably acts at a hydrophobic s i t e between some of the compon-ents of the oxidase chain and so prevents e f f i c i e n t electron transport between them. 6. Extraction and p u r i f i c a t i o n of soluble SDI! from acetone powder Tt i s possible that the presence of l i p i d s w i l l prevent the s o l -u b i l i z a t i o n of ind i v i d u a l enzymes and r e s u l t in the separation of the enzyme together with other proteins i n a large molecular weight com-plex. To avoid t h i s problem an acetone powder of E. co1i c e l l s was prepared and SUIT extracted from t h i s with aqueous buffer i n the presence or absence of succinate. 62 The presence of succinate in the phosphate buffer increased the SDH activity (Table X), probably by preventing the inactivation of the enzyme after its separation from the membrane. This is probably an example of allotopy (267), that is, a difference in the properties of the soluble enzyme when compared to the original membrane-bound enzyme. As will be discussed later, the effect of succinate is not due to the "activation" phenomenon. In the presence of succinate about 20-30% of total SDH was isolated in the first extraction of the acetone powder. The acetone powder extract contains only trace amounts of cyt bj. To remove cyt bj adsorption chromatography on a column of calcium phosphate column was performed. The enzyme was eluted with phosphate buffer only to avoid possible interference by the presence of succinate. As shown in Fig. 9, two peaks of SDH were found. The minor peak was eluted at concentrations below 0.01 M phosphate and the major peak be-tween 0.02 M to 0.15 M phosphate. No cyt bx was detected in the SDH of both peaks. Cyt bt was found in the material eluted above 0.2 M phosphate. The second peak of SDH had a specific activity of 0.0934, while the sample which had been applied to the column had a specific activity of 0.0633. The specific activity of the latter had dropped to 0.0246 on the day when the activity of SDH in the column eluant was analyzed. Therefore, it was not possible to calculate its degree of purification. Further purification studies on this enzyme were not carried out because the enzyme was extremely labile and i t was not possible to stabilize i t . Succinate or dithiothreitol, alone or in combination, did not stabilize the enzyme. TABLE X Extraction of soluble SDH from acetone powder with phosphate buffer, pH 7.5, in presence or absence of succinate (A) Extraction with 0.01 M phosphate buffer Total Control Heat-treated Preparation protein (mg) Specific Total Recovery of Specific Total Recovery of activity activity activity (%) activity activity activity (%) Acetone powder 350 0.017 6.47 100 0.013 4.9 100 suspension First extract 93 0.046 4.35 67 0.025 2.3 47/7 Second extract 13 0.021 0.28 4.3 Very low 0 0 Residue suspension 150 0.020 3.00 46.2 0.020 3.0 61.6 (B) Extraction with phosphate-succinate buffer Acetone powder 300 0.099 29.8 100 0.060 17.8 100 suspension First extract 91.4 0.068 6.2 21. 0.029 2.7 15 Second extract 10.3 0.200 2.1 .6.9 0.051 0.52 3 Residue suspension 106 0.118 12.5 42 0.111 11.82 66.2 0.5 g acetone powder was extracted twice with 20 ml of deaerated buffer, as described in Materials and Methods. Fig. 9. Purification of acetone powder extract on a column of calcium ^phosphate . 7 The soluble SDH was extracted from the acetone powder and chromatographed on a column of calcium phophate, as described in Materials and Methods. SDH activity is expressed as units per 0.2 ml eluant using 1.2mM PMS in the assay. 64 O io d CD ^- CM O " J U 0 8 S IB e o u e q j o s q v — — 65 7. Ammonium sulfate fractionation of acetone powder extract and its purification by gel filtration Ammonium sulfate fractionation of the acetone powder extract was performed as shown in Table XI. Most of the SDH activity was recovered in the 0.5F fraction. Therefore, this fraction was used for further purification. When this fraction was applied to a column of calcium phosphate and eluted with a linear gradient of phosphate buffer concen-tration from 0.01 M to 0.3 M, SDH was eluted below 0.2 M phosphate. Cyt bi was not found. Only one peak of SDH, which was eluted at a similar position to the second peak of SDH in Fig. 9, was observed. It is assumed that both are the same species of SDH. Because of the lability of SDH obtained from chromatography on calcium phosphate, its purification was attempted on a column of Sepharose 4B. As shown in Fig. 10, the peaks of SDH and cyt bx were not coincident and the molecular weight of SDH is larger than that of cyt bj. This may indicate that there is polymerization of the SDH protein. When the same preparation was applied to a column of Biogel P-300 the SDH activity was not excluded in the void volume. The molecular weight of SDH would then be less than 6 x 105. Therefore i t was purified on a Sephadex G-200 column (Fig. 11). Under these conditions there were two peaks of SDH. The minor peak had a molecular weight of about 3.3 x 105 (specific activity, 0.045) and a major peak around 1 x 10s (specific activity, 0.073). The spec-i f i c activity of the cell-free extract was 0.125 but since the activity 66 TABLE XI Distribution of SDH activity in fractions from ammonium sulfate fractionation of acetone powder extract Fraction Total protein (mg) Specific activity Total activity Yield (%) Purifi-cation Cyt bj Suspension of acetone powder 430.5 0.043 18.3 100 1 + Acetone powder -extract 121.6 0.069 8.4 46 1.6 + 0.5 F 49.3 0.105 5.2 28 2.5 + 0.7FF 39.5 0.014 0.56 3 0.3 + 0.9 F 4.8 0 0 0 0 -The procedure is described in Materials and Methods. The SDH activity was determined without prior activation. 67 50 1 0 0 El ut io n Volume, m l 0.1 O 0.05 Fig. 10. Purification of acetone powder extract by chromatography on a column of Sepharose 4B. The extraction of the acetone powder and the chromatography of the extract were performed as described in Materials and Methods. SUM activity is expressed as absorbance units -per ml eluant, using 0.3 mM PMS assay mixture, where one unit equals an absorbance change at 600 nm of 1.0 per minute. Cytochrome b-^  is expressed as the height of the Soret band in the difference spectrum. 68 Fig. 11. Purification of acetone powder extract by chromatography on a column of Sephadex G-200. The extraction of the enzyme and the conditions of chromatography are described in Materials and Methods. SDH activity is expressed as absorbance units per ml eluant using 1.2 mM PMS, where one unit equals an absorbance change at 600 nm of 1.0 per minute. Vo, void volume a,b,c,d and e, are elution volumes of y-globulin, bovine serum albumin, cyt c, SDH with lower molecular weightr and SDH. with higher molecular weight, respectively. The inset shows the calibration curve for the above proteins. E L U T I O N VO L U M E , ml Fig. 1 1 . Purification of acetone powder extract by chromatography on a column of Sephadex G-200. 70 of SDH on the same day when the column eluant was analyzed was not det-ermined i t is not possible to state the extent of purification. 8. The role of cyt b x in SDH The function of cyt by in the respiratory chain of E. coli grown either aerobically or anaerobically has been described in the Intro-duction of this thesis (160, 162, 163, 218, 353). It has been suggested that cyt b occurs as a structural component of SDH in the mitochondrial systen (42, 89, 360, 361, 370), and poss-ibly also in bacterial systems (C. diptheriae and P. pentosaceum: see Introduction of this thesis) because cytochrome was found in the partially purified dehydrogenases. In E. coli cyt bt.could not be easily removed from formic dehydrogenase (218, 353). These facts suggest that there may be more than one variety of cyt bL; one in-volved in the redox system under aerobic conditions and another fun-ctioning under the anaerobic conditions or acting as a structural com-ponent. This point will be discussed later. The author tried to remove cyt bx from SDH-containing preparations. Only the SDH from the acetone powder extract after chromatography on calcium phosphate showed no cyt b l t although a small amount could s t i l l have been present. However, the molecular weight of 100,000 would suggest that cytochrome bi was absent. It is possible that cyt b l f NHI and the flavin moiety of SDH con-stitute and are required for a functional unit for SDH to react with the cytochrome chain but are not "Required for the reduction of PMS-DCIP 71 system. Removal of cyt bj would increase the l a b i l i t y of SDH i f i t was an es s e n t i a l component of the enzyme. (2) PROPERTIES OF SDH 1. Determination of SDH a c t i v i t y In mammalian systems soluble or p a r t i c u l a t e SDH shows the a c t i v -i t i e s of DCIP reductase, quinone reductase, f e r r i c y a n i d e reductase and PMS-DCIP reductase (289). Among these the PMS-DCIP reductase a c t i v i t y i s the highest. Therefore, succinate PMS-DCIP was rou t i n e l y employed to measure SDH a c t i v i t y . The reaction i s as follows: Succinate + SDH ^==* fumarate + reduced SDH reduced SDH + 2PMS SDH + 2H*PMS 2H - P M S + DCIP 5===^ 2PMS + DCIPH2 Net: Succinate + DCIP 5===—• fumarate + DCIPH2 PMS thus acts as a c a t a l y s t to transfer hydrogen between reduced f l a v o -p r o t e i n and DCIP. The fractionated preparations from c e l l - f r e e extracts of E. c o l i have very low succinate-ferricyanide or -DCIP reductase a c t i v i t i e s (Table VI) but the combined PMS-DCIP system was a c t i v e l y reduced by succinate.. Therefore, t h i s method has been employed to determine the SDH a c t i v i t y of E. c o l i . In the presence of excess DCIP and succinate, the re a c t i o n rate i s dependent on the l e v e l of PMS. Therefore, i t was necessary to use d i f f e r e n t concentrations of PMS to determine the Vmax f o r PMS. The 72 value so obtained for the activity of SDH v/as considered to be the Vmax for SDH. The presence of KCN prevents hydrogen peroxide from causing oxi-dation of PMS (187). It also shunts the electron flow, which is normally to cytochrome oxidase, towards PMS. In the determination of NADH dehydrogenase, PMS-DCIP reduction could not be measured because NADH itself chemically reduces PMS. Since either menadione or ferricyanide are reduced by this enzyme either reductase activity is conventionally represented as the bacterial NADH dehydrogenase. It was not determined whether the electron donating site of the dehydrogenase to the next natural electron acceptor is the same as that for reduction of the artif i c i a l electron acceptor. The site may be different since succinate PMS-DCIP reductase is unstable while succinate-cyt bx reductase is stable. 2. Kinetics of SDH For the determination of Km, the initial rate of DCIP reduction was chosen since the preparations employed gave a hyperbolic increase in activity with time (see below). Despite this, the relationship of initial SDH activity against substrate concentration gave a typical hyperbolic curve (Fig. 12A and inset of Fig. 13). Thus the reaction may be described by classical Michaelis-Menten kinetics. Determination of the Km of the "activated enzyme" (see the next section) was not attempted because the exact mechanism of the activation phenomenon is not known. Moreover, succinate must be added for the activation. It 73 is possible that the succinate added for the activation procedure may be bifunctional. One function may be the modification of the SDH molecule itself, and the other to act as substrate for the enzyme. From this study i t was found that SDH is not an allosteric enzyme because it did not show S-type kinetics. Therefore the control of succinate oxidation is not mediated directly by substrate but can be controlled via SDH since the activity of this enzyme is inhibited com-petitively by the product, fumarate. Malonate was also a competitive inhibitor, as shown in Fig. 13-16. As shown in Table XII, both the Km for succinate and the Ki for malonate are lower than those of mitochondrial SDH. The Km of the enzyme of the cell-free extract is very similar to that reported by Hirsch et al_. (148). As shown in Table XIII, the presence of KC1 decreased the Km by half without affecting Ki significantly. There was no marked in-crease in Vmax in the presence of KC1. KC1 was included in the eluting buffer during separation and purification of the soluble respiratory complex by gel filtration because the salt was reported to facilitate elution by decreasing the effects of ionic bonding. Actually, the pattern of chromatography in the presence or absence of KC1 did not show any marked change in the elution position of SDH. The reason for the effect of KC1 on the Km of SDH is not clear. The effect on Km but not on Vmax should suggest that the salt is effecting the binding of substrate. But it is difficult to reconcile this with the absence of effect on the Ki for malonate, since malon-ate as a competitive inhibitor should bind at the same site as succinate. 74a Fig. 12. Determination of Km for succinate with a whole cell suspension. A. Plot of the SDH (V) versus succinate concentration (S) B. Lineweaver-Burk plot of data from Fig. A Determination of SDH activity was performed as described in Materials and Methods, except that the final concentration of succi-nate was varied. V is expressed in absorbance units using 1.2 mM PMS in the assay mixture, where one unit equals an absorbance change at 600 nm of 1.0 per minute. 0.2 ml (1.8 mg protein) whole cell suspension: was. assayed without activation. The whole cell suspension was prepared by suspending 3 g packed cells in 52 ml 0.01 M phosphate buffer, pH 7.0. 74 Fig. 12 (B) 75 Fig. 13. Determination of Km and Ki with cell-free extract. A suspension of 4 g packed cells in 50 ml 0.01 M phosphate buffer, pH 7.5, was sonicated for 15 minutes and the cell-free extract obtained as described in Materials and Methods. Aliquots (0.20 mg protein) were employed to determine SDH activity in the absence (a) or in the presence of malonate (b, 0.005 mM; c, 0.015 mM), as described in Materials and Method using 1.2 mM PMS in the assay mixture. The enzyme was not activated, V is expressed in absorbance units, where one unit equals an absorbance change at 600 nm of 1.0 per min. S, concentration of succinate. The inset is the substrate-activity plots with or without malonate. 77 Fig. 14. Determination of Km and Ki in the presence of KC1 with the cell-free extract. A suspension of 4 g packed cells in 50 ml 0.01 M phosphate buffer, pH 7.5, was sonicated for 15 minutes and the cell-free extract obtained as described in Materials and Methods. The same buffer was added to give a final concentration of 0.25 M KC1. Aliquots (0.12 mg protein) were used for the determination of SDH activity in the absence (a) or in the presence of malonate (b, 0.005 mM; c, 0.015 mM), as described in Materials and Methods. V is expressed in absorbance units, where one unit equals an absorbance change at 600 nm of 1.0 per minute. S, concentration of succinate. 78 Fig. 14. Determination of Km and Ki with cell-free extract. 79 Fig. 15. Determination of Km and Ki with the soluble respiratory complex . A suspension of 4 g packed cells in 52 ml 0.01 M phosphate buffer, pH 7.5, was sonicated for 15 minutes and the cell-free extract obtained as described in Materials and Methods. The soluble respiratory complex was prepared from the extract as described in Materials and Methods. Elution of the enzyme from a Sepharose 4B column (40 x 2.8 cm) was carried out with the phosphate buffer. The eluant containing the highest SDH activity was used to determine Km and Ki. Aliquots (0.044 mg protein) were removed to assay SDH activity in the absence (a) or in the presence of malonate (b, 0.005 mM; c, 0.015 mM), as described in Materials and Methods, using 1.2mM PMS in the assay mixture. The enzyme was not activated. V is expressed in absorbance units, where one unit equals an absorbance change at 600 nm of 1.0 per minute, S, concentration of succinate. 1 / S , mM Fig. 15. Determination of Km and Ki with the soluble respiratory-complex. 81 Fig. 16. Determination of Km and Ki in the presence of KC1 with the soluble respiratory complex. A suspension of 4 g packed cells in 54 ml 0.01 M phosphate buffer, pH 7.5, was sonicated for 15 minutes and the cell-free extract obtained as described in Materials and Methods. The sol-uble respiratory complex was obtained from the extract as described in Materials and Methods. Elution of enzyme from a Sepharose 4B column (41 x 2.8 cm) was performed with 0.01 M phosphate containing 0.5 M KC1, pH 7.5. The eluant containing the highest SDH activity was used for the determination of Km and Ki. Aliquots (0.013 mg protein) were removed to determine SDH activity in the absence (a) or in the presence of malonate (b, 0.005 mM; c, 0.015 mM), as des-cribed in Materials and Methods, using 1.2 mM PMS in the assay mixture. The enzyme was not activated. V is expressed in absorbance units, where one unit equals an absorbance change at 600 nm of 1.0 per minute. S, concentr-ation of succinate. 1 / S, mM Fig. 16. Determination of Km and Ki in the presence of KC1 with with the soluble respiratory complex. TABLE XII Solubilized or purified SDH from various sources Km for Ki for Ki for Molecular Source succinate fumarate malonate Reference (mM) (mM) (mM) weight Beef heart 1.3 1.9 0.041 200,000 288 Yeast (aerobic) 1.0 1.03 0.01 200,000 203 M. lactilyticus 5.3 0.22 0.23 460,000 339,341 P. pentosaceum 2.2 0.7 - - 213 M. edulis (mussel) 2.0 0.15 0.06 - 276 C. purpurea (ergot fungus) 3.3 0.93 0.03 - 231 E. coli (whole cells) 0.1 - - - Author E. coli (cell-free (.extract) p. 55% - 0.01 - Author E. coli (soluble respiratory complex) 0.24 - 0.002 1,600,000 Author E. coli (soluble SDH) - - - 100,000 Author E. coli (cell-free extract) 0.26 - - - 148 TABLE XIII Km and Ki for different preparation of SDH from E. coli Km (Succinate) Ki (Malonate) Preparation Addition mM mM Reference Whole cell suspension Cell-free extract Cell-free extract None None KC1 Soluble respiratory complex None Soluble respiratory complex KC1 Cell-free extract None 0.10 0.55 0.26 0.24 0.12 0.26 0.008 0.011 0.0021 0.018 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 148 85 The lower Km of the soluble respiratory complex compared to that of the SDH of cell-free extract may indicate that the active site of SDH is masked. Solubilization may make i t more accessible to substrate. The Km of the soluble respiratory complex was greater than that of a whole cell suspension. The latter preparation is assumed to give the true Km of the native, membrane-bound SDH. Since the effect of KC1 on the Km of the SDH of the soluble respiratory complex and the cell-free extract is to make these values more closely approximate that of the whole cell suspension, it may be that KC1 provides an ionic environment around the enzyme similar to that found in the intact cell. 3. Activation of SDH Activation of mitochondrial SDH by succinate, malonate and phos-phate (187, 188, 201, 202, 325, 289, 290), and of succinate oxidase by heating (294, 325) has been reported. However, there are no reports of the activation of these enzymes in bacterial systems. If succinate was added to the whole cell suspension, cell-free extract, particulate fraction, or the soluble respiratory complex, and the enzyme system kept at 0°, SDH activity remained unchanged as in the absence of succinate (Fig. 17B-b). However, if these systems were then heated at 38° in the presence of the added succinate, there was marked increase in SDH activity (Fig. 17 and 18). Heating with-out addition of succinate either did not change the activity or there was a slight increase (Fig. 18). If the enzyme has not been previously activated, then activat-ion may occur during the determination of its activity. Thus, as 86 Fig. 17. Time-course of DCIP reduction by succinate dehydrogenase. A Whole cells were suspended in 0.01 M phosphate buffer, pH 7.0. Aliquots (0.045 mg and 0.09 mg protein for a and b, respectively) were removed and assayed for SDH activity in the presence of 0.3 mM PMS. Activity was determined before (b) and after (a) activation with 30 mM succinate at 38° for 15 minutes. B 0.2 F fraction, obtained from cell-free extract as described in Materials and Methods, was suspended in 0.01 M phosphate buffer, pH 7.0. Aliquots (0.03 mg protein) were removed for SDH assay in the presence of 0.6 mM PMS. Activity was determined before (c) and after (a) activation with 15 mM succinate at 38°. b, incubated with 15 mM succinate for 5 minutes without heating. The reduction of DCIP is expressed as the absorbance change at 600 nm with time. 87 Fig. 17. Time-course of DCIP reduction by succinate dehydrogenase. 88 Fig. 18 (a) (Left) # Conditions for activation of SDH activity. The cell-free extract was prepared in phosphate-succinate buffer, pH 7.0 (9.8 mg protein per ml). The extract was kept at 0°. The extract was preincubated as below. Aliquots (0.96 mg protein for (A) and 0.24 mg protein for (B) and (C) were removed and assayed for SDH activity in the presence of 1.2 mM PMS. (A) extract kept at 0° or after further addition of 0,075 M disodium succinate (B) extract incubated at 38° for 15 minutes without further addition of succinate (C) extract incubated for 15 minutes at 38° after further addition of 0.075 M disodium succinate. (b) (Right). Effect of flavin on SDH activity. Flavin was added to the cell-free extract prepared in 0.01 M Tris buffer, pH 7.5, as below. The enzyme was tested before and after activation. Aliquots (0.07 mg protein for activated extract and 0.53 mg protein for non-activated extract) were assayed for specific activity of SDH. (A) no addition; (B) 1.25 mM FMN added to non-activated extract and 0.83 mM FMN added to activated extract; (C) 1.25 mM FAD added to non-activated extract and 0.83 mM FAD added to activated enzyme. Speci f ic CD O S P E C I F I C ACTIVITY p p P p o -* io cn o cn Q O T 1 r [ggcontrol, • act i vated 68 90 shown in Fig. 17A-b, the whole cell suspension in phosphate-succinate buffer (25 mM succinate) showed gradually increasing SDH activity during the course of the assay. Heating abolished the hyperbolic kinetics and gave a straight line response of DCIP reduction with time and at a greater rate than before. The 0.2F fraction obtained from the cell-free extract also gave similar behaviour (Fig. 17B). The presence of succinate did not increase the catalytic activity un-less heat-treatment was done. For the measurement of the activity of SDH of "control" enzymes the reduction of DCIP occurring between the second and third minutes after addition of enzyme was used so as to avoid the contributions brought about by modification of the enzyme itself. For the deter-mination of the activity of the activated enzyme the initial rate of DCIP reduction as in the control was generally used. The optimal time of heating for activation of SDH was measured. As shown in Fig. 19A, complete activation required 15 minutes incubat-ion at 38° in the presence of succinate. There was no activation of NADH or lactate dehydrogenases by heating the enzyme either with succ-inate or lactate. Therefore, it is assumed that the SDH flavoprotein is separate from that of NADH or lactate dehydrogenases. The minimal level of succinate required for the activation was not measured, but there was no difference in the extent of activation with concentrations of succinate above 15 mM. With mammalian SDH, increasing the incubation temperature de-creases the incubation time necessary for f u l l activation (e.g. 15 minutes at 25° or 7 minutes at 38°). The incubation temperature 91 Fig. 19A# Determination of the optimal incubation time for the activation of SDH and succinate oxidase activities at 38° . For the experiment with SDH a cell-free extract (4.9 mg pro-tein per ml) prepared in phosphate-succinate buffer, pH 7.5, was used. For succinate oxidase a suspension of particulate fraction (9 mg protein per ml) in 0.01 M phosphate buffer, pH 7.5, was employed. The enzyme preparations were activated as described in Mat-erials and Methods, except that the time of incubation was varied. Aliquots of 0.1 ml (0.12 mg protein) and 1 ml (9 mg protein) were assayed for specific activities of SDH and succinate oxidase, respectively. Fig. 19B . Spontaneous inactivation of activated SDH. Enzyme solution was activated as described in Materials and Methods. The activated enzyme was kept at room temperature. Ali-quots (0.15 mg protein) were removed at intervals and assayed for SDH activity in the presence of 0.6 mM PMS. The activity is ex-pressed in units. For this experiment the suspension of 0.2F fraction (9.5 mg protein) obtained from the cell-free extract as described in Materials and Methods (also see Table IX) was used. 92 .L 10 20 30 M I N U T E S Fig. 19A 30 Minutes 60 p i g . 19B 93 affected the rate rather than the extent of a c t i v a t i o n . At 0° the a c t i v -ation process was very slow (187, 289). During t h i s work the slow a c t i v a t i o n of SDH of the c e l l - f r e e ex-t r a c t at 0° in the presence of succinate was not observed. As shown i n F i g . 19A, succinate oxidase a c t i v i t y was also complete-l y a c t i v a t e d f o r 15 minutes heating at 38° in the presence of succinate, as observed with SDH. This indicates that the a c t i v a t i o n of succinate oxidase i s mainly due to the a c t i v a t i o n of the SDH molecule. When a c e l l - f r e e extract was heated at 38° f o r 10 minutes with 30 mM l a c t a t e , no a c t i v a t i o n of succinate or lactate oxidase a c t i v i t i e s was observed. Heating with 30 mM fumarate or malonate completely i n h i b i t e d succinate oxidase a c t i v i t y . If the c e l l - f r e e extract was kept overnight with 30 mM succinate or l a c t a t e at 0°, and then heated at 38° f o r 10 minutes, the l a c t a t e -incubated preparation had the same succinate oxidase a c t i v i t y as the control but the succinate-incubated enzyme showed one ha l f of the succ-inate oxidase a c t i v i t y of the c o n t r o l . This i s in agreement with the repeated observation that prolonged incubation of SDH with succinate was i n h i b i t o r y (187, 188, 202). Thus, the a c t i v a t i o n phenomenon appears to be l i m i t e d to the SDH enzyme of the succinate oxidase system. But, when the p u r i f i e d p a r t i c u l a t e f r a c t i o n obtained by gel f i l t -r a t i o n in a phosphate buffer containing 0.025 mM succinate was incubated with further succinate at 0° there was an increased SDH a c t i v i t y . Heating at 38° f o r 15 minutes fur t h e r increased the a c t i v i t y markedly (Table XIV). Thus i t i s possible that a purer enzyme can be activated i n the presence of succinate, to some extent, without heating. It i s TABLE XIV Activation of purified particulate fraction Preincubation Specific Activity of SDH ~ ~ Control with Ratio condition Control a d d e d 3 ^ , . ^ ^ (0.25M) Not heated 0.0288 0.075 2.6 Heated at 38° 0.0346 0.553 16.0 for 15 minutes The purified particulate fraction obtained by chromatography on Sepharose 4B (see Fig. 6) was kept at 0° for 40 minutes with or without the addition of an equal volume of 0.5 M disodium succinate, pH 7.5. The SDH activity was then determined before and after preincubation at 38° for 15 minutes. 95 not known why further addition of succinate is required for the complete activation, since the enzyme should have had adequate exposure to succ-inate during purification. The further requirement of succinate was also observed in Fig. 18A. The level of succinate in the preparation before further addition of succinate was enough to activate the enzyme fully i f i t was heated at 38°. When the activated enzyme was kept at room temperature, deactivat-ion occurred progressively with time (Fig. 19B). Once the activated en-zyme was deactivated it could not be activated again. In a similar manner when activated SDH was kept in the cold room, the SDH activity was lost and could not be reactivated by heating at 38° (Fig. 20).. These results indicate that heat activation was not a reversible process. Presumably the enzyme was irreversibly modified by heating at 38°, such that i t could not return to its original unactiv-ated form. In the experiments at 0° the enzyme was deaerated, so that oxidat-ion is unlikely to be the cause of deactivation. It is possible that some fumarate may have accumulated and that this caused denaturation, but no attempt was made to determine the level of either succinate or fumarate in the solution. A contrast to the previously described behaviour was observed with the SDH activity of either the Triton X-100-treated enzyme (Table XV) or or the acetone powder suspension or extract (Table X). As shown in Fig. 21, the reduction of DCIP by the SDH of the acetone powder suspen-sion or extract progressively decreased slowly with time. Heating at 96 Fig. 20. Irreversibility of activation of SDH. The cell-free extract in phosphate-succinate buffer (4.1 mg protein per ml) was prepared as described in Materials and Meth-ods. Part of the extract was kept at 0° and the SDH activity determined daily (curve a). Another portion of the extract was activated as described in Materials and Methods and then stored at 0°. The activity of the activated extract was then determined daily either without (curve b) or with (curve c) preincubation at 38° for 15 minutes. 97 TABLE XV Effect of Triton X-100 on SDH activity SDH Total Control Activated Preparation Protein (mg) Specific Activity Total Activity Specific Activity Total Activity Cell suspension 306 0.074 22.8 0.556 170.0 Cell-free extract 257 0.114 29.4 0.357 91.8 Triton X-100 treated enzyme 63 0.370 23.4 0.320 20.2 To a cell-free extract prepared from 2 g cells in 50 ml phosphate-succinate buffer, pH 7.5, as described in Materials and Methods, solid ammonium sulfate was added to 0.4 saturation. The mixture was centrifuged at 10,000 x g for 15 min-utes. The sedimented pellet was resuspended in 25 ml of the same buffer. 1% Triton X-100 was added to this suspension to give a final concentration of 0.27%. After the solution had been stirred for 30 minutes at 4°, ammonium sulfate was added to 0.4 saturation, and the mixture centrifuged at 10,000 x g for 15 min-utes. The precipitate was suspended in the same buffer (final volume, 6 ml), applied to a Sephadex G-25 column (2 x 26 cm), and eluted with the same buffer. The SDH activity of the elutant, and of the cell-free extract and the original cell suspension, was determined as described in Materials and Methods. Fig. 21. Time-course of DCIP reduction by succinate dehydrogenase solubilized from acetone powder. A . The SDH was a suspension of acetone powder in phosphate-succinate buffer, pH 7.0. Aliquots (0.175 mg protein) were removed for SDH assay. Activity was determined before (a) and after (b) heating at 38° for 15 minutes. B. The acetone powder extract in phosphate-succinate buffer, pH 7.0, was prepared as described in Materials and Methods. Aliquots (0.33 mg protein) were removed for SDH assay. Activity was determined before (a) and after (b) heating at 38° for 15 minutes. The reduction of DCIP is expressed as the change in absorbance at 600 nm with time. 99 38° for 15 minutes in the presence of succinate gave linear reduction with time but the activity was less than that of the initial activity of the non-heated enzyme. This phenomenon was not reported for the SDH extracted from the acetone powder of mammalian mitochondria (288). The decreasing activity of SDH was not due to simple denaturation of the enzyme, since enzyme kept under the same conditions in the absence of the assay re-agents did not lose activity. It is probable that a component of the assay mixture may have inhibited the enzyme or facilitated its denatur-ation. It is possible that the cyanide present in the assay mixture may react with the nonheme iron of the enzyme (117). This iron might be more exposed in the lipid-free acetone powder enzyme than in the lipid containing membrane fragments. The similar behaviour of the Triton-treated enzyme (Table XV) would be understandable since this detergent can solubilize lipid and would also presumably make the enzyme protein more accessible to denaturants or inhibitors. 4. Lability of SDH Since SDH is unstable the effect of the composition of the buffer during sonication was examined. As shown in Fig. 22, SDH activity was stable for a week when sonic-ation was carried out either in phosphate or Tris buffer containing succinate or containing both succinate and glycerol. SDH was labile when enzyme was prepared in buffers not containing succinate. Deaeration of buffer before use did not affect the lability of SDH in the absence of succinate. In other experiments, after sonication the cell-free extract was flushed with nitrogen gas to remove air from the solution. As shown in Fig. 23, removal of oxygen stabilized SDH 100 Fig. 22 . Effect of buffer composition on ageing of SDH activity of the cell-free extract. The cell-free extract was prepared as described in Materials and Methods in 0.01 M phosphate buffer, pH 7.5 (left-hand graph), or in 0.01 M Tris buffer, pH 7.5 (right-hand graph). The sonica-tion buffer also contained the following components: A and a, 20% glycerol; B and b, 25 mM succinate; C and c, 20% glycerol with 25 mM succinate; D and d, no addition. Sonication was for 5 min-utes using an 0.03% (w/v) cell suspension. The extracts were assayed for SDH activity before (solid line) or after activation (broken line). SDH activity is expressed as umole succinate oxidized per min per 10 ml extract. 101 ig. 22. Effect of buffer composition on ageing of SDH activity of the cell-free extract. 102 Fig. 23. Effect of oxygen on ageing of SDH activity. The cell-free extract was prepared in phosphate-succinate buffer (left-hand graph) or 0.01 M Tris buffer containing 25 mM succinate, pH 7.5 (right-hand graph). Part of each extract was stored under air at 0° and part was gassed with nitrogen and stored under nitrogen gas. Aliquots were removed at intervals and the SDH activity determined before (solid line) and after activation (broken line). B, D, b, d extract stored under air; A, C, a, c, extract stored under nitrogen. SDH activity is expressed as umole succinate oxidized per min per ml extract. Fig. 23. Effect of oxygen on ageing of SDH activity. 104 slightly but not significantly. Therefore, removal of oxygen during the preparation of SDH was not routinely carried out. Unexpectedly, the inclusion of succinate in Tris buffer caused complete activation of the enzyme at 0°. Heating at 38° for 15 minutes after further addition of succinate did not increase SDH activity although the activity of the enzyme in succinate and glycerol could be further in-creased by heating. Thus, this activation phenomenon is possibly differ-ent from the activation produced by heating the enzyme in the presence of succinate (Fig. 24). The presence of glycerol seemed to inhibit the spontaneous activation of SDH by succinate. The mechanism for the spon-taneous activation of SDH by succinate and Tris at 0° is not known. The acetone powder extract has the least stable SDH enzyme (Table XVI). Since King (189, 192, 193) had shown that the presence of succin-ate was necessary to give an active enzyme, and SDH is known to contain essential sulfhydryl groups (188, 288, 289). 0.1 M succinate, 1.5 mM dithiothreitol or succinate with dithiothreitol were added to the buff-ers during preparation of the enzyme. These compounds did not protect the SDH of the acetone powder extract from spontaneous inactivation. Deaeration of buffer did not stabilize the enzyme. Furthermore, the soluble SDH obtained from the acetone powder extract by calcium phosphate column chromatography or Sepharose 4B column chromatography was even more labile. The lability of the acetone powder enzyme is probably due to the lack of acetone-soluble lipids or modification of the electron-donating site of SDH toward PMS. This explanation is favoured since extraction of lipids and quinone with petroleum ether from the particulate fraction did not effect succinate-cyt b x reductase activity (see Part III of this 105 100 I > § 50 x Q tn A Pi Buf fer pH 7.5 1 0 0 -Fig. 24. Activation of SUM under different condition. This figure was drawn from the results of Fig. 22. See the legend to Fig. 22 for symbols. SDH activity is expressed as per cent of maximal activity after activation given by the enzyme prepared in phosphate buffer only (Fig. 24 A-D). 106 TABLE XVI Lability of SDH activity in acetone powder extract First Day Second Day Preparation Total Specific Total Specific Total Activity Protein (mg.) Activity Activity Activity Activity Remaining Si sp ens ion of Acetone Powder 531 0.105 56.3 0.068 36.6 64.5% Extract 174 0.067 11.7 0.019 3.3 28.3% Suspension of Residue 282 0.131 37.1 0.079 22.4 60.3% The acetone powder (1 g) was suspended in 50 ml phosphate-succinate buffer, pH 7.5, and the extract prepared as described in Materials and Methods and stored at 0°. The activity of SDH was determined without prior activation. 107 thesis). Cerletti et al. (60, 61) have shown that the addition of phos-pholipids to a soluble SDH from mitochondria greatly increased the act-ivity and stability of the enzyme. Gutman et al. (115, 116) indicated that the lability of isolated NADH dehydrogenase from E. coli was due to modification of the NHI of NADH dehydrogenase. Since, the presence of NHI in the soluble SDH was not established this possible explanation cannot be verified. 5. Effect of PCMB on activation of SDH Since PCMB inhibited SDH activity (Fig. 25A) probably by reacting with sulfhydryl groups of the SDH protein, an attempt was made to see if PCMB would effect the activation of the enzyme. The effect of the addition of PCMB to SDH before and after activ-ation was compared (Fig. 25B). Addition of PCMB after activation of SDH resulted in a lower degree of inhibition. Thus, incubation with substrate at 38° may have changed the conformation of SDH to protect its sulfhydryl groups from reacting with PCMB. Since complete protection was not obtained, i t is possible that the heat treatment facilitated the. penetrance of PCMB into the SDH molecule so that some of the protected sulfhydryl groups could react. The sulfhydryl group(s) of SDH is probably essential for the en-zyme activity, as in mitochondrial SDH (289, 290). 6. Mechanism of activation of SDH The SDH of soluble purified preparations, or of succinate oxidase from the mitochondria of beef heart or aerobic yeast, has been found to 108 Fig. 25 . Effect of PCMB on SDH activity of cell-free extract. Cell-free extract (3.9 mg protein per ml) was incubated with PCMB for 10 minutes at 4° before assay. SDH activity was determined as described in Materials and Methods, without activation. (a) Cell-free extract (0.78 mg protein per ml) was activ-ated as described in Materials and Methods. Then PCMB was added and incubation continued at 38° for 5 minutes before assay. (b) Cell-free extract (0.78 mg protein per ml), after addi-tion of PCMB, was heated for 5 minutes at 38°. The extract was then activated as described in Materials and Methods prior to assay of SDH activity. The cell-free extract was prepared as described in Materials and Methods from 0.45 g packed cells suspended in 20 ml of 0.01 ml phosphate buffer, pH 7.5, which had been sonicated for 5 minutes. 0.1 0.2 0.3 Fig. 25 C PC M B • , mM 110 be activated by various compounds such as succinate, phosphate, malonate and fumarate (186, 187, 188, 202, 294, 325). The activation of mitochondrial SDH was inhibited when sulfhydryl groups of SDH were modified by PCMB (189, 188) or PCMS (228), so that sulfhydryl group(s) may be involved in activation. The activation was independent of pH in the range of pH 6.0 to 9.0, which suggests that an ionizable group is not important in activation (187, 188, 202). The activation energy of mitochondrial SDH was 35.6 (187, 188) or 34.7 (325) Kcal per mole, calculated from the Arrhenius plot. It has been suggested that activation involves a molecular change around the NHI of SDH (188, 325). It has also been proposed that there is an intramolecular change in the whole!SDH molecule initiated by com-bination of the enzyme with activators capable of binding at the active site with a resulting increased turnover rate (201). The high activat-ion energy and change in absorption spectrum support the latter proposal. Activation and deactivation caused by addition or removal of the activator were found to be reversible with the mitochondrial SDH (201), Deactivation was reported to be independent of temperature (201). In addition, i t was stated that the effect of incubation temperature on SDH activation in the presence of activator was on the rate but not on the extent of activation (187, 188). During this study with the SDH of E. coli, activation of SDH was not observed with competitive inhibitors (phosphate or malonate) but only with succinate. Inclusion of succinate in the phosphate buffer solution during the preparation of SDH stabilized the enzyme for at least one week, I l l but did not activate SDH activity at 0°. The enzyme was activated by heating. The presence of succinate in Tris buffer not only stabilized SDH but also activated it completely at 0° and further heating at 38° did not increase enzyme activity unless glycerol was present. Glycerol appeared to inhibit this spontaneous activation process. Addition of phosphate to the enzyme solution in Tris buffer was without effect. The heat-independent activation phenomenon is probably different from heat-activation, since the non-activated enzyme of the glycerol-succinate system can be further activated by heating. Activation of the heat-a activated enzyme is irreversible because i f the activated enzyme is once deactivated it cannot be reactivated by activation procedures. Since the heat-independent activated enzyme was so stable the spontan-eous inactivation of the enzyme was not examined. It is probable that the two activation phenomena do not involve the same mechanism. Kearney (188) suggested that activation process was as follows: E + A EA > E* + A where E* is the active form of the enzyme and A, the activator. Later Kimura et al. (202) proposed the following mechanism: Eu^i^EuC ^ —^EaC -c Eu: unactivated enzyme Ea: activated enzyme C: activator and that the conversion of the unactive to the active form of SDH does not involve flavin directly since the reduction of fumarate by FMNH^  was unaffected during activation. 112 A further extension of this scheme was made by McDonald-Gibson and Thorn (230), who proposed an activation-deactivation mechanism for SDH as follows: T EaC +c Eu i Ei -S» c +c -c i Ei Ei: inactivated enzyme which cannot be activated T : temperature sensit-ive process The author proposes that the mechanism for activation is as shown in Fig. 26. The unactivated enzyme (Eu) reacts first of all with succ-inate to form a complex (Eu»S) which, when enzyme is prepared in Tris buffer, spontaneously forms an activated enzyme-succinate complex (Ea*»S), The complex now reacts in the presence of electron acceptors to give products and regenerate the activated enzyme (Ea*). It is not known i f Ea* will break down to a form of the enzyme which cannot be reactivated. The complex, Eu«S, i f prepared in phosphate buffer, can be activ-ated by heat to give an activated enzyme complex (Ea»S). This, in the presence of electron acceptors, will react to give products and the ac-tivated enzyme Ea. The activated enzyme can break down to a form Ei which cannot be reactivated. It is also assumed that the unactivated enzyme can break down to give inactive enzyme (Ei). It is not known i f conversion of Eu to Ea or Ea* involves conform-ational changes but this seems likely because of the work on the mito-chondrial enzyme (188). 113 S ; S u c c i n a t e . F ; F u m a r a t e . Eu ; U n a c t i v a t e d enzyme w i t h l o w e r a c t i v i t y . Ea ; Enzyme a c t i v a t e d by h e a t i n g a t 3 8 ° i n t h e p r e s e n c e o f s u c c i n a t e . E a * ; H e a t - i n d e p e n d e n t a c t i v a t e d enzyme. E i ; I n a c t i v a t e d enzyme w h i c h c a n n o t be r e a c t i v a t e d . F i g . 26. P r o p o s e d m e c h a n i s m f o r a c t i v a t i o n o f SDH. 114 PART II - THE ROLE OF NHI IN E, COLI Despite numerous reports concerning NHI proteins which are widely distributed in animals, plants, photosynthetic and anaerobic bacteria, l i t t l e work has been carried out on the role of nonheme iron found in aerobic bacteria. Iron in E. coli is mainly distributed as heme proteins and nonheme proteins. Heme iron is a minor component of the total iron, the bulk of which is nonheme iron (35, 76, 77). Some of this nonheme iron may be associated with NADH dehydrogenase (31, 115, 116) or with other sites in respiratory chain (30) of E. coli. However, it is possible that a large percentage of the NHI is not associated with the bacterial respiratory chain. Therefore,in the second part of this thesis, the nature, proper-ties, role and location of nonheme iron in the cell envelope of E. coli were investigated. RESULTS 1. Spectrophotometric determination of ferrous iron using o-phenanthroline o-Phenanthroflne is a very strong chelating agent for ferrous iron. The chelate, or ferrous-o-phenanthrolinate, has a red color, and is res-istant to degradation by heating for more than a few hours in the pres-ence of concentrated sulfuric acid. Harvey (136) has given detailed accounts of the spectrophotometric determination of ferrous-o-phenanthrolinate. Since then this method has 115 been widely employed to estimate the inorganic ferrous iron content of biological materials. In particular, the iron of iron-containing enzymes has been determined by Mahler and Elowe (225), Massey (228, 229), Yonetani (365), King et_ a l ^ (200), Kurup and Brodie (209, 210), and others. (i) Absorption spectrum of ferrous-o-phenanthrolinate As shown in Fig. 27, ferrous-o-phenanthrolinate has a definite ab-sorption spectrum, with an absorption maximum at 510 nm which is propor-tional to the concentration of ferrous iron i f enough chelating agent is present. If the concentration of ferrous iron is low (around 0.005 mM), the absorption spectrum shows a broad peak rather than sharp peak at 510 nm. (ii) Determination of the extinction coefficient of ferrous-o-phenanth-rolinate in transparent solutions Since the reaction of o-phenanthroline with ferrous iron provides a sensitive method for the determination of iron, the measurement of the extinction coefficient of ferrous o-phenanthroline was carried out. On the basis of the absorption at 510 nm or the absorbance difference at 510 nm relative to 550 nm, extinction coefficients of 11.0 and 7.8 liter mmoles'^m-1, respectively, were determined (Fig. 28). The former value is in good agreement with the reported values of 11.4 by Yonetani (365) and 11.1 by Massey (228, 229). An attempt was made to calculate the extinction coefficient after the addition of dithionite. No difference of absorbance was found 116 Fig. 27. Absorption spectrum of ferrous-o-phenanthrolinate. 25 yl 0.05 M o-pheiianthroline solution was added to the sample cuvette which contained 1 ml ferrous ammonium sulfate (2.5 uM for curve b and 5.0 uM for curve c). The spectrum was recorded against a cuvette which contained the iron solution only, a, base-line. 117 £ c O) c > C O • H 8 0 u e q J o s q v 118 0.6 Fig. 28. Determination of the extinction coefficient of ferrous-o-phenanthrolinate. The spectrum of ferrous-o-phenanthrolinate was measured using several concentrations of ferrous iron, as described in Materials and Methods. Curve A, absorbance measured at 510 nm. Curve B, absorbance at 510 nm relative to 550 nm. The extinction coefficients from A and B were 11.0 and 7.8 lit e r mmoles - 1cnfrespectively. 119 immediately a f t e r adding d i t h i o n i t e but then the solution became turbid and the spectrum could not be scanned. ( i i i ) Determination of the ex t i n c t i o n c o e f f i c i e n t of ferrous-o-phenanthrolinate i n a transparent solution i n the presence of TCA To estimate the nonheme iron content of enzymes the nonheme iron has u s u a l l y been extracted with TCA from the enzyme (209, 210, 225, 228, 233). During the estimation of the nonheme iron released from the parties ulate f r a c t i o n by treatment with 5-8% TCA, i t was found that there was a slow progressive development of red color i f the same conditions were used as had been employed f o r the estimation of iron i n absence of TCA. Incubation of a mixture of ferrous iron i n TCA with 1.25 mM o-phenanth-r o l i n e at 38° f o r an hour, as described by Kurup and Brodie (209) did not give rapid development of the c o l o r . This interference i n chelate formation by TCA could be overcome by adding a ten tiroes higher concen-t r a t i o n of o-phenanthroline than that used i n the absence of TCA. As shown in F i g . 29, with 4 yM ferrous i r o n , at le a s t 0.2 mM and 17 mM of chelating agent was required to form the chelate i n s t a n t l y i n the ab-sence and the presence of TCA, r e s p e c t i v e l y . Provided that s u f f i c i e n t c h e l a t i n g agent was added the e x t i n c t i o n c o e f f i c i e n t of ferrous-o-phenanthrolinate was the same i n the presence or absence of TCA. 120 0.04 o 0.0 3 o c f 0.0 2 o co < 0.01 a • / fa " » • -11 i n i i t i • i i i i i i " i *' • I I ' '» .* II i I— i i m\ in 11 t •+1 t •# i c * / d / * I i i *' 1 1 -1 Log C 0 - PhH, mM Fig. 29. Determination of minimum concentration of o-phenanthroline required' to react with 4 uM ferrous iron solution. Different concentrations of o-phenanthroline were added to 4 uM ferrous ammonium sulfate solution, in the absence (a) or in the presence (b, c, d) of 3% trichloroacetic acid. The absorbance at 510 nm relative to 550 nm was measured at 5 minutes (d), 15 minutes (c) and 35 minutes (b) after the addition of o-phenanth-roline. In (a) the colour development was instantaneous. 121 (iv) Determination of bound ferrous iron in the particulate fraction When there was strong absorption by the turbid particulate fraction it was not possible to use the extinction coefficient of ferrous o-phen-anthrolinate based on the absorbance at 510 nm to estimate endogenous ferrous iron bound to the particulate fraction. The main reason for this is the non-specific absorption at 510 nm by substances in the particulate fraction. In addition, the turbid protein solution shows light-scattering at this wavelength. To avoid erroneous absorption at 510 nm, the extinc-tion coefficient calculated from the absorbance difference between an ab-sorption maxima at 510 nm and 550 nm was adopted in this thesis to cal-culate the particulate-bound ferrous iron content. When o-phenanthrolinc' was added to the particulate fraction to which dithionite had been added (Fig. 30), there was an increase in the absorption at about 510 nm. The spectrum was similar to that in Fig. 27, thus indicating that ferrous-o-phenanthrolinate had been formed. The peak at 510 nm was proportional to the level of particle-bound ferrous iron present as had also been found by Kurup and Brodie (210). In order to measure particle-bound ferrous iron the extinction co-efficient of ferrous-o-phenanthrolinate in a turbid solution was deter-mined as described in Materials and Methods and in Fig. 31. After the addition of o-phenanthroline to the particulate fraction, the absorbance at 510 to 550 nm increased slowly. It became stable at 20 to 40 minutes depending on the level of iron. The particle-bound iron which had so reacted was termed "directly-reacting" iron. If various concentrations of ferrous iron were now added, further formation of 122 Fig. 30. The absorption spectrum of ferrous-o phenanthrolinate in the particulate, fraction. The particulate fraction in 0.01 M phosphate buffer, pH 7.5, (1 ml; 2.06 mg protein) was reduced with dithionite. The spectrum was recorded versus the particulate fraction in the reference cuvette (curve b). 25 yl of o-phenanthroline was then added to the dithionite-reduced cuvette and the spectrum recorded as before (curve c). Curve a, base-line. 0.05 h o o c n o CO n < 0.025 400 Fig. 30 500 Wavelength,nm 600 124 ferrous o-phenanthrolinate occured due to the presence of the added iron. The absorbance differences at 510 nm relative to 550 nm after correcting for the absorbance obtained in the absence of added iron were a linear function of the concentration of the added iron (inset of Fig. 31). From these values an extinction coefficient of 6.4i0.01 (t standard deviation of results with four separate experiments) was obtained. This value was used to quantitate "directly-reacting" (i.e. iron present in the particulate fraction which reacted with o-phenan-throline in the absence of reducing agent) succinate-reducible and NADH-reducible iron. This value does not agree with the value of 7.8 for the absorbance difference at 510 nm relative to 540 nm in the particulate preparation used by Kurup and Brodie (210). (v) Determination of the extinction coefficient of ferrous-o-phenanthrolinate in a turbid solution in the presence of dithionite Addition of dithionite not only reduced reducible substances con-tained in the particle, but also caused some denaturation of protein. Addition of o-phenanthroline to the dithionite-reduced particulate fraction increased the absorbance difference at 510 nm relative to 550 nm, partly due to the increase in ferrous iron by the reduction of ferric iron by dithionite (i.e. dithionite-reducible iron) but also due to the increase in turbidity. Thus, the determination of the extinction coefficient of ferrous-o-phenanthrolinate in a turbid solution with dithionite present was 125 Fig. 31. Determination of the extinction coefficient of ferrous-o-phenanthrolinate in the pres-ence of the particulate fraction. 25 yl 0.05 M o-phenanthroline was added to the particulate fraction (1.3 mg protein per ml) in 0.01 M phosphate buffer, pH 7.5. The absorbance difference at 510 nm relative to 550 nm was measured as described in Materials and Methods. At the arr-ow the following additions were made: a, no addition; b, 2.25 yM ferrous iron; c, 5.0 uM ferrous iron; d, 10 yM ferrous iron; 4, 20 yM ferrous iron. In the inset the absorbance of curves b to e corrected for the absorbance of curve a is plotted versus ferrous iron concentration. 3ONvauosav 127 performed as described in Materials and Methods, and Fig. 32. To avoid the contribution of a-band of cytochrome b1, the particulate preparation was diluted so that this peak was less than 0.01 absorbance units. Thus the contribution by cyt bj was not considered to be important in calcul-ations of the amount of iron in the particulate fraction. The extinction coefficient calculated from the linear graphs (inset of Fig. 32) was 7.1±0.5 (±standard deviation with seven separate preparations). This value was used to estimate dithionite-reducible iron. 2. Spectrophotometric determination of ferric iron using Tiron. Inorganic ferric iron forms a colored ferric-Tiron complex which may be used to determine ferric iron. Extensive work on the spectro-photometric determination of ferric iron by this means has been reported by Yoe and Jones (367). The color of the ferric-Tiron complex is changed depending on the pH used. At pH values below 5.0, the color of the complex is deep blue. If the blue solution is made alkaline the color changes into violet at pH 6.0 to 7.5, and to red above pH 8.0. The color, and so the absor-bance and the absorption maxima, vary considerably at pH values below 8.0. There is no change in the absorbance or the absorption maxima above pH 9.0. The exact mechanism of the color change is not known, but it may be due to change in the ratio of molecules of Tiron reacting with an atom of iron as the pH is changed (367). The changing absorption maxima of the ferric-Tiron complex under different pH conditions is shown in Fig. 33C. 128 Fig. 32. Determination of the extinction coefficient of ferrous o-phenanthrolinate in the presence of the particulate fraction and dithionite. 25 yl 0.05 M o-phenanthroline was added to the particulate fraction (1.3 mg protein per ml) in 0.01 M phosphate buffer, pH 7.5, to which a few crystals of sodium dithionite had been added. The absorbance at 510 nm relative to 550 nm was measured as des-cribed in Materials and Methods. At the arrow the following additions of ferrous iron were made: (a) none, (b) 2.5 yM, (c) 5.0 yM, (d) 10 yM, and (e)" 20 yM. In the inset the absorbance of curves b to e, corrected for the absorbance of curve a, is plotted versus ferrous iron concentration. o o u e q j o s q v 130 The order of addition of Tiron or phosphate buffer to the ferric iron solution was found to be a critical step which determined if there was instant or progressive formation of ferric-Tiron complex. We did not attempt to see i f there was a similar effect on color development if non-phosphate buffers were used. However, Yoe and Jones (367) re-ported that there was no interference by phosphate with color development. When Tiron was added to ferric ammonium sulfate at pH 3 to 4, there was instant formation of a deep blue color, and successive addit-ion of the alkaline phosphate to it changed the color into violet and red in order. These colors formed instantly and were stable. There was no serious color increase after several hours, but the absorbance was increased by increasing the concentration of phosphate buffer at alkaline pH values. If Tiron was added to the ferric iron which was previously buff-ered with phosphate buffer, pH 7.7, the development of the color from colorless to a red color was slow and progressive. It took several hours to reach maximal color development. The absorbance was also pro-portional to the concentration of phosphate at this pH (Fig. 33B). The slow color development may be due to the formation of polymeric ferric hydroxide complexes at this pH (380, 381, 382). These would probably react only slowly with Tiron. Since the particulate fraction was prepared in phosphate buffer, attempts were made to determine the optimal conditions for the forma-tion of a complex using both phosphate buffer, pH 7.7, and phosphate-NaOH buffer, for pH values above 9.0. 131 (i) Reaction of Tiron with ferric iron at pH 7.7 Since Massey (228) used phosphate buffer, pH 7.6, for the deter-mination of ferric iron content of mammalian SDH, phosphate buffer of pH 7.7 was used in this study. As described previously, if Tiron was added to an acidic ferric iron solution (pH 3 to 4) a deep blue color formed rapidly. Addition of phosphate buffer to adjust the pH to 7.7 changed the color to red, and the absorbance was stable and proportional to the concentration of phosphate at constant ferric iron content (Fig. 33A). The absorption maxima was around 484 nm. When Tiron was added to the buffer solution containing ferric iron, however, the color developed very slowly from colorless to a red color, as shown in Fig. 33B. The absorbance increased both with time and with the concentration of phosphate. There was a slight difference in absorbance when the same kind of experiment was repeated. This suggests that the formation of the complex was not stable and was subject to slight changes depending on the individual reaction conditions. As shown in Fig. 33C, it is fair to state that above pH 9.0 the complex is very stable and not subject to further changes. Below pH 8.0, however, the complex is an inter-mediate complex and sensitive to changes in pH. This complex can be used as a pH indicator (367). It was not feasible to estimate ferric iron reproducibly in the particulate fraction which was prepared in phosphate buffer, pH 7.5. 132 Fig. 33. Conditions for the reaction of Tiron with ferric iron. (Part 1) A. 5 yl 0.05 M Tiron was added to 50 yl ferric iron solution. This solution was diluted to 5 ml with different concen-trations of phosphate buffer, pH 7.7. Final concentration ferric iron was 0.02 M. B. The procedure is similar to that in A, except that Tiron was added to the 0.02 M ferric iron solution in 5 ml phosphate buffer, pH 7.7. a, 0.4 M phosphate buffer; b, 0.1 M phosphate buffer; c, p.01 M phosphate buffer. Absorbance is expressed as the absorbance difference at 510 nm relative to 550 nm. C. Absorption maxima of ferric-Tiron complex versus pH. This figure is adapted from the paper by Yoe and Jones (367), 133 Fig. 33. Conditions for the reaction of Tiron with ferric iron (Part 1) . 134 (ii) Reaction of Tiron with ferric iron at pH 9.2 Since the color formation was not reproducible at pH 7;7, and since Yoe and Jones (367) had used phosphate-NaOH buffer of pH 9.2, we employed it to observe the reaction of Tiron with ferric iron. The reaction was similar to that at pH 7.7 (Fig. 34). After addit-ion of Tiron to acidic ferric iron solution of pH 3 to 4, and adjustment of the pH to 9.2 with phosphate-NaOH buffer, the color changed from deep blue to deep red. The color was stable and was formed rapidly. The ab-sorption was proportional to the concentration of ferric iron but the linear curve did not pass through the origin (Fig. 34C). Therefore, no attempt was made to calculate the extinction coefficient under these reaction conditions. When Tiron was added to buffer containing ferric iron, the color developed very slowly and the absorbance increased with increasing phos-phate concentration, as seen in Fig. 34A, 34B and 35B. To investigate the effect of the concentration of phosphate buffer on the formation of the complex which formed instantly, the blue complex was adjusted to pH 9.2 with different concentration of phosphate-NaOH buffer. As shown in Fig. 34C and 35A, above 0.05 M phosphate concen-tration, the formation of the red complex was rapid and there was no significant difference in absorbance in 0.05 to 0.04 M phosphate buffer. Below 0.05 M phosphate buffer less complex was formed. This may have been due to inadequate control of the pH by this buffer. When Tiron was added to phosphate-NaOH buffer containing 0.02 mM ferric iron, the red color developed more quickly than at pH 7.7 (Fig. 33B and 35B). As shown in Fig, 35B, there was a slight difference in 135 Fig. 34. Conditions for the reaction of Tiron with ferric iron (Part 2) . A,B. 5 ul 0.05 M Tiron was added to 0.1 ml 0.05 M (A) or 0.4 (B) phosphate-NaOH buffer containing a series of different concentations of ferric iron and then diluted to 1.0 ml with the same buffer solution. The absorbance was recorded at (a) 30 minutes, (b) 10 minutes and (c) 5 minutes after the addition of Tiron After the addition of 5 yl 0.05 M Tiron to 0.1 ml of a series of standard ferric iron solution the mixture was diluted to 1 ml with 0.4 M(a), or 0.05 M (b) phosphate-NaOH buffer, pH 9.2 Absorbance is expressed in the absorbance change at 480 nm relative to 550 nm. 136 Fig. 34. Conditions for the reaction of Tiron with ferric iron (Part 2). 137 absorbance when 0.4 and 0.1 M buffer was used. Therefore the author decided to use 0.1 M phosphate-NaOH buffer of pH higher than 9.2, rather than biological pH values, to determine ferric iron with Tiron, since complex formation was quite rapid and quantitative at this pH or above. (iii ) Determination of extinction coefficient of ferric-Tiron complex in a turbid particulate preparation As shown in Fig. 36, the red complex had an absorption maximum at 480 nm at pH values above 8.2. The absorbance was proportional to the concentration of phosphate (Fig. 35B and 36). Tiron was added to a suspension of particulate fraction in 0.1 M phosphate-NaOH buffer, pH 9.4, in the absence or presence of added iron. The formation of the red complex was progressive, as shown in Fig. 37-2. The extinction coefficient was calculated from the plot of absorbance against concentration of added ferric iron by correcting for the absorbance obtained in the absence of added iron at 20 and 60 minutes (Fig. 37-3). A linear curve was obtained. Since the absorbance was slightly variable because of the prog-ressive development of the red color, it was not possible to obtain a reproducible extinction coefficient. Thus, it was necessary to cal-culate the extinction coefficient with each individual preparation which was assayed. From Fig. 37-3, extinction coefficients of 1.23 and 1.7 mM^cnT1 were calculated at 20 and 60 minutes, respectively, after the addition of Tiron. 138 0.50-0.25 B pH 9.2 r e - - -•L ®-e a 1 10 0 H O U R S 10 Fig. 35. Conditions for the reaction of Tiron with ferric iron (part 3). A. 5 ul.0.05 M Tiron was added to 0.1 ml of ferric iron solution, and the solution was diluted with buffer to 5 ml to make a final concentration of 20 uM ferric iron in 0.4 M (d), 0.1 M (c), 0.05 M (b) or 0.01 M (a) phosphate-NaOH buffer, pH 9.2. B. The same conditions as in A, except that Tiron was added to 5 ml buffer solution containing 20 uM ferric iron. The absorbance in these figures is the absorbance difference at 480 nm relative to 550 nra. 139 Fig. 36. The absorption spectrum of the ferric-Tiron complex. 5 yl 0.05 M Tiron was added to different concentrations of phosphate-NaOH buffer, pH 9.2, containing 0.02 M ferric iron. Curve a, base-line; b, 0.01 M; c, 0.05 M; d, 0,1 M phosphate-NaOH buffer, pH 9.2. The spectrum was recorded a few minutes after addition of Tiron to the iron solution. Absorbance is expressed as the ab-sorbance difference at 480 nm relative to 550 nm. Fig. 36 Wavelength, nm 141 Fig. 37. Determination of ferrous and ferric iron levels in the particulate fraction. 1. 25 yl 0.05 M o-phenanthroline was added to 1 ml particulate fraction (1.13 mg protein) prepared in 0.1 M Na2HP04-Na0H buffer, pH 9,4, either with (C) or without (A) the prior addition of a few crystals of sodium dithionite, The ab-sorbance at 510 nm relative to 550 nm was measured, as described in Materials and Methods. At D, dithionite was added to sample A. 2. 5 yl 0,05 M Tiron was added to 1 ml particulate fraction (1.13 mg protein) in 0.1 M phosphate-NaOH buffer', pH 9,4 (curve a). In curves b, c and d, 5 yM, 10 yM and 20 yM ferric iron, respectively, were present. The absorbance at 480 nm relative to 550 nm was recorded, 3. The absorbance of the ferric-Tiron complex from the exp-eriment of figure a (above) at 20 minutes (curve a) and at 60 minutes (curve b) was plotted versus ferric iron concentration. The absorbance was corrected for the absorbance obtained in the presence of added iron. The sample of the particulate fraction used in these ex-periments contained 10.4 mg natoms iron per mg protein, as determined by the method of King et al. (200). LLi O z < CO cc o CO CQ < 0.050 ~ 0.025 -4 0 60 0 20 M I N U T E S 4 0 60 80 O Z < CO cc o CO CO < 0.0 25 3 y b „.»* ** • • • 0 10 20 30 Fig. 37. Determination of ferrous and ferric iron levels in the particulate fraction. 143 3. Measurement of nonheme iron bound to the particulate fraction (i) Total iron Chemical analysis for total iron in the particulate fraction by King's method (200) gave variable values from preparation to preparat-ion, ranging from 9 to 15 n atoms iron per mg protein (Table XVII). (ii) o-Phenanthroline-reacting iron When o-phenanthroline was added to the particulate preparation there was a slow increase in the absorbance difference at 510 nm to 550 nm. This became stable at 20 to 40 minutes after addition of o-phenanthroline. Addition of dithionite at this point increased the amount of complex markedly. The first phase of complex formation was termed "Directly-reacting iron" and the second phase "Dithionite-reducible iron" (Fig. 38). Both directly-reacting and dithionite-reducible iron reacted rapidly with o-phenanthroline when o-phenanthroline was added to the dithionite-reduced particulate fraction, as shown in Fig. 39-d. In Table XVII, the levels of cyt bx, and o-phenanthroline-reacting iron and total iron in particulate fractions are compared. The level of cyt bx was relatively constant, but the concentration of various species of iron varied markedly. However, the ratio of directly-reacting plus dithionite-reducible iron to total iron was fairly constant. For twenty different samples of particulate fraction the following values, expressed as % total iron, were obtained for 144 Fig. 38. Effect of PCMS on the reaction of o-phenanthroline with the partic-ulate fraction. o-Phenanthroline (25 yl; 0.05 M) was added at zero time to the particulate fraction (1 ml; 1.86 mg protein per ml, 0.01 M phosphate buffer, pH 7.5) with PCMS present as indic-ated. The absorbance at 510 nm relative to 550 nm of the solution ("iron reacted") was compared to that of a cuvette containing the particulate fraction and PCMS only. D, dith-ionite added. 145 0 2 0 4 0 6 0 Minutes Fig. 38. Effect of PCMS on the reaction of o-phenanthroline with the particulate fraction. 146 TABLE XVII Relationship between o-phenanthroline-reacting iron and total iron Prepara- Cytochrome o-Phenanthroline-reacting iron Total o-Rhen. Fe tion V Directly-reactingb Dithionite-reducible & iron 1 3 Total Pe x i U U 1 0.352 1.94 1.36 8.8 38 2 0.368 2.54 2.81 14.5 37 3 0.256 3.07 3.33 15.0 43 4 0.320 2.90 2.97 _____ 14.0 42 5 - 2.26 2.32 10.5 44 6 0.330 2.70 1.50 9.0 47 7 0.343 2.72 1.48 10.2 41 *1 Values expressed as n moles per milligram of protein. b Values expressed as n atoms per milligram of protein. 147 directly-reacting iron, dithionite-reducible iron and the sum of these two forms: 21+6%, 21±6% and 42±4%. (i i i ) Tiron-reacting iron From the data shown in Fig. 37, an average of 3.1 n atoms Fe per mg protein was calculated. The Tiron-reacting iron was 29.5% of total iron content of the particulate fraction. 4. Nature of iron in the particulate fraction The reaction of about half of the o-phenanthroline-reacting iron directly with the chelating agent at pH 7.5 suggested that ferrous iron itself might be present in the particulate fraction in a bound form. Addition of PCMS as a thiol inhibitor to the particulate fraction decreased the directly-reacting iron. The higher the concentration of PCMS added, the smaller the amount of directly-reacting iron which was found (Fig. 38). Concentrations of PCMS less than 0.5 mM were without effect. This result is similar to that obtained by Massey with mammal-ian SDH (229). Therefore, this species of iron was probably originally present as ferric iron but was reduced by the sulfhydryl groups of the protein either before or after it had reacted with o-phenanthroline. Ferrous and ferric iron of the directly-reacting iron could exist in the equilibrium where ferric iron is the favored species: Fe""1"'' + SH-Protein —^=- Fe + + + H+ + Protein-S Fe + + + 3 o-Ph ^= Fe + + - (o-Ph)3 o-Ph: o-Phenanthroline 148 When o-phenanthroline is added, the equilibrium would be displaced toward the formation of ferrous iron due to the formation of ferrous-o-phenanthrolinate. This reaction would be expected to give the directly-reacting iron curve shown in Fig. 36 or Fig. 37-1-(C). Therefore, the formation of directly-reacting iron may be limited by available sulfhydryl groups. The fact that directly-reacting iron is a constant percentage (20%) of total iron indicates that the directly-reacting ferric iron and associated sulfhydryl groups must be in a unit, and must be in close physical proximity in the particulate fraction. Since assay of sulfhydryl groups of of acid-labile sulfur was not attempted, it is premature to suggest that the directly-reacting iron occurs as the typical ferric iron-sulfur nonheme iron structures of compounds like ferredoxin (322), The effect of PCMS on the reaction dithionite-reducible iron with o-phenanthroline could not be measured since the solution became black (release of mercury from PCMS) and more turbid in the presence of PCMS above 1 mM. There was no difference in the dithionite-reducible iron level when the PCMS was present at concentrations of less than 0.5 mM. The above results suggest that the dithionite-reducible iron must be located such that i t cannot be readily reduced by protein SH groups, (probably at the surface of the protein). Part of dithionite-reducible iron can be reduced by NADH or substrate to react with o-phenanthroline (see below). ' When iron was assayed in a particulate fraction prepared in 0.1 M phosphate-NaOH buffer, pH 9.4, (Fig. 37-1), an average of 1.45 and 3.22 149 n atoms per mg protein, respectively, of directly-reacting and dithionite-reducible iron was obtained. The Tiron-reacting and total iron were 3.06 and 10.4 n atoms iron per mg protein, respectively. The value of dir-ectly-reacting ferrous iron is very low compared to those given in Table XVII. This could be due to extensive denaturation of protein at this pH or the SH groups might have been oxidized, or the structural proximity of iron and SH groups disarranged. As expected, dithionite-reducible iron was increased, and the total o-phenanthroline-reacting iron (44%) was the same as that found at pH 7.5. The ferric iron (Tiron-reacting iron), 3.06 n atoms, was equal to the dithionite-reducible iron, 3.22 n atoms, which may indicate that these are the same species. Thus, Tiron may have been unable to penetrate to the place where both ferric iron-reducible SH groups and SH-reducible iron (i.e. directly-reacting iron) were located because of hydrophilic properties of Tiron. Addition of glutathione (GSH) to the particulate fraction or PCMS-treated particulate fraction did not effect the established ratio of directly-reacting and dithionite-reducible iron. This indicates that the added GSH must be oxidized immediately by particulate bound oxidant(s) (biologically or chemically) or was not able to penetrate to the locus where directly-reacting iron is located. Since less than half the total nonheme iron reacted with o-phenanthroline even in the presence of dithionite, attempts were made to increase the amount of iron accessible to the reagent by treating the particulate fraction with chaotropic agents (83). It has been shown that urea (6.8 M) increased the exposure of iron in SDH (228) and, more 150 recently, it has been found that treatment with 6M urea or 0.01 M sodium dodecyl sulfate was necessary to expose a l l the iron of cysteamine oxidase to the chelating agent (60). Treatment of the particulate fraction with high concentrations of Triton X-100, ranging from 1% to 5%, gave only a slight increase in o-phenanthroline-reacting iron (Fig. 39-a,b). Triton treatment of the particulate preparation almost abolished the directly-reacting iron (Fig. 39-C), which suggests that denaturation of protein by Triton X-100 is so extensive that most of the sulfhydryl groups which are in-volved in the reduction of ferric iron are removed from the proximity of the ferric iron. Accordingly, there was the expected increase in the dithionite-reducible iron. The possible oxidation of ferrous iron to ferric iron by oxygen during stirring was ruled out, since there was no change in the iron distribution under the same conditions in a con-trol experiment (Fig. 39-f). No significant change in the amount of directly-reacting or dithionite-reducible iron was found in the partic-ulate fraction treated with 6 M urea (Fig. 40). Extensive experiments with the particulate preparation were per-formed to see i f Triton X-100 could release or increase iron which is able to react with o-phenanthroline. The action of several different concentrations of detergent on the release from the particulate frac-tion of cyt b_, SDH, protein, directly-reacting and dithionite-reducible iron was measured (Fig. 41). Stirring the particulate fraction over-night (control sample) even in the absence of detergent resulted in the solubilization of 35% of the protein and SDH. About 20% of the 151 Fig. 39. Effect of Triton X-100 on the reaction of NHI of the particulate fraction with o-phenanthroline. Particulate fraction (0.73 mg protein per ml) in 0.01 M phos-phate buffer, pH 7.5, was stirred for four hours with (a, b, c) or without (d, e, f) Triton X-100 at a final concentration of 5%. To 1 ml of this solution 25 ul 0.05 M o-phenanthroline was added and the change in absorbance at 510 nm relative to 550 nm followed as described in Materials and Methods. Curves a and d, sodium dith-ionite added before o-phenanthroline; curves c and f, no dithionite added. At the arrows a few crystals of sodium dithionite were added to samples c and f to give • curves b and e, respectively. 0.050 0 25 50 75 M I N U T E S Fig. 39. Effect of Triton X-100 on the reaction of NHI of the particulate fraction with o-phenanthroline. 153 Fig. 40. Effect of urea on the reaction of NHI of the particulate fraction with o-phenanthroline. Particulate fraction (2.34 mg protein per ml) in 0.01 M phosphate buffer, pH 7.5, was stirred overnight at 4° in the presence of (a, b, c, g) or in the absence of (d, e, f, h) 6 M urea. To 1 ml aliquots of these solutions was added 25 yl 0.05 M o-phenanthroline and the absorbance difference at 510 nm relative to 550 nm was measured, as described in Materials and Methods. Dithionite (at arrow) was added to the control and the urea-treated enzymes at 10 min (a, d), 20 min (b, e), or 30 minutes (c, f) after addition of o-phenanthroline at zero minutes. Dithionite was not added to the samples giving curves g and h. 0 . 2 0 H I o z < CQ CC o CO < 0 . 1 0 h 20 40 M INUTES 60 80 tn Fig. 40. Effect of urea on the reaction of NHI of the particulate fraction with o-phenanthroline. 155 Fig. 41. Liberation of succinate dehydrogenase, cyt b_, protein, directly-reacting (D) and dithionite-reducible (R) iron from the particulate fraction by Triton X-100. Particulate fraction (1,40 mg protein per ml) was stirred overnight with the indicated concentration of Triton X-100 at 4°, and then centrifuged at 110,000 x g for 2 hours. The pellet was resuspended to the original volume in 0.01 M phosphate buffer, pH 7.5. The pellet suspension (P) and supernatant (S) were an-alyzed for cytochrome b^ SDH, protein, directly-reacting and dithionite-reducible iron as described in Materials and Methods. The values are expressed as the total amount of substance in the fraction. Units of SDH activity are expressed as umoles DCIP reduced per minute. Fig. 41 157 directly-reacting and dithionite-reducible iron, and 9% of the cyt b_ were also liberated in a form which was not sedimented by centrifuging at 100,000 x g for 2 hours. In the presence of increasing concentrations of detergent the dehydrogenases and cyt b were progressively released until at 0.1% Triton X-100 most of these components had been solubil-ized. Dithionite-reducible iron was also progressively released by detergents into the soluble fraction, while the level of directly-reacting iron remained almost constant in the supernatant. The de-crease of directly-reacting iron in the particulate fraction may be due to the same cause as described for Fig. 39. However, despite the large release of dithionite-reducible iron into the soluble fraction, the amount in the detergent-treated particulate fraction was not dim-inished. It is clear that the bulk of the iron in the particulate fraction was not associated with SDH, although it does not prove that the solubilized SDH was associated with iron. The recovery of protein, cyt b_, SDH, and o-phenanthroline-reacting iron in this experiment is shown in Fig. 42. There was no loss of protein although the slight decrease in SDH and cyt bj indic-ate that some denaturation had occurred. The sum of the directly-reacting ferrous iron of the particulate fraction and supernatant decreased with increasing concentration of detergent in agreement with the result of Fig. 39. The increase in dithionite-reducible iron for the sum of values for the particulate fraction and supernatant sugg-ested that some of the hidden iron had been exposed. The total sum of directly-reacting and dithionite-reducible iron for both particulate 158 Fig. 42. Recovery of SDH, cytochrome bi, protein and NHI after treating the particulate fraction with Triton X-100. The data are from Fig. 41. To calculate total recovery, the values from Fig. 41 are expressed as the sum of the values for the particulate and the supernatant fractions. (A) Recovery of SDH, cytochrome bi and protein. (B) Recovery of NHI, which reacts with o-phenanthroline. a, directly-reacting NHI. b, dithionite-reducible NHI. c, combined values of a and b (i.e. total o-phenanthroline-reacting NHI). The total iron present is 55.5 natoms. T o t a l Cytochrome b-i,nmoles & SDH, units ( » - * - * ) (•"•-•> . ro era n o 3 X I o o u Tot a I Prote i n , m g Total i r o n Reacted, nmoles (D), ^(R), (D+R) 6ST 160 fraction and supernatant (Fig. 42-B) shows a total increase of 4.5 n atoms iron from a control value of 22.5 n atoms. This is an increase of about 8% of the value for total iron (55.5 n atoms iron). Heme iron (ca. 2 n atoms iron) is a minor component. Despite the treatment with Triton X-100, i t was not possible to expose the hidden 45% total iron (ca. 25.5 n atoms iron) such that it would react with o-phenanth-roline. 5. Binding of iron by the particulate fraction Ferrous salts were added to the particulate fraction and, after incubation for various lengths of time, o-phenanthroline was added (Fig. 43). If the chelating agent was added immediately after the ferrous iron there was f u l l and immediate development of the color of the chelate compound in the absence of dithionite. However, even i f only 3 minutes elapsed before the o-phenanthroline was added the rate and extent of reaction was lower. If the difference in time between additions was extended to 36 or 160 minutes the rate and extent of re-action was further diminished. These results indicated that the added ferrous iron may have been bound by the particulate fraction such that its ability to react with o-phenanthroline was impaired. It is not known whether the bound iron can be oxidized to ferric iron in the particulate fraction, but i f oxidation occured then iron hydroxide polymers might be formed (380, 381, 382). These would probably react slowly with the chelating agent. Thus, iron would appear to have been bound by the particulate fraction. In the presence of dithionite, any bound ferrous iron (or ferric iron if part of i t was oxidized) was released and could then readily 161 Fig. 43, Binding of Fe**" to particulate fraction in the presence and absence of dithionite. Ferrous ammonium sulfate solution was added to particulate fraction (1 ml; 1.4 mg protein per ml 0.01 M phosphate buffer, pH 7.5), to give a final concentration of 5 yM Fe-H-. After 3 minutes (•), 36 minutes (o) or 160 minutes (A) 25 yl 0.05 M o-phenan-throline was added and the absorbance at 510 nm relative to 550 nm ("iron reacted") was compared to that of an identical solution from which o-phenanthroline had been omitted (lower graph). In the upper graph, sodium dithionite was added just prior to the addition of o-phenanthroline. The theoretical absorbance values are shown by the broken line. 162 0045 0 10 20 Minutes Fig. 43 163 react with the chelating agent. 6. Solubilization of o-phenanthroline-reacting iron Mahler and Elowe (225) developed a method for the direct deter-mination of nonheme iron by treating iron-containing enzymes with TCA. This treatment released nonheme iron into a soluble form. Massey has used this method to determine the iron content of mammalian SDH (197, 228, 229). Kurup and Brodie (209) reported that there was quantitative release of nonheme iron from the particulate fraction i f there was prior addition of dithionite. Therefore, to see if the dithionite-reducible iron of the partic-ulate fraction was released by dithionite, the following experiment was performed (Table XVIII). The particulate fraction was allowed to react with succinate, NADH, or dithionite for 45 minutes. In one experiment TCA was added after the reduction and ferrous iron was measured in the supernatant following removal of the denatured protein by centrifug-ation. In the other experiment after reduction with succinate, NADH or dithionite, the particulate fraction was sedimented by ultra-centrifugation and again ferrous iron was determined in the supernatant. In the absence of reducing agent, neither ferrous nor ferric iron was released from the particulate fraction with the supernatant by washing or by TCA treatment. This suggested that the iron was strongly bound to the protein, and was not released by extensive denaturation. Dithionite treatment of the particulate fraction released ferrous iron (30.4 n atoms Fe) probably by reducing ferric iron to ferrous iron but also by labilizing the resultant ferrous iron by the secondary de-164 TABLE XVIII Liberation of soluble iron compounds from particulate fraction by substrate3 Total o-phenanthroline-reacting iron in soluble fraction Substrate -TCA +TCA n atoms m atoms None P 0 Succinate 0 11.3 NADH 0 6.3 Dithionite 30.4 47.4 a Particulate fraction ( 6 ml in 0.01 M phosphate buffer, pH 7.5: 10.1 mg' protein, total 104 n atoms Fe) was incubated for 45 minutes with 0.02 M succinate, 0.005 M NADH or with sufficient sodium dithionite to give ful l reduction of the cytochromes. To half of each solution, TCA was added to give 8 % final concentration. Both TCA treated and untreated solutions were centrifuged at 110,000 x g for 2 hours. The iron content of the supernatant was measured after addition of o-phenanthroline (0.5 ml 0.05 M o-phenanthroline per ml TCA supernatant or 25 yl o-phenanthroline per ml non-acidified supernatant). This particulate fraction contains a total of 46.7 n atoms Fe which is reactable with o-phenanthroline; 19.6 n atoms, directly-reacting Fe; 27.1 n atoms, dithionite-reducible Fe. In addition, the total amount of heme iron is not more than 5.0 n atoms Fe at most. 165 naturating effect of dithionite itself. In the presence of TCA addit-ional ferrous iron (17.4 n atoms Fe) was released. Further experiments with the same preparation gave the value of total directly-reacting and dithonite-reducible iron of 19.6 and 27.1 natoms Fe, respectively. The heme iron content was less than ca. 5 natoms Fe. Thus, the values for directly-reacting and dithionite-reducible iron were close to those for iron released by dithionite in the absence or in the presence of TCA, respectively. This suggests that directly-reacting iron is not located on the surface of the en-zyme and that extensive denaturation is required to release i t . As shown in Fig. 44, succinate oF NADH as substrate reduce ferric iron. From Fig. 45 i t is seen that substrate reduced a part of the dithionite-reducible iron. After treating the particulate fraction with substrate, no iron was released in the absence of TCA. This suggests that the substrate-reduced iron is s t i l l strongly bound to protein and requires denatur-ation to expose i t . 7. Effect of succinate on iron in the particulate fraction The result of the previous experiment indicated that about 40% of dithionite-reducible iron (or 25% of o-phenanthroline-reacting iron, or 10% of total iron) responded to the presence of succinate. As shown in Table XX the flavin, cyt bx and cyt o of the partic-ulate fraction were reducible by succinate and NADH. Whereas these components were almost completely reduced by NADH, succinate only re-duced them to 55% to 70% of the dithionite-reduced value. 166 Fig. 44 . Reduction of NHI by substrate . 7,5 mM succinate or 2.5 mM NADH was added at 0 minutes to 1 ml particulate fraction (2.06 mg protein per ml) in 0,01 M phosphate buffer, pH 7.5. At 70 minutes, -25 yl 0.05 M o-phenan-throline was added to the cuvette and the absorbance at 510 nm relative to 550 nm was recorded as described in Materials and Methods. a, without substrate; b, addition of NADH; c, addition of succinate. 168 Fig. 45, Effect of HQNO, succinate and dithionite on the reaction of particulate fraction with o-phenanthroline. The absorbance of the particulate fraction (1 ml; 1.95 mg protein per ml, 0.01 M phosphate buffer, pH 7.5) at 510 nm rel-ative to 550 nm ("iron reacted") was compared after the indicated additions to that of an identical solution to which nothing had been added. Symbols: • , particulate fraction without HQNO; o, partic-ulate fraction to which HQNO (0.7 mM final concentration) had been added; S, addition of 25 yl 0.05 M o-phenanthroline; D, addition of a few crystals of sodium dithionite. 169 Fig. 45„ 170 Fig. 45 (lower graph) shows the effect of adding succinate or dith-ionite to the particulate fraction after almost a l l of the directly-reacting iron had reacted with o-phenanthroline. In a similar way to dithionite, succinate caused additional formation of ferrous o-phenanth-rolinate. This additional increment was termed "succinate-reducible iron". The amount of succinate-reducible iron was found to be 34±7% (6 determinations) of the dithionite-reducible iron. This value is in good agreement with that released by TCA in the presence of succinate (Table XVIII). If HQNO- was present during the reaction of the succinate-reduced particulate fraction with o-phenanthroline (Fig. 45, upper graph) the rate of formation of the chelate was increased. From the lower graph of Fig. 45 it can be seen that the presence or the absence of HQNO did not influence the reaction of directly-reacting iron with d-phenanthroline. Thus, HQNO must have some effect on the succinate-induced reaction. To determine what the effect of HQNO was on this reaction, the reduction of cytochrome b t in the particulate fraction (Fig. 46, lower graph) and the reaction of iron with o-phenanthroline (Fig. 46, upper graph) were measured in the same experiment with and without HQNO. Cyt-ochrome bx was reduced by succinate to only 50% of full reduction in the anaerobic steady state. Addition of HQNO at this point caused the cyt bx to become fully reduced when anaerobic conditions had been re-established after addition of the inhibitor. Thus HQNO must act in the sequence of the respiratory chain between oxygen and at least 50% of the cytochrome b x. When the effect of HQNO on the reaction of iron was examined in this experiment, it was found that the inhibitor had also 171 Fig. 46. Effect of HQNO and succinate on reduction of cyt b_ and reaction of o-phenanthroline with the particulate fraction. In the lower graph the reduction of cytochrome b_ (ex-pressed as absorbance at 428 nm relative to 410 nm in the diff-erence spectrum) is followed after the addition of 25 yl 0.3 M succinate to particulate fraction (1 ml; 1.95 mg protein per ml, 0,01 M phosphate buffer, pH 7.5). Symbols: Q, addition of HQNO (final concentration, 0.7 mM) to +QN0 sample; P, 25 yl 0.05 M o-phenanthroline added to both samples; D, addition of sodium dithionite crystals to -QNO sample. Following addition of o-phenanthroline the reaction of iron was also followed with the same samples (upper graph) by measuring the change in absorbance at 510 nm relative to 550 nm ("iron reacted"). 173 increased the amount of iron able to react with o-phenanthroline in the presence of succinate to the level found with dithionite. The analogous behavior of iron and cytochrome b_ suggests that electrons must pass along the respiratory chain at least as far as the HQNO-inhibition site for this iron to become fully reactive with o-phenanthroline. Electron paramagnetic resonance spectroscopy has been used to in-vestigate the function of metals in enzymes (23, 27, 198, 199). By use of this technique a signal at g=1.94 has been found in a preparation from E. coli (134). This signal has been attributed to the presence of NHI in certain enzymes but it is probable that only a few percent of the total NHI is involved in giving this signal (87). The EPR spectrum of the particulate fraction was measured to see i f reduction of iron could be detected by this method. Under the con-ditions described in Materials and Methods (sensitivity 6 x 10" spins) no electron paramagnetic resonance signals were detected in the g=1.94 region even in the presence of substrate or dithionite. 174 DISCUSSION Iron is an essential trace element in the biological kingdom. There are two kinds of iron in biological materials: the heme proteins (hemoglobin, myoglobin, cytochromes and others) and the nonheme proteins. The latter group is comprised of transferrin for iron transport, ferritin for iron storage, and other nonheme iron proteins including iron-sulfur proteins which are well-known electron transport carriers, and possibly iron proteins not containing sulfur. This thesis work is centered on NHI, therefore the discussion of the iron of heme protein is beyond its scope. NHI proteins of the ferredoxin class from the anaerobic and photo-synthetic bacteria and plants have been reviewed by Buchanan (46), NHI compounds isolated from various aerobic microbial sources have been reviewed by Neilands (241). In the bacterial systems iron metabolism is not clearly understood. Ferrous iron forms strong chelate compounds with ferroverdin from Streptomyces or with ferropyrimine from Pseudomonas. These compounds have been isolated from them but their function is not known. Hydrox-amic acids from various microbial sources form strong, stable complexes with ferric iron. In addition to them-', citrate, 2, 3, -dithydroxy benzoylserine (DHBS), or 2, 3, -dihydroxy benzoylglycine (DHBG) are known biological chelating compounds for ferric iron in bacteria. These chelating compounds are involved in the iron transport in bacteria (43, 258, 342). In E. coli, enterochelin (77, 244) or DHBS (43, 342), a degradat-ion product of enterochelin, are involved in active transport of ferric 175 iron by forming stable, soluble chelate compounds with i t . These chelates are at a concentration of 2,000 times higher inside the cell than in the outside medium (342). Also, if citrate is included in the growth medium it may also possibly be involved in iron transport (258, 342). DHBG is reported to be involved in the active transport of ferric iron in Bacillus subtilis (258). There is no report concerning the metabolism and transport of ferrous iron in aerobic bacteria. Probably ferric iron is the same source of both ferric and ferrous iron found in the cell. The ferrous iron would be produced by reduction within the cell. The reduced form of iron complexed to a chelating compound is involved in the biosynth-esis of various iron-requiring proteins including heme (241) and non-heme proteins. The E. coli which the author used were grown aerobically in the absence of added iron. Iron uptake is expected to occur from the trace amounts present in the chemicals of the medium. The citrate present in the medium, as well as iron deficiency of the medium, probably stimulated the active transport of iron. The cytochrome content of the particulate fraction was slightly higher than that reported by Bragg (35) and by Cox et al. (76). Despite the higher level of cytochromes in this preparation the amount of heme iron is at most not more than 5% of the total iron. Nonheme iron also predominates over heme iron in the particulate fraction of M. phlei (209). Therefore, the bulk of iron in the E. coli envelope is NHI or other unspecified iron species. Since there is no appropriate method to investigate NHI proteins in the particulate fraction, the formation of ferrous-o-phenanthrolinate 176 following the addition of the chelating agent to substrate or dithionite-reduced particulate fraction was employed. This method has previously been used by Kurup and Brodie (210) and Bragg (35) to investigate the re-duction of respiratory chain-linked NHI. As a physical method, EPR spectroscopy can be used, but only a relatively small amount of the total nonheme iron appears to be detectable by this method (88). Moreover, we failed to observe the EPR signal at g=1.94 in our preparations which has been reported to be due to nonheme iron (25). If o-phenanthroline is added to this particulate fraction, three distinct types of nonheme iron can be detected: (a) nonheme iron which reacts slowly with the chelating agent in the absence of substrate or dithionite ("directly-reacting iron); (b) nonheme iron which will react with o-phenanthroline in the presence of substrate or dithionite after al l of the directly-reacting iron has reacted ("substrate- or dithionite-reducible iron"); and (c) iron which will not react with e-phenanthroline even in the presence of reducing agents ("non-reacting nonheme iron"). The amount of these species were 21%, 21% and 53% of the total iron, respectively. The heme iron constituted 5% of the total iron. The reaction of the directly-reacting iron with o-phenanthroline indicates the progressive formation of ferrous-o-phenanthrolinate which is probably due to the reduction of ferric iron by sulfhydryl groups located near the iron in the particulate fraction. This progressive color development was also observed with pure cysteamine oxidase which also contains iron (60). The requirement of the reducing agent, dithionite, to produce the dithionite-reducible iron suggests that this iron probably exists in 177 the ferric state. The formation of the chelate was not as rapid as would be expected if dithionite-reducible ferric iron was completely exposed for reaction with o-phenanthroline. However, since Massey (228) found that dithionite increased the rate of reaction of the ferric iron of SDH with Tiron, we cannot exclude the possibility that dithionite caused exposure of hidden iron as well as reduction. An attempt to apply Massey's procedure was not successful because dith-ionite instantly decolorized the blue or red color of the ferric-Tiron complex. It is not understood how the complex retained its color after its reduction in his experiment. Added ferrous iron can be bound by the particulate fraction and dithionite will release this bound iron. Thus, a large part of the dithionite-reducible iron might be held by similar bonds to those which wiil bind added ferrous iron. When Tiron was added to the particulate fraction at pH 9.4, about 30% of total iron formed a complex with Tiron, of which 10% is possibly contributed by the iron of directly-reacting iron exposed or released at this pH. This suggests that the Tiron-reacting iron is the same iron species as the dithionite-reducible iron. As discussed previously, TCA treatment in the absence or pres-ence of substrate or dithionite indicates that dithionite-reducible iron is easily released while directly-reacting iron is not. This clearly indicates that this ferric iron with associated SH groups is located where Tiron cannot penetrate, but where o-phenanthroline will react. This iron is only released by dithionite in .the presence of TCA. Succinate and NADH appear to reduce part of the dithionite-reducible iron. It is not clear whether NADH-reducible iron is 178 different from succinate-reducible iron. The amount of iron released by NADH in the presence of TCA is less than that released by succinate (Table XVIII). This suggests that some of the iron may be located differently. The results of cyt bx reduction suggest that the succin-ate and NADH chains may be different, since succinate can only reduce part of the cytochrome bj in.contrast to NADH which will reduce a l l of this cytochrome. It is possible that these substrates reduce ferric iron enzymatic-ally or they could cause the iron to be placed in a location accessible to reduction by sulfhydryl groups. Failure to detect the g=1.94 signal in the presence of succinate, NADH or dithionite does not prove that iron has not been reduced by substrate in this system. Reduction of ferric iron to ferrous iron would not give a detectable signal. Thus, this cannot be used as evidence that substrate does not reduce ferric iron. In support of true reduction of ferric iron by substrate is the fact that the properties of the succinate-reducible iron show some kin-etic similarities to cytochrome bx reduction. Both cytochrome bj of succinate-reduced chain and succinate-reducible iron reach steady-state values under anaerobic conditions which are less than total reaction of these components. In the presence of HQNO both components fully react to give values equivalent to those given by dithionite. Thus the be-havior of this form of iron is closely linked to that of cytochrome bj. This clearly suggests that HQNO blocks on the oxygen site of NHI and cyt bt in the succinate oxidase chain. Succinate oxidase is inhib-ited by the presence of o-phenanthroline (35% inhibition of oxidase ac-tivity at 0.5 mM of chelating agent) which suggests that NHI is located in the main chain of the succinate oxidase system. SDH is less sensitive 179 to o-phenanthroline (15% inhibition of SDH activity at 5.0 mM of the chelating agent) which indicates that most of the NHI of the succinate oxidase chain is located on the oxygen side of SDH. Therefore, the succinate-reducible iron is probably located between the flavoprotein of SDH and cyt b_ as has been found in M. phlei (209, 210). The succ-inate reducible iron which is found only in the< presence of HQNO may be on a branch chain, as has been proposed by Bragg (35) . The cyt b^  which which is reducible further by the presence of HQNO may also be located in this position. It is of interest that some of the cyt b of the mammalian mitochondria is reduced only when antimycin, which has a similar action to MONO, is present (298). It has been reported that NHI is bound to NADH dehydrogenase of E. coli (31, 115) and to SDH of beef heart mitochrondria (371) and P.  pentosaceum (213). There is no report concerning NHI in SDH of E. coli yet. This study indicates that probably only a very small part, if any, of the NHI is involved in succinate PMS-DCIP reductase and so may be not an essential component of SDH. However, i t could be a component of an SDH complex containing cyt b_. The close linkage of SDH and cyto-chrome b in the mitochondrial respiratory chain has been suggested by the work of Doeg et al. (89, 369) Cox et al. (76) postulated that a complex of UQ with NHI was lo-cated before and after cyt b1 in the NADH chain of E. coli but was not on the direct pathway of electrons. HQNO was suggested to attack at these sites. From this study i t was not possible to prove this sugg-estion. No biphasic reduction of NHI, due to two locations, was ob-served. HQNO did not appear to block before NHI and cytochrome b x. Moreover, the ratio of UQ to substrate-reducible iron in the partic-ulate fraction is at least more than 20. 180 The major part of the iron of the particulate fraction is not accessible to o-phenanthroline even in the presence of reducing agents. Treatment of the particulate fraction with urea revealed a slight in-crement of o-phenanthroline-reacting iron and inhibited completely both the succinate-linked reduction of flavin and of cyt bx (Table XXII) Treatment of the particulate fraction with Triton X-100 also exposed some more o-phenanthroline-reacting iron but retained very low activ-ity of flavin and cyt bx reduction. The manner of denaturation of the particulate fraction by both denaturants may be quite different, and possibly the sources of iron revealed by them is also different. The redox state and function of this iron is unknown. The iron re-vealed by denaturation of the particulate fraction by Triton X-100 or urea is very small (less than 10% of total iron, Fig. 39 and 42-B). There is s t i l l at least 40% of the total NHI of the particulate fraction which will not react with o-phenanthroline or with Tiron. We have not been able to expose or react this with chelating agents under the conditions we have employed. Kurup and Brodie (210) have called the NHI of M. phlei envelope fragments which is not reducible by sub-strate "structural NHI", but there is no proven example of the poss-ible existence of NHI as a structural material. Thus, it is not clear whether our non-reacting iron f u l f i l l s a structural role, is a storage form of iron, or has some other funct-ion. The exact nature, including the redox state, of the bulk of the NHI, remains to be found. To determine the exact role of NHI in the respiratory chain probably requires isolation of the individual electron carriers and the reconstitution of the chain from them. 181 The rate of appearance or disappearance of the EPR signal of mammalian SDH is commensurate with the overall rate of oxidation, so that nonheme iron plays some role in the respiratory chain (88). The role of NHI in oxidative phosphorylation in bacterial systems is un-likely to be proven until the mechanism of oxidative phosphorylation itself is more clearly understood. 182 PART III - COMPOSITION OF THE RESPIRATORY CHAIN IN THE PARTICULATE FRACTION RESULTS The composition and the concentration of the respiratory components were determined as described in Materials and Methods. A reduced minus oxidized difference spectrum of a suspension of the particulate fraction showed a trough at 460 nm indicative of flavin, and three absorption peaks of cyt b_ at 558 nm (a-band), 530 nm (3-band) and 427 nm (Soret band) as shown in Fig. 47. A similar spectrum was also observed by Bragg et al. (31, 35). No cyt aj or a 2 were observed in this prepar-ation. The concentrations of respiratory components in the author's prep-aration are compared to those obtained by other workers in Table XIX. In Table XX the extent of reduction of flavin, cyt b_ and cyt o in the particulate fraction by succinate, NADH and dithionite is shown. (1) The role of flavoprotein Experimentally the level of flavoprotein was calculated from the absorbance of the flavin trough at 460 nm - 470 nm relative to 510 nm in the reduced minus oxidized difference spectrum. Changes in the flavoprotein trough reflect also absorbance changes due to cytochrome as well as to flavoprotein itself (69, 243). The problems concerning spectrophotometric determination of flavin (or the substrate-reducible level of flavin) in mitochondrial preparations has been discussed in detail by Nicol and Malviya (243) and also by Singer et al. (296). 183 Fig. 47. Reduced minus oxidized difference spectrum of particulate fraction. The spectra were recorded as described in Materials and Methods. Each cuvette contained particulate fraction in 0.01 M phosphate buffer, pH 7.5 (6.64 mg protein per ml). Curves a, b and c were obtained in the presence of sodium succinate (4.5 mM), NADH (1.5 mM) and sodium dithionite (a few crystals), respectively, d, base-line. 0.1 • • • • 0.0 8 O.0 2r UJ o < OQ CC o co m < 0.0 2! 410 500 W A V E L E N G T H , nm 6 0 0 Fig. 47. Reduced minus oxidized difference spectrum of particulate fraction. 185 TABLE XIX Concentration of respiratory components in the particulate fraction of E. coli Bragg (35) Cox et al. (76) Author Flavin _ 0.25 0.54 ± 0.12 Cyt bx 0.19 ± 0.03 0.19 0.34 ± 0.02 Cyt o 0.045 0.073 0.124± 0.015 UQ 1.84 ± 0.17 4.7 3.91 ± 0.29 K2 0 0.67 0 Cyt a.2 0 0.027 0 Values are expressed as nmoles per mg of protein. 186 TABLE XX Reducibility of electron transport components of unwashed particulate fraction Extent of reduction3  Substrate Flavin Cyt bj Cyt o n moles per ^ n moles per o, n moles per mg protein ° mg protein ° mg protein Succinate 0.293 55 0.125 60 0.053 71 NADH 0.482 90 0.191 92 0.108 144 Dithionite 0.533 100 0.208 100 0.075 100 a Mean of three separate determinations. 187 In addition to the effect by cytochrome, i t was reported that NHI also contributes absorption at 460 nm in the difference spectrum (69, 264) because SDH and NADH dehydrogenase are well known metalloflavo-proteins. But in the F,. coli system the addition of o-phenanthroline to the reduced particulate fraction did not make a significant differ-ence in the absorbance between 427 and 460 nm although the absorption peak at 510 nm of ferrous-o-phenanthrolinate showed that iron was being chelated (see the second part of this thesis). The absorption contrib-ution by NHI at 450 nm will be very small, if any, in the particulate fraction which the author has used. Fluorescence techniques have been used to measure flavoprotein by Chance et al. (69) and also by Singer (296). But these were not applicable to isolated SDH and NADH dehydrogenase which are non-fluorescent (70). By using this method, Chance et al. (68) identified -two species of flavoprotein in mitochondrial NADH dehydrogenase, one of which was highly fluorescent (Fdx) and the other of low fluorescence (Fd2). This work has been criticized by Ragan and Garland (265), who found that both fluorescent flavoproteins were not involved in the respiratory chain. Therefore, the author preferred to use conventional absorption methods to quantitate flavoproteins in spite of the object-ions discussed above. In the mitochondrial system NADH dehydrogenase is an FMN-containing protein, while SDH has tightly bound FAD. With E. coli it is not known which flavin is the prosthetic group of SDH and NADH dehydrogenase, since neither enzyme has been highly purified. Moreover, it is not possible to differentiate FMN and FAD-containing enzymes by spectro-188 photometric techniques. The NADH dehydrogenase of E. coli may contain either or both flavins (see Table V), but there have been no reports concerning the prosthetic group of SDH from bacterial sources. The identification of the flavin of the soluble SDH from the ace-tone powder extract was not attempted, although this would have been the most suitable fraction to have examined. However, the effect of adding flavin on the SDH activity of the cell-free extract was measured. As shown in the right figure of Fig. 18B, addition of FMN had a slightly inhibitory effect on the SDH activity. In a similar experiment using the enzyme which had been precipitated at the isoelectric point addit-ion of FMN also slightly decreased the activity below that of the con-trol (Table XXI). In contrast, FAD only slightly stimulated SDH with both preparations. The absence of a marked effect of flavin may be due to the pros-thetic group being covalently linked to the apoenzyme. This would be in agreement with the result found for the mitochondrial enzyme where FAD is covalently linked to the protein of SDH (190). (2) The role of UQ It has been reported that UQ is necessary for cyt b_ reduction in the formate-nitrate reductase system of anaerobically grown E. coli (160, 161). With aerobically grown E. coli cells, there is a controversy con-cerning the location and function of UQ in E. coli. Kashket and Brodie (178) suggested that UQ and MK were specifically involved in the succi-nate and NADH oxidase systems, respectively. Jones (174) suggested that 189 TABLE XXI Effect of flavin on SDH activity of proteins obtained from cell-free extract by isoelectric precipitation Addition Specific Activity None FMN FAD 0.056 0.032 0.064 The enzyme precipitated from the cell-free extract at the isoelectric point was prepared as described in Materials and Methods. Flavin (0.83 mM) was incubated with the enzyme at 38° for 15 minutes. Aliquots (0.22 mg protein) were removed for determination of SDH activity after activation as described in Materials and Methods. 190 UQ functions between flavin and cyt bl in the NADH oxidase system, while Bragg (31) has shown that UQ may not be involved directly in the NADH oxidase chain but may be on a minor pathway. Menaquinone did not fun-ction in the NADH chain of the preparations studied by either of these workers. Recently Cox et_al.(76) reported, using an E. coli mutant lacking the UQ biosynthetic system, that UQ was involved in the NADH, lactate and malate oxidation systems, and suggested that UQ functions at two sites, one before and one after cyt b_ in the NADH oxidase chain, but not directly on the electron transfer pathway. Evidence for this has also been reported by Baillie, Hou and Bragg (16). Therefore, the author examined the role of UQ in E. coli. First, the nature of the quinones in E. coli cells was analyzed as described in Materials and Methods. Quinone was directly extracted from freezer-dried cells with petroleum ether and the extract was chromatographed on a column of Magnesol-Celite. The elution was carried out with isooctane and then with 1% ether in isooctane. No materials absorbing in the ultraviolet range were detected (Fig. 48-1, 2, 3). When the eluant with 1% ether in isooctane was evaporated to dryness under reduced pressure and the residue dissolved in alcohol, the absorption spectrum before and after reduction showed compound (s) which absorbed in the ultraviolet (Fig. 48-5a § b). These compound (s) were not identified but they may be lipid containing trace amounts of different isoprenol-ogues of UQ. No MK was detected. When the column was eluted with 1% alcohol in isooctane an absor-ption peak at 275 nm was observed in spectra of the eluant, (Fig. 48-4). The eluant was evaporated to dryness. The dry residue dissolved in ethanol had the same absorption spectrum as that of UQ (Fig. 49). It 191 Fig. 48. Spectrum of fractions obtained during separation of quinones from E. coli cells. The extraction of the quinones from whole cells and the methods.of chromatography is described in Materials and Methods Curve 1, isooctane eluants of column before application of sample; 2a and 2b, isooctane fractions from column after the application of the sample; 3, fraction eluted by 1% (V/V) ether in isooctane; 4, fraction eluted by 1% (V/V) ethanol in isooctane; 5a, material from fraction 3 after this fraction had been evaporated to dryness under reduced pressure and the residue dissolved in 2 ml ethanol; 5b, the same as 5a but after the addition of a few crystals of potassium borohydride. 193 Fig. 49. The absorption spectrum of ubiquinone. Ubiquinone was extracted and purified from the particulate fraction as described in Materials and Methods. The quinone was dissolved in ethanol to give a 0.018 M solution, a, base-line; c, absorption spectrum of oxidized form; d, absorption spectrum of quinone after reduction with K B H 4 ; b, KBH^-reduced minus oxidized difference spectrum. 194 Wave l e n g t h , n m Fig. 49. The absorption spectrum of ubiquinone. 195 is probable that this species of UQ is UQ8 because UQ8 is predominant in E. coli (215). It has been reported that there are various UQ's and MK's in E. coli (56, 82) but these occur in trace amounts which would not have been detected by our method. The amount of ubiquinone was est-imated as described in Materials and Methods (Table XIX). In order to investigate the function of UQ the following experi-ments were performed. In a suspension of freeze-dried cells in 0.01 M phosphate buffer, pH 7.5, cyt b_ was reduced 65 to 70% by succinate or NADH. This indicates that the substrate-cyt b_ reductase system was fairly stable. The freeze-dried cells were extracted twice with petrol-eum ether to remove UQ, as described in Materials and Methods, and the extracted residue suspended in the same buffer solution. With this suspension 70% of the cyt b_ was reduced by succinate or NADH. These results indicate that UQ is probably not directly involved in the cyt b_ reductase system. A possible location of UQ after cyt bt (76) cannot be ruled out. A possible factor which was not examined was to determine if trace amounts of functioning UQ were s t i l l present after extraction with isooctane. Addition of UQ in alcoholic solution back to the quinone extracted preparation decreased the reduction of cyt bx (57% and 52% of cyt b_ was reduced by succinate and NADH, respectively). This inhibition may be due to the inhibitory effect by alcohol itself. Treatment of the particulate fraction with methanol-petroleuro ether, as described in Materials and Methods, destroyed cyt bx reduction by substrate completely. Addition of UQ in alcoholic solution was with-out effect. It is likely that in this experiment there was extensive denaturation of protein by methanol. 196 (3) The role of cytochrome o This carbon-monoxide-binding cytochrome was originally observed by Castor and Chance (58, 59) and was designated cytochrome o. Cyto-chrome o was also observed in F,. co 1 i by Bragg (35) and by Rev sin and Brodie (268). The presence of cyt o in the particulate fraction used in the author's experiments is shown in Fig. 50. The CO-cyt o complex has an absorption maximum at 416 nm. This cytochrome has not been isolated from E. coli so its nature is unknown. As shown in Table XIX, cyt o was reduced by substrate. The increased level of cyt o reduction given by NADH over that pro-duced by dithionite was unexpected, since the latter reducing agent would be expected to reduce a l l of the cytochrome. The reason for this is not known. The amount of cyt o is small compared to the other respiratory carriers. This nay indicate that one of the rate limited steps of the respiratory chain is at the level of cytochrome o, unless the turnover rate of this cytochrome is very high. No information is available on this point. (4) The role of NHI The role of NHI has already been described in Part II of this thesis. (5) Effect on the succinate oxidase system of detergents, ultraviolet irradiation and inhibitors. 197 Fig. 50. Carbon monoxide difference spectrum of particulate fraction. The spectra were determined as described in Materials and Methods. Each cuvette contained 1 ml particulate fraction (5.5 mg protein per ml 0.01 M phosphate buffer, pH 7.5). Curve A, B and C were obtained in the presence of sodium dithionite ( a few crystals), NADH(1.5 mM), and sodium succinate (4.5 mM), respectively. Curve D, base-line. 198 0.04h 0.0 2h o co CQ 0.0 2 h 0 r~ 450 550 WAVELENGTH , nm Fig. 50. Carbon monoxide difference spectrum of particulate fraction. 199 (a) Effect of detergents or denaturing agents The presence of urea or Triton X-100 seriously damaged both the reduction of flavin and cyt b x by succinate (Fig. 51 and Table XXII), while cholate had l i t t l e effect. Triton X-100 probably acts at a site between SDH and cyt bj because SDH activity was unaffected by the pres-ence of the detergent. Since SDH is liberated to some extent by treat-ing the particulate fraction with Triton X-100 (Fig. 41) it is probable that at least part of the inhibition produced by Triton was due to loss of the enzyme from the membrane. Therefore, it seems that the lack of effect of cholate is because it did not disaggregate the bacterial respiratory system. It should be noted that the release of the soluble respiratory complex from the particulate fraction by cholate requires the presence of ammonium sulfate as well. (b) UV-irradiation Ultraviolet irradiation of the cell-free extract did not inhibit SDH activity markedly but longer irradiation seemed to denature the SDH enzyme itself (Table XXIII). To see if the UV-labile component was present in the particulate or the supernatant fraction of the cell-free extract, the following experiment was performed. After the supernatant had been irradiated with ultraviolet light, it was combined with non-irradiated particulate fraction. As shown in Table XXIV, the succinate oxidase activity was l i t t l e affected, although longer treatment did result in some loss of activity. Irradiation of the particulate fraction followed by the sub-sequent addition of non-irradiated supernatant showed serious impair-200 Fig. 51. Effect of detergents on cytochrome bj reduction by succinate , f Particulate preparation in 0.01 M phosphate buffer, pH 7. (1.85 mg protein per ml), was treated with 0.5 % Triton X-100, 0.5 % sodium cholate or diluted with equal volume of buffer, and stirred for 20 minutes at 4°. The reduction of cyt b_ by succinate was recorded as a reduced minus oxidized difference spectrum as described in Materials and Methods. The graph shows the change of absorbance of the Soret band following addition of 50 yl 0.15 M succinate (S). D, a few crystals of dithionite added. a, control enzyme; b, cholate-treated enzyme; c, Triton X-100-treated enzyme. 201 202 TABLE XXII Effect of detergents and urea on succinate-linked reduction of flavin and cytochrome The procedure is described in the legend to Fig. 51. Treatment3 Reduction of flavin 0 Reduction of cyt b] C Succinate*3 Dithionite Succinate*3 Dithionite None 0.45 0.85 0.27 0.48 0.5 % Triton X-100 Trace 0.95 0.048 0.45 0.5 % Cholate 0.55 0.95 0.22 0.48 6M Urea 0 1.23 0 0.43 Triton-urea^ 0 1.14 0 0.48 Cholate-ureae 0 0.90 0 0.48 a The particulate fraction (1.75 mg protein per ml) was stirred with the detergents or urea for 1 hour. b 50 pi 0.15 M succinate was added to 1 ml ^particulate preparations (treated or non-treated). c Calculated from the absorbance change in the reduced minus oxidized difference spectrum. Units are in n moles per mg protein. d 0.25 % Triton X-100 and 3 M urea. e 0.25 % cholate and 3 M urea. 203 TABLE XXIII Effect of irradiation with near-ultraviolet light on SDH of cell-free extract Irradiation Specific Activity Time (min) Activity (%) 0 0.079 100 10 0.073 92 20 0.060 77 30 0.032 41 The cell-free extract (7.65 mg protein per ml 0.01 M phosphate buffer, pH 7.0) was irradiated for various times as described in Materials and Methods. Aliquots were tested for SDH activity without activation. 204 TABLE XXIV Effect of irradiation with ultraviolet light on succinate oxidase activity of combined particulate and supernatant fractions Time of Succinate oxidase Experiment Irradiation Specific (mins.) Activity % Particulate fraction 0 0.024 + irradiated supernatant 0 0.038 100 + irradiated supernatant 10 0.040 106 + irradiated supernatant 20 0.036 94 + irradiated supernatant 30 0.031 81 Supernatant 0 0 + irradiated particles 0 0.030 100 + irradiated particles 10 0.016 54 + irradiated particles 20 0.008 27 + irradiated particles 30 0.005 17 The particulate fraction was obtained as described in Materials and Methods, but with the modification that it was not washed with 0.01 M phosphate buffer, pH 7.0. The supernatant fraction was also kept. Irradiation was performed as described in Materials and Methods. Equal volumes of particulate and supernatant fractions in 0.01 M phosphate buffer, pH 7.0, were mixed after the indicated treatment and incubated at 0° for an hour. Aliquots were then removed for assay of succinate oxidase without prior activation. Experiment A: particulate fraction 39.2 mg protein per ml; supernatant fraction 7.65 mg protein per ml. Experiment B: particulate fraction, 30 mg protein per ml; supernatant,12.2 mg protein per ml. 205 ment of succinate oxidase activity. This indicated that there was a certain compound (s) present in the particulate-bound succinate oxidase chain, not at the level of SDH, which ultraviolet irradiation inactiv-ated or decomposed with the resultant loss of oxidase activity. When the particulate fraction was irradiated, cyt bj reduction by succinate and NADH was impaired markedly (ca. 20 - 25%) (Table XXV). Therefore, the inhibition of succinate oxidase by UV-irradiation is due to the impairment of a site before cyt b_.. The UV-labile compound could be either a quinone or another UV-sensitive compound (212.) The possibility of the former is excluded because quinone does not seem to be involved in this system since extraction of UQ did not im-pair cyt b reduction by substrate. Recently, Bragg (36) has found that the destruction of UQ by UV irradiation was not the major cause for loss of activity in the NADH oxidase chain of E. coli although Kashket and Brodie (37, 177) have reported that UQ was destroyed by UV-irradiation. The UV-sensitive compound is probably similar to that found in M. phlei, where a light-sensitive component, which is not a quinone, is located before cyt b in the succinate oxidase chain (10, 11). Since the inhibition of succinate oxidase was more drastic than the effect on the cyt b_ reduction by succinate (Tables XXIV and XXV), it is also possible that UV light also inhibits between cytochrome b_ and oxygen, as also found by Bragg (36). The non-irradiated and the ultraviolet-treated particulate frac-tions were mixed to see whether there was any interaction between the respiratory chains in the two preparations (54). The extent and rate 206 TABLE XXV Effect of ultraviolet irradiation on the reduction of cyt b_ by substrate in particulate fraction Succinate NADH Dithionite 30-UV 60-UV 30-UV 60-UV 30-UV 60-UV Control 0.24 0.25 0.18 0.16 0.41 0.37 UV-irradiated 0.093 0.063 0.082 0.006 0.36 0.31 Mixed 0.24 0.18 - 0.079 0.39 0.33 (expected values) 0.167 0.157 0.131 0.083 0.39 0.34 The particulate fraction (1.95 mg protein per ml; total 7.5 ml) in 0.01 M phosphate buffer, pH 7.5, was irradiated with ultra-violet light as described in Materials and Methods for 30 (30-UV) and for 60 (60-UV) minutes. Control samples were kept at 0° with-out irradiation for these times. The "mixed" sample was obtained by mixing equal volumes of irradiated and control enzyme. After irradiation the reduced minus oxidized difference spectrum was recorded 20 minutes after the addition of 30 yl 0.15 M succinate, 30 yl 0.05 M NADH or a few crystals of sodium dithionite to 1 ml enzyme. The results are expressed as nmoles reduced per mg protein. 207 of the reduction of cyt bl in this mixed preparation was the same as in the control preparation and higher than would have been expected from the sum of the reduction of cyt b^  in the two separate preparations. This indicates that "succinate-reducible" cyt b1 of the irradiated par-ticulate fraction can accept electrons from either flavoprotein, ubi-quinone or succinate-reducible cyt bi of the other intact succinate oxidase chain. It is assumed that there was close interaction among the same species, that is, succinate-reducible cyt bL of the cyt bx pool, but not between this pool of cyt bL and the NADH reducible cyt bj. Since no difference in the rate of reduction of cyt bx was obser-ved in the mixed preparation, the rate of interaction between the res-piratory chains must be rapid. (c) Effect of inhibitors on SDH and succinate oxidase activities Bacterial systems are generally less sensitive to inhibitors com-pared to the mitochondrial systems (299, 300). Sodium azide which binds at cytochrome o, HQNO which inhibits the oxidation of reduced cyt bx (see Part II of this thesis), PCMB which binds to sulfhydryl groups, and o-phenanthroline which binds ferrous NHI, were the most effective inhibitors of the succinate oxidase system (Table XXVI). Since o-phenanthroline did not give significant inhibition of SDH, it is assumed that NHI is not involved or not an important group for succinate PMS-DCIP reductase activity. TABLE XXVI Effect of inhibitors on SDH and succinate oxidase activities Concentration SDH Succinate oxidase Inhibitor (mM) % Control Preparation % Control Preparation Sodium azide 0.5 0 c 5.0 107 a Amytal 0.5 90 a 5.0 84 a 2,4-D in itropheno1 0.5 90 c Dipyridyl 0.5 106 c o-Phenanthro1ine 0.5 94 b 65 c 5.0 85 b EDTA 0.5 85 b 1.4 78 c 5.0 79 b HQNO 0.23 130 a 0.54 0 c 2.3 107 a TFA 0.5 107 5.0 107 a CCCP 0.54 60 c Dicumarol 0.5 100 a 100 c 5.0 62 a PCMB 0.5 67 a 0 c 5.0 17 a The enzymes were preincubated with the inhibitors for ca. 5 minutes prior to assay. SDH activity was measured with non-activatecT~enzymes, but for succinate oxidase activity the enzyme was activated as described in Materials and Methods and then incubated with the inhibitor. Preparation a: Cell-free extract (1.8 mg protein per ml) was prepared in 0.01 M phosphate buffer, pH 7.5, as described in Materials and Methods. Sonication time, 15 minutes. Specific activity of SDH, 0.057. Preparation b: Cell-free extract (3,2 mg protein per ml) was prepared in 0.01 M phosphate buffer, pH 7,5, Sonication time, 10 minutes. Specific activity of SDH, 0.072, Preparation c: Unwashed particulate fraction (6,8 mg protein per ml) was prepared in 0.01 M Tris-0.04 M MgSOi, buffer, pH 7,5, as described in Materials and Methods. Sonication time, 5 minutes. Specific activity of succinate oxidase, 0.082. 209 Sulfhydryl groups are important for both SDH and succinate oxidase activities, therefore it is likely that the inhibitions of succinate oxidase by PCMB is due to blocking the essential sulfhydryl groups of SDH. As expected from the known sites of action of azide and HQNO (see above) these inhibitions did not effect SDH. The absence of an effect of TFA on SDH is expected since, although this substance inhibits succinate oxidase of mitochondria, it is without effect on the purified soluble SDH from mitochondria. Its site of ac-tion is between SDH and cyt b (372) in mitochondria. 210 DISCUSSION From the experiments employing specific inhibitors or techniques it is possible to arrange the sequence of the respiratory components of E. coli. Cox et al. (76) proposed that the respiratory sequence of the NADH oxidase of E. coli was as in Fig. 52. In this scheme UQ cannot act as an individual respiratory carrier but only as a complex with NHI. The complex is located both before and after cyt b^ (76). Moreover, the UQ is not directly situated on the respiratory pathway and so may not be essential for oxidase activity. They did not describe the se-quence of the succinate oxidase system. Hendler et al. (147) also proposed a scheme for the respiratory chain of both succinate and NADH oxidase systems, as shown in Fig. 53, In this scheme, and that of Cox et al., the same cytochromes function both for the oxidation of succinate and NADH, since either succinate or NADH reduced a l l or nearly a l l of the cytochromes present. In add-ition, the possible participation of UQ and cyt o was excluded. The author proposed, on the basis of the work described in this thesis, that the respiratory chain for the succinate oxidase system is as in Fig. 54. In this scheme three species of NHI are introduced: Succinate-reducible iron (NHIS), NADH-reducible iron (NHI^ ) and the other NHI which can be reduced only in the presence of HQNO by succi-nate. The location of the NADH-reducible iron (NHId) and that of the third species of iron (NHI) was not examined. This species may not be important for the respiratory chain-linked electron transport. 211 Lactate Amytal NADH > Fd $• NHI > Cyt bj —>NHI > K-UQ [ HQNO Piericid in — > Cyt o Cyt a 2 ->o2 UQ <r —; Inhibition Fig. 52. The NADH and lactate oxidase systems of E. coli proposed by Cox et al. (76). NADH -> Fd Succinate ^ Fs Cyt b, > Cyt a, > Cyt a 9 > 0. Fig. 53. The respiratory chain of E. coli as proposed by Hendler et a l . (147). F u m a r a t e Mai on.ate S u c c i n a t e NADH -> Fd SDH UV i i i O-Ph -»[X • N H I s ] PCMB PCMS PMS > DCIP > [ X • NH I d ( ? ) ] C y t b i P o o l / / // 'Cyt' C y t b : d .Cyt b ! d . HQNO C y t b i d P o o l CO A z i d e c y a n i d e -> C y t o > 0; to y-i ^ ; i n h i b i t i o n s i t e . X ; U V - s e n s i t i v e s i t e . o-Ph ; o - P h e n a n t h r o l i n e . NHI" ; N A D H - r e d u c i b l e NHI. N H I S ; S u c c i n a t e - r e d u c i b l e NHI. C y t b x s ; S u c c i n a t e - r e d u c i b l e c y t b _ . C y t b . d ; N A D H - r e d u c i b l e NHI c y t b i . F i g . 54. P r o p o s e d s e q u e n c e o f t h e s u c c i n a t e o x i d a s e c h a i n o f E. c o l i . 213 The sequence between the ultraviolet sensitive component (X) and succinate-reducible iron (NHIS) was not defined, but the location of NHIS is probably very close to cyt b^  since Bragg (36) found that UV-irradiation impaired the reduction of iron in the NADH oxidase system. Since about 60 % of the total cyt b^  was reducible by succinate in the particulate fraction (Table XX and Fig. 47) and since most of cyt bj was reducible by NADH, it is likely that succinate can only re-duce certain species of cyt b^  (cyt bj s), while NADH can reduce a l l of the cyt bj. A similar result has been obtained with submitochond-ria l preparations (97, 374, 375, 376). If the redox potential of cyt b^  (NADH-reducible cyt b^ which is not reducible by succinate) is lower than that of succinate-reducible cyt b^, NADH can reduce a l l of cyt b^. Both species may have the same spectral properties, at least when the spectra are recorded at room temperature. When oxidation of re-duced cyt bj is blocked with HQNO, chemical equilibration will allow the reduction of NADH-reducible cyt b^  from succinate-reducible cyt bj provided that the redox potential of the NADH-reducible cyt bi is not too low. Modification of the redox potential of cyt b^  may be poss-ible i f it could bind with redox potential modifiers. Recently Ruiz-Herrera and DeMoss (275) suggested that the two kinds of cyt b^  with different redox potentials function sequentially in the transfer of electrons from formate to nitrate in anaerobically grown E. coli. They stated that the cyt b^ of the nitrate reductase system is distinct from that formed under aerobic conditions. They questioned whether the same species of cyt b^ was associated with the different types of respiratory chains which may be found in E. coli, and suggested that there was a specific cyt b^ for the various electron transport chains (275). 214 Therefore, it may be that the succinate and NADH oxidase systems of E. coli have two different types of cyt bj, or that the same cyt b^  with different modifiable properties may be involved in each system. There also appears to be close interactions between the same spec-ies of cyt bj_, possible as a cyt b^s pool, but possibly due to mobile carriers like UQ, since there was electron transfer from the intact substrate-reduced respiratory chain to the cyt b^  of a UV-irradiated respiratory chain in which cyt bj could not be reduced by substrate. The exact sequence s i l l remains to be determined. It is necess-ary to develop inhibitors which will react with specific respiratory carriers in bacterial systems. 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