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The organization and properties of the cytochromes of the respiratory chain of Escherichia coli Pudek, Morris Romuald 1976

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THE ORGANIZATION AND PROPERTIES OF THE CYTOCHROMES OF THE RESPIRATORY CHAIN OF ESCHERICHIA COLI by MORRIS ROMUALD PUDEK B. S c , Simon Fraser U n i v e r s i t y , 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1976 Morris Horcuald Pudek In presenting th is thesis in par t ia l fu l f i lment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shal l make i t f reely avai lable for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publ icat ion of th is thesis for f inancia l gain shal l not be allowed without my writ ten permission. Department of / O / o c i r The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i ABSTRACT E s c h e r i c h i a c o l i contains cytochromes a-p b ^ g , 0553, d, and o w i t h smaller amounts of a c-type cytochrome(s) and cy t o -chrome b^^2 *-n t n B membrane bound e l e c t r o n t r a n s p o r t system. The l e v e l s of these components vary w i t h the growth, c o n d i t i o n s . Cytochromes d and o f u n c t i o n as te r m i n a l oxidases. Membrane p a r t i c l e s prepared from c e l l s grown to the s t a t i o n a r y phase c o n t a i n higher l e v e l s of cytochrome d and lower l e v e l s of cy t o -chrome o than c e l l s grown to the exponential phase. The NADH oxidase a c t i v i t y of p a r t i c l e s which contain the ghi.gh.er l e v e l s of cytochrome d aise l e s s s e n s i t i v e to i n h i b i t i o n by cyanide. The second order r a t e constant f o r the formation of cyanocyto-chrome d i n p a r t i c l e s o x i d i z i n g NADH i s found to c o r r e l a t e w i t h the r a t e constant determined f o r the i n h i b i t i o n of NADH oxidase a c t i v i t y by cyanide. The magnitude of the second order r a t e constant f o r the formation o f cyanocytochromesd i s d i r e c t l y p r o p o r t i o n a l to the r a t e of e l e c t r o n f l u x through cytochrome d. The o x i d i z e d form of cytochrome d w i t h an alpha absorp-t i o n peak at 648 nm and the reduced form w i t h an alpha absorp-t i o n peak at 628 nm are not d i r e c t o x i d a t i o n - r e d u c t i o n prod-ucts of each other. Cytochrome d goes through an intermediate form (d*) i n the normal o x i d a t i o n - r e d u c t i o n c y c l e . This form, which has no apparent alpha absorption peak, i s the species which r e a c t s w i t h cyanide. Cytochrome d can be trapped as t h i s intermediate at subzero temperatures i n p a r t i c l e s o x i d i z -i i ing ascorbate i n the presence of phenazine methosulfate. The nature of t h i s intermediate i s discussed i n r e l a t i o n to the cytochrome oxidase r e a c t i o n mechanism. The greater s e n s i t i v i t y of NADH oxidase a c t i v i t y to i n h i -b i t i o n by cyanide i n c e l l s c o n t a i n i n g low l e v e l s of cytochrome d i s due to the higher steady s t a t e l e v e l of the intermediate species r e a c t i v e w i t h cyanide caused by the greater r a t e of e l e c t r o n f l u x through the smaller cytochrome d pool i n these c e l l s . The c o n t r i b u t i o n of cytochrome d and cytochrome o to the NADH and succinate oxidase a c t i v i t y appears to be d i r e c t l y pro-p o r t i o n a l to t h e i r r e s p e c t i v e concentrations i n the membrane bound r e s p i r a t o r y system. The midpoint oxidation-reductionspotentialsro'f: the cyto-chromes of the r e s p i r a t o r y chain of E s c h e r i c h i a c o l i were det-ermined. Cytochromes d and->a-^  have midpo-int o x i d a t i o n - r e d u c -t i o n p o t e n t i a l s of +260 mV and +147 mV, r e s p e c t i v e l y . Cyto-chrome b can be re s o l v e d i n t o two major components by d i f f e r -ence spectroscopy at 77°K and by po t e n t i o m e t r i c t i t r a t i o n s . Cytochrome b ^ g and cytochrome b r ^ g have midpoint o x i d a t i o n -r e d u c t i o n p o t e n t i a l s of +165 mV and +35 mV, r e s p e c t i v e l y , i n membranes from exponential phase c e l l s . The p o t e n t i a l s of the cytochrome b components may vary w i t h the growth phase. Cyto-chrome b r ^ g i n c r e a s e s i n amount r e l a t i v e to cytochrome b r ^ g i n the t r a n s i t i o n from the exponential to the s t a t i o n a r y phase of growth. P a r t i a l compartmentalizatiori of the NADU, and succinate oxidase systems i s i n d i c a t e d by the k i n e t i c s of r e d u c t i o n of the b cytochromes and by the greater i n h i b i t i o n by 2-heptyl-4 hydroxyquinoline N-oxide of NADH oxidase compared w i t h s u c c i -nate oxidase. This i n h i b i t o r appears to blo c k e l e c t r o n trans port before and a f t e r both cytochromes and b r ^ g . A scheme f o r the arrangement of the cytochromes i n the NADH and succinate oxidase pathways i n r e s p i r a t o r y p a r t i c l e s of E. c o l i i s presented. i v TABLE OF CONTENTS Page ABSTRACT. . . . .'. . ..... . . . . . . ...... . ..... .......... . . i TABLE OF CONTENTS..........., .................... ... i v LIST OF TABLES....................................../ v i i LIST OF FIGURES......................................... v i i i ABBREVIATIONS ............. x i i ACKNOWLEDGEMENTS . . . . . . . . x i i i INTRODUCTION ........... 1 Cytochrome a-^  4 Cytochrome o 5 Cytochrome d 8 Cytochrome b 10 Cytochrome c 14 Nonheme i r o n 16 Quinones 18 The dehydrogenases 21 Factors a f f e c t i n g the composition and nature of the r e s p i r a t o r y chain of E. c o l i 22 The o r g a n i z a t i o n of the r e s p i r a t o r y chain of E. c o l i . . . 26 Ou t l i n e of t h e s i s problem 31 MATERIALS AND METHODS 33 Chemicals . ......... . 33 Growth and maintainance of c e l l s . . . . . . . . . . . . . . . . . . 33 Harvesting of c e l l s v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 S t a r v a t i o n of c e l l s . . . . . 36 V Page Pr e p a r a t i o n of membrane p a r t i c l e s . . . . . . . . . . . . . . . . . 36 Measurement of sp e c t r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Determination of the cytochrome c o n t e n t . . . . . . . . . . . 38 Determination of succinate and NADH oxidase ac-t i v i t i v i t y 38 E f f e c t of KCN on NADH oxidase . . . . . . . 39 E f f e c t of KCN. on the spectrum of cytochrome d 40 The r e l a t i o n s h i p between the absorption peaks at at 648 nm and 628 nm of the o x i d i z e d and r e -duced forms of cytochrome d 41 P o t e n t i o m e t r i c t i t r a t i o n s 42 A n a l y s i s of po t e n t i o m e t r i c data 45 Low temperature d i f f e r e n c e spectroscopy 46 Trapping of an intermediate species mm the o x i -d a t i o n - r e d u c t i o n c y c l e of cytochrome d 47 Reduction k i n e t i c s 48 Determination of p r o t e i n . 49 PART I . THE INHIBITION BY CYANIDE OF THE RESPIRATORY t 'A CHAIN OF EL COLI AND THE PROPERTIES OF CYTOCHROME d. . . .'. 50 RESULTS. . . . . . . . . . . . . . . . . . . . . 50 P r o p e r t i e s of p a r t i c l e s prepared from c e l l s grown to the exponential and s t a t i o n a r y phases of growth 50 K i n e t i c s of i n h i b i t i o n of NADH oxidase by cya§ nide 54 S p e c t r a l s t u d i e s of the r e a c t i o n of cyanide w i t h the r e s p i r a t o r y chain. 66 P o t e n t i o m e t r i c t i t r a t i o n , of cytochrome d. 74 K i n e t i c s of r e a c t i o n of cyanide w i t h c y t o -chrome d 76 v i v Page The r e a c t i o n of cyanide w i t h cytochrome d i n r e s p i r a t o r y p a r t i c l e s from exponential phase E. c o l i 91 Trapping of an intermediate i n the o x i d a t i o n -r e d u c t i o n c y c l e of cytochrome d 97 DISCUSSION 105 PART I I . THE ORGANIZATION OF THE CYTOCHROMES IN •EHE RESPIRATORY CHAIN OF E. COLI 120 RESULTS 120 Low temperature (77%K) d i f f e r e n c e spectra of p a r t i c l e s prepared from c e l l s grown to the ex-p o n e n t i a l and s t a t i o n a r y phase of growth 120 Pot e n t i o m e t r i c t i t r a t i o n s of the cytochromes 126 Reduction k i n e t i c s of cytochrome b and the e f f e c t of HOQNO 139 Low temperature d i f f e r e n c e spectra of the d i f -f e r e n t redox st a t e s of cytochrome b 147 Examination of the com p a r t m e n t i l i z a t i o n of cy-tochrome b 151 DISCUSSION .. 155 BIBLIOGRAPHY 169 v i i LIST OF TABLES Table Page 1 1 P r o p e r t i e s of p a r t i c l e s prepared from c e l l s grown to the s t a t i o n a r y and exponential phases of growth 53 2 K i n e t i c constants f o r e f f e c t of KCN on NADH oxidase 65 3 Midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l s and amount of the major b cytochromes of r e s p i -r a t o r y p a r t i c l e s prepared from c e l l s at d i f f e r e n t phases of growth 136 v i i i LIST OF FIGURES Figure Page 1. Proposed schemes f o r e l e c t r o n t r a n s p o r t i n - E. c o l i 27 2. D i f f e r e n c e spectra of p a r t i c l e s prepared from c e l l s grown to the exponential and s t a t i o n a r y phases of growth 51 3. E f f e c t of concent r a t i o n of KCN on the o x i -d a t i o n of NADH by p a r t i c l e s prepared from c e l l s grown to the exponential or s t a t i o n -ary phase 55 4. Lineweaver-Burk a n a l y s i s of the k i n e t i c s of i n h i b i t i o n of NADH oxidase a c t i v i t y 57 5. F i r s t order a n a l y s i s of the o x i d a t i o n of NADH by p a r t i c l e s i n the presence and ab-se sence of d i f f e r e n t concentrations of KCN 58 6. DixoDixon p l o t of the k i n e t i c s of i n h i b i t i o n by cyanide of the i n i t i a l r a t e s of NADH o x i -dase a c t i v i t y 59 7. E f f e c t of concent r a t i o n of KCN on the i n i t -i a l r a t e of o x i d a t i o n of NADH by p a r t i c l e s prepared from c e l l s grown on succinate med-ium 61 8. E f f e c t of concent r a t i o n of KCN on the oxida-t i o n of NADH by p a r t i c l e s prepared from c e c e l l s grown to the exponential or s t a t i o n a r y phase of growth 62 9. E f f e c t of concentration of KCN on the oxida-t i o n of NADH by p a r t i c l e s prepared from c e l l s grown on glucose, l a c t a t e , s u c c i n a t e , or complex medium 63 10. E f f e c t of KCN on reduced cytochromes of m membrane p a r t i c l e s prepared from c e l l s grown to the exponential or s t a t i o n a r y phase of growth 67 i x F i g u r e Page LI . E f f e c t .of KCN on the reduced minus o x i d i z e d d i f f e r e n c e spectrum of p a r t i c l e s prepared from c e l l s - grown to the .stationary- phase... . . 69 12. Reduction of the cytochromes i n membrane p a r t i c l e s by a l i m i t i n g amount of sub s t r a t e . . . 71 13. Reduction of the cytochromes i n membrane p a r t i c l e s by a l i m i t i n g amount of su b s t r a t e . . . 73 14. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome d 75 15. Behaviour of the trough at. 648 nm during 'the p o t e n t i o m e t r i c t i t r a t i o n of cytochrome d 77 16. Potentiometric. t i t r a t i o n of the chr.omophore absorbing at 675-680 nm i n the o x i d i z e d form ... 78 17. E f f e c t of cyanide on the absorption spec-trum of o x i d i z e d cytochrome d 79 18. Reaction of cyanide w i t h o x i d i z e d c y t o -chrome d . 81 19. E f f e c t of cyanide on o x i d i z e d cytochrome d under turnover c o n d i t i o n s . .... ... 83 20. Reaction of cyanide w i t h cytochrome d under turnover c o n d i t i o n s 84 21. Reaction of cyanide w i t h cytochrome d under turnover c o n d i t i o n s 86 22. Reaction of cyanide w i t h cytochrome d at d i f f e r e n t turnover r a t e s 87 23. E f f e c t of r a t e of turnover of the r e s p i r a -t o r y chain on the second-order r a t e constant .Jw q f o r the formation of cyanocytochrome d 89 24. Reaction of cyanide w i t h cytochrome d i n a pr e p a r a t i o n o x i d i z i n g succinate showing a d e v i a t i o n from the expected k i n e t i c beha-v i o u r 90 25. Reaction of cyanide w i t h cytochrome d of membrane p a r t i c l e s from exponential phase c e l l s i n the presence of su c c i n a t e . . . . . . . . . . . . 93 X F i g u r e Page 26. Reaction of cyanide with, cytochrome d i n the presence of succin a t e . . . . ................. 94 27. Reaction of cyanide w i t h cytochrome d of membrane p a r t i c l e s from exponential phase c e l l s i n the presence of NADH . . 96 28. Low temperature C77°K) d i f f e r e n c e spectra of the aerobic steady s t a t e s of r e d u c t i o n of p a r t i c l e s o x i d i z i n g d i f f e r e n t s u b s t r a t e s . . . 98 29. D i f f e r e n c e spectra of r e s p i r a t o r y p a r t i c l e s of E. c o l i . 100 30. E f f e c t of temperature (°C) on the amount of cytochrome d i n the intermediate form i n the aerobic steady s t a t e i n p a r t i c l e s o x i d -i z i n g ascorbate w i t h PMS 101 31. Absolute spectra of o x i d i z e d cytochrome d and cytochrome d of p a r t i c l e s o x i d i z i n g ascorbate i n the presence of PMS 103 32. E f f e c t of H00N0 on the aerobic steady s t a t e l e v e l of r e d u c t i o n of cytochromes i n p a r t i -c l e s o x i d i z i n g ascorbate i n the presence of PMS 104 33. Low temperature (77°K) reduced versus o x i -d i z e d d i f f e r e n c e spectra of p a r t i c l e s pre-pared from c e l l s grown to the exponential and s t a t i o n a r y phase of growth 121 34. Low temperature d i t h i o n i t e reduced versus o x i d i z e d d i f f e r e n c e spectra of the cyt o -chrome b re g i o n of p a r t i c l e s grown under d i f f e r e n t c o n d i t i o n s . . - 124 35. Reduction of cytochrome b by succinate and NADH 125 36. Reduction of cytochrome bybysascorbate i n the presence of PMS 127 37. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome a^ 128 38. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome b 130 39. S p e c t r a l a n a l y s i s of the cytochrome b com-ponents reduced at d i f f e r e n t p o t e n t i a l s . . 131 x i F i g u r e Page 40. P o t e n t i o m e t r i c . t i t r a t i o n of cytochrome b. i n p a r t i c l e s prepared from e a r l y exponen-t i a l c e l l s . . . . . . . . 133 41. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome b i n p a r t i c l e s prepared from s t a t i o n a r y c e l l s 134 42. E f f e c t of d i f f e r e n t mixtures of mediators on the p o t e n t i o m e t r i c t i t r a t i o n curve of the cytochrome b components 138 43. Reduction of cytochrome b by succinate and NADH. i n p a r t i c l e s prepared from c e l l s grown on succinate to the s t a t i o n a r y phase of growth 140 44. Reduction of cytochrome b by succinate and NADH i n p a r t i c l e s prepared from c e l l s grown on succinate to the exponential phase of growth 142 45. Reduction of cytochrome b by NADH i n the presencepresenbee'and afesehefeuof H0QN0 143 46. Reduction of cytochrome b by succinate i n the presence and absence of HOQNO.v 144 47. E f f e c t of concent r a t i o n of HOQNO on NADH and succinate oxidase a c t i v i t i e s and on the aerobic steady s t a t e l e v e l s of reduc-t i o n of cytochrome b 146 48. E f f e c t of HOQNO on the steady s t a t e l e v e l s of r e d u c t i o n of cytochrome b. . . . . . 148 49. E f f e c t of HOQNO on the steady s t a t e l e v e l s of r e d u c t i o n of cytochrome b i n p a r t i c l e s from exponential c e l l s - 150 50. Reduction of cytochrome b i n the presence of both NADH and succinate. . 152 51. Reduction of cytochrome d i n the presence of both NADH and succ i n a t e . 154 52. Model proposed f o r the arrangement of the cytochromes of the r e s p i r a t o r y chain of E. c o l i . . . 166 X l l ABBREVIATIONS - adenosine-5'-triphosphate - 3',5'-adenosine monophosphate - cytochrome - 2,6-dichlorophenolindophenol - oxidatiorij?reduction p o t e n t i a l mea-sured r e l a t i v e to the hydrogen e l e c t r o d e - midpoint o x i d a t i o n - r e d u c t i o n po-t e n t i a l - e l e c t r o n paramagnetic resonance - f l a v i n adenine d i n u c l e o t i d e - f l a v i n mononucleotide - 2-hepty1-4-hydroxyquinoline-N-ox-ide - menaquinone d e f i c i e n t mutant - nicotinamide adenine d i n u c l e o t i d e - nicotinamide adenine d i n u c l e o t i d e , reduced - nicotinamide adenine d i n u c l e o t i d e phosphate - o x i d i z e d - phenazine e t h o s u l f a t e - phenazine methosulfate - reduced - t r i c a r b o x y l i c a c i d c y c l e - 0.1 M T r i s - H 2 S 0 4 b u f f e r , pH 7:5, 0.01 M MgCl 2 - thenoyl t r i f l u o r o a c e t o n e - ubiquinone d e f i c i e n t mutant - ubiquinone-8 - u l t r a v i o l e t x i i i ACKNOWLEDGEMENT S L am most g r a t e f u l to my sup e r v i s o r , Dr. P.D. Bragg, f o r h i s guidance and encouragement during my graduate s t u d i e s and h i s c o n s t r u c t i v e c r i t i c i s m throughout the pre-p a r a t i o n of t h i s t h e s i s . I would l i k e to thank Dr. M. Smith.,. Dr. G. Tener and Dr. A. Addison f o r s e r v i n g on my re s e a r c h committee and fo r t h e i r comments on the i n i t i a l d r a f t of t h i s t h e s i s . I would a l s o l i k e to thank Mr. Reinhart R e i t h -meier, Mrs? Cynthia Hou., Dr. A.P. Singh and my other c o l -leagues f o r t h e i r u s e f u l d i s c u s s i o n s and help during my stay i n Dr. Bragg's l a b o r a t o r y . Thanks are a l s o due to Dorothy Buckland f o r the typi n g of t h i s t h e s i s . I am indebted to the. Medical Research. C o u n c i l f o r f i n a n c i a l support through an MRC Studentship awardeddto me. t o S u s a n 1 INTRODUCTION The cytochromes are the e l e c t r o n c a r r i e r s of the t e r m i n a l p o r t i o n of the r e s p i r a t o r y system which e i t h e r i n -d i r e c t l y or d i r e c t l y provides the means by which most l i v i n g c e l l s o b t a i n the energy to c a r r y on t h e i r e s s e n t i a l l i f e pro-cesses. E s c h e r i c h i a c o l i can form a v a r i e t y of cytochromes '• and e l e c t r o n t r a n s p o r t systems which can generate through the o x i d a t i o n of substrates a hig h energy intermediate or s t a t e able to d r i v e a c t i v e t r a n s p o r t (1), energy dependent trans'hydrogenation of NADP+ by NADH (2) , c e l l m o t i l i t y (3) , and ATP formation (4). I have chosen to examine the orga n i -z a t i o n of the cytochromes i n the membrane-bound r e s p i r a t o r y system of E. c o l i . K e i l i n i n 1925 (5) rediscovered cytochromes a f t e r the " i n i t i a l o b servation by .McMunri i n 1886 (6) . K e i l i n found that the t y p i c a l four-banded v i s i b l e absorption spec-trum of a v a r i e t y of c e l l s and t i s s u e s was due to f e r r o -and ferrihaemochromes which he named cytochromes a, b, and c. He proved that the change i n i r o n valency of these mole-cules was r e l a t e d to t h e i r f u n c t i o n as e l e c t r o n c a r r i e r s be-tween substrate and molecular oxygen. K e i l i n and Yaoi and Tamiya (7) observed that the wave-lengths of the absorption bands i n many b a c t e r i a were not the 2 same as those i n higher organisms. This suggested that the cytochromes i n a l l organisms were not i d e n t i c a l . This work has been followed by r e l a t i v e l y s u p e r f i c i a l observations of b a c t e r i a l systems. M i t o c h o n d r i a l e l e c t r o n t r a n s p o r t has been examined i n much more depth. The study of b a c t e r i a l cyto-chromes has only r e c e n t l y emerged from i t s q u a l i t a t i v e phase and attempts are now being made to e l u c i d a t e the organiza-t i o n and the p r o p e r t i e s of these cytochromes. B a c t e r i a l systems provide an i n t e r e s t i n g a l t e r n a t i v e system f o r study w i t h some advantages such as the a b i l i t y to o b t a i n mutants i n e l e c t r o n t r a n s p o r t and o x i d a t i v e phosphorylation. I t i s now r e a l i z e d that b a c t e r i a can form membrane-bound e l e c t r o n t r a n s -port systems which may be more complex than i n mitochondria. M u l t i p l e dehydrogenases feed e l e c t r o n s i n t o the e l e c t r o n t r a n s p o r t system which interconnects and branches to v a r y i n g degrees to feed e l e c t r o n s through the d i f f e r e n t t e r m i n a l o x i -dases to oxygen or to other acceptors such as n i t r a t e , n i t r i t e or fumarate. The composition of t h i s system can vary c o n s i -derably depending on-the co n d i t i o n s employed to grow the b a c t e r i a . This has l e d to d i f f i c u l t y i n e l u c i d a t i n g the com-p l e x network of p o s s i b l e e l e c t r o n c a r r i e r sequences. With the development of s o p h i s t i c a t e d spectroscopic techniques i t i s now p o s s i b l e to approach some of these problems. I t i s not always p o s s i b l e to draw p a r a l l e l s between 3 m i t o c h o n d r i a l and b a c t e r i a l r e s p i r a t o r y systems because there are some major d i f f e r e n c e s . The e l e c t r o n t r a n s p o r t sys-tem of b a c t e r i a i s not organized i n d i s c r e t e o r g a n e l l e s such as the mitochondria but i s l o c a l i z e d i n the cytoplasmic mem-brane. Generally b a c t e r i a l e l e c t r o n t r a n s p o r t systems are l e s s s e n s i t i v e to i n h i b i t o r s commonly used i n the study of m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t . The components of bacter-i a l species can not be e a s i l y s o l u b i l i z e d i n t o d i s c r e t e com-plexes. R e s p i r a t o r y c o n t r o l i s g e n e r a l l y not observed i n b a c t e r i a l systems. The s t o i c h i o m e t r y of the components of b a c t e r i a l systems can vary considerably w i t h the growth con-d i t i o n s . E a r l y spectroscopic observations of E. coli? revealed the presence of cytochromes a^, b-^ , d, and a carbon monoxide b i n d i n g pigment, cytochrome o, as the only cytochromes pre-sent ( 8 ) . More r e c e n t l y , w i t h the a p p l i c a t i o n of low tem-perature d i f f e r e n c e spectroscopy techniques, Shipp ( 9 ) has r e s o l v e d the cytochrome b-^  absorption peak i n t o f i v e compo-nents, t e n t a t i v e l y i d e n t i f i e d as two "c-type" cytochromes, two "b-type" cytochromes, and cytochrome o. Because of t h i s complexity the o r g a n i z a t i o n i s d i f f i c u l t to examine. In the f o l l o w i n g s e c t i o n s the known r o l e of the com-ponents of the r e s p i r a t o r y chain of E. c o l i w i l l be d i s -cussed. 4 Cytochrome The e x i s t e n c e of cytochrome a-^  and i t s r o l e as a t e r -minal oxidase was f i r s t suggested by studies w i t h Acetobacter  pasteurianum (10). The pigment i n t h i s organism had an ab-s o r p t i o n peak at 589 nm, was .auto x i d i z able and reacted w i t h carbon monoxide and cyanide. Warburg's choice of organism was fortu n a t e since i n other b a c t e r i a where cytochrome a^ has been observed, other t e r m i n a l oxidases such as cytochrome o and d are a l s o found (11). The presence of these other t e r m i n a l oxidases i n organisms such as E. c o l i g r e a t l y com-p l i c a t e s the demonstration that cytochrome a-^  f u n c t i o n s as a te r m i n a l oxidase. Cytochrome a-^  probably has the same heme as does cyto-chrome a. The noncovalently bound heme can be removed by e x t r a c t i o n w i t h acetone-HCl and converted to the p y r i d i n e ferrohemochrome w i t h an e£= peak at 580 - 590 nm (12) . The spectrum of reduced cytochrome a^ has absorption peaks at 590 and 440 nm. The p h o t o d i s s o c i a t i o n spectrum of the carbon monoxide-cytochrome a-^  complex i n A. pasteurianum (13-15, 11) and Beneckea natriegens (16) confirmed that cytochrome a-^  func-t i o n s as a t e r m i n a l oxidase i n these organisms. Because n i t r a t e could o x i d i z e cytochrome a-^  to a greater extent than the other cytochromes i n Haemophilus p a r a i n f l u e n z a e (17) and the b i o s y n t h e s i s of cytochrome a-^  was st i m u l a t e d by the pre-sence of n i t r a t e (18) , cytochrome a-^  has been suggested to 5 p a r t i c i p a t e i n n i t r a t e r e d u c t i o n . A r o l e i n n i t r i t e metabol-ism has a l s o been suggested f o r cytochrome a-^  i n c e r t a i n N i t r o b a c t e r species. In N i t r o b a c t e r winogradskyi, cytochrome a-^  was found to reduce cytochrome during n i t r i t e oxida-t i o n (19, 20) by reversed e l e c t r o n flow which was d r i v e n by ATP generated i n the t e r m i n a l p o r t i o n of the chain (21-23). The cytochrome a-^  absorption peak at 590 nm of N i t r o b a c t e r  a g i l i s was r e s o l v e d i n t o two components w i t h midpoint oxida-t i o n - r e d u c t i o n p o t e n t i a l s of +352 mV and +100 mV (24). I t was suggested that both of these components functioned i n n i t r i t e metabolism r a t h e r than i n the r e s p i r a t o r y chain. In b a c t e r i a such as Azotobacter v i n e l a n d i i (25) and E. c o l i i t i s d i f f i c u l t to assay a c c u r a t e l y f o r cytochrome a-^ . Cytochrome a-^ , absorbing at 594 nm, i s only a minor component of the r e s p i r a t o r y chain of E. c o l i and i t s r o l e as a t e r m i n a l oxidase i s d o u b t f u l . There i s no evidence f o r a r e a c t i o n of cytochrome a-^  w i t h carbon monoxide or cyanide i n E. c o l i . Cytochrome o Cytochrome oxidases are g e n e r a l l y c h a r a c t e r i z e d by t h e i r r a p i d a u t o j x i d i z a b i l i t y and by t h e i r r e a c t i v i t y w i t h cyanide and carbon monoxide. Carbon monoxide binds to the cytochrome oxidase causing a s h i f t i n the p o s i t i o n of the absorption peak. The cytochrome oxidase, cytochrome o, was f i r s t i d e n t i f i e d by i t s carbon monoxide spectrum (13, 14). 6 I t s alpha absorption band overlaps w i t h the cytochrome b^ ab-s o r p t i o n peak and th e r e f o r e could not be re s o l v e d from cyto-chrome b^ i n reduced versus o x i d i z e d d i f f e r e n c e spectra. The carbon monoxide-cytochrome o complex can be d i s s o c i a t e d by l i g h t . The p h o t o d i s s o c i a t i o n spectrum f o r a carbon monoxide-oxidase complex has become a d i a g n o s t i c technique i n the i d -e n t i f i c a t i o n of cytochrome oxidases (11, 15). Even today, however, few p r o v i s i o n a l cytochromes o have been r i g o r o u s l y c h a r a c t e r i z e d by t h e i r photochemical a c t i o n spectra. The carbon monoxide complex of cytochrome o has a, 6 , and tyyabsorption bands at approximately 565 - 570 nm, 535 nm, and 416-418 nm r e s p e c t i v e l y (27). Peaks at 567 nm, 538 nm, and 416 nm w i t h a trough at 558 nm i s observed f o r the com-p l e x i n E. c o l i (28, 29, 13). The absolute spectrum of cytochrome o i s unknown. The p r o s t h e t i c group of cytochrome o has been t e n t a t i v e l y c h a r a c t e r i z e d as protoheme, which i s the same as the p r o s t h e t i c group of cytochrome b (30). Attempts have been made to i d e n t i f y an absorption peak i n the 555-565 nm range w i t h cytochrome o. The te n t a -t i v e l y i d e n t i f i e d o-type cytochromes can be d i v i d e d i n t o two groups, one w i t h the a. peak at 555-558 nm and the other w i t h the a peak at 564-565 nm (27) Taber and Morrison -i(30) have r e s o l v e d the cytochrome b-region of Staphylococcus aureus at 77°K i n t o an absorption peak at 555 and one at 557 nm. The 555 nm component has been i d e n t i f i e d as cytochrome o because of i t s greater r e d u c i b i l i t y by ascorbate i n the presence of dichlorophenolindophenol (DCIP) and by the a b i l i t y to form a 7 carbon monoxide complex i n the presence of t h i s s ubstrate. Taniguchi and Kamen (31) a s s o c i a t e d cytochrome o w i t h a com-ponent absorbing at 565 nm i n Pvhodospir i l i u m rub rum. In near-l y a l l cases only i n d i r e c t evidence has been obtained f o r the i d e n t i t y of cytochrome o. Attempts have been made to p u r i f y cytochrome o from V i t r e o s c i l l a (32, 33), Halobacterium cutirubrum (34), Acetobac-t e r suboxydens (35, 36) Mycobacterium p h l e i (29) and B a c i l l u s  megaterium (37). In each case the p u r i f i e d component has shown a l t e r e d p r o p e r t i e s such as a lowered midpoint o x i d a t i o n -r e d u c t i o n p o t e n t i a l , slow a u t o x i d i z a b i l i t y and displacement of absorption bands. This suggests that the a c t u a l cytochrome o has not been i s o l a t e d or that the p r o p e r t i e s of cytochrome o have been a l t e r e d because of i t s environment. In the process of p u r i f i c a t i o n cytochrome b may be modified and become aut-o x i d i z a b l e and may l e a d to the f a l s e c o n c l u s i o n that cytochrome o has been p u r i f i e d . The cytochrome o p u r i f i e d from V i t r e o -s c i l l a has a carbon monoxide d i f f e r e n c e spectrum w i t h absorp-t i o n maxima at 569, 535, and 419 nm. An "oxygenated" species w i t h absorption maxima at 577, 544, and 420 nm was formed i n i n the aerobic phase upon a d d i t i o n of NADH. The reduced form of cytochrome o has absorption maxima of 560 and 435 nm (38, 39). The formation of the "oxygenated" species was i n h i b i t e d by cyanide. These r e s u l t s suggest that the p u r i f i e d cyto-chrome was indeed cytochrome o and could f u n c t i o n as a termin-a l oxidase. The formation of a cyanide complex w i t h cyto-chrome o d i d not r e s u l t i n obvious s p e c t r a l changes. 8 In b a c t e r i a such as Acetobacter suboxydans cytochrome o appears to be the only t e r m i n a l oxidase (27). In other bac-t e r i a i t may occur together w i t h cytochrome oxidases a + a^, a-p and/or d. A problem a r i s e s i n attempting to determine the r e l a t i v e c o n t r i b u t i o n to the oxidase a c t i v i t y of these various oxidases when they are present together. Photoaction spectra of the d i s s o c i a t i o n of oxidase-carbon monoxide complexes have been, used f o r t h i s purpose w i t h a number of b a c t e r i a (11). The r e s u l t s of t h i s type of experiment i n d i c a t e that cyto-chrome o i s the primary t e r m i n a l oxidase i n E. c o l i c e l l s grown to the exponential phase of growth. In the s t a t i o n a r y phase of growth cytochrome d and cytochrome o both f u n c t i o n as t e r m i n a l oxidases. However, because of the d i f f e r i n g sen-s i t i v i t i e s of cytochrome o and other oxidases to carbon mono-xide the photoaction spectrum may not a c c u r a t e l y r e f l e c t the r e l a t i v e a c t i v i t y of these oxidases (11, 40). Cytochrome d Y a o i and Tamiya (7) showed that c e l l s of E. c o l i and S h i g e l l a dysenteriae possessed an absorption band at 630 nm which was d i f f e r e n t from any other cytochromes reported. K e i l i n (41) named i t cytochrome d. I t was found to be aut-o x i d i z a b l e and could combine w i t h carbon monoxide and cyan-ide . Another i n t e r e s t i n g feature was that the o x i d i z e d form had a strong absorption band at 645 nm (42-44). Negelein and Gerisher (42) showed that cytochrome d 9 reacted w i t h carbon monoxide and the absorption band of r e -duced cytochrome d was s h i f t e d from 632 to 637 nm. The addi-t i o n of cyanide under anaerobic c o n d i t i o n s produced no changes i n the absorption spectrum but cyanide i n the presence of oxy-gen caused the band at 632 nm to disappear. They were the f i r s t to suggest that cytochrome d functioned as a te r m i n a l oxidase. The p r o s t h e t i c group of t h i s cytochrome i s an i r o n -dihydroporphyrin ( c h l o r i n ) d e r i v a t i v e not an ir o n - p o r p h y r i n as found i n "a-type" cytochromes. Therefore i t i s no longer c a l l e d cytochrome a 2 , but ra t h e r cytochrome d. The s t r u c t u r e of heme d i n Aerobacter aerogenes i s a modified form of proto-heme, w i t h one v i n y l group converted to a hydroxyethyl side chain and porphyrin r i n g IV p a r t i a l l y reduced It45) .,.. Attempts have been made to p a r t i t i o n and e n r i c h cyto-chrome d w i t h l i t t l e success because of i t s l a b i l i t y (46). Membrane preparations c o n t a i n i n g t h i s cytochrome always con-t a i n e d other cytochromes as w e l l (25, 26). Cytochrome d i n n e a r l y a l l organisms i s found together w i t h the t e r m i n a l oxidases cytochrome a-^  and o. Before the concept of branching e l e c t r o n t r a n s p o r t systems w i t h m u l t i p l e t e r m i n a l oxidases, the f u n c t i o n of cytochrome d as a te r m i n a l oxidase was doubted. Castor and Chance (11) showed that cyto-chrome d was a te r m i n a l oxidase by o b t a i n i n g photoaction spec-t r a of the r e l i e f of carbon monoxide i n h i b i t i o n of r e s p i r a t i o n i n c e l l s c o n t a i n i n g cytochrome d. However, as mentioned ear-10 l i e r , no simple r e l a t i o n s h i p e x i s t s between the oxidase a c t i -v i t y of a pigment and the height of the bands i n the photoac-t i o n spectrum. Also there i s l i t t l e c o r r e l a t i o n between the amount of cytochrome d i n b a c t e r i a and t h e i r r e s p i r a t o r y ac-t i v i t y (47, 48). Cytochrome d has been a s s o c i a t e d w i t h cyanide r e s i s t a n t r e s p i r a t i o n i n Achromobacter (49). This r e s i s t a n c e was a t t r i -buted to the induced formation of cytochrome d which has a low a f f i n i t y f o r cyanide when the b a c t e r i a were grown i n the pre-sence of cyanide (50). Cytochrome d synthesis was a l s o i n -duced i n E . c o l i when grown i n the presence of low concentra-t i o n of cyanide (51). I t has a l s o been suggested, but w i t h no c o n c l u s i v e evidence, that cytochrome d may a l s o be i n v o l v e d w i t h the n i -t r a t e reductase system. Low concentrations of n i t r a t e (10 mM) s t i m u l a t e cytochrome d synthesis i n E . . c o l i when grown anaero-b i c a l l y (52, 53). However, higher concentrations repress cytochrome d syntheses (54), More recent work has shown that cytochrome d i s not i n i t m a t e l y a s s o c i a t e d w i t h the n i t r a t e r e -ductase system (55). Cytochrome d i s i n v o l v e d i n the r e s p i r a t o r y chain of E i c o l i but i t s c o n t r i b u t i o n to oxidase a c t i v i t y has not been f i r m l y e s t a b l i s h e d . Cytochrome b K e i l i n demonstrated the presence of a b-type cyto-chrome i n E . c o l i i n 1927 (56) and w i t h Harpley (57) showed 11 that crushed c e l l s possessing t h i s cytochrome along w i t h cyto-chromes d and a-^, but w i t h no apparent cytochrome c, could o x i -d i z e succinate and l a c t a t e . The p r o s t h e t i c group of the b a c t e r i a l cytochrome b i s protoheme. I t i s not c o v a l e n t l y l i n k e d to the p r o t e i n . The absorption maxima may range from.557 to 564 nm. Those w i t h an alpha absorption peak from 560 to 563 nm are u s u a l l y designated cytochrome b w h i l e those w i t h an alpha peak from 557 to 560 nm are r e f e r r e d to as cytochrome b-^  (46). Cytochrome o because i t has noncovalently-bound protoheme as i t s p r o s t h e t i c group i s al s o c l a s s i f i e d as a cytochrome b. I t has been suggested (58) that there may be a gradation of oxidase a c t i v i t y between the c l a s s i c a l n on-autoxidizable cytochrome b and the r a p i d l y aut-o x i d i z a b l e cytochrome o. In p a r t i a l l y p u r i f i e d dehydrogenases from E. c o l i and other b a c t e r i a l systems, cytochrome b^ has o f t e n been found a s s o c i a t e d w i t h the dehydrogenases. Linnane and Wrigley (59) s o l u b i l i z e d a p r e p a r a t i o n of cytochrome b-^  a s s o c i a t e d w i t h f o r -mate dehydrogenase. Kim and Bragg (60) i s o l a t e d a succinate dehydrogenase f r a c t i o n c o n t a i n i n g cytochrome b-^ . The homogen-e i t y of these preparations i s not known. Therefore i t would be d i f f i c u l t to i n t e r p r e t the o r g a n i z a t i o n a l r e l a t i o n s h i p between cytochrome b-^  and the dehydrogenase. MacGregor (55) has p u r i -f i e d n i t r a t e reductase to homogeneity and has found cytochrome b-^  (molecular weight - 19,500) a s s o c i a t e d as one of the sub-u n i t s . Whether the cytochrome b-, c l o s e l y a s s o c i a t e d w i t h the 12 n i t r a t e reductase system i s i d e n t i c a l w i t h that i n v o l v e d i n the r e s p i r a t o r y chain i s not known. Other attempts have been made to p u r i f y cytochrome b^ and to measure some of the p r o p e r t i e s of these preparations. F u j i t a et al.(61) obtained a 50% pure p r e p a r a t i o n of cyto-chrome b^ which had a molecular weight of 600,000 - 800,000. The midpoint p o t e n t i a l of cytochrome b-^  i n t h i s p r e p a r a t i o n was -20 mV. This p a r t i a l l y p u r i f i e d p r e p a r a t i o n was a l s o s t r o n g l y a u t o x i d i z a b l e . Deeb and'Hager- (62) have a l s o p u r i -f i e d cytochrome b-^  of E. c o l i r e l e a s e d by s o n i c a t i o n of the b a c t e r i a l membranes. The cytochrome b-^.was as s o c i a t e d as a 500,000 molecular weight complex w i t h e i g h t hemes. At h i g h pH t h i s complex could be d i s s o c i a t e d i n t o monomeric species of molecular weight 60,000. The midpoint p o t e n t i a l of the o c t a -meric form of cytochrome b-^  was -340 mV w h i l e the monomeric form had a midpoint p o t e n t i a l of 0 mV. A c o l o u r l e s s p r o t e i n , which separated at an e a r l y stage of p u r i f i c a t i o n of the cytochrome, when added to the c r y s t a l l i n e cytochrome b-^ . caused i t to assume a more p o s i t i v e midpoint p o t e n t i a l . The environ-ment the r e f o r e appears to be a strong determinant of the po-t e n t i a l of t h i s cytochrome. Another b-type cytochrome has been p u r i f i e d from E. c o l i . A s o l u b l e cytochrome b w i t h an absorption peak at 562 nm i s formed i n E. c o l i grown under a number of c o n d i t i o n s (61, 63). This cytochrome was present at a t h i r d of the concentra-t i o n of the cytochrome b-^  (64). A h i g h l y p u r i f i e d p r e p a r a t i o n , obtained by F u j i t a (65) had a redox p o t e n t i a l of + 113 mV and 13 a molecular weight, of 12,000. The amino a c i d sequence has a l s o been determined (66). Although cytochrome b ^ ^ w a s Iso-l a t e d from the s o l u b l e f r a c t i o n of the c e l l i t may a l s o be a component of the membrane-bound e l e c t r o n t r a n s p o r t system as a peak at t h i s wavelength has been observed i n p a r t i c l e prepara-t i o n s . The s o l u b l e and membrane-bound forms, however, may be u n r e l a t e d components. I t had been g e n e r a l l y considered that there was only one cytochrome b^ present i n the membrane bound e l e c t r o n t r a n s -p o r t system of E, c o l i . However, r e s u l t s of B a i l l i e et a l (67), Kim and Bragg (68), and Ruiz-Herrera and DeMoss (69) suggested the presence of two d i s t i n c t cytochromes b. Anaero-b i c growth i n the presence of n i t r a t e r e s u l t e d i n the synthe-s i s of a b-type cytochrome w i t h an alpha band at 555 nm (77°K) wh i l e i n the absence of n i t r a t e there was an alpha band at 558 nm (77°K) (69). The cytochrome b 5 5 5 (77°K) produced i n c e l l s grown on n i t r a t e c o n s i s t e d of two k i n e t i c a l l y d i f f e r e n t i a b l e species which appeared to be d i s t i n c t from components pro-duced under aerobic growth c o n d i t i o n s . With the a p p l i c a t i o n of a f o u r t h order f i n i t e d i f f e r -ence a n a l y s i s technique to low temperature (77°K) d i f f e r e n c e s p e c t r a (9) the cytochrome b region of E. c o l i was r e s o l v e d i n t o f i v e components (cytochromes £543 > c 5 5 3 » ^556' ^559' a n c ^ ^565^ ' ^ e ^ v n c ^ o n a n d o r g a n i z a t i o n these components i n the r e s p i r a t o r y chain has not been determined. The m u l t i p l e nature of the cytochrome b components was a l s o shown, by. Hendler et. aL. (70) who c a r r i e d out p o t e n t i o -14 me t r i c t i t r a t i o n s o f the membrane-bound cytochrome b-^  of E. c o l i . The absorption peak of cytochrome b^ was r e s o l v e d i n t o three redox components w i t h midpoint p o t e n t i a l s of -50 mV, +110 mV, and +220 mV. Cytochrome c Yamagutchi (71) f i r s t obtained evidence f o r a c-type cytochrome i n E. c o l i . I t was observed as a shoulder at 552 nm on the absorption peak of reduced cytochrome b^. When the spectrum was examined at 77°K the shoulder due to the c-type cytochrome appeared much sharper w i t h a peak at 551 nm (57). P u r i f i e d cytochrome c from mitochondria absorbs at 550 nm and i s s h i f t e d to 547 nm at low temperatures i n d i c a t i n g t h a t the c-type cytochrome present i n E. c o l i i s not i d e n t i c a l w i t h that from mitochondria or some other sources. In f a c t , u n l i k e B a c i l l u s s u b t i l i s and many other aerobic c e l l s , E. c o l i i s incapable of o x i d i z i n g p-phenylenediamine or the "Nadi" rea-gent. This has been explained by the absence of cytochrome c (72). Cytochrome C552, found as a s o l u b l e form, was induced maximally when E. c o l i was grown a n a e r o b i c a l l y i n the presence of 0.1% sodium n i t r a t e . This cytochrome was p u r i f i e d w i t h d i f -f i c u l t y because o f i t s tendency to e x i s t as a polymer (65). The molecular weight was estimated to be 12,000 and the redox p o t e n t i a l was between -194 and -220 mV. This i s much lower than the redox p o t e n t i a l o f m i t o c h o n d r i a l cytochrome c (+265 mV) 15 (73). The low o x i d a t i o n - r e d u c t i o n p o t e n t i a l of cytochrome °552 a n c^ i t s r e p r e s s i o n by oxygen p o i n t e d to a r o l e i n anaero-b i c e l e c t r o n t r a n s p o r t (74). Because of i t s simultaneous i n -duction w i t h hydrogenase and hydrogenlyase a c t i v i t i e s i t was suggested to be i n v o l v e d i n the formic hydrogenlyase r e a c t i o n of E. c o l i . Other evidence suggested an involvement i n n i t r i t e r e -ductions (75-78) as seen by i t s i n d u c t i o n i n the presence of n i t r i t e and the concomitant development of n i t r i t e reductase. As cytochrome c<j$2 ^ s l o c a t e d i n the c e l l w a l l or outer cyto-plasmic membrane and reduced cytochrome can be r e o x i d i z e d d i -r e c t l y by n i t r i t e F u j i t a and Sato (79) suggested that the p h y s i o l o g i c a l f u n c t i o n of cytochrome might be to d e t o x i -f y n i t r i t e . The r a t e of re d u c t i o n of n i t r i t e to ammonia by E. c o l i when glucose i s the e l e c t r o n donor was found to be p r o p o r t i o n a l to the cytochrome present (80). F u j i t a and Sato (61) al s o reported the existence of a second c-type cytochrome, cytochrome C^Q, i n a n a e r o b i c a l l y grown E. c o l i . The f u n c t i o n of t h i s cytochrome i s unknown but may be i n v o l v e d i n s u l f i t e metabolism (26). Shipp (9) has reported the existence of small amounts of two c-type cytochromes from low temperature d i f f e r e n c e spec-t r a of p a r t i c u l a t e membranes. These membrane-bound cytochromes, f o r which the f u n c t i o n i s not known, may be d i f f e r e n t from the s o l u b l e c-type cytochromes present when the c e l l s are grown an-, a e r o b i c a l l y . Haddock and S c h a i r e r (81) have suggested they are 16 not e s s e n t i a l f o r r e s p i r a t o r y a c t i v i t y . Nonheme i r o n A s s o c i a t e d w i t h the e l e c t r o n t r a n s p o r t chain of E. c o l i i s a second type of i r o n - c o n t a i n i n g e l e c t r o n c a r r i e r , the i r o n -s u l f u r p r o t e i n s or nonheme i r o n p r o t e i n s . Very l i t t l e i s known about i t s f u n c t i o n i n E. c o l i . The r o l e of nonheme i r o n i n var i o u s microorganisms has been reviewed by Bragg (82). In E. c o l i there i s much more nonheme i r o n than heme i r o n (82). Most i n v e s t i g a t i o n s of i t s r o l e have been i n d i r e c t , mainly through the use of metal c h e l a t o r s . O x i d a t i o n of NADH and succinate i s i n h i b i t e d by the i r o n c h e l a t o r s s a l i c y l a l d -oxime, ;8-hydroxyquinoline, 2 , 2 1 - d i p y r i d y l , t h e n o y l t r i f l u o r o -acetone (TTFA) , and o-phenanthroline (4, 28, 60, 83). Nonheme i r o n i s i n v o l v e d i n the dehydrogenase segment of the NADH oxidase system of E. c o l i . The i n h i b i t i o n by 2 , 2 ' - d i p y r i d y l was competitive w i t h substrate suggesting that a metal, p o s s i b l y nonheme i r o n , was cl o s e to the bi n d i n g s i t e f o r NADH (28). Gutman et a l . (84) found approximately equal amounts of nonheme i r o n and a c i d l a b i l e s u l f i d e i n a prepara-t i o n of NADH dehydrogenase suggesting the presence of Fe-S centres as found i n m i t o c h o n d r i a l dehydrogenases (85). This dehydrogenase-associated nonheme i r o n was r e d u c i b l e by ascor-bate (84) which would not be expected f o r components i n v o l v e d at s i t e s before the cytochromes i n sequence. The i n d i c a t e d h i g h p o t e n t i a l of t h i s nonheme i r o n would place i t c l o s e r to 17 oxygen i n the sequence. Although nonheme i r o n appears to be i n v o l v e d w i t h NADH dehydrogenase i t i s not as i n t i m a t e l y i n v o l v e d w i t h succinate dehydrogenase (68). Bragg (86) and Kim and Bragg (60) have demonstrated i n E. c o l i p a r t i c l e s a species of nonheme i r o n which reacts r a p i d l y ( w i t h i n one minute) w i t h o-phenanthroline. . I f NADH, ascorbate-PMS or d i t h i o n i t e i s used approximately 20% of the t o t a l nonheme i r o n r e a c t s . I f succinate i s used as the e l e c t r o n donor only TU of the t o t a l nonheme i r o n r e a c t s w i t h o-phenanthroline. Bragg (86) found that the re d u c t i o n of non-heme i r o n by NADH was i n h i b i t e d by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and although the r a t e of re d u c t i o n of cyto-chrome b-^  was slowed complete r e d u c t i o n of the cytochrome could occur without r e d u c t i o n of nonheme i r o n . I t was suggested that NADH-reducible o-phenanthroline-reacting nonheme i r o n was not on the main r e s p i r a t o r y pathway but s t i l l f u nctioned i n some manner between cytochrome b-^  and oxygen. Using e l e c t r o n paramagnetic resonance (EPR) spectros-copy Hamilton et al.(87) and Hendler (83) have made d i r e c t mea-surements of nonheme i r o n . Hendler reported that TTFA i n h i b i -t e d both the generation of the EPR s i g n a l at g = 1.94 a s s o c i a -ted w i t h reduced nonheme i r o n and the red u c t i o n of cytochrome b-^  when NADH was used as the substrate. TTFA d i d not prevent the r e o x i d a t i o n of these components. Therefore, i t was con-cluded that nonheme i r o n occurs between NADH dehydrogenase and cytochrome b n (83). 18 These r e s u l t s suggest that p a r t of the nonheme i r o n i s l i n k e d to the r e s p i r a t o r y chain probably at two s i t e s , be-fore and a f t e r cytochrome b-, i n the sequence, but i t i s impos-s i b l e to designate the exact r o l e of nonheme i r o n i n the e l e c -t r o n t r a n s p o r t chain at present. Quinones Much work has been done on the r o l e of ubiquinone i n the r e s p i r a t o r y chain of E. c o l i . L e s t e r and Crane (88) f i r s t r eported the presence of ubiquinone-8 and a naphthoquinone, l a t e r i d e n t i f i e d as v i t a m i n K 2 (40) (89), i n a e r o b i c a l l y grown E. c o l i . More r e c e n t l y low l e v e l s of demethyl v i t a m i n K 2 (40) (90) and the isoprenologues, ubiquinone-5, -6, and -7 have also been detected i n E. c o l i - , (91) . Kashket and Brodie (92) f i r s t i n d i c a t e d the importance of these lipoquinones i n the r e s p i r a t o r y chain of E. c o l i by r e -c o n s t i t u t i n g NAD +-linked substrate o x i d a t i o n by a d d i t i o n of ub-iquinone and naphthoquinone to membrane p a r t i c l e s i n which the lipoquinones had been i n a c t i v a t e d by n e a r - u l t r a v i o l e t i r r a d i a -t i o n . Subsequently they showed (93) that U V - i r r a d i a t e d c u l -tures o f E. c o l i grow p o o r l y on fermentable carbon sources and not at a l l on non-fermentable carbon sources even though the carbon source was o x i d i z e d . These r e s u l t s suggested a r o l e f o r lipoquinones i n the coupling of energy,production to the r e s p i -r a t o r y chain. There remained considerable confusion over the s p e c i f i c 19 involvement of ubiquinone.and v i t a m i n . i n the r e s p i r a t o r y chain. The use of mutants unable to synthesize ubiquinone (ubi~) or vi t a m i n (men-) r a t h e r than U V - i r r a d i a t i o n or s o l -vent e x t r a c t i o n to deplete the membrane o f these quinones has proved mo.r,e u s e f u l i n dec i d i n g t h e i r p a r t i c u l a r r o l e s . Thus, U V - i r r a d i a t i o n may r e s u l t i n e f f e c t s other than d e s t r u c t i o n of quinones. Bragg (94) has shown that U V - i r r a d i a t i o n of E. c o l i r e s p i r a t o r y p a r t i c l e s r e s u l t e d i n marked d e s t r u c t i o n of cyto-chrome d. Jones (95), the f i r s t to e x p l o i t the use of u b i q u i -nones d e f i c i e n t mutants, and Gibson, Cox and coworkers, f o l l o w -i n g these i n i t i a l observations w i t h more extensive i n v e s t i g a -t i o n s , have shown that ubiquinone-8,.not v i t a m i n . w a s essen-t i a l f o r growth on malate, succinate and l a c t a t e (96„ 97) and was th e r e f o r e r e q u i r e d f o r e l e c t r o n t r a n s p o r t and o x i d a t i v e phosphorylation i n v o l v i n g these s u b s t r a t e s . Examination of the steady s t a t e l e v e l of re d u c t i o n of the r e s p i r a t o r y components o x i d i z i n g l a c t a t e or NADH showed th a t ubiquinone may f u n c t i o n both before and a f t e r cytochrome b-^  i n the r e s p i r a t o r y chain (97). The f u n c t i o n of v i t a m i n (40) i s s t i l l unclear but New-ton et al.(98) have demonstrated that v i t a m i n f u n c t i o n s i n the anaerobic o x i d a t i o n of dihydroorotate therefore?, suggesting a r o l e i n the b i o s y n t h e s i s of pyr i m i d i n e s . In w i l d type s t r a i n s , ubiquinone-8 occurs at a concen-t r a t i o n 25 times that of cytochrome b-^ . Ubiquinone i s r e q u i r e d at these high l e v e l s f o r normal e l e c t r o n t r a n s p o r t as demonstra-ted by the markedly lowered NADH oxidase a c t i v i t y i n a mutant of E. c o l i c o n t a i n i n g only 4 times as much ubiquinone as cytochrome 20 b (99). This quinone i s found about 50% i n the reduced form even when the r e s t of the r e s p i r a t o r y chain i s f u l l y o x i d i z e d . Cox et al.(97) suggested that t h i s was due to the dispropor-t i o n a t i o n of the quinone i n ,'the semiquinone form to the o x i -dized and reduced forms when extract e d . EPR measurements (87) i n d i c a t e d that only 2% of the quinone was i n the semiquinone form. However, i t was suggested t h a t the low EPR s i g n a l may be due to the bi n d i n g of ubisemiquinone to a metal to give a chelate complex devoid of an EPR s i g n a l (100). Nonheme i r o n was suggested as the most l i k e l y c a r r i e r to form a complex w i t h ubiquinone since nonheme i r o n can f u n c t i o n at d i f f e r e n t redox p o t e n t i a l s and th e r e f o r e a l l o w f o r the involvement of ubiquinone both before and a f t e r cytochrome b-^  (97) . In the absence of ubiquinone, u b i " mutants are unable to c a r r y out r e s p i r a t o r y chain d r i v e n energy-dependent proces-ses . Thus, l a c k of ubiquinone reduced the r a t e of tr a n s p o r t of galactose, s e r i n e , and phosphate. The r a t e of tran s p o r t of s e r i n e and phosphate was found to be approximately p r o p o r t i o n -a l to the r a t e of e l e c t r o n t r a n s p o r t (101). Ubiquinone d e f i -c i e n t mutants are a l s o impaired i n t h e i r a b i l i t y to energize the membrane as i n d i c a t e d by the reduced l e v e l of e l e c t r o n t r a n s p o r t dependent quenching of a t e b r i n fluorescence. The quenching of a t e b r i n fluorescence which i s an i n d i c a t i o n of the a b i l i t y of the r e s p i r a t o r y chain to t r a n s l o c a t e protons can be r e c o n s t i t u t e d i n these mutants by the a d d i t i o n of u b i -quinone- 1 (102) . Even though i t i s known that ubiquinone i s e s s e n t i a l 21 f o r e l e c t r o n t r a n s p o r t and o x i d a t i v e phosphorylation there are a number of questions concerning ubiquinone f u n c t i o n s t i l l to be answered. The dehydrogenases There are a la r g e number of dehydrogenases i n v o l v e d i n g l y c o l y s i s and the TCA cy c l e i n E. c o l i . Most of these are lo c a t e d i n the cytoplasm. The r e s p i r a t o r y c h a i n - l i n k e d dehy-drogenases, which contain f l a v i n , FAD, or FMN as the prosthe-t i c group, are f i r m l y bound to the membrane. In general the ra t e l i m i t i n g process i n b a c t e r i a l e l e c t r o n t r a n s p o r t i s the feeding of e l e c t r o n s i n t o the e l e c t r o n t r a n s p o r t system by the membrane-bound dehydrogenases (103). Formate dehydrogen-ase (104, 105, 59, 106, 107), L-a-glycerolphosphate dehydro-genase (104, 108, 109), D- and L - l a c t a t e dehydrogenase (104, 84, 111), succinate dehydrogenase (104, 4, 106, 84, 107, 68, 83, 112), malate dehydrogenase (84, 97), and NADH dehydrogen-ase (28, 83, 84, 92, 104, 114-116) are a l l able to feed e l e c -trons d i r e c t l y i n t o the r e s p i r a t o r y chain of E. c o l i . These dehydrogenases can be f o r m e d ' d i f f e r e n t i a l l y during d i f f e r e n t growth c o n d i t i o n s (103). I t i s thought that e l e c t r o n s fed i n t o the r e s p i r a t o r y chain by these dehydrogenases converge on a common cytochrome pathway at the l e v e l of cytochrome b^ (83). 22 Factors a f f e c t i n g the composition and nature of the r e s p i r a -t o r y chain of E. c o l i Oxygen i s perhaps the most imp o r t a n t . f a c t o r determining the composition of the ''e^leetron t r a n s p o r t system of b a c t e r i a which are capable of growing both a e r o b i c a l l y and an a e r o b i c a l -l y . Under c o n d i t i o n s of vigorous a e r a t i o n , or i n the e a r l y exponential phase of growth, the e l e c t r o n t r a n s p o r t chain of E. c o l i c o n s i s t s p r i m a r i l y of .cytochrome b ^ g (77 K), u b i q u i -none, and cytochrome o. When the oxygen l e v e l s are low or i n the t r a n s i t i o n to the s t a t i o n a r y phase of growth the l e v e l s of cytochromes a^, (771>K) and d increase (9, 81). Moss (117) was the f i r s t to demonstrate that cytochrome d l e v e l s were maximal at low oxygen tension. However, under s t r i c t l y anaerobic c o n d i t i o n s the l e v e l s of a l l cytochromes (118), and the l e v e l s of other r e s p i r a t o r y enzymes such as the TCA enzymes (119, 120), are lower than i n a e r o b i c a l l y grown c e l l s . Oxygen appears to be necessary to enhance the a c t i v i t y and/or l e v e l s of enzymes i n v o l v e d i n heme formation. Using continuous c u l t u r e techniques Wimpenny and Neck-l e n (121) examined the e f f e c t of the t r a n s i t i o n from anaero-b i o s i s to a e r o b i o s i s on the l e v e l s of the r e s p i r a t o r y compo-nents. Instead of monitoring the oxygen l e v e l i n the media the redox p o t e n t i a l was measured to i n d i c a t e the degree of ae-r a t i o n . At p o t e n t i a l s below 0 mV, i n d i c a t i n g a n a e r o b i o s i s , the c e l l s e x h i b i t e d low l e v e l s of TCA enzymes and cytochromes as w e l l as a low growth y i e l d . At p o t e n t i a l s near +100 mV 23 (barely detectable oxygen l e v e l s ) cytochrome synthesis was maximal. The l e v e l of the TCA enzymes and the growth y i e l d was only s l i g h t l y increased. At higher p o t e n t i a l s , +200 to +300 mV, TCA enzyme synthesis and growth, y i e l d were maximal, but cytochrome l e v e l s were low. At even higher p o t e n t i a l s , i n d i c a t i n g s a t u r a t i n g oxygen c o n d i t i o n s TCA enzymes, growth y i e l d , and cytochrome l e v e l s were a l l lowered. I t i s obvious that r e g u l a t i o n of the l e v e l s of the cytochromes and the TCA enzymes i s complex and i s not coordinated. H a r r i s o n and Loveless (122) found that the s p e c i f i c r e s p i r a t i o n r a t e (qC^) i n E. c o l i increased at low oxygen ten sions probably as a r e s u l t of the increased l e v e l s of cyto-chrome b^ and d. There was a lower growth y i e l d under these c o n d i t i o n s supporting the evidence of Wimpenny and Necklen (121). Cytochrome d has a higher a f f i n i t y f o r oxygen as i n d i -cated by the lower c r i t i c a l oxygen concentration of c e l l s of Haemophilus par a i n f l u e n z a e c o n t a i n i n g elevated l e v e l s of t h i s cytochrome (123). Thus, the increased l e v e l of cytochrome d formed at low oxygen t e n s i o n i n t h i s organism has been sugges ted to be an adaptive measure. Presumably H. par a i n f l u e n z a e and E. c o l i enlarge and branch t h e i r e l e c t r o n t r a n s p o r t chain to maintain maximal r e s p i r a t o r y r a t e s at low oxygen concentra t i o n s . A s h c r o f t and Haddock (51) found that w e l l aerated c e l l of E. c o l i grown i n the presence of low concentrations of cy-24 anide.have increased l e v e l s of cytochromes b ^ g , a^, and d resembling the p a t t e r n of cytochromes of c e l l s grown w i t h poor a e r a t i o n . The suggestion that the a v a i l a b i l i t y of oxygen i n the growth medium i s the only c o n t r o l l i n g f a c t o r i s too simple, and i t i s more l i k e l y a combination of f a c t o r s which a f f e c t s the i n t e r n a l redox p o t e n t i a l of the c e l l s that may be invo l v e d . Together w i t h the changes i n composition of the r e s p i r -a t o r y chain there are changes i n the r e s p i r a t o r y e f f i c i e n c y as seen by the changes i n the growth y i e l d . Attempts have been made.to.determine the phosphorylation e f f i c i e n c y , P/0 r a -t i o (moles phosphate e s t e r l f i e d to y i e l d ATP/ g atoms of oxy-gen reduced) , of b a c t e r i a l r e s p i r a t o r y systems (12.4-126) . The methods employed to measure P/0 r a t i o s i n mitochondria are not a p p l i c a b l e i n b a c t e r i a l systems because of the many ATPase r e -ac t i o n s ;.o;ccuring. i n i n t a c t c e l l s and the impermeability of the c e l l membrane to coenzymes. Hempfling (125) employed a d i r e c t method f o r determin-i n g phosphorylation e f f i c i e n c y i n i n t a c t E. c o l i c e l l s . He 32 determined the r a t i o of P e s t e r i f i e d to the amount of NADH o x i d i z e d (P/2e~), equivalent to the P/0 r a t i o . Hempfling (125) found that the phosphorylation e f f i c i e n c y of E. c o l i v a r i e d w i t h the phase of growth. A P/2e" of 0.33 f o r e a r l y exponential phase c e l l s increased to over 3.0 i n the l a t e e xponential phase of growth. However, the d i r e c t methods em-ployed by Hempfling depend on s e v e r a l assumptions which may not be v a l i d . 25 Meyer and Jones.(126) determined the molar growth y i e l d w i t h respect to oxygen u t i l i z e d ( Y A T p ) and from t h i s c a l c u l a t e d P/0 r a t i o s * * f o r E. c o l i . When c e l l s contained h i g h l e v e l s of cytochrome o (grown w i t h high aeration) P/0 r a t i o s were about 3. This i m p l i e s that there are three s i t e s of phosphorylation i n the r e s p i r a t o r y chain of E. c o l i , s i m i l a r to those found i n mitochondria. The synthesis of cytochromes a^ and d i n E. c o l i (oxygen l i m i t e d c o n d i t i o n s ) was accompan-ied" by a sharp decrease i n t h i s value suggesting that cyto-chrome d may be as s o c i a t e d w i t h a non-phosphorylating path-way. According to the chemiosmotic hypothesis (127) the r a -t i o of the number of protons t r a n s l o c a t e d across the membrane to the number of atoms of oxygen reduced (H +/0 quotient) should a l s o r e f l e c t the e f f i c i e n c y of energy conservation. Employing t h i s method to determine r e s p i r a t o r y e f f i c i e n c y i n E. c o l i only two s i t e s of energy conservation were found (128). One s i t e was l o c a t e d between NADH dehydrogenase and cytochrome b-^  and the other between cytochrome b^ and the t e r -minal oxidase. Meyer and Jones (126) and B r i c e et a l . (129) found that there was a drop i n the H+/0 r a t i o concomitant w i t h the development of the te r m i n a l oxidase, cytochrome d. I t would appear from these r e s u l t s that E. c o l i s a c r i f i c e s ' growth e f f i c i e n c y at low oxygen tensions w i t h the development of t h i s a l t e r n a t e pathway of r e s p i r a t i o n . Another f a c t o r i n c o n t r o l l i n g the l e v e l s of cytochromes i n the r e s p i r a t o r y chain of E. c o l i i s c y c l i c AMP. Broman et 26 al.(130) suggest that the r o l e c y c l i c AMP plays i n the meta-bolism of E. c o l i i s not r e s t r i c t e d to c a t a b o l i t e r e p r e s s i b l e systems i n v o l v e d i n carbohydrate metabolism.since t h i s nucleo-t i d e may al s o be r e q u i r e d f o r increased cytochrome s y n t h e s i s . The o r g a n i z a t i o n of the r e s p i r a t o r y chain of E. c o l i The number and arrangement of the components of the r e s p i r a t o r y chain i n the membrane, of E. c o l i has not been f i r m l y e s t a b l i s h e d . A number of models of the r e s p i r a t o r y chain have been proposed based on the e a r l y spectroscopic e v i -dence that the only cytochromes present were cytochromes b-^ , a x , d;--arid o (82, 83, 97, 107, 131). These schemes ( F i g . 1) inco r p o r a t e some common features and contain some d i s t i n c t d i f f e r e n c e s . One of the e a r l i e r schemes ( F i g . l a ) proposed that UQ-8 functioned i n the succinate oxidase pathway whi l e v i t a m i n p a r t i c i p a t e d i n the NADH oxidase pathway ('3-3.1) . This i d e a was r e t a i n e d i n the model proposed by B i r d s e l l and Cota-Robles ( F i g . l c ) (107). However, w i t h the use of u b i q u i -none d e f i c i e n t mutants (97) i t was shown that UQ-8 functioned i n the NADH oxidase system as w e l l as other e l e c t r o n t r a n s p o r t pathways. Hendler's (83) scheme ( F i g . l b ) based on re d u c t i o n and o x i d a t i o n k i n e t i c s of the cytochrome components includes cytochrome a-^  not as a termi n a l oxidase but as a cytochrome f u n c t i o n i n g between cytochrome b^ and cytochrome d. There i s noyclear evidence f o r i t s p o s i t i o n i n the r e s p i r a t o r y system and even Hendler had r e s e r v a t i o n s about i t s l o c a t i o n . The 27 FIGURE 1. a) NADH dehydrogenase - > V i t . K, S u c c i n a t e dehydrogenase > UQ-c y t bj—cyt b) NADH -> F S u c c i n a t e >• F c y t b - ^ — c y t a - ^ — ^ c y t d —^ 0, c) HIGH SINSITIVITr F O R M A T E - > r D H -SUCCINATE>SOH NADH > NOH KOQHO AUTIHTCIH A LOW IE A 6ITIVITT F i g . 1 c o n t i n u e d n e x t page 28 FIGURE 1. (continued) d) NADH > f Fe P L a c t a t e f P V -> c y t b , — > Fe 1 I UQ-8 JQ-8 c y t o c y t d e) NADH PD Fe S u c c i n a t e PS F S D UQ-8 c y t b 1 D Fe S UQ-8. c y t b l s A c y t a. c y t d c y t o f) S u b s t r a t e ——> dehydrogenase / \ c y t b 55i - > 0 , c y t b 5 5 6 > c y t o >0, -> c y t d >• 0, FIG. 1. Proposed schemes f o r e l e c t r o n t r a n s p o r t i n E . c o l i , a) Kashket and Brodie (131) b) Hendler (83). c) B i r d s e l l and Cota-Robles (107).d) Cox e t a l . ( 9 7 ) . e) Bragg (82). f) Haddock and S c h a i r e r (81). A b b r e v i a t i o n s : YK^ - v i t a m i n K 2 ( 4 0 ) ; UQ-8, Q-8, - ubiquinone-8; ? , NDH, F p D , - NADH dehydrogenase; SDH, F p g , Fg, - Su c c i n a t e dehydrogenase; FDH - formate dehydrogenase; f - f l a v o p r o t e i n s ; NER -non- e n z y m a t i c a l l y r e d u c i b l e . 29 l i n e a r sequence of components as proposed by Cox et al.(97) shown i n F i g . I d i s based on experiments u s i n g ubiquinone de-f i c i e n t mutants and i n h i b i t o r s . By studying the aerobic steady s t a t e l e v e l s of red u c t i o n they proposed that HOQNO i n -h i b i t s e l e c t r o n t r a n s p o r t at s i t e s both before and a f t e r cy-tochrome b-^  as the aerobic steady s t a t e l e v e l of re d u c t i o n of t h i s cytochrome remained e s s e n t i a l l y unchanged i n the presence of t h i s i n h i b i t o r even when r e s p i r a t i o n was g r e a t l y i n h i b i t e d . I t was suggested that HOQNO d i s r u p t s the p o s t u l a t e d ubisemiqui-none-nonheme i r o n complex which they suggested was l o c a t e d on both sides of cytochrome b-^  (97). In many of these schemes e l e c t r o n s from d i f f e r e n t sub-s t r a t e s are assumed to converge on a common e l e c t r o n t r a n s p o r t pathway p r i o r to the l e v e l of cytochrome b-^ . Evidence f o r branching i n the e l e c t r o n t r a n s p o r t system i n membrane f r a g -ments derived from E. c o l i has been presented by B i r d s e l l and Cota-Robles (97) ( F i g . l c ) . The d i f f e r e n t s e n s i t i v i t i e s of the NADH oxidase, formate oxidase, and succinate oxidase sys-tems to azide and cyanide i n h i b i t i o n suggested t h a t these sys-tems were compartmentalized. The idea that there was overlap between the systems was i n d i c a t e d by the f a c t that when the substrates were added together l e s s cytochrome was reduced at anaerobiosis than the t o t a l amount reduced at anaerobiosis w i t h each substrate added i n d i v i d u a l l y . B i r d s e l l and Cota-Robles a l s o found a small p o r t i o n of non-enzymatically redu-c i b l e cytochrome b-^ . Their r e s u l t s should be accepted w i t h 30 ca u t i o n because the fragments employed were derived from deter-gent treatment of E. c o l i spheroplasts. The detergent ( B r i j 58) may have d i s r u p t e d some of the e l e c t r o n t r a n s p o r t pathways lea d i n g to the r e s u l t s found. However, branched and compart-mentalized e l e c t r o n t r a n s p o r t systems have been suggested f o r other b a c t e r i a l species such as Haemophilus p a r a i n f l u e n z a e (103), Azotobacter v i n e l a n d i i (25), Halobacterium cutirubrum (132), and Rhodopseudomonas p a l u s t r i s (133). Bragg (82) i n a recent review a r t i c l e has presented the scheme shown i n F i g . l e . There i s i n s u f f i c i e n t data and con-t r a d i c t o r y evidence f o r p l a c i n g cytochrome b-p ubiquinone, and nonheme i r o n i n a defined sequence. Therefore i t was c o n s i -dered best to represent them as a s i n g l e grouping .of 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 . Note that the cytochrome b pools are a l s o compartmentalized i n t h i s scheme w i t h e l e c t r o n s converging at the l e v e l of the t e r m i n a l oxidases. Cytochrome a-^  i s grouped together w i t h cytochromes d and o as a t e r m i n a l oxidase but i t i s doubtful that cytochrome a-^  f u n ctions as an oxidase i n E. c o l i . The cytochrome b absorption peak has been r e s o l v e d i n t o m u l t i p l e components at 77°K (9). Only the work by Haddock and S c h a i r e r (81) takes i n t o account the r e s o l v e d cytochromes i n formulating a sequence ( F i g . I f ) . They employed a mutant of E. c o l i unable to form cytochromes unless supplemented w i t h 5-amino-levulenic a c i d and examined the cytochromes produced i n r e c o n s t i t u t i o n experiments. Haddock and S c h a i r e r suggested that two membranea bound e l e c t r o n t r a n s p o r t systems using oxy-31 gen as a t e r m i n a l acceptor can c o e x i s t . Under c o n d i t i o n s of vigorous a e r a t i o n cytochrome b ^ g together w i t h the t e r m i n a l oxidase, cytochrome o, form one pathway. In the s t a t i o n a r y phase of growth or under con d i t i o n s of poor a e r a t i o n cyto-chrome b ^ g and the t e r m i n a l oxidase cytochrome d form the other pathway. The c o n t r i b u t i o n of these i n d i v i d u a l pathways to the oxidase pathways f o r the d i f f e r e n t substrates was not examined. The p h y s i c a l p r o p e r t i e s ( i . e . the midpoint oxida-t i o n - r e d u c t i o n p o t e n t i a l s ) of these cytochrome components were not known at the time I commenced my t h e s i s work. Ou t l i n e of t h e s i s problem In my t h e s i s work I have attempted to answer a number of questions concerning the o r g a n i z a t i o n and p r o p e r t i e s of the cytochromes of the r e s p i r a t o r y chain of E. c o l i . I have ex-amined the r e l a t i v e c o n t r i b u t i o n of the t e r m i n a l oxidases, cytochromes d and o, to the NADH oxidase and succinate oxidase a c t i v i t i e s i n membrane p a r t i c l e s , prepared from ; c e l l s grown to contain d i f f e r e n t l e v e l s of these cytochromes. Cyanide, an i n h i b i t o r commonly used to block e l e c t r o n t r a n s p o r t at the l e v e l of the t e r m i n a l oxidase has an unknown mechanism of ac-t i o n i n E. c o l i . The mechanism and the k i n e t i c s of i n h i b i -t i o n by cyanide of cytochrome d were studied. I have a l s o ob-served other p r o p e r t i e s of cytochrome d which may shed l i g h t on the oxidase r e a c t i o n mechanism. The p o t e n t i o m e t r i c t i t r a -t i o n methods of Wilson and Dutton (134) were employed to det-3 2 ermine.the o x i d a t i o n - r e d u c t i o n p o t e n t i a l s of the cytochromes. The cytochrome b-^  peak was re s o l v e d i n t o m u l t i p l e components by low temperature spectroscopic methods and by potentiomet-r i c techniques . The f u n c t i o n of these r e s o l v e d components i n the NADH oxidase and succinate oxidase pathways was exa-mined by measuring the r e due a k i n e t i c s of r e d u c t i o n of these cytochromes i n membrane p a r t i c l e s o x i d i z i n g NADH and succin a t e , and by the use of low temperature (77°K) spectro-scopic techniques to trap the cytochromes i n d i f f e r e n t redox s t a t e s . A branched and p a r t i a l l y compartmentalized cytochrome system i s proposed on the b a s i s of t h i s work. 33 MATERIALS AND METHODS Chemicals The f o l l o w i n g companies su p p l i e d the m a t e r i a l s i n d i c a -ted. F i s h e r S c i e n t i f i c Co.: Disodium suc c i n a t e , f e r r i c c i t -r a t e , 37o H2O2 s o l u t i o n , sodium h y d r o s u l f i t e (sodium d i t h i o -n i t e ) , potassium cyanide, and as c o r b i c a c i d ; Sigma Chemical Co.: D L - l a c t i c a c i d ( l i t h i u m s a l t ) , reduced nicotinamide ad-enine d i n u c l e o t i d e (NADH), and 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO); M a l l i n c k r o d t Chemical Co.: glucose and suc-rose; J.T. Baker Chemical Co.: potassium f e r r i c y a n i d e ; BBL : t r y p t i c a s e soy bro t h ; Eastman Organic Chem-i c a l s : dichlorophenolindophenol (DCIP) and 2-hydroxy-1,4-naphthoquinone; Calbiochem: phenazine methosulfate (PMS) and adenQsine-5'-triphosphate (ATP); N u t r i t i o n a l Biochemical Corp.: phenazine e t h o s u l f a t e (PES); A l d r i c h Chemical Co.: duroquinone; K and K L a b o r a t o r i e s : pyocyanine; Canadian L i -quid A i r : n i t r o g e n ; Matheson: carbon monoxide. Growth and Maintenance of c e l l s Stock c u l t u r e s of E. c o l i NRC 482 were maintained at 4°C on minimal s a l t s - g l u c o s e - a g a r s l a n t s i n screw capped tubes. The c u l t u r e s were t r a n s f e r r e d every two to three months. E. c o l i NRC 482 was a s e p t i c a l l y removed from the stock c u l t u r e s l a n t w i t h a platinum or Nichrome wir e i n n o c u l a t i n g 34 loop and innoculated i n t o a tube c o n t a i n i n g 10 ml of minimal s a l t s medium w i t h a 'glucose concentration of 0 .47 o (W/V) . The c u l t u r e was incubated at 37°C f o r 24 hours without shaking, and then used to inn o c u l a t e a 500 ml erlenmeyer f l a s k c o n t a i n -i n g 100 ml of the same medium as was used subsequently. This was shaken at 37°C i n a r e c i p r o c a t i n g water bath shaker (New Brunswick S c i e n t i f i c Co., Model R76) f o r approximately 16 hours. The f i n a l growth medium was innoc u l a t e d using a volume of t h i s c u l t u r e equivalent to one-tenth to one-twentieth of the volume of the f i n a l c u l t u r e . B a c t e r i a were e i t h e r grown on minimal s a l t s p l u s 0 . 4 % glucose, 0 . 4 % D L - l a c t i c a c i d ( l i t h i u m s a l t ) or 0 . 8 % or 1 .4% disodium s u c c i n a t e ; or t r y p t i c a s e soy broth (complex medium). The minimal s a l t s medium (135) c o n s i s t i n g of 4 0 . 2 mM K^HPO^, 2 2 . 0 mM KH 2 P 0 4 , 0 . 8 mM M g S 0 4 , 7 . 6 mM (NH 4 ) 2 S 0 4 , and 1 . 7 mM so-dium c i t r a t e was supplemented w i t h 12 jiM f e r r i c c i t r a t e . The c u l t u r e s were grown a e r o b i c a l l y i n a v a r i e t y of ways. C e l l s grown on glucose, l a c t a t e , and succinate to be harvested i n the exponential phase of growth were grown i n a 6 1 f l o r e n c e f l a s k c o n t a i n i n g 2 - 3 1 of c u l t u r e medium, i n c u -bated at 37°C i n a water bath and aerated by bubbling f i l t e r e d a i r i n t o the c u l t u r e through a sparger. Evaporation was m i n i -mized by bubbling the a i r through d i s t i l l e d water at 37°C p r i o r to i t s entry i n t o the c u l t u r e v e s s e l . Growth was moni-tored by f o l l o w i n g the change i n the absorbance of the c u l t u r e at 420 nm read on a Coleman 124 spectrophotometer using a one-tenth d i l u t i o n bar i n the sample cuvette. The c e l l s were har-3 5 vested at an absorbance of 5.0 to 7.0. The pH of the c u l t u r e growing on succinate was monitored p e r i o d i c a l l y using a F i s h e r Accumet pH meter and was maintained w i t h i n the range of 6.8 to 7.8 by a d d i t i o n s of small amounts of 2M H^SO^. C e l l s grown to the s t a t i o n a r y phase of growth on suc-c i n a t e or t r y p t i c a s e soy broth ("complex" medium) were c u l -t u red i n 1 l i t e r f l a s k s c o n t a i n i n g 250 ml of the c u l t u r e me-d i a . These f l a s k s were shaken at 120 rpm i n a r e c i p r o c a t i n g water bath at 37°C f o r 18-20 hours and the c e l l s were har-vested i n the s t a t i o n a r y phase of growth. To o b t a i n a e r o b i c a l l y grown c e l l s i n the e a r l y exponen-t i a l , phase of growth a 1 l i t e r f l a s k c o n t a i n i n g 250 ml of suc-c i n a t e medium was inn o c u l a t e d w i t h a 1% innoculum and aerated v i g o r o u s l y i n a c o n t r o l l e d environment incubator shaker (New Brunswick S c i e n t i f i c Co., Model G-25) at 300 rpm at 37°C. The c e l l s were harvested when the absorbance at 420 nm was approximately 1.0.i Harvesting of c e l l s The c u l t u r e was c e n t r i f u g e d i n 500 ml polycarbonate b o t t l e s at 5,000 rpm (4,400 x g) f o r 15 minutes at 4°C i n a Beckman J-21 c e n t r i f u g e using a JA10 r o t o r . The medium was decanted and the c e l l s resuspended i n e i t h e r 0.9% NaCl or 0.1M •potassium phosphate b u f f e r , pH 7.0, to a concentration of 1 g of c e l l s (wet weight) per 20 ml. The c e l l s were resedimented using the same co n d i t i o n s as described above. 36 S t a r v a t i o n of c e l l s This procedure was c a r r i e d out to deplete the endogen-ous stores of substrate w i t h i n the c e l l s . The washed c e l l s were resuspended i n 0.1M potassium phosphate b u f f e r , pH 7.0, to a concentration of 1 g c e l l s (wet weight) per 50 ml. The suspension (250 ml) was placed i n a 1 l i t e r f l a s k and aerated at 37°C i n a shaking water bath or an incubator shaker (250 rpm) f o r 2-4 hours. The c e l l s were resedimented u s i n g the same co n d i t i o n s described above. The supernatant was decan-ted and the c e l l s were st o r e d at 4°C f o r no more than 24 hours. In a l l cases except where mentioned, f r e s h l y grown c e l l s which had not been st o r e d frozen were employed i n my experiments. P r e p a r a t i o n of membrane p a r t i c l e s The washed and sometimes starved c e l l s were resuspen-ded i n e i t h e r 0.1M T r i s - H 2 S 0 4 b u f f e r , pH 7.5, c o n t a i n i n g 0.01 M MgCl 2 ("TM b u f f e r " ) or 0.1M potassium phosphate b u f f e r , pH 7.0, at a concentration of 1 g c e l l s (wet weight) per 5 ml b u f f e r and d i s r u p t e d i n a French press (Aminco) at 20,000 p s i . The ruptured c e l l m a t e r i a l i n the supernatant was c a r e f u l l y separated from the p e l l e t e d unbroken c e l l debris a f t e r c e n t r i -f u g a t i o n at 12,000 rpm (17,600 x g) f o r 15 minutes at 4°C u s i n g a Beckman JA20 r o t o r i n the Beckman J21 c e n t r i f u g e . The supernatant was then c e n t r i f u g e d at 100,000 x g f o r 2 hours at 37 4 C i n a Beckman L2-65B u l t r a c e n t r i f u g e using a 50.1 r o t o r . The membrane p a r t i c l e s c o n t a i n i n g the membrane-bound r e s p i r a -t o r y system of E. c o l i appeared as a reddish-brown opalescent p e l l e t . The supernatant was decanted and the p e l l e t s t o r e d at 4°C f o r no more than 24 hours. I t was never stored f r o z -en. The r e s p i r a t o r y p a r t i c l e s were then suspended i n e i t h e r "TM b u f f e r " or 0.1M potassium phosphate b u f f e r , pH 7.0, to a concentration of 3-10 mg p a r t i c l e p r o t e i n per ml. Measurement of s p e c t r a D i f f e r e n c e s p e c t r a were taken w i t h a P e r k i n Elmer 356 spectrophotometer. For the reduced versus o x i d i z e d d i f f e r -ence spe c t r a 1.5 ml of the r e s p i r a t o r y p a r t i c l e suspension was placed i n both the reference and sample cuvettes. The p a r t i -c l e suspension i n the sample cuvette was reduced w i t h a few c r y s t a l s of sodium d i t h i o n i t e (approximately 1 mg) and the suspension i n the reference cuvette was o x i d i z e d w i t h 10 y l of 3% H2O2, or a few c r y s t a l s of potassium f e r r i c y a n i d e (ap-proximately 1 mg), or by shaking i n a i r . The spectrum of the reduced components was scanned from 400-700 nm against the ox-i d i z e d sample at a scan speed or 120 nm/min using a bandwidth of 1.0 nm. The pathlength of the quartz cuvettes was 1 cm. For reduced plus KCN versus reduced d i f f e r e n c e spectra the r e s p i r a t o r y p a r t i c l e s i n both cuvettes were reduced w i t h o sodium d i t h i o n i t e and 100 ; t l of f r e s h l y prepared 0.15M KCN was then added to the sample cuvette. The spectrum was 38 scanned as above. To measure carbon monoxide d i f f e r e n c e s p e c t r a the r e s -p i r a t o r y p a r t i c l e s i n both cuvettes were reduced w i t h sodium d i t h i o n i t e and.then carbon monoxide was bubbled slowly through the s o l u t i o n i n the sample cuvette u n t i l a constant spectrum was obtained. Determination of the cytochrome content The content of cytochrome d was c a l c u l a t e d by measur-i n g one-half the height i n absorbance u n i t s from the trough at 648 nm to the peak at 628 nm i n the reduced versus o x i d i z e d d i f f e r e n c e spectrum and using the e x t i n c t i o n c o e f f i c i e n t of 8.51 mM'^ cm""'" f o r t h i s absorbance d i f f e r e n c e (136). The con-tent of cytochrome b-^  was determined from the peak height i n absorbance u n i t s at 558 nm above the b a s e l i n e . The e x t i n c t i o n -1 -1 . c o e f f i c i e n t used f o r cytochrome b-^  was 16.0 mM cm (62). The cytochrome a-^  content was determined from the absorbance above the b a s e l i n e at 594 nm using the e x t i n c t i o n c o e f f i c i e n t f o r cytochrome a^ of 4.6 mM~"'"cm~"'" (137). Cytochrome o can only be determined from the carbon monoxide d i f f e r e n c e spectrum us-in g the e x t i n c t i o n c o e f f i c i e n t of 170 mM~'''cm~^  f o r the absor-bance d i f f e r e n c e between the peak at 415 nm and the trough at 430 nm (67). Determination of succinate and NADH oxidase a c t i v i t y NADH and succinate oxidase a c t i v i t i e s were determined 39 from the time, r e q u i r e d f o r 1.5 ml of a f u l l y aerated p a r t i c l e suspension to become anaerobic a f t e r the a d d i t i o n of 2 mM NADH or 1.67 mM succi n a t e , r e s p e c t i v e l y . Anaerobiosis was i n d i c a t e d by the r a p i d phase of re d u c t i o n of cytochrome b^ which occurred on t r a n s i t i o n from the aerobic to the anaero-b i c steady s t a t e . The redox s t a t e of cytochrome b^ was f o l -lowed w i t h a dualwavelength spectrophotometer ( P e r k i n Elmer/ H i t a c h i 356) using the wavelength p a i r 558-540 nm. The oxy-gen concentration i n the suspension when f u l l y aerated was assumed to be 0.26 mM at 22°C. NADH oxidase was al s o deter-mined s p e c t r o p h o t o m e t r i c a l l y as described in. the next s e c t i o n . A l l assays were c a r r i e d out at 22°C. E f f e c t of KCN on NADH oxidase The r e s p i r a t o r y p a r t i c l e suspension (50-100 y l ) was ad-ded to a cuvette c o n t a i n i n g "TM b u f f e r " , followed by the addi-t i o n of 0-250 yil f r e s h l y prepared 0.15 M KCN to give a f i n a l c o n c e n t r a t i o n of 0-25 mM KCN i n a t o t a l volume of 1.5 ml. Then 50 p l of 9.83 mM NADH was added e i t h e r immediately or af-t e r a p r e i n c u b a t i o n p e r i o d at 22°C. The decrease i n absorp-t i o n at 340 nm due to the o x i d a t i o n of NADH was fol l o w e d at 22°C using a Coleman 124 spectrophotometer equipped w i t h a l i n e a r recorder. The e x t i n c t i o n c o e f f i c i e n t at 340 nm f o r NADH of 6.22 mM~''"cm~''" was used to c a l c u l a t e the con c e n t r a t i o n of NADH. 40 E f f e c t of KCN on the spectrum of cytochrome d D i r e c t spectra of E. c o l i membrane p a r t i c l e s i n the 600-700 nm range were taken at 22 C .using as a reference sample a suspension of E. c o l i membrane p a r t i c l e s which had no absorption bands i n t h i s range. The reference sample was prepared e i t h e r by re d u c t i o n of the membrane p a r t i c l e s w i t h d i t h i o n i t e i n the presence of 15 mM KCN to cause e l i m i n a t i o n of the absorption band of cytochrome d or by using p a r t i c l e s prepared from E. c o l i N-^^ c e l l s grown on glucose to the ex-p o n e n t i a l phase of growth which l a c k cytochrome d. The ef-f e c t o f KCN on the o x i d i z e d form of cytochrome d was examined by adding f r e s h l y prepared 0.3 M KCN to the sample cuvette c o n t a i n i n g a suspension of membrane p a r t i c l e s to give a f i n a l c o n centration of 0-25 mM i n a t o t a l volume of 1.5 ml. The spectrum was rescanned at timed i n t e r v a l s . The e f f e c t of KCN on the disappearance of the o x i d i z e d form of cytochrome d under turnover c o n d i t i o n s was followed at 22°C by measuring the change i n absorbance at 648 nm ( o x i d i z e d cytochrome d) r e l a t i v e to 607 nm ( i s o s b e s t i c p o i n t ) using a Pe r k i n Elmer/Hitachi Model 356 spectrophotometer. The spec-trophotometer was operated i n the dual wavelength mode w i t h the f u l l s c a l e d e f l e c t i o n set at 0.03 absorbance u n i t s . Elim-i n a t i o n of the absorption band due to o x i d i z e d cytochrome d occurred too f a s t to be fol l o w e d by rescanning the t o t a l spec-trum (600-700 nm). NADH ( f i n a l c o n c e n t r a t i o n , 2 mM) or s u c c i -nate ( f i n a l c o n c e n t r a t i o n , 1.67 mM) was added to the suspen-41 s i o n of r e s p i r a t o r y p a r t i c l e s c o n t a i n i n g v a r y i n g amounts of KCN (0-25 mM) i n a t o t a l volume of 1.5 ml. Succinate dehydrogenase a c t i v i t y was c o m p e t i t i v e l y i n -h i b i t e d to v a r y i n g degrees by the a d d i t i o n of malonate to a concentration of 0-0.33 mM. This allowed the e l e c t r o n f l u x to be regulated. C e l l s grown to the exponential phase of growth con-t a i n e d much lower l e v e l s of cytochrome d. Therefore i t was necessary to f o l l o w the k i n e t i c s of the disappearance of the o x i d i z e d form of cytochrome d using a f u l l s c a l e d e f l e c t i o n of 0.01 absorbance u n i t s . Oxidation rates f o r NADH and s u c c i -nate were determined from the time r e q u i r e d f o r the system to become anaerobic as measured by the r a p i d disappearance of the absorption peak at 648 nm at anaerobiosis. The r e l a t i o n s h i p between the absorption peaks at 648 and 628  nm of the oxidized-and reduced forms of cytochrome d The r e d u c t i o n of the cytochromes at anaerobiosis i n the presence of s a t u r a t i n g l e v e l s of substrate occurred too quick-l y to d i f f e r e n t i a t e between the time of disappearance of the o x i d i z e d form of cytochrome d and, the appearance of the r e -duced form of cytochrome d. To examine the k i n e t i c s of reduc-t i o n 1.5 ml of membrane p a r t i c l e s prepared from starved c e l l s grown to the s t a t i o n a r y phase of growth to contain h i g h l e v e l s of cytochrome d were placed i n a sealed cuvette* and n i t r o g e n was bubbled through the suspension f o r 15" minutes to make the 42 system anaerobic. The spectrum was scanned at 22°C from 700. nm - 500 nm at timed i n t e r v a l s to ensure that no r e d u c t i o n of the cytochromes had occurred. A l i m i t i n g amount of an anaero-b i c s o l u t i o n of substrate (ascorbate (16 uM) plus PMS (5 uM), NADH (25 uM), or succinate (25 ]M)) was then added v i a a Hamil-ton syringe under p o s i t i v e n i t r o g e n pressure to i n i t i a t e the r e d u c t i o n of the cytochromes. The spectrum of the alpha band region of the cytochromes was scanned against an o x i d i z e d r e f -erence sample at timed i n t e r v a l s . The appearance of the ab-s o r p t i o n bands due to the reduced cytochromes d (628 nm) , a-^  (594 nm), and b^ (558 nm) and the disappearance of the absorp-t i o n band due to the o x i d i z e d form of cytochrome d (648 nm) were fo l l o w e d w i t h time a f t e r the a d d i t i o n of the substrate. The percent r e d u c t i o n of each component was r e l a t e d to the l e -v e l of r e d u c t i o n obtained a f t e r the addition>of sodium d i t h i o -n i t e . P o t e n t i o m e t r i c t i t r a t i o n s The methods of Wilson and Dutton (134) were employed. A cuvette assembly was constructed to c a r r y out the potentiomet-r i c t i t r a t i o n s . A 1 cm pathlength cuvette (pyrex) was a t -tached w i t h r e s i n to the tapered end of a 1 i n c h diameter glass (pyrex) tube (2 inches i n length) which had an attached ground g l a s s j o i n t : ( S i z e 24/40) at the top end. The top end was sealed w i t h a rubber stopper equipped w i t h an i n l e t and o u t l e t f o r bubbling n i t r o g e n gas through the cuvette assembly 43 and a F i s h e r platinum combination e l e c t r o d e (13-369-82) to measure the redox p o t e n t i a l i n the cuvette. A glas s sidearm was attached to the assembly at a 30° angle and sealed w i t h a serum cap. T o t a l volume of the cuvette assembly was 12 ml. The reagents were added to the cuvette v i a a Hamilton syringe through the sidearm and the contents were s t i r r e d p e r i o d i c a l -l y w i t h an 8 mm s t i r r i n g bar using a small' magnetic s t i r r e r . The Ag +/AgCl reference e l e c t r o d e or the combination redox e l e c t r o d e was c a l i b r a t e d w i t h a standard calomel e l e c -trode and found to be +202 mV r e l a t i v e to the standard hydro-gen e l e c t r o d e (0 mV). The mixture of membrane p a r t i c l e s suspended i n 0.1 M potassium phosphate b u f f e r , pH 7.0, and the redox mediators were added to the cuvette assembly. To e s t a b l i s h anaerobiosis n i t r o g e n was f l u s h e d through the system and small amounts of NADH (0.2 M), ascorbate (0.5 M) or succinate (0.1 M) were ad-ded to u t i l i z e the r e s i d u a l oxygen. Further r e d u c t i o n was ac-complished by repeated a d d i t i o n s of small volumes (1-10 ul) of the substrates f o l l o w e d by i n t e r m i t t e n t s t i r r i n g . O xidation was accomplished by the a d d i t i o n of small volumes (1-10 u l ) of 0.2 M potassium f e r r i c y a n i d e . The t i t r a t i o n s were c a r r i e d out at 22 °C. -During the o x i d a t i v e and r e d u c t i v e t i t r a t i o n s the spec-trum of the contents i n the cuvette assembly was scanned at appropriate measured redox p o t e n t i a l s against an o x i d i z e d r e f -erence sample. In general a s i n g l e r e d u c t i v e t i t r a t i o n was 44 \ c a r r i e d out followed by a s i n g l e o x i d a t i v e t i t r a t i o n ; the en-t i r e procedure t a k i n g approximately 2 hours. The absorbance increments at 628 nm, 594 nm, and 558 nm above the b a s e l i n e were p l o t t e d versus the corresponding redox p o t e n t i a l of the system to obtai n the t i t r a t i o n curves f o r cytochromes d, a p and b-^ , r e s p e c t i v e l y . S i m i l a r l y , the s i z e of the trough at 648 nm i n the reduced versus o x i d i z e d d i f f e r e n c e spectrum due to the o x i d i z e d form of cytochrome d and at 680 nm due to the o x i d i z e d form of an u n i d e n t i f i e d chromophore were also p l o t t e d versus the p o t e n t i a l to determine t h e i r midpoint o x i d a t i o n -r e d u c t i o n p o t e n t i a l s . P a r t i c l e s prepared from c e l l s grown to the s t a t i o n a r y phase of growth were employed to o b t a i n the p o t e n t i o m e t r i c t i t r a t i o n s curves f o r cytochromes d and a p However, the po-t e n t i o m e t r i c data f o r cytochrome b-^  was obtained from p a r t i c -l e s prepared from c e l l s grown to the e a r l y e x p o n e n t i a l , l a t e e x p o n e n t i a l , and s t a t i o n a r y phases of growth. The f o l l o w i n g mediators were used i n . t h e various exper-iments to e s t a b l i s h e q u i l i b r i u m between the redox e l e c t r o d e and the membrane bound cytochrome system: potassium f e r r i c y -anide ( E q, +430 mV), quinhydrone ( E q , +280 mV), dichlorophen-olindophenol (DCIP). ( E Q , +217 mV), phenazine methosulfate (PMS) ( E Q , +80 mV), phenazine e t h o s u l f a t e (PES) ( E q, +55 mV), duroquihone ( E Q , +10 mV), pyocyanine ( E q, -34 mV), and 2-hy-droxy-1,4-naphthoquinone (E , -145 mV). 45 A n a l y s i s of p o t e n t i o m e t r i c data The r e l a t i o n s h i p between the p o t e n t i a l of the medium and the r a t i o of the o x i d i z e d to the reduced form of any o x i d -i z a b l e component i s (138) : E h = E m + (RT/nF)ln<ox/red.. where E^ i s the e l e c t r i c a l p o t e n t i a l measured r e l a t i v e to the -hydrogen e l e c t r o d e ; E m i s the midpoint p o t e n t i a l of the o x i d -i z a b l e component; R i s the u n i v e r s a l gas constant; F i s Far-aday's constant; T i s the absolute temperature; n i s the number of e l e c t r o n s t r a n s f e r r e d from the reduced (red) to the o x i d i z e d form. (ox). For a one e l e c t r o n t r a n s f e r at 22°G, when the p o t e n t i a l s are expressed i n m i l l i v o l t s t h i s expression s i m p l i f i e s to E, = E + 5 9 l o g ox/red . h m & i Therefore f o r a s i n g l e homogeneous component a p l o t of E^ ver-sus l o g ox/red should give a s t r a i g h t l i n e with:.; .a slope of 59 mV and an i n t e r c e p t on the E^ a x i s at the value of E^ where l o g ox/red =0. In t h i s manner the midpoint oxidation-reduc-t i o n p o t e n t i a l s of the cytochrome components of E. c o l i were determined. When there were two components w i t h d i f f e r e n t p o t e n t i a l s absorbing at the same wavelength the composite curve was s i g -moidal i n nature. The p l o t of E^ versus percent r e d u c t i o n i n t h i s case demonstrated two sigmoidal steps and i f each compo-nent d i f f e r e d s u f f i c i e n t l y i n t h e i r midpoint oxidation-reduc-t i o n p o t e n t i a l s these curves could be r e s o l v e d a r i t h m e t i c a l l y . 46 The p l o t of versus l o g ox/red f o r the r e s o l v e d components now showed a l i n e a r r e l a t i o n s h i p and the i n d i v i d u a l midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l s could be determined. Low Temperature d i f f e r e n c e spectroscopy A P e r k i n Elmer/Hitachi Model 356 spectrophotometer was used w i t h the cryogenic u n i t designed f o r t h i s instrument. P a r t i c l e s were suspended i n 0.05 M potassium phosphate b u f f e r , pH 7.0, c o n t a i n i n g 1.0 M sucrose and placed i n two 3 mm path-length cuvettes arranged side by side i n a metal cuvette h o l -der. Each compartment h e l d approximately 1.0 ml of the p a r t i -c l e suspension. To obta i n the reduced versus o x i d i z e d d i f f e r -ence spectrum NADH (10 p l of 0.2 M), succinate (10 p l of 0.1 M) , ascorbate (10 P l of 0.5 M) plus PMS (1 p l of 2 mM), or d i -t h i o n i t e (approximately 1 mg) was added to the sample compart-ment of the cuvette. A few c r y s t a l s (approximately 1 mg) of potassium f e r r i c y a n i d e were sometimes added to the reference compartment of the cuvette to maintain the o x i d i z e d s t a t e , otherwise the reference compartment contained the a i r - o x i d i -zed sample. The cuvette holder was immersed i n l i q u i d N 2 (77°K) a f t e r s u f f i c i e n t time had been allowed f o r the p a r t i -c l e suspension i n the sample compartment to become anaerobic. A f t e r 3-4 minutes, i n which time the sample had cooled to the temperature of l i q u i d N 2, the cuvette was removed and heated w i t h a 200 watt incandescent bulb f o r 3 minutes to d e v i t r i f y the sample (139),. replaced-in the cryogenic compartment of the 47 spectrophotometer, and again cooled to the temperature of l i -q uid The spectrum was scanned at 60 nm/min using a band width of 1.0 nm. To o b t a i n higher r e s o l u t i o n the spectrum was rescanned using a band width of 0.4 nm and a scanning speed of 10 nm/min. The cytochromes were trapped i n the aerobic steady s t a t e by immersing the cuvette holder i n l i q u i d N2 immediately a f t e r the a d d i t i o n of the substrate. Trapping-of an intermediate species i n the o x i d a t i o n - r e d u c t i o n  c y c l e of cytochrome d A P e r k i n Elmer/Hitachi model 356 spectrophotometer w i t h the cryogenic attachment was employed f o r the spectroscopic a n a l y s i s at subzero temperatures. R e s p i r a t o r y p a r t i c l e s pre-pared from c e l l s grown on succinate to the s t a t i o n a r y phase were suspended i n 0.05 M potassium phosphate b u f f e r , pH 7.0, and 50% ethylene g l y c o l . Ethylene g l y c o l , which had been shown not to i n t e r f e r e w i t h e l e c t r o n transport even at higher concentrations (140) was present to prevent f r e e z i n g of the samples at temperatures as low as -38°C. The p a r t i c l e sus-pension was aerated by mixing i n a i r at room temperature and 1.0 ml was then placed i n both the reference and the sample compartment of the cuvette holder. The pathlength of the cu-v e t t e employed was 3 mm. The cuvette holder was immersed i n the dewar f l a s k of the cryogenic system which contained ethan-o l cooled w i t h dry i c e to give the d e s i r e d temperature of 1°C to -38°C. The temperature of the sample was measured d i r e c t -l y using a Yellow Springs Instrument Co. model 42SC t e l e - t h e r -43 mometer. A f t e r e q u i l i b r a t i o n f o r 10-15 min at the d e s i r e d temperature, 1 5 i u l of 0.25 M ascorbate i n 50% ethylene g l y c o l and 10 y l of 1 mM phenazine methosulfate i n 50% ethylene g l y -c o l were added to the sample cuvette. The d i f f e r e n c e spectra of the aerobic steady s t a t e were scanned against the o x i d i z e d reference compartment immediately and L'C. at 2-3 min i n t e r v a l s t h e r e a f t e r to ensure that a steady s t a t e had been reached. The temperature i n the sample cuvette during the experiment was s t a b l e to w i t h i n + 1°C. At temperatures above 1°C the system became anaerobic too q u i c k l y so that the aerobic stea-dy s t a t e d i f f e r e n c e spectrum could not be obtained. To o b t a i n absolute spectra of the o x i d i z e d cytochrome d the o x i d i z e d sample was scanned against a sample reduced w i t h d i t h i o n i t e and co n t a i n i n g 20 mM KCN. The cyanocytochrome d so formed i n the reference sample has no absorption bands i n the 600-700 nm range (141, 142). When the i n h i b i t o r HOQNO (11 mM i n EtOH) was used i t was added p r i o r to c o o l i n g the samples. A l l s pectra were normalized to the b a s e l i n e . Reduction k i n e t i c s The redox s t a t e of the b-type cytochromes was followed using the Pe r k i n - E l m e r / H i t a c h i spectrophotometer i n the dual wavelength mode of opera t i o n at a wavelength p a i r of 558-540 hm. To f o l l o w the re d u c t i o n k i n e t i c s of cytochrome d the wavelength p a i r of 628-605 nm was used. NADH ( f i n a l concen-49 t r a t i o n , 2 mM) or succinate ( f i n a l c o n centration, 1.67 mM) was added to the cuvette c o n t a i n i n g 1.5 ml of membrane p a r t i -c l e suspension i n the presence or absence of the i n h i b i t o r , HOQNO (0-110 uM). The time course of the r e d u c t i o n of the cytochrome was followed at 22°C w i t h a Perkin-Elmer chart recorder operating at 60 nm or 120 nm/min. The 100% reduc-t i o n l e v e l of the cytochromes was determined by adding a few c r y s t a l s of sodium d i t h i o n i t e to the cuvette. In studying the e f f e c t of a d d i t i o n of two substrates c o n s e c u t i v e l y on the r e d u c t i o n of the cytochromes the second substrate was ad-ded by a r a p i d plunger device ( H i t a c h i ) . HOQNO was d i s s o l v e d i n 0.01 N NaOH at a concentration of 1.1 mM. Determination of p r o t e i n P r o t e i n was determined by the method of Lowry et al„ (143). 50 PART I. THE INHIBITION BY CYANIDE OF THE RESPIRATORY CHAIN OF E. COLI AND THE PROPERTIES OF CYTOCHROME d. RESULTS P r o p e r t i e s of p a r t i c l e s prepared from c e l l s grown, to the expo-n e n t i a l and s t a t i o n a r y phases of growth. I t i s known that i n the t r a n s i t i o n from exponential to s t a t i o n a r y growth the l e v e l s of the cytochromes i n E. c o l i change (9). The upper curves i n F i g . 2 show the t y p i c a l r e -duced versus o x i d i z e d d i f f e r e n c e spectra of p a r t i c l e s pre-pared from c e l l s grown to the exponential and s t a t i o n a r y phas-es of growth. The peaks at 628 nm, 594 nm, 560 nm, 530 nm, and 428 nm are due to the alpha absorption bands of reduced cytochromes d, a-^ , b^, the beta band of reduced cytochrome b^, and the composite Soret band of these cytochromes, r e s p e c t i v e -l y . The trough at 648 nm i s due to the absorption of the o x i -dized form of cytochrome d and that at 456 nm i s due to the absorption of the o x i d i z e d f l a v i n s and nonheme i r o n . I t should be noted that the l e v e l s of cytochromes d, a-^ , and b^ a l l increased i n the t r a n s i t i o n to s t a t i o n a r y growth, w i t h the greatest s p e c t r a l l y observable increase i n cytochrome d. In the exponential phase of aerobic growth the nature of the growth substrate d i d not a f f e c t the r e l a t i v e amounts of the cytochromes. However, when glucose was used as the growth substrate the o v e r a l l l e v e l of the cytochromes were s l i g h t l y lower, perhaps through c a t a b o l i t e r e p r e s s i o n (130). 51 FIGURE 2. D i f f e r e n c e spectra of p a r t i c l e s prepared from c e l l s grown to the exponential (L) and s t a t i o n a r y (S) phases of growth. The two upper curves are d i t h i o n i t e reduced versus a i r o x i d i z e d d i f f e r e n c e spectra. The two lower curves are the d i t h i o n i t e reduced plus carbon monoxide versus d i t h i o n i t e r e -duced d i f f e r e n c e spectra. The c e l l s were grown on succinate and harvested i n e i t h e r the exponential or s t a t i o n a r y phase of growth. P a r t i c l e s prepared from these c e l l s grown to the ex-p o n e n t i a l phase of growth and the s t a t i o n a r y phase of growth were suspended i n phosphate b u f f e r , pH 7.0, to a conce n t r a t i o n of 6.0 mg protein/miland 4.8 mg p r o t e i n / m l , r e s p e c t i v e l y . Spectra were scanned at 120 nm/min. 51a 4 0 0 5 0 0 6 0 0 7 0 0 W a v e I e n a t h » n m 52 Cytochrome o can only be detected by i t s carbon monox-ide d i f f e r e n c e spectrum as shown i n the lower curves of F i g . 2. A considerable d i f f e r e n c e can be seen i n the carbon mono-xid e d i f f e r e n c e spectra of p a r t i c l e s from exponential and s t a t i o n a r y phase c e l l s . The absorption peaks at 567 nm and 415 nm and the troughs at 555 nm and 430 nm are due to the reduced plus carbon monoxide versus reduced d i f f e r e n c e spec-trum of cytochrome o. The absorption peak at 641 nm, and p o s s i b l y 537 nm, and the troughs at 620 nm and 443 nm are due to the reduced plus carbon monoxide versus reduced d i f f e r -ence spectrum of cytochrome d. I t i s obvious that cytochrome o dominates the carbon monoxide d i f f e r e n c e spectrum of p a r t i -c l e s from c e l l s harvested i n the exponential phase of growth and cytochrome d i s most prominent i n the carbon monoxide d i f -ference spectrum of p a r t i c l e s prepared from c e l l s grown to the s t a t i o n a r y phase. The l e v e l s of the cytochromes and the NADH and su c c i n -ate oxidase a c t i v i t i e s of these p a r t i c l e s are summarized i n Table 1. I t was c l e a r that the content of cytochrome d i n s t a t i o n a r y c e l l s was much greater than cytochrome o, whereas i n exponential c e l l s they were at s i m i l a r concentrations. The l e v e l s of the cytochromes, other than cytochrome o, i n -creased i n the t r a n s i t i o n to s t a t i o n a r y growth; however the NADH and succinate oxidase a c t i v i t i e s measured r e l a t i v e to the p a r t i c l e p r o t e i n content were approximately the same as those i n exponential phase c e l l s . 53 TABLE 1 PROPERTIES OF PARTICLES PREPARED FROM CELLS GROWN TO THE STAT-IONARY AND EXPONENTIAL PHASES OF GROWTH. Oxidase a c t i v i t i e s (ng atom 0 sec "*"/mg p r o t e i n ) NADNADH succinate Exponential 0.038 0.36 0.076 0.066 7.1 0.41 St a t i o n a r y 0.25 0.96 0.64 0.0'?/ 5.7 0.53 Cytochrome content P a r t i c l e s (nmoles/mg p r o t e i n ) a l a l ^1 ^ c 54 K i n e t i c s of i n h i b i t i o n of NADH oxidase by cyanide. There are two p o t e n t i a l cytochrome oxidases, cyto:r~ chromes d and o, i n E. c o l i . According to Castor and Chance (11), cytochrome o i s the predominant f u n c t i o n a l oxidase formed during the exponential phase of growth, whereas cyto-chrome d becomes s i g n i f i c a n t during the s t a t i o n a r y phase. As an i n i t i a l step i n the study of the o r g a n i z a t i o n of the cyto-chromes, p a r t i c u l a r l y cytochromes d and o, ""I examined the k i n -e t i c s of i n h i b i t i o n by cyanide of the NADH oxidase system i n E. c o l i c e l l s grown to contain d i f f e r e n t l e v e l s of cytochromes d and o. The mechanism of i n h i b i t i o n by cyanide, which acts at the l e v e l of the te r m i n a l oxidase, i s not known. I t was thought that the v a r i a t i o n i n the l e v e l of the te r m i n a l o x i -dases would a l t e r the s e n s i t i v i t y to i n h i b i t i o n by cyanide. The progress of NADH o x i d a t i o n i n the presence of d i f -f e r e n t concentrations of cyanide f o r p a r t i c l e s prepared from both exponential and s t a t i o n a r y phase c e l l s i s shown i n F i g . 3. The o x i d a t i o n of NADH by r e s p i r a t o r y p a r t i c l e s from expo-n e n t i a l c e l l s was more s e n s i t i v e to i n h i b i t i o n by KCN than was the oxidase a c t i v i t y from c e l l s grown to the s t a t i o n a r y phase. The curves i n d i c a t e d that there was progre s s i v e i n h i -b i t i o n of NADH oxidase a c t i v i t y w i t h time. P r e i n c u b a t i o n of the p a r t i c l e s w i t h KCN d i d not e l i m i n a t e t h i s progressive i n -h i b i t i o n . I t i s known that cyanide reacts w i t h NAD + to form a complex which a l s o absorbs at 340 nm (110). Under the con-d i t i o n s of my experiments t h i s occurs too slow l y to i n t e r f e r e 55 FIGURE 3. E f f e c t of concentration of KCN on the o x i d a t i o n of NADH by p a r t i c l e s prepared from c e l l s grown to the exponen-t i a l (L) or s t a t i o n a r y phase of growth (S). The absorbance (A) due to NADH was measured at 340 nm. In the curves marked -p the r e s p i r a t o r y p a r t i c l e s were preincubated at 22°C w i t h KCN f o r 5 minutes before'NADH was added. P a r t i c l e p r o t e i n ; L, 0.34 mg/ml; S, 0.49 mg/ml. 55a 56 w i t h the apparent k i n e t i c s of HADH o x i d a t i o n . The k i n e t i c s of i n h i b i t i o n were examined by a number of methods. Lineweaver-Burk a n a l y s i s provided no i n s i g h t i n -to the mechanism. F i g . 4 shows the type of p l o t obtained, where V was determined by drawing tangents to the slopes i n F i g . 3 at various p o i n t s during the progress of o x i d a t i o n . Substrate c o n c e n t r a t i o n , S, was c a l c u l a t e d at each of these p o i n t s from the corresponding absorption at 340 nm due to NADH. There was i n c r e a s i n g i n h i b i t i o n w i t h i n c r e a s i n g concen-t r a t i o n . However, the breaks i n the curves and the changing i n t e r c e p t s were not r e a d i l y i n t e r p r e t a b l e . When the curves i n F i g . 3 were analysed f o r a f i r s t or-der dependence of the r a t e of r e a c t i o n on the concentration of NADH by p l o t t i n g the lo g a r i t h m of the absorbance at 340 nm versus time, s t r a i g h t l i n e s were not obtained ( F i g . 5). These curves again d i d not provide any i n s i g h t i n t o the mechanism of i n h i b i t i o n so an a l t e r n a t i v e method of a n a l y s i s was adop-ted. The i n i t i a l rates of NADH o x i d a t i o n were determined a f t e r the r e s p i r a t o r y p a r t i c l e s had been preincubated f o r f i v e minutes w i t h KCN. The Dixon p l o t of the data (1/V versus concentration of KCN) f o r two d i f f e r e n t concentrations of NADH i s shown i n F i g . 6. P a r a l l e l l i n e s were observed i n d i c a t i n g that cyanide combines w i t h the NADH oxidase system during the pre i n c u b a t i o n phase and acts as an uncompetitive i n h i b i t o r . The number of b i n d i n g s i t e s f o r cyanide i n the pr e i n c u b a t i o n 57 FIGURE 4. Lineweaver-Burk a n a l y s i s of the k i n e t i c s of i n h i b i -t i o n of NADH oxidase a c t i v i t y i n p a r t i c l e s grown on glucose to the exponential phase of growth. V e l o c i t i e s (V, nmoles/min) were determined from the tangents to the curves s i m i l a r to those i n F i g . 3 obtained at d i f f e r e n t concentrations of cyan-i d e . (mM) . The corresponding c o n c e n t r a t i o n of NADH (S, mM) at the tangent was c a l c u l a t e d from the absorbance at 340 nm. P r o t e i n c o ncentration, 0.36 mg/ml. 57a 1 0 2 0 1 / S 58 FIGURE 5. F i r s t order a n a l y s i s of the o x i d a t i o n of N A D H by p a r t i c l e s i n the presence and absence of d i f f e r e n t concentra-t i o n s of cyanide. The logarithm of the absorbance at 3 4 0 nm ( A ^ ^ Q) was c a l c u l a t e d from t r a c e s s i m i l a r to those i n F i g . 3 . The p a r t i c l e suspension ( 0 . 3 6 mg protein/ml) was preincubated f o r 5 minutes w i t h the concent r a t i o n of cyanide i n d i c a t e d (mM) p r i o r to the a d d i t i o n of N A D H . 58a 0 , 2 . 0 ' 1 1 ' 1 L 1 2 3 4 5 M i n u t e s 59 FIGURE 6. Dixon p l o t of the k i n e t i c s of i n h i b i t i o n by cyanide of the i n i t i a l r a t e s of NADH oxidase a c t i v i t y . The p a r t i c l e s were prepared from c e l l s grown on succinate to the exponential phase of growth and stored at -20°C. P a r t i c l e s i n "TM" b u f f e r suspended to a concent r a t i o n of 0.40 mg protein/ml were p r e i n -cubated w i t h cyanide p r i o r to the a d d i t i o n of 0.082 mM NADH ( s o l i d squares) or 0.164 mM NADH (open c i r c l e s ) . The i n i t i a l v e l o c i t i e s (V) (umoles/min) were c a l c u l a t e d from the tangent to the NADH o x i d a t i o n curve immediately a f t e r a d d i t i o n of NADH. 59a 60 p e r i o d was determined from a H i l l p l o t of the i n i t i a l r a t e s ( F i g . 7). The t h e o r e t i c a l slope of n=l could be drawn through the p o i n t s f o r both concentrations of NADH i n d i c a t i n g that one type of bi n d i n g s i t e could account f o r the i n h i b i t i o n by cyan-ide of NADH oxidase a c t i v i t y during the pr e i n c u b a t i o n p e r i o d . These These analyses d i d not e x p l a i n the pro g r e s s i v e i n h i b i -t i o n by cyanide of NADH oxidase a c t i v i t y during the o x i d a t i o n of NADH. A p o s s i b l e explanation f o r t h i s p r o g r e s s i v e i n h i b i -t i o n might be that the r a t e of r e a c t i o n of cyanide with;the NADH oxidase system to form an i n h i b i t e d complex was r a t e l i m -i t i n g and could occur r a p i d l y only during the o x i d a t i o n of NADH. I f t h i s hypothesis i s c o r r e c t the r a t e of NADH oxida-t i o n at any p a r t i c u l a r time (e.g., tangent to the curve i n F i g . 3 at that time) would be dependent on the amount of un-i n h i b i t e d enzyme which was present at that time. For a p a r t i -c u l a r concentration of KCN a graph of l o g (rate of NADH oxida-t i o n ) versus time should y i e l d a s t r a i g h t l i n e i n which slope of l i n e = [KCN] + k_ 1 where k^ and k ^ are the v e l o c i t y constants f o r the r e a c t i o n of u n i n h i b i t e d NADH oxidase (E) w i t h cyanide E + HCN — ^ E.HCN k - l The v e l o c i t y constants k-^  and k_-^  can be evaluated by p l o t t i n g the slope of the l i n e versus KCN . The data of F i g . 3 were analysed i n t h i s manner ( F i g . 8 and 9). As shown i n F i g . 8 f o r p a r t i c l e s prepared both from exponential (grown on l a c t a t e , succinate and glucose) and s t a -61 FIGURE 7. E f f e c t of concentration of KCN on the i n i t i a l r a t e of o x i d a t i o n of NADH by p a r t i c l e s prepared from c e l l s grown on succinate medium. S o l i d p o i n t s , 0.16 mM NADH; open p o i n t s , 0.32 mM NADH. v , i n i t i a l r a t e of NADH o x i d a t i o n (change i n absorbance at 340 nm/min) i n absence of KCN^ ; - v, i n i t i a l r a t e of NADH o x i d a t i o n i n presence of KCN. The l i n e s are the c a l -c u l a t e d l i n e s f o r the bi n d i n g of 1 molecule c y a n i d e / s i t e . P a r t i c l e p r o t e i n , 0.42 mg/ml. 61a L o g [ K C N ] 62 FIGURE 8. E f f e c t of concentration of KCN on the o x i d a t i o n of NADH by p a r t i c l e s prepared from c e l l s grown to the expo-n e n t i a l (L) or s t a t i o n a r y (S) phase of growth. From the da-t a of F i g . 3 the ra t e NADH o x i d a t i o n (change i n absorbance at 340 nm/min) was determined at various times. In the curves marked -p the r e s p i r a t o r y p a r t i c l e s were preincubated at 22°C w i t h KCN f o r 5 minutes before NADH was added. 62a 63 FIGURE 9. E f f e c t of concentration of KCN on the o x i d a t i o n of NADH by p a r t i c l e s prepared from c e l l s grown on glucose (G), l a c t a t e ( L ) , succinate (S) or complex (C) medium. The slopes of the l i n e s shown i n F i g . 8, or from data graphed i n a s i m i -l a r manner, are p l o t t e d versus c o n c e n t r a t i o n of KCN present. The s o l i d p o i n t s were obtained from experiments i n which the r e s p i r a t o r y p a r t i c l e s were preincubated at 22 SC w i t h KCN f o r 5 minutes before NADH was added. 63a K C N ( m M ) O 10 20 K C N ( m M ) 64 t i o n a r y phase c e l l s (grown on complex media), the curves shown i n F i g . 3 could be explained by a time-dependent r e a c t i o n of the i n h i b i t o r , cyanide, w i t h the enzyme to give an i n h i b i t e d complex. The l i n e s i n F i g . 8 deviated from l i n e a r i t y once 80-90?o of the oxidase a c t i v i t y had been i n h i b i t e d . This could be due to the presence of an a l t e r n a t e , c y a n i d e - i n s e n s i t i v e pathway. I f the r e s p i r a t o r y p a r t i c l e s were preincubated at 22°C w i t h cyanide f o r 5 to 20 minutes p r i o r to a d d i t i o n of NADH there was some i n h i b i t i o n of NADH oxidase a c t i v i t y . How-ever, the r a t e of r e a c t i o n of the enzyme w i t h cyanide i n the presence of NADH was unaffected by the pr e i n c u b a t i o n ( F i g . 8 and 9). That i s , the v e l o c i t y constants f o r t h i s r e a c t i o n were unaffected. The v e l o c i t y constants, k^uand k ^ f o r the r e a c t i o n of cyanide w i t h the u n i n h i b i t e d NADH oxidase system of c e l l s grown to the exponential and s t a t i o n a r y phases on glucose, l a c t a t e , s u c c i n a t e , and complex media were evaluated from the slope and i n t e r c e p t , r e s p e c t i v e l y , of the type of p l o t shown i n F i g . 9 and were used to c a l c u l a t e an i n h i b i t o r constant, (Table 2). Summarizing the data, c e l l s grown to the expo--1 -1 n e n t i a l phase of growth demonstrated a o f 1,3 + 0.3 M s , a^&_1 of 1.8 x 10" 3 + 0,2 x 1 0 " 3 s - 1 , and a K. of 1.4 + 0.3 mM, while c e l l s grown to the s t a t i o n a r y phase of growth e x h i b i t e d a k x of 0.26 + 0.05 M" 1s" 1, a k_± of 2.0 x 10" 3 4- 0.5 x 10" 3 s-''", and a of 7.8 + 0.6 mM f o r the r e a c t i o n of cyanide w i t h the NADH oxidase system. The lower s e n s i t i v i t y to cyanide i n -65 TABLE 2 KINETIC CONSTANTS FOR EFFECT OF KCN ON NADH OXIDASE 3 Growth Cytochrome c o n t e n t 0 k ] k _ i k - l medium 3 b l a 2 o o J (M - 1£ L (s ) x l 0 ~ n 3 (mM) Glucose 0. ,35 0. 080 0. 081 1. 0 1. ,13 1 .6 1 .4 Glucose 0, ,23 0. 056 0. 046 1. 3 1. ,03 1 .7 1 .7 Glucose*^ 0. ,25 0. 062 0. 047 1. 3 1, ,12 1 .8 1 .6 Eacfcate 0, .46 0. 14 0. 0834 1. 7 1, ,56 1 .7 1 .1 1. ,28 2 .1 1 .6 Succinate 0, .38 0. 092 0. 065 1. 4 1. ,82 1 .7 0 .92 Complex 1, ,08 0. 69 0. 009 81 0. ,27 2 .2 8 .2 0. ,22 1 .6 7 .5 Complex 0. ,84 0. 55 0, ,22 1 .4 6 .9 Complex 0. ,96 0. 68 0 0. ,33 2 .7 8 .5 K i n e t i c constants were determined by the g r a p h i c a l a n a l -y s i s shown i n FIG. 3, 8, 9. Cytochromes were measured as des-c r i b e d i n MATERIALS AND METHODS. ^ C e l l s grown on glucose and l a c t a t e were harvested at mid exponential phase and on succinate at l a t e exponential phase, and on complex medium during the s t a t i o n a r y phase of growth. nmoles/mg p r o t e i n No f e r r i c c i t r a t e i n growth medium. 66 i n h i b i t i o n of c e l l s grown to the s t a t i o n a r y phase of growth appeared to c o r r e l a t e w i t h the much higher p r o p o r t i o n of cy-tochrome d r e l a t i v e to cytochrome o i n these c e l l s i n con-t r a s t w i t h the almost equal l e v e l s of these cytochromes i n c e l l s grown to the exponential phase of growth. S p e c t r a l studies of the r e a c t i o n of cyanide w i t h the r e s p i r a -t o r y chain. Since the k i n e t i c experiments had suggested that there was a q u a n t i t a t i v e l y d i f f e r e n t response to cyanide de-pending on the r e l a t i v e amounts of cytochrome d or of cyto-chrome O j we examined the reduced plus cyanide minus reduced d i f f e r e n c e s p e c t r a of r e s p i r a t o r y p a r t i c l e s prepared from exponential and s t a t i o n a r y phase c e l l s to see i f a r e a c t i o n of these cytochromes w i t h cyanide could be demonstrated ( F i g . 10). In the presence of 13.6 mM cyanide d i t h i o n i t e - r e d u c e d r e s p i r a t o r y p a r t i c l e s from c e l l s grown to the s t a t i o n a r y phase showed a l o s s of absorption bands due to reduced cyto-chrome d (troughs at 628 ,nm and 442 nm) and of a b or c-type cytochrome (troughs at 555 nm and 423 nm). When NADH was used as the reductant the absorption due to the b or c-type cytochrome was not e l i m i n a t e d by cyanide although cyto-chrome d behaved as before. The s o l u t i o n had to be shaken i n a i r f o r the absorption band of reduced cytochrome d to be completely e l i m i n a t e d i n the presence of cyanide. I f the spectra were measured under anaerobic c o n d i t i o n s a d d i t i o n of 67 FIGURE 10. E f f e c t of KCN on reduced cytochromes of membrane p a r t i c l e s prepared from c e l l s grown to the exponential (A,B) or s t a t i o n a r y (C,D) phase of growth. Curves A and C, base-l i n e s curves B and D, d i t h i o n i t e - r e d u c e d minus d i t h i o n i t e -reduced d i f f e r e n c e spectrum f o l l o w i n g a d d i t i o n of 13.6 mM KCN to sample cuvette. The s o l u t i o n i n the sample cuvette was then gently shaken i n a i r to f u l l y develop the spectrum. P a r t i c l e p r o t e i n : curves A and B, 7 mg/ml; curves C and D, 5. 9 mg/ml. 4 0 0 5 0 0 6 0 0 7 0 0 W a v e l e n g t h - n m 68 cyanide d i d not discharge the absorption band of reduced cyto-chrome d. When the same experiment was c a r r i e d out using par-t i c l e s prepared from exponential phase c e l l s the absorption band of cytochrome d e l i m i n a t e d i n the presence of cyanide was much smaller than w i t h s t a t i o n a r y phase c e l l s . This would be expected from the much lower amount of t h i s cytochrome present i n the former c e l l s . The absorption bands of a b-cytochrome (trough at 428 nm) a l s o was l e s s i n the presence of cyanide but the decrease was smaller than w i t h the s t a t i o n a r y phase c e l l s . Moreover, the absorption maximum was d i s p l a c e d towards higher wavelengths. C a r e f u l examination of the Soret peak of t h i s cytochrome revealed a shoulder at about 423 nm w i t h the exponential phase c e l l s , w h i l e there was a shoulder at about 428 nm w i t h the s t a t i o n a r y phase c e l l s . Thus, the Soret band probably contains two components w i t h maximum absorption at 423 and 428 nm, the r e l a t i v e amounts of which d i f f e r w i t h the growth phase. Figure 11 shows the reduced minus o x i d i z e d d i f f e r -ence spectrum i n the a-band region of membrane p a r t i c l e s from s t a t i o n a r y phase E. c o l i . Absorption peaks at 559 nm, 593 nm, and 628 nm showed that cytochromes b^, a-^ , and d were present. When cyanide was added to the open cuvette c o n t a i n i n g the d i -t h i o n i t e - r e d u c e d suspension there was a p a r t i a l disappearance of the absorption band at 628 nm due to reduced cytochrome d. When the system became anaerobic there was no f u r t h e r decrease 69 FIGURE 11. E f f e c t of KCN on the reduced minus o x i d i z e d d i f -ference spectrum of p a r t i c l e s prepared from c e l l s grown to the s t a t i o n a r y phase. Curve A, d i t h i o n i t e reduced minus o x i d i z e d d i f f e r e n c e spectrum; curve B, f o l l o w i n g a d d i t i o n of 13.6 mM •KCN to reduced p a r t i c l e s ; curve C, same as B but the subsequent shaking of s o l u t i o n i n a i r ; curve D, b a s e - l i n e . P a r t i c l e pro-t e i n , 5.9 mg/ml. 69a 70 i n the s i z e of t h i s . peak. A e r a t i o n was re q u i r e d to cause com-p l e t e e l i m i n a t i o n of t h i s band. I f cyanide was added to the reduced cytochromes i n an anaerobic cuvette no e f f e c t was seen on the absorption spectrum, even a f t e r prolonged i n c u b a t i o n . However, when the cuvette was subsequently aerated there was a r a p i d disappearance of the 628 nm band without the formation of the 648 nm band. The r e l a t i o n s h i p between the o x i d i z e d and reduced absorption  bands of cytochrome d The r e l a t i o n s h i p between the absorption band at 648 nm i n the spectrum of the o x i d i z e d cytochrome and that at 628 nm found a f t e r r e d u c t i o n w i t h substrate or d i t h i o n i t e was not c l e a r . When a l i m i t i n g amount of substrate (ascorbate i n the presence of phenazine methosulfate, NADH, or succinate) was added to an anaerobic suspension of membrane p a r t i c l e s pre-pared from s t a t i o n a r y phase c e l l s which had been starved to deplete endogenous s u b s t r a t e s , the 648 nm band disappeared w e l l i n advance of the appearance of the 628 nm band. Figure 12 shows the appearance of the reduced cytochrome components scanned against an o x i d i z e d reference sample at i n t e r v a l s af-t e r the a d d i t i o n of ascorbate i n the presence of phenazine methosulfate. The i n c r e a s i n g s i z e of the trough at 648 nm i n -d i c a t e s the disappearance of t h i s absorption i n the sample be-in g reduced. When s a t u r a t i n g l e v e l s of substrate were used the r e d u c t i o n of the Cytochromes occurred too q u i c k l y at an-71 FIGURE 12. Reduction of the cytochromes i n membrane p a r t i c l e s by a l i m i t i n g amount of substrate. Ascorbate (16 uM) i n the presence of PMS (5jiyM) was added to a sealed cuvette c o n t a i n -i n g an anaerobic suspension of p a r t i c l e s (6.4 mg protein/ml) prepared from c e l l s grown to the s t a t i o n a r y phase on succinate medium. The c e l l s were starved p r i o r to p r e p a r a t i o n of the p a r t i c l e s to l i m i t r e d u c t i o n by endogenous substrates. The spectrum of the alpha band re g i o n was scanned against an o x i -d i z e d reference sample at timed i n t e r v a l s a f t e r a d d i t i o n of the substrate. 71a 72 a e r o b i o s i s f o r the r a t e of appearance and disappearance of the two forms of cytochrome d to be d i f f e r e n t i a t e d . Figure 13 shows the r e l a t i v e s t a t e s w i t h time a f t e r the a d d i t i o n of ascorbate-PMS, NADH, and succinate of the trough at 648 nm ( o x i d i z e d cytochrome d), and the absorption peaks at 628 nm (reduced cytochrome d), 594 nm (reduced cyto-chrome a^) , and 560 nm (reduced cytochrome b-^ ) . With a l l sub-s t r a t e s the 648 nm band disappeared w e l l i n advance of the ap-pearance of the 628 nm band. This suggested that the 628 nm band was not a d i r e c t r e d u c t i o n product of the component w i t h the 648 nm absorption bands and that there may be an intermedi-ate species i n the normal o x i d a t i o n - r e d u c t i o n c y c l e of cyto-chrome d which may l a c k a d i s t i n c t absorption band i n the a l -pha band region. Under the c o n d i t i o n s used i n the experiments shown i n Fig.112 and F i g . 13 e l e c t r o n s are fed i n t o the cytochrome sys-tem very slowly t h e r e f o r e e q u i l i b r i u m r a t h e r tftantRinetor ' c i vfactors determine the d i s t r i b u t i o n of e l e c t r o n s i n the r e s p i r a t o r y chain. The r e l a t i v e l e v e l s of r e d u c t i o n should be an i n d i c a -t i o n of the r e l a t i v e o x i d a t i o n - r e d u c t i o n p o t e n t i a l s of the i n -d i v i d u a l cytochrome components assuming t h i s s i t u a t i o n . The r e d u c t i o n step i n v o l v i n g the disappearance of the 648 nm ab-s o r p t i o n band appeared to have a mid-point p o t e n t i a l of 40-80 mV higher than the r e d u c t i o n step r e s u l t i n g i n the appearance of the 628 nm absorption band. This d i f f e r e n c e was c a l c u l a t e d using the Nernst equation, on the assumption that there were two one-electron r e d u c t i o n steps i n the o x i d a t i o n - r e d u c t i o n 73 FIGURE 13. Reduction of the cytochromes i n membrane p a r t i c l e s by l i m i t i n g amounts of substrate. Ascorbate (16 uM) i n the presence of PMS (5 {iM) , NADH (25 uM) , and succinate (25 uM) were added at 0 min to an anaerobic suspension of membrane par-t i c l e s i n a sealed cuvette and the spectra were scanned as i n F i g . 14. The percent r e d u c t i o n of each component was r e l a t e d to the l e v e l of r e d u c t i o n obtained a f t e r the a d d i t i o n of d i -t h i o n i t e . Ascorbate plus PMS, A; NADH, N; Succinate, S. 73a M i n u t e s 74 c y c l e of cytochrome d. The apparent midpoint p o t e n t i a l s of cytochromes a-^  and b-^  were much lower. The higher r e l a t i v e s t a t e of r e d u c t i o n of cytochrome a-^  seen i n F i g . 13 when ascor-bate^PMS was employed i n d i c a t e d that e l e c t r o n s from t h i s sub-s t r a t e are probably more a c c e s s i b l e to cytochrome a-^  than those from NADH or succinate. The absorption band at 675 nm due to an u n i d e n t i f i e d chromophore i n the o x i d i z e d form (see F i g . 12) disappeared upon re d u c t i o n l a t e r than the 648 nm ab-s o r p t i o n band but p r i o r to the appearance of the 628 nm absorp-t i o n band (data not shown). The nature of t h i s component i s not known. However, i t i s u n l i k e l y to be d i r e c t l y r e l a t e d to cytochrome d because i t s apparent l e v e l i n r e l a t i o n to the l e v e l of cytochrome d v a r i e d from p r e p a r a t i o n to pr e p a r a t i o n . P o t e n t i o m e t r i c t i t r a t i o n of cytochrome d The p o t e n t i o m e t r i c t i t r a t i o n of cytochrome d, shown i n F i g . 14, was c a r r i e d out employing the methods of Wilson and Dutton (134). The spectrum at various p o t e n t i a l s was scanned against an o x i d i z e d sample and the height above the b a s e l i n e f o r reduced cytochrome d at 628 nm was p l o t t e d ver-sus the p o t e n t i a l . The r e s u l t i n g sigmoidal curve was r e s o l -ved by the Nernst equation to give a midpoint o x i d a t i o n - r e -duction p o t e n t i a l of +260 mV. The t h e o r e t i c a l slope of n=l can be drawn through the r e s o l v e d p o i n t s i n d i c a t i n g that the r e a c t i o n i n v o l v e d the t r a n s f e r of one e l e c t r o n . The o x i d i z e d form w i t h an absorption band at 648 nm d i d not f o l l o w normal redox behaviour. When the redox p o t e n t i a l of the system was 75 FIGURE 14. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome d. P a r t i -c l e s were prepared from c e l l s grown on succinate to .the s t a -t i o n a r y phase of growth and suspended to a conce n t r a t i o n of 6.4 mg protein/ml i n 0.1 M phosphate b u f f e r , pH 7.0. The med-i a t o r s used were 50 yM PMS and 50 yM quinhydrone. Succinate (0.1 M) and f e r r i c y a n i d e (0.2 M) were used to reduce and o x i d -i z e the system, r e s p e c t i v e l y . The redox s t a t e of cytochrome d was determined from the height of the absorbance peak at 628 nm. The t h e o r e t i c a l l i n e f o r n=l i s drawn through the p o i n t s i n the lower f i g u r e . 75a 76 taken from 0 mV to higher p o t e n t i a l s , even at p o t e n t i a l s of over +400 mV the absorption band f o r t h i s o x i d i z e d form d i d not appear ( F i g . 15). Only f o l l o w i n g a d d i t i o n of pulses of water saturated w i t h oxygen or of ^2^2 t b e n m ^and appear. I t may be that the formation of t h i s form of cyto-chrome d from reduced cytochrome d re q u i r e s oxygen. The u n i d e n t i f i e d chromophore absorbing at 675 nm i n the o x i d i z e d form appeared to have a midpoint p o t e n t i a l of approximately +435 mV ( F i g . 16). The p o t e n t i o m e t r i c t i t r a t i o n data f o r the other cy-tochrome components of the r e s p i r a t o r y chain of E. c o l i are presented i n Part I I . K i n e t i c s of r e a c t i o n of cyanide w i t h cytochrome d The f o l l o w i n g experiments were done to c o r r e l a t e the k i n e t i c s of i n h i b i t i o n of the NADH oxidase system by cyanide w i t h the e f f e c t of the i n h i b i t o r on the absorption spectrum of cytochrome d. In a recent paper Kauffman and Van Gelder (144) r e -ported that cyanide reacted w i t h the o x i d i z e d form of cyto-chrome d i n A. v i n e l a n d i i . In E. c o l i we found that cyanide caused the 648 nm absorption band to disappear at a slow r a t e but without the appearance of another absorption band ( F i g . 17). From t h i s i t would appear that cyanide reacted w i t h o x i -d i z e d cytochrome d to give cyanocytochrome d which had no, or very l i t t l e , a bsorption i n the a-band region. The disappear-77 FIGURE 15. Behaviour of trough at 650 nm during the p o t e n t i o -metric t i t r a t i o n of cytochrome d. The same co n d i t i o n s as i n F i g . 14 were used. 77a 78 FIGURE 16. P o t e n t i o m e t r i c t i t r a t i o n of chromophore absorb-i n g at 675 - 680 nm i n the o x i d i z e d form. The same condi^ t i o n s as i n F i g . 14 were used. 78a . 0 0 8 6 8 0 . 0 0 4 79 FIGURE 17. E f f e c t of cyanide on the absorption spectrum of o x i d i z e d cytochrome d i n E. c o l i membrane p a r t i c l e s . The sam-pl e cuvette contained membrane p a r t i c l e s from E. c o l i NRC482 grown to the s t a t i o n a r y phase suspended to a concent r a t i o n of 6.0 mg protein/ml i n 0.1 M phosphate b u f f e r , pH 7.0, con t a i n -i n g 0.01 M MgC^. The reference cuvettedcpntained a suspen-s i o n of membrane p a r t i c l e s from E. c o l i grown to the ex-p o n e n t i a l phase to contain no absorption bands i n the 600-700 nm regi o n . The concentration of the p a r t i c l e s i n the r e f e r -ence cuvette was adjusted to c o r r e c t f o r t u r b i d i t y . The curves show the absorption spectrum of o x i d i z e d cytochrome d at 1, 4, 7, 10, 13, 16, 20, and 25 min a f t e r a d d i t i o n of 20 mM KCN to the sample cuvette. Curve B, b a s e - l i n e . Scan . speed, 120 nm/min. 79a 80 ance of the 648 nm band followed f i r s t - o r d e r k i n e t i c s f o r d i f f e r e n t cyanide concentrations ( F i g . 18). A second-order r a t e .constant (k-^) of 0.011 M~^ s~''" was determined from the slope of the s t r a i g h t l i n e obtained by p l o t t i n g the pseudo-f i r s t - o r d e r constants versus cyanide concentration. The v e l -o c i t y constant, k_-^ , f o r the d i s s o c i a t i o n of cyanocytochrome -5 -1 d was estimated to be 6.7 x 10 s from the i n t e r c e p t of . t h i s p l o t w i t h the ord i n a t e . Kauffman and Van Gelder (144) determined these constants f o r the r e a c t i o n of cyanide w i t h cytochrome d i n A. v i n e l a n d i i . Although the value f o r k_^ was s i m i l a r (5 x 10 s^""*") , a higher value f o r k^ was obtained (0.7 M'^s""*"). However, t h e i r experiments were c a r r i e d out at 33°C. The r a t e constant determined f o r the r e a c t i o n of cyanide w i t h the o x i d i z e d form of cytochrome d i s much too low to account f o r the i n h i b i t i o n of NADH oxidase. As shown i n a previous s e c t i o n , k-^  was found to be 0.26 M'^s-^. Since" our k i n e t i c studies of i n h i b i t i o n by cyanide were c a r r i e d out under c o n d i t i o n s of e l e c t r o n f l u x through the r e s p i r a t o r y chain, we examined the k i n e t i c s of cyanocyto-chrome d formation under turnover c o n d i t i o n s using NADH as substrate. An aerobic suspension of membrane p a r t i c l e s was used. Under these c o n d i t i o n s the disappearance of the 648 nm absorption band f o l l o w i n g the a d d i t i o n of cyanide was too f a s t to f o l l o w by rescanning of the spectrum. Therefore, the r e a c t i o n was measured by f o l l o w i n g the change i n absorption at 648 nm r e l a t i v e to 607 nm using a P e r k i n Elmer 356 spectro-81 FIGURE 18. Reaction of cyanide w i t h o x i d i z e d cytochrome d. Log of the absorbance at 648 nm (Ag^g) from the type of data shown i n F i g . 17 was p l o t t e d versus the time a f t e r the addi-t i o n of 0 - 20 mM cyanide (upper graph). The slopes (pseudo-f i r s t order constants) of the l i n e s i n the upper graph were p l o t t e d versus the concentration of cyanide (lower graph). The same co n d i t i o n s as i n F i g . 17 were used. 81a 1 0 2 0 [ K C N ] m M 82 photometer operating i n the dual wavelength mode. When cyan-ide was added to the aerobic suspension w i t h no substrate present there was a very slow decrease i n absorption at 648 nm. However, when 2 mM NADH was added to the suspension of p a r t i c l e s c o n t a i n i n g cyanide the 648 nm absorption band d i s -appeared r a p i d l y ( F i g . 19). The lower t r a c e shows that there was l i t t l e change i n the absorption at 648 nm i n the absence of cyanide u n t i l the system became anaerobic when cytochrome d was r a p i d l y reduced. In the traces where cyanide was pre-sent there were two phases of disappearance of the 648 nm absorption band. The f i r s t phase was due to the formation of cyanocytochrome d and was c h a r a c t e r i z e d by the disappear-ance of the 648 nm band without concomitantt appearance of the bandhat 628 nm due to reduced cytochrome d. The second phase occurred when the system became anaerobic through de-p l e t i o n of oxygen by NADH oxidase a c t i v i t y . In t h i s phase there was a cconrcomifantt appearance of the absorption band of reduced cytochrome d at 628 nm. The s i z e of the 628 nm band which appeared was d i r e c t l y r e l a t e d to the amount of o x i d i z e d cytochrome d unreacted w i t h cyanide present ataanaerofoiosis. P s e u d o - f i r s t order constants f o r the disappearance of o x i d i z e d cytochrome d were obtained at each concentration of. cyanide by p l o t t i n g l o g (absorbance increment at 648 nm r e l a t i v e to 607 nm) versus time f o r the r e a c t i o n o c c u r r i n g during the f i r s t phase of the curves shown i n F i g . 19 ( F i g . 20). A graph of these values against the concentration of 83 FIGURE 19. E f f e c t of cyanide on o x i d i z e d cytochrome d under turnover c o n d i t i o n s . E. c o l i membrane p a r t i c l e s were suspend-ed i n 0.1 M phosphate b u f f e r , pH 7.0, c o n t a i n i n g 0.01 M MgC^, to a concen t r a t i o n of 1.5 mg pro t e i n / m l . The absorbance at 648 nm r e l a t i v e to 607 nm was measured to f o l l o w the disappear-ance of o x i d i z e d cytochrome d. 2 mM NADH was added i n the pre-sence of 0, 12, 15, 17, 19, and 22 mM KCN. Progress of spec-t r a l change followed from r i g h t to l e f t . 83a 1 minute N A D H 2 2 K C N 1 9 m M 1 7 m M 15mSV1 1 2 m M 0 m M 84 FIGURE 20. Reaction of cyanide w i t h cytochrome d under t u r n -over c o n d i t i o n s . Log (absorbance increment at 648 nm r e l a t i v e to 607 nm) from the data shown i n F i g . 19 was p l o t t e d versus time a f t e r a d d i t i o n of 2 mM NADH. 85 - 1 - 1 cyanide gave a second-order r a t e constant (k^) of 0.58 M s f o r the r e a c t i o n of o x i d i z e d cytochrome d w i t h cyanide ( F i g . 21). From the i n t e r c e p t a value f o r k ^ of 5.0 x 10~^s ^ was c a l c u l a t e d . The value f o r k^ i s near the value (o.26 M-"*" s-''") obtained f o r the i n h i b i t i o n by cyanide of NADH oxidase a c t i v i t y . When experiments s i m i l a r to those above were c a r r i e d out using succinate as subs t r a t e a much lower r a t e constant was found f o r the formation of cyanocytochrome d. By compar-in g the r a t e constants determined using NADH and succinate as substrates i t was found that they were approximately propor-t i o n a l to the r a t e of o x i d a t i o n of these substrates. T h i s , together w i t h the evidence that the r a t e of formation of cy-anocytochrome d was very slow i n the absence of subst r a t e , suggested that the r a t e of turnover of the r e s p i r a t o r y chain i n f l u e n c e d the steady s t a t e l e v e l of an intermediate species i n the o x i d a t i o n - r e d u c t i o n c y c l e of cytochrome d. This i n t e r -mediate species would be that which reacted w i t h cyanide. To examine t h i s hypothesis the r a t e of e l e c t r o n f l u x through the r e s p i r a t o r y chain from succinate was con-t r o l l e d by the competitive i n h i b i t o r malonate (60) . These ex-periments were c a r r i e d out i n a s i m i l a r manner to those using NADH as substrate to determine the second-order r a t e cons-tants f o r the formation of cyanocytochrome d ( F i g . 22). With 1.67 mM succinate as substrate i n the presence of 0, 0.167 mM, 0.267 mM and 0.33 mM malonate the r a t e of o x i d a t i o n of s u c c i -nate i n the absence of cyanide was 0.088, 0.048, 0.042 and 86 FIGURE 21. Reaction of cyanide w i t h cytochrome d under t u r n -over c o n d i t i o n s . The slopes of the l i n e s shown i n F i g . 20 were p l o t t e d versus the concen t r a t i o n of KCN present. 86a 87 FIGURE 22. Reaction of cyanide w i t h cytochrome d at d i f f e r -ent turnover r a t e s . Experiments were c a r r i e d out i n a s i m i -l a r manner to those shown i n F i g . 19-21 using membrane p a r t i -c l e s at a concentration of 3.4 mg prot e i n / m l . Succinate (1.67 mM) was added as substrate i n presence of the i n d i c a t e d con-c e n t r a t i o n s of malonate. 87a 88 0.026 yg atoms 0/min/mg p r o t e i n . As the o x i d a t i o n r a t e de-creased the second order r a t e constant f o r the formation of cy-anocytochrome d also decreased. From the i n t e r c e p t on the or-dinate i t can be concluded that the r a t e of d i s s o c i a t i o n of the cyanide complex a l s o decreased w i t h the decreasing r a t e of e l e c -t r o n f l u x through the r e s p i r a t o r y chain. The r e l a t i o n s h i p between the r a t e of formation of cyanocytochrome d (k-^) and the r a t e of e l e c t r o n f l u x through cytochrome d i s demonstrated i n F i g . 23. The data shown i n t h i s f i g u r e was obtained from two separate preparations of mem-brane p a r t i c l e s . The o x i d a t i o n r a t e s were r e l a t e d to the l e v e l of cytochrome d as a measure of the r a t e of e l e c t r o n f l u x through cytochrome d. Since the c e l l s used i n these e x p e r i -ments contained n e g l i g i b l e amounts of cytochrome o t h i s assump-t i o n was probably v a l i d . The r a t e constant f o r the formation of cytochrome d was p r o p o r t i o n a l to the r a t e of e l e c t r o n f l u x through t h i s cytochrome. The p l o t of the p s e u d o - f i r s t - o r d e r r a t e constants f o r cyanocytochrome d formation versus the concentration of cy-anide f o r p a r t i c l e s o x i d i z i n g succinate deviated from l i n e a r i t y at high concentrations of cyanide i n some preparations ( F i g . 24). In these cases i f the k i n e t i c constants were p l o t t e d ver-sus the square of the concentration of cyanide a b e t t e r s i • s t r a i g h t l i n e was obtained f o r the higher concentrations of cy-anide. The l i n e deviated from l i n e a r i t y at lower concentra-t i o n s of cyanide. This r e s u l t suggested that i n some prepara-89 FIGURE 23. E f f e c t of r a t e of turnover of the respiratory-chain on the second-order r a t e constant f o r the formation of' cyanocytochrome d. The r a t e constants were-determined from two separate experiments c a r r i e d out i n a s i m i l a r manner to F i g . 22 using succinate (1.67 mM) as substrate i n the pre-sence of malonate (0 - 0.33 mM) to r e g u l a t e the o x i d a t i o n r a t e . The r a t e of e l e c t r o n f l u x through cytochrome d i s ex-pressed as g atoms oxygen consumed by the system per mole cy-tochrome d present. S o l i d c i r c l e s , 4.7 mg p r o t e i n / m l ; open c i r c l e s 3.4 mg pr o t e i n / m l . 89a 90 FIGURE 24. Reaction of cyanide w i t h cytochrome d i n a prepara-t i o n o x i d i z i n g succinate (1.67 mM) showing a d e v i a t i o n from the expected k i n e t i c behaviour. The experiments were c a r r i e d ;out_. i n a s i m i l a r manner to those shown i n F i g . 19-21 using membrane p a r t i c l e s grown on succinate to the s t a t i o n a r y phase of growth at a concentration of 3.1 mg p r o t e i n / m l . In the upper graph the Log (absorbance increment at 648 nm r e l a t i v e to 607 nm) i s p l o t t e d versus time a f t e r the a d d i t i o n of succinate. Lower l e f t graph shows the p l o t of the slope of the l i n e s i n the up-per graph versus the c o n c e n t r a t i o n of cyanide. Lower r i g h t hand graph shows the same slopes p l o t t e d versus the square of the concentration, of cyanide. 90a M i n u t e s 91 t i o n s o x i d i z i n g s uccinate the mechanism of cyanocytochrome d formation might be a bimolecular process at higher concentra-t i o n s of cyanide. However, i t has been shown by Kasahara and 'An'rakusl.i (145) that i n the presence of succinate cyanide can a c t i v a t e u n a c t i v a t e d succinate dehydrogenase a c t i v i t y up to three f o l d at the concentrations of cyanide used i n our exper-iments. The r a t e of cyanocytochrome d formation under these c o n d i t i o n s would have increased w i t h i n c r e a s i n g f l u x of e l e c -trons from the a c t i v a t e d succinate dehydrogenase and could have caused the observed d e v i a t i o n i n the k i n e t i c s . The marked d e v i a t i o n from l i n e a r i t y f o r the r a t e of cyanocytochrome d f o r -mation versus concentration of KCN p l o t shown i n F i g . 24 may have been due to the high degree of unac t i v a t e d succinate de-hydrogenase present i n that p r e p a r a t i o n . The l i n e a r i t y obser-ved i n the p l o t where the concentration of cyanide was squared may be f o r t u i t o u s and not because of a bimolecular r e a c t i o n of cyanide w i t h cytochrome d. The r e a c t i o n of cyanide w i t h cytochrome d i n r e s p i r a t o r y par-t i c l e s from exponential phase E. c o l i As p r e v i o u s l y shown membrane p a r t i c l e s prepared from E s c h e r i c M a e c o l i grown'? to the s t a t i o n a r y phase contained much higher l e v e l s of cytochrome d than of cytochrome o. In exponential phase c e l l s cytochromes d and o were present i n a-bout equal amounts. I n these c e l l s NADH oxidase a c t i v i t y was more s e n s i t i v e to i n h i b i t i o n by cyanide than i n the s t a t i o n a r y 92 phase c e l l s . Thus, the increased s e n s i t i v i t y to cyanide ap-peared to c o r r e l a t e w i t h the presence of cytochrome o. However, the t o t a l amount of the cytochrome oxida-ses present i n membrane p a r t i c l e s from s t a t i o n a r y phase c e l l s was about f i v e times greater than exponential phase c e l l s a l -though the NADH oxidase a c t i v i t i e s were s i m i l a r . Thus, a more r a p i d e l e c t r o n f l u x must occur through the smaller pool of t e r m i n a l oxidases i n c e l l s grown to the exponential phase. Since cyanide does not re a c t w i t h the o x i d i z e d or the reduced forms of cytochrome d but w i t h an intermediate form generated i n the o x i d a t i o n - r e d u c t i o n c y c l e , the increased s e n s i t i v i t y of exponential phase c e l l s to cyanide need not be e n t i r e l y due to the possession of cytochrome o but could also r e s u l t from the higher aeorbic steady s t a t e l e v e l of t h i s intermedi-ate. The r e a c t i o n of cyanide w i t h cytochrome d i n exponential phase c e l l s was examined to c l a r i f y t h i s problem. The logarithm of the absorbancy change due to the disappearance of o x i d i z e d cytochrome d p l o t t e d versus time af-t e r the a d d i t i o n of various concentrations of cyanide to mem-brane p a r t i c l e s from exponential phase c e l l s i n the presence of 1.67 mM succinate i s shown i n F i g . 25. The p s e u d o - f i r s t order r a t e constants determined from the slope of these l i n e s was p l o t t e d versus the concentration of cyanide ( F i g . 26). From the slope of t h i s l i n e a second order r a t e constant of 0.22 M'^s-^" was c a l c u l a t e d f o r the r e a c t i o n of cyanide w i t h cy-tochrome d i n the presence of 1.67 mM succinate. The r a t e of succinate o x i d a t i o n w i t h t h i s c oncentration of succinate was 93 FIGURE 25. Reaction of cyanide w i t h cytochrome d of membrane p a r t i c l e s from exponential phase c e l l s i n the presence of 1.67 mM succinate. The absorbance at 648 nm r e l a t i v e to 607 nm was measured to f o l l o w the disappearance of o x i d i z e d cytochrome d. Log (absorbance increment at 648 nm r e l a t i v e to 607 nm) i s p l o t t e d versus time a f t e r a d d i t i o n of succinate. Membrane p a r t i c l e s , 8.3 mg p r o t e i n per ml. 94 FIGURE 26. Reaction of cyanide with, cytochrome d i n the pre-sence of 1.67 mM succinate. The slopes of the l i n e s shown i n F i g . 25 are p l o t t e d versus concentration of cyanide present. 94a 95 0.41 ng atom 0 s mg p r o t e i n " . In a s i m i l a r way using 2 mM NADH as substrate the second order r a t e constant f o r the formation of cyanocyto-chrome d was found to be 4.0 M'^ s""*" and the o x i d a t i o n r a t e was 7.1 ng atom 0 s""'" mg protein""'" ( F i g . 27). Comparing these data i t i s seen that i n membrane p a r t i c l e s from exponential phase c e l l s the r a t e of formation of cyanocytochrome d i s d i r e c t l y p r o p o r t i o n a l to the r a t e of e l e c t r o n f l u x through t h i s cytochrome as was p r e v i o u s l y shown w i t h membrane p a r t i c l e s from s t a t i o n a r y phase c e l l s . More-over, i t i s evident that e l e c t r o n s from both NADH and su c c i n -ate must pass through the same cytochrome d pool. From the data p r e v i o u s l y presented f o r station.? ary phase c e l l s the r a t e of formation of cyanocytochrome d r e -l a t i v e to the ra t e of f l u x through the cytochrome d pool was c a l c u l a t e d by d i v i d i n g the second order r a t e constant f o r the formation of cyanocytochrome d by the r a t e of e l e c t r o n f l u x through cytochrome d expressed as g atom 0,.:reduced per second per mole cytochrome d (slope of l i n e i n F i g . 23). A value of 0.096 mole cytochrome d M cyanide-''" g atom O"''" was obtained f o r these c e l l s which contain cytochrome d as the only termin-a l oxidase. A s i m i l a r c a l c u l a t i o n from the r e s u l t s f o r expo-n e n t i a l phase c e l l s c o n t a i n i n g 0.066 and 0.076 nmoles of cyto-chromes o and d per mg p r o t e i n , r e s p e c t i v e l y , gave values of 0.043 and 0.042 mole cytochrome d M cyanide"^ g atom 0""'" f o r the i n h i b i t i o n by cyanide of NADH and succinate oxidase a c t i -96 FIGURE 27. Reaction of cyanide with, cytochrome d of membrane p a r t i c l e s from exponential phase c e l l s i n the presence of 2 mM NADH. The same experimental c o n d i t i o n s and the same p a r t i c l e p r e p a r a t i o n were employed as i n F i g . 25 and 26. 9 6 a 97 v i t i e s . This c a l c u l a t i o n was made assuming that the e l e c t r o n s passed through cytochrome d only. However, i f the e l e c t r o n s are assumed to be p a r t i t i o n e d e q u a l l y between the almost equal pools of cytochromes o and d then these values become about 0.085 mole cytochrome d M cyanide-"'" g atom O-''". This value i s of the same order of magnitude as that found w i t h the s t a t i o n -ary phase c e l l s and suggests that cytochrome d and cytochrome o are both f u n c t i o n a l cytochrome oxidases f o r NADH and s u c c i -nate o x i d a t i o n i n exponential phase c e l l s of E. c o l i . Trapping of an intermediate i n the o x i d a t i o n - r e d u c t i o n c y c l e  of cytochrome d. The aerobic steady s t a t e l e v e l of redu c t i o n of term i n a l oxidases i s u s u a l l y very low because of the r a p i d ox-i d a t i o n r e a c t i o n w i t h oxygen. Thus, only a small f r a c t i o n of the cytochrome d e x i s t s as the intermediate species i n the aerobic steady s t a t e at room temperature i n p a r t i c l e s o x i d i z -i n g NADH. The reduced form, absorbing at 628 nm, does not ap-pear u n t i l anaerobiosis. In d i f f e r e n c e spectra of the aerobic steady s t a t e of p a r t i c l e s o x i d i z i n g ascorbate i n the presence of PMS measured at 77°K 60-70% of the cytochrome d could be trapped as the intermediate species. This d i d not occur w i t h p a r t i c l e s o x i d i z i n g succinate or NADH ( F i g . 28). The s i z e of the trough at 648 nm i n reduced versus o x i d i z e d d i f f e r e n c e spectra i s i n d i c a t i v e of the degree of conversion of the o x i d -i z e d cytochrome d to another form. The f a c t that there was no 98 FIGURE 28. Low temperature (77°K) d i f f e r e n c e spectra of the aerobic steady s t a t e s of r e d u c t i o n of p a r t i c l e s o x i d i z i n g d i f -f e r e n t s u b s t r a t e s . NADH (2Tr,mM) (N) , succinate (1 mM) (S) , and ascorbate (5 mM) (A) plus PMS were added to p a r t i c l e s from s t a -t i o n a r y phase c e l l s suspended to a c o n c e n t r a t i o n of 5.2 mg protein/ml i n 0.05 M phosphate b u f f e r , pH 7.0, c o n t a i n i n g 1 M sucrose and the r e a c t i o n mixture was immediately frozen i n l i q u i d N^ to trap the cytochromes i n the aerobic steady s t a t e of r e d u c t i o n . HOQNO (HO yM) was added p r i o r to the a d d i t i o n of ascorbate plus PMS. 98a 99 concommitant appearance of the reduced cytochrome d absorbing at 628 nm i n the aerobic steady s t a t e spectrum at 77°K of p a r t i c l e s o x i d i z i n g ascorbate i n the presence of PMS suggest-ed that a l a r g e f r a c t i o n of cytochrome d e x i s t e d as an i n t e r -mediate species w i t h no a-absorption band. This enhancement of the steady s t a t e l e v e l of the intermediate species at 77°K was i n h i b i t e d by HOQNO. In order to examine t h i s s i t u a t i o n -under more c o n t r o l l e d c o n d i t i o n s at subzero temperatures par-t i c l e s suspended i n 50% ethylene g l y c o l to prevent f r e e z i n g of the sample were employed. Ethylene g l y c o l has been shown not to i n t e r f e r e w i t h e l e c t r o n t r a n s p o r t (140). Aerobic steady s t a t e d i f f e r e n c e spectra of p a r t i -c l e s o x i d i z i n g ascorbate i n the presence of PMS showed an i n -crease i n the s i z e of the trough at 648 nm w i t h decreasing temperature due to the disappearance of the absorption peak of o x i d i z e d cytochrome d i n the sample cuvette ( F i g . 29). The absorption peak at 628 nm of reduced cytochrome d d i d not ap-pear ccah:cjDmiittantd"5y w i t h the disappearance of the o x i d i z e d cytochrome i n d i c a t i n g that cytochrome d was i n the intermedi-ate form. The aerobic steady s t a t e l e v e l of r e d u c t i o n of cy-tochrome b-^  decreased s l i g h t l y w i t h the decrease i n tempera-ture. The upper curve shows the reduced versus o x i d i z e d d i f -ference spectrum of the r e s p i r a t o r y p a r t i c l e s employed i n these experiments. F i g . 30 shows the r e l a t i o n s h i p between the amount of cytochrome d i n the intermediate form and temperature i n 100 FIGURE 29. D i f f e r e n c e s p e c t r a of r e s p i r a t o r y p a r t i c l e s of E. c o l i . The upper curve shows the d i t h i o n i t e - r e d u c e d versus a i r -o x i d i z e d d i f f e r e n c e spectrum of the r e s p i r a t o r y p a r t i c l e s em-ployed i n these experiments. The lower curves represent the d i f f e r e n c e spectra at-l°C and -29°C of the aerobic steady s t a t e of r e s p i r a t o r y p a r t i c l e s o x i d i z i n g ascorbate (3.75 mM) i n the presence of PMS (0.01 mM) versus the a i r - o x i d i z e d r e s -p i r a t o r y p a r t i c l e s . The spectra were determined as described i n MATERIALS Arid METHODS. A 1 cm pathlength cuvette was em-ployed f o r the upper spectrum w h i l e a 3 mm pathlength cuvette was employed f o r the lower spectra. The membrane p a r t i c l e s were prepared from c e l l s grown to the s t a t i o n a r y phase of growth and suspended i n 0.05 M phosphate b u f f e r , pH 7.0, and 50% ethylene g l y c o l to a c o n c e n t r a t i o n of 14.0 mg p r o t e i n / m l . — I ; , — ; 1 » . 1 5 5 0 6 0 0 6 5 0 7 0 0 Wavelength nm 101 FIGURE 30. E f f e c t of temperature (°C) on the amount of cyto-chrome d i n the intermediate form i n the aerobic steady s t a t e i n p a r t i c l e s o x i d i z i n g ascorbate w i t h PMS. The experiment was c a r r i e d out as i n F i g . 29. The amount of cytochrome d i n the intermediate form was r e l a t e d to the peak height of o x i d -i z e d cytochrome d at 648 nm i n the absolute spectrum shown i n F i g . 31. 101a 102 p a r t i c l e s o x i d i z i n g ascorbate i n the presence of PMS. The a-mount of cytochrome d i n the intermediate form i s expressed r e l a t i v e to the peak height of o x i d i z e d cytochrome d i n the absolute spectrum. The steady s t a t e l e v e l of the intermedi-ate form increased from 28% at 1°C to a l e v e l of 57% at -38°C. To determine i f t h i s p l a t e a u l e v e l was due to conversion of o x i d i z e d cytochrome d to a form w i t h no a-ab-s o r p t i o n band, or to formation of a species w i t h a new absorp-t i o n peak overlapping at 648 nm, the absolute spectra of cyto-chrome d i n the o x i d i z e d form and i n the presence of ascorbate w i t h PMS were measured at -18°C ( F i g . 31). Substrate decreas-ed the s i z e of the peak of o x i d i z e d cytochrome d without f o r -mation of a new peak or displacement of the absorption maxi-mum. This provides f u r t h e r evidence that there i s p a r t i a l conversion of cytochrome d to an intermediate form w i t h no ex-absorption band at low temperatures. The steady s t a t e l e v e l of t h i s intermediate form at -20 °C was decreased from 517c to 40% and 30% i n the presence of 55 and 165 yM HOQNO, r e s p e c t i v e l y ( F i g . 32). The steady s t a t e l e v e l of redu c t i o n of cytochrome b-^  increased from 4.7% to 9.7% and 9.1%, r e s p e c t i v e l y , i n the presence of HOQNO under these c o n d i t i o n s . I t was also p o s s i b l e to enhance the steady s t a t e l e v e l of the intermediate form at low temperatures using NADH i n the presence of PMS. 103 FIGURE 31. Absolute s p e c t r a of o x i d i z e d cytochrome d (upper curve) and cytochrome d of r e s p i r a t o r y p a r t i c l e s o x i d i z i n g ascorbate i n the presence of PMS (lower curve). The spect r a were obtained as described i n MATERIALS AND METHODS at -18°C using the same p a r t i c l e p r e p a r a t i o n as i n F i g . 29. 103a 6 0 0 6 5 0 7 0 0 Wavelength nm 104 FIGURE 32. E f f e c t of HOQNO on the aerobic steady s t a t e l e v e l of r e d u c t i o n of cytochromes i n p a r t i c l e s o x i d i z i n g ascorbate w i t h PMS. The reduced minus o x i d i z e d d i f f e r e n c e s p e c t r a were measured at -20°C as i n F i g . 29. HOQNO (55 uM and 165 uM) was added p r i o r to c o o l i n g . 104a 5 5 0 6 0 0 6 5 0 7 0 0 Wavelength nm 105 DISCUSSION Cytochromes d, a-^ , and b-^  a l l increase i n con-c e n t r a t i o n i n the membrane-bound r e s p i r a t o r y system of E. c o l i i n the t r a n s i t i o n from exponential to s t a t i o n a r y growth whi l e the l e v e l of cytochrome o decreases. The increase i n the content of the cytochromes does not r e f l e c t an increase i n NADH and succinate oxidase a c t i v i t y because the dehydro-genases are g e n e r a l l y the r a t e l i m i t i n g components of e l e c -t r o n t r a n s p o r t (103). I t has been suggested that the purpose of the i n d u c t i o n of these cytochromes i s to all o w the r e s p i r -a tory chain to f u n c t i o n at low oxygen tensions (123). Low oxygen tensions a r i s e i n aerobic growth through high bacter-i a l c e l l d e n s i t i e s i n the growth medium p r i o r to s t a t i o n a r y growth which causes more r a p i d u t i l i z a t i o n of oxygen present. Cytochrome d apparently has a higher a f f i n i t y f o r oxygen than cytochrome o (146). Castor and Chance (11) determined the a c t i o n spectra f o r the p h o t o d i s s o c i a t i o n of carbon monoxide - cyto-chrome oxidase complexes and found that cytochrome o i s the primary oxidase i n c e l l s grown to the exponential phase wh i l e cytochromes d and o f u n c t i o n i n c e l l s grown to the s t a t i o n a r y phase of growth. There have been o b j e c t i o n s as to whether t h i s method of determination of oxidase f u n c t i o n a c c u r a t e l y r e f l e c t s the c o n t r i b u t i o n of the d i f f e r e n t cytochrome oxidas-es to the oxidase a c t i v i t i e s (25). The s e n s i t i v i t y to cyanide i n h i b i t i o n of c e l l s 1 0 6 grown to contain d i f f e r e n t l e v e l s of these cytochrome oxidas-es was examined i n t h i s t h e s i s . NADH o x i d a t i o n i n r e s p i r a -t o r y p a r t i c l e s from c e l l s grown to the exponential phase of growth was much more s e n s i t i v e to cyanide i n h i b i t i o n than NADH o x i d a t i o n i n r e s p i r a t o r y p a r t i c l e s from c e l l s grown to the s t a t i o n a r y phase of growth. Cyanide acted as an uncompe-t i t i v e i n h i b i t o r of the i n i t i a l r a t e of NADH o x i d a t i o n a f t e r p r e i n c u b a t i o n of the r e s p i r a t o r y p a r t i c l e s w i t h cyanide sug-ge s t i n g that cyanide combines w i t h the enzyme-substrate com-pl e x to y i e l d a s t a b l e o x i d a s e - i n h i b i t o r complex. The nature of the enzyme-substrate complex was not known at t h i s p o i n t . I t appeared, from the k i n e t i c s , that cyanide combined w i t h the NADH oxidase system at one type of binding s i t e . I t was evident however that turnover of the r e s p i r a t o r y chain was re q u i r e d f o r a more r a p i d r a t e of i n h i b i t i o n by cyanide. The r e a c t i o n of cyanide w i t h the u n i n h i b i t e d NADH oxidase to form an i n h i b i t e d complex was r a t e l i m i t i n g . k l E + HCN T * E.HCN k - i I have i n d i c a t e d that HCN r a t h e r than CN~ i s the r e a c t i v e form of cyanide, but t h i s has not been proven. Second order r a t e constants (k^) could be determined from the a n a l y s i s of the k i n e t i c data. The magnitude of the i n h i b i t o r constant, K^, determined from the r a t i o of the r a t e constants k_-^  and k-^  f o r the r e a c t i o n could be r e l a t e d to the r e l a t i v e proportions of the two oxidases i n the r e s p i r a t o r y p a r t i c l e s . The r e s p i r a t o -r y p a r t i c l e s having a higher r e l a t i v e concentration of cyto-107 chrome d ( c e l l s grown to the s t a t i o n a r y phase) reacted l e s s s t r o n g l y w i t h cyanide than those w i t h a higher r e l a t i v e amount of cytochrome o ( c e l l s grown to the exponential phase). Jones and Redfearn (25) examined the r e l a t i v e s e n s i t i v i t y to cyanide of substrate o x i d a t i o n v i a cytochrome o and cytochrome d i n Az-otobacter v i n e l a n d i i . In t h i s organism these oxidases are on d i f f e r e n t branches of the main pathway and, u n l i k e E. c o l i , e l e c t r o n s can be s p e c i f i c a l l y s u p p l i e d to e i t h e r of the two cytochromes. In agreement w i t h our r e s u l t s they found that ox-i d a t i o n proceeding v i a cytochrome o was more s e n s i t i v e to cyan-ide than that v i a cytochrome d. S i m i l a r conclusions have been made f o r the cytochrome oxidases of Achromobacter (49, 50). In f a c t , the cyanide r e s i s t a n t r e s p i r a t i o n i n these c e l l s when grown i n the presence of cyanide has been a t t r i b u t e d to the i n -duction of increased l e v e l s of cytochrome d. Subsequently to my work, cytochrome d has a l s o been shown to be r e s p o n s i b l e f o r cyanide r e s i s t a n t r e s p i r a t i o n at low concentrations of cy-anide i n E. c o l i (51). Before the e f f e c t of cyanide on the spectrum of cyto-chrome d can be i n t e r p r e t e d , the r e l a t i o n s h i p between the o x i d -i z e d and the reduced absorption bands of cytochrome d at 648 nm and 628-nm, r e s p e c t i v e l y , must be examined. I t has been as-sumed that these absorption bands belong to cytochrome d i n d i f -f e r e n t redox s t a t e s but t h i s has never been shown. I t was found that the disappearance of the band absorbing at 648 nm, during slow r e d u c t i o n w i t h l i m i t i n g amounts of substrate ( n e a r - e q u i l i -108 brium c o n d i t i o n s ) , d i d not co i n c i d e w i t h the appearance of the reduced band at 628 nm. I t appeared that the two spectroscop-i c a l l y observed forms of cytochrome d were not d i r e c t oxida-t i o n - r e d u c t i o n products of each other. The same behaviour has been observed f o r cytochrome d i n A. v i n e l a n d i i (147). This suggests e i t h e r that these absorption bands are due to two d i f f e r e n t u n r e l a t e d components or that cytochrome d goes through an intermediate species i n the normal oxidation-reduc-t i o n c y c l e which has no apparent alpha absorption band. In the f i r s t e x p l a n a t i o n , the 648 nm band would be due to a high-er p o t e n t i a l component as i t was reduced p r i o r to the reduc-t i o n of the other components. The other explanation would sug-gest t h a t i t represents another form of o x i d i z e d cytochrome d which occurs i n the presence of oxygen. Bot'entiometric t i t r a t i o n s were c a r r i e d out w i t h the membrane-bound cytochrome d and a value of +260 mV was deter-mined f o r the component absorbing at 628 nm. The absorption band at 648 nm d i d not f o l l o w normal redox behaviour. At pot-e n t i a l s as high as +450 mV t h i s peak d i d not appear. Only a f-t e r a d d i t i o n of pulses of oxygen or ^ 2^2 con^-a *-he 648 nm form be seen. The p o t e n t i a l was not increased any f u r t h e r by these a d d i t i o n s , thus, oxygen appeared to be e s s e n t i a l to induce the formation of t h i s form. I t has been suggested that the high absorbance of the 648 nm component i n the presence of oxygen i s due to a high e l e c t r o n d e n s i t y on the heme-iron (147) .It'iyjas- proposed that a 109 charge t r a n s f e r from the l i g a n d to the i r o n occurs i n the presence of oxygen r e s u l t i n g i n a conformation of cytochrome d which absorbs at 648 nm. The p o t e n t i o m e t r i c t i t r a t i o n data revealed that the u n i d e n t i f i e d chromophore which absorbs at 675 - 680 nm i n the o x i d i z e d form had a midpoint o x i d a t i o n - r e d u c t i o n pot-e n t i a l of +435 mV (n=l). This component appears only as a minor component i n reduced minus o x i d i z e d d i f f e r e n c e spectra which has hampered study of i t . However, i t might have some important, as yet undetermined, f u n c t i o n i n e l e c t r o n t r a n s -p o r t . I t may be a copper p r o t e i n s i n c e some of these pro-t e i n s also absorb at about t h i s wavelength i n the o x i d i z e d form (148). Evidence f o r the r e a c t i o n of cyanide w i t h the cy-tochromes was obtained from reduced plus cyanide minus r e -duced d i f f e r e n c e s p e c t r a . Cyanide reacted w i t h the d i t h i o -n i t e or NADH reduced system to e l i m i n a t e the absorption peaks of reduced cytochrome d at 442 and 628 nm without reappear-ance of the absorption bands of the o x i d i z e d species. Oxygen was necessary to cause e l i m i n a t i o n of these absorption bands; cyanide d i d not react d i r e c t l y w i t h the reduced cytochromes. With p a r t i c l e s from c e l l s grown e i t h e r to the exponential or s t a t i o n a r y phases a d d i t i o n of cyanide i n the presence of d i -t h i o n i t e also decreased absorption bands at 428 nm and 423 nm which are due to b and/or c-type cytochromes. The r e l a t i o n -ship of these cytochromes to the two c-type and three b-type cytochromes found i n E. c o l i by Shipp\j|9>^as.i'.Q6^-*dfetermin^d-. 110 The b or c-type cytochromes do not appear to be cytochrome o sinc e these bands were smaller i n p a r t i c l e s from exponential phase c e l l s which have higher l e v e l s of cytochrome o than those from s t a t i o n a r y phase c e l l s . Moreover, they d i d not r e -act w i t h cyanide when NADH was the reductant or, more probab-l y , were not reduced by NADH. This i s f u r t h e r evidence that these cytochromes are not cytochrome o since cytochrome o i s reduced by NADH i n E. c o l i . In none of our experiments d i d we o b t a i n spectroscopic evidence f o r the formation of a com-p l e x of cytochrome o w i t h cyanide. The absorption spectrum of such a complex has not been reported yet. Cyanide appears to react s l o w l y w i t h the o x i d i z e d form of cytochrome d to cause e l i m i n a t i o n of the absorption band of the cytochrome at 648 nm without the appearance of an-other absorption band. This agrees w i t h the observations made by Kauffman and Van Gelder (144). The disappearance of t h i s absorption band i n the presence of cyanide occurred w i t h a second-order r a t e constant (k^) of 0.011 M'^ s-'''. This value i s too low to account f o r the i n h i b i t i o n of NADH oxidase a c t i v i t y by cyanide (k^, 0.26 M- "^ s ^ ) . However, i n the presence of NADH a second-order r a t e constant of 0.58 M'^ s""*" f o r the disappearance of the 648 nm band was found. This value i s c l o s e r to that observed f o r the fo r the i n h i b i t i o n of NADH oxidase a c t i v i t y and supports the hypothesis that the i n h i b i t i o n of NADH oxidase by cyanide i n s t a t i o n a r y phase c e l l s was due to the r e a c t i o n of the i n h i b i t o r I l l with, cytochrome d. The r a t e of d i s s o c i a t i o n ( k ^ ) of cyanide from cyan-ocytochrome d al s o appeared to be greater under turnover con-d i t i o n s . The f i r s t order r a t e constant f o r d i s s o c i a t i o n was 5.0 x I0~^s~^~ i n p a r t i c l e s o x i d i z i n g NADH whereas i t was only 6.7 x lO'^s"''" when cyanide was reacted w i t h the enzyme i n the o x i d i z e d s t a t e . This could p o s s i b l y be explained by a con-format i o n a l change of cytochrome d caused by the red u c t i o n of the other components of the r e s p i r a t o r y chain, thus inducing more r a p i d d i s s o c i a t i o n of cyanocytochrome d and subsequent reduc t i o n . This has been suggested f o r the more r a p i d d i s s o -c i a t i o n of cyanide from cyanocytochrome d i n A. V i n e l a n d i i (144), and from cyanocytochrome aa^ i n mitochondria (149) when under reducing c o n d i t i o n s . When the k i n e t i c s of the formation of cyanocyto-chrome d were st u d i e d using s u c c i n a t e , which was more slowly o x i d i z e d than NADH, lower second-order r a t e constants were found suggesting that the magnitude of the r a t e constant was p r o p o r t i o n a l to the o x i d a t i o n r a t e . By s y s t e m a t i c a l l y r e g u l a -t i n g the o x i d a t i o n r a t e of succinate using malonate as a com-r p e t i t i v e i n h i b i t o r we found that the r a t e constant f o r the f o r -mation of cyanocytochrome d was d i r e c t l y p r o p o r t i o n a l to the r a t e of e l e c t r o n f l u x through cytochrome d. This suggests that cyanide r e a c t s w i t h the intermediate form, of cytochrome d which i s generated i n the normal o x i d a t i o n - r e d u c t i o n c y c l e . Thus, higher r a t e s of e l e c t r o n f l u x through the r e s p i r a t o r y chain r e -112 s u i t s i n higher steady s t a t e l e v e l s of t h i s intermediate spec-i e s . As p r e v i o u s l y discussed the existence of t h i s i n termedi-ate species (d*) was al s o suggested by the re d u c t i o n s t u d i e s , and by the need f o r the r e s p i r a t o r y chain to tu r n over f o r i n -h i b i t i o n of NADH o x i d a t i o n to occur. I t i s l i k e l y that i t i s the intermediate species which binds cyanide to form cyanocy-tochrome d. ox red d648 ^ s d628 A HCN d*.HCN The nature of the intermediate species i s unknown. The greater s e n s i t i v i t y of NADH oxidase a c t i v i t y to i n h i b i t i o n by cyanide i n c e l l s grown to the exponential phase of growth was also r e f l e c t e d i n the higher rates of cyanocyto-chrome d formation i n r e s p i r a t o r y p a r t i c l e s prepared from these c e l l s when compared to the rates of cyanocytochrome d formation i n p a r t i c l e s from s t a t i o n a r y c e l l s . I t should be noted that the t o t a l cytochrome oxidase concentration i n exponential c e l l s i s approximately 5 - f o l d l e s s than i n s t a t i o n a r y c e l l s . The s e n s i t i v i t y could t h e r e f o r e be r e l a t e d to the greater e l e c -t r o n f l u x through the smaller pool of cytochrome oxidase. The steady s t a t e l e v e l of the intermediate species i n the oxida-t i o n - r e d u c t i o n c y c l e of cytochrome d which i s r e a c t i v e w i t h cyanide would be higher under these c o n d i t i o n s . Therefore, cy-anocytochrome d formation would occur at a f a s t e r r a t e . In 113. f a c t , the constants c a l c u l a t e d f o r the r a t e of cyanocytochrome d formation i n r e s p i r a t o r y p a r t i c l e s of exponential and sta-^ t i o n a r y phase c e l l s r e l a t i v e to the e l e c t r o n f l u x through cyto-chrome d were s i m i l a r . In p a r t i c l e s where there i s approxi-mately an equal amount of cytochrome o and cytochrome d i t ap-peared that e l e c t r o n s from both NADH and succinate were d i s t r i -buted e q u a l l y to both t e r m i n a l oxidases. This i s not i n agree-ment w i t h the p h o t o d i s s o c i a t i o n spectroscopic data of Castor and Chance (11) who underestimated the c o n t r i b u t i o n of cyto-cyrome d to the oxidase a c t i v i t y i n exponential c e l l s . Kauffman and Van Gelder (142) have also demonstrated that turnover of the r e s p i r a t o r y chain increases the r a t e of cyanocytochrome d formation i n A. V i n e l a n d i i . They have a l s o proposed that an intermediate i n the o x i d a t i o n - r e d u c t i o n c y c l e of cytochrome d i s the species r e a c t i v e towards cyanide. How-ever, the r e l a t i o n s h i p between the r a t e of e l e c t r o n f l u x and the r a t e of cyanocytochrome d formation was not demonstrated as c l e a r l y w i t h t h e i r system. Much lower concentrations of cyan-ide (100-500yuM cyanide) were r e q u i r e d to i n h i b i t r e s p i r a t o r y a c t i v i t y or to i n i t i a t e cyanocytochrome d formation i n A. v i n e -l a n d i i than i n E. c o l i (1-20 mM) . The reasons f o r t h i s d i f f e r -ence are not obvious but may be due to some s l i g h t s t r u c t u r a l d i f f e r e n c e s between the cytochromes of these two organisms or to d i f f e r e n c e s i n a c c e s s i b i l i t y of cyanide to the membrane bound cytochrome d. Turnover a l s o f a c i l i t a t e s the r e a c t i o n of cyanide w i t h cytochrome c oxidase, cytochrome aa-, of mi t o c h o n d r i a l sys-114 terns (150-152). Cyanide i n h i b i t s cytochrome c oxidase a c t i v i -t y at concentrations f a r below that r e q u i r e d f o r complex f o r -mation w i t h e i t h e r the s t a t i c o x i d i z e d or reduced form of the enzyme (153). Yoshikawa and O r i i (154) have shown that a k i n -e t i c a l l y i n a c t i v e complex i s formed between cyanide and an i n -termediate species of cytochrome oxidase which occurs only i n the f u n c t i o n i n g s t a t e . This s i t u a t i o n i s very s i m i l a r to that w i t h cytochrome d even though the s t r u c t u r e s of the hemes are d i s s i m i l a r . A number of intermediates may e x i s t i n the ox i d a -t i o n - r e d u c t i o n c y c l e of t e r m i n a l cytochrome oxidases (155). The oxidase r e a c t i o n i s of n e c e s s i t y a complex process by which four e l e c t r o n s are donated to oxygen to y i e l d two mole-cules of water. The cytochromes are only one e l e c t r o n c a r -r i e r s . Therefore the r e d u c t i o n would probably occur through a number of s h o r t - l i v e d intermediate steps. This process must occur i n thermodyriaimically favourable stages without the r e -lease of any intermediate reduced st a t e s of oxygen which may be d e l e t e r i o u s to the organism. The existence of an "oxygenated" intermediate i n the c a t a l y t i c c y c l e of cytochrome aa^ was suggested by O r i i and Okunuki (156). They suggested that the "oxygenated" spec-i e s was formed by the r a p i d r e a c t i o n of oxygen w i t h the r e -duced cytochrome w i t h a subsequent slow r e a c t i o n to form the o x i d i z e d cytochrome and rL^O. However, Wharton and Gibson (157) found that the "oxygenated" form was slowly formed from 115 the o x i d i z e d form. I t should be cautioned that not every r e -a c t i o n demonstrable w i t h an enzyme i s n e c e s s a r i l y p a r t of i t s c a t a l y t i c mechanism. whether or not the "oxygenated" species i s a r e q u i r e d intermediate i n the c a t a l y t i c c y c l e , i t can be formed by a e r a t i o n of the reduced p u r i f i e d oxidase. The "ox-ygenated" form of cytochrome aa^ d i f f e r s c o n f o r m a t i o n a l l y from the o x i d i z e d form of t h i s cytochrome (158) yet both forms can accept four e l e c t r o n s from a reductant presumably by r e d u c t i o n of the two hemes and the two copper atoms (159). An "oxygenated" form of p u r i f i e d cytochrome o from V i t r e o s -c i l i a has a l s o been t e n t a t i v e l y i d e n t i f i e d (39). I t i s formed i n aerobic s o l u t i o n s i n the presence of a reducing source such as NADH. Cyanide can bind to the o x i d i z e d form of t h i s cytochrome but has no e f f e c t on the "oxygenated" form. There i s some s i m i l a r i t y i n the behaviour of t h i s ."ox-ygenated" form to the p r o p e r t i e s of the 648 nm form of cyto-chrome d which i s al s o not very r e a c t i v e w i t h cyanide. How-ever, the 648 nm form of cytochrome d i s the s t a b l e conforma-t i o n of o x i d i z e d cytochrome d when oxygen i s present i n s o l u -t i o n . The presence of subs t r a t e i n the aerobic steady s t a t e appears to generate the intermediate d* form. I t may be that the d* form represents the o x i d i z e d form of cytochrome d and the 648 nm form i s the "oxygenated" form of cytochrome d which i s formed by the r e a c t i o n of oxygen w i t h the o x i d i z e d form. The t r a n s i t i o n s i n the oxidase r e a c t i o n occur too 116 q u i c k l y at room temperature to study the nature of the i n t e r -mediate s t a t e s . The study of e l e c t r o n t r a n s p o r t processes at subzero temperatures has proven u s e f u l f o r t h i s purpose (140, 160). The amount of the intermediate form of cytochrome d, cytochrome d*, could be enhanced at subzero temperatures i n p a r t i c l e s o x i d i z i n g ascorbate i n the presence of PMS. The e l -ectron mediator feeds e l e c t r o n s from ascorbate i n t o the r e s p i -r a t o r y chain at a s i t e c l o s e r to oxygen than the temperature-s e n s i t i v e dehydrogenases to reduce components of the t e r m i n a l p o r t i o n of the chain. E l e c t r o n s enter at a po i n t near the l e v e l of cytochrome b-^  but p r i o r to a s i t e of i n h i b i t i o n by HOQNO and subsequently are t r a n s f e r r e d to oxygen v i a cyto-chrome d. This was i n d i c a t e d by the increase i n the steady s t a t e l e v e l of red u c t i o n of cytochrome b-^  and the decrease i n the steady s t a t e l e v e l of d* at subzero temperatures i n p a r t i -c l e s o x i d i z i n g ascorbate plus PMS when HOQNO was present. The a b i l i t y to increase the aerobic steady s t a t e l e v e l of the i n -termediate species d* at subzero temperatures i n d i c a t e s e i t h e r i ) that the t r a n s i t i o n from the 648 nm form to the intermedi-ate form, d*, i s l e s s temperature s e n s i t i v e e t h a n the subse-quent r e d u c t i o n step to y i e l d the reduced 628 nm form, or i i ) that the 648 form can be reduced d i r e c t l y to y i e l d the 628 nm form which i s then r a p i d l y r e o x i d i z e d to give the o x i d i z e d form, d*, which only slowly reforms the 648 nm component i n a temperature s e n s i t i v e step. The f i r s t case above would imply that a re d u c t i o n step might occur i n the t r a n s i t i o n from the 648 nm form to the 117 d* form of cytochrome d. This may i n v o l v e r e d u c t i o n of anoth-er group (X) as s o c i a t e d w i t h the cytochrome r a t h e r than the heme i t s e l f s ince r e d u c t i o n of the heme has been shown f o r the subsequent step. There i s no precedent, other than f o r the copper atoms attached to cytochrome aa^, f o r such a r e a c t i o n (161), but cytochrome d may be unique i n t h i s respect. The t r a n s i t i o n of d* to the 628 nm (reduced) form would be the tem-p e r a t u r e - s e n s i t i v e step. This r e a c t i o n r e s u l t s i n a change i n 4-3 4-2 the redox s t a t e of the heme of cytochrome d (d to d ) and can occur i n e i t h e r d i r e c t i o n . d + 2 x-a 6 2 8 ' X *+3 d .X temperature s e n s i t i v e 4 H + + ©, 2 H 20 The second case r e q u i r e s that the 648 nm form or the d* form can be reduced d i r e c t l y to y i e l d the 628 nm (r e -duced) form. This i s then very r a p i d l y r e o x i d i z e d by oxygen, which may have already been present attached to the 648 nm (Voxygenated") form, to y i e l d the d* (oxidized) form of cyto-chrome d. The reformation of the "oxygenated" 648 nm species, r e q u i r i n g oxygen, then occurs slowly at low temperatures. Thus, the d* form w i l l predominate at low temperatures i n the aerobic steady s t a t e . The r a p i d r e o x i d a t i o n of the reduced 118 form absorbing at 628 nm would e x p l a i n why i t i s not detec-t a b l e i n the aerobic steady s t a t e even at low temperatures. This scheme r e q u i r e s that the "oxygenated" form of cytochrome d i s p r e f e r e n t i a l l y reduced or that oxyg'en can very r a p i d l y b i n d to the reduced form, even at low temperatures to r e o x i d -i z e i t back to the o x i d i z e d form. d+2 • d*+3 s d.+ 3 ' a 6 2 8 7- X . ' a- ^ " 7 " d648-°2 (reduced) / \ (Oxidized) t e m P e r a t u r e (oxygenated) / \ j s e n s i t i v e " 4 H + ( + 0 2 ) 2 H 2 0 In the schemes presented above no attempts have been made to i n d i c a t e the number of molecules of heme r e q u i r -ed because i t i s not known which components supply the four e l e c t r o n s i n v o l v e d i n the re d u c t i o n of dioxygen to water. Perhaps other redox components are i n t i m a t e l y i n v o l v e d i n t h i s mechanism such as the p o s t u l a t e d component "X", or the 675 nm component which has an apparent midpoint o x i d a t i o n - r e -duction p o t e n t i a l of +435 mV. There are mechanistic problems i n both of the schemes presented. In the f i r s t scheme there i s p r e s e n t l y no evidence f o r two o x i d a t i o n - r e d u c t i o n steps as suggested. The +3 - +3 argument that the o x i d a t i o n step (d* .X to dg^g.X) req u i r e s oxygen i s not too l i k e l y s ince oxygen would more l i k e l y be 119 reduced at the stage where the cytochrome oxidase i s i n the +2 f u l l y reduced (<^^28'^ ) f ° r m n o t where i t i s i n the p a r t i a l l y reduced (&£28'^ ^ form. Therefore, the second scheme i s f a -voured. However, there i s presently no evidence that the "oxygenated" form contains oxygen. This i s also true for the "oxygenated" mitochondrial cytochrome oxidase (46). The sys-tem i n which we followed electron transport was i n the aero-b i c steady state. Therefore, the spectrum indicates the e-quilibrium rather than the k i n e t i c a l l y important transient states. In order to distinguish between the two cases d i s -cussed the k i n e t i c s would have to be more f u l l y examined us-ing other techniques as well as spectroscopic techniques. There are no doubt other s h o r t - l i v e d transient states which are not trapped by my method.. It i s premature to speculate on a scheme for the reduction of oxygen by cyto-chrome d u n t i l more i s known concerning the nature of the i n -termediate species or of the 648 nm form of cytochrome d. It should be possible to use the low temperature techniques I have discussed to trap the cytochrome i n i t s i n -termediate state or states and examine other biophysical para-meters such as electron paramagnetic properties to gain more information concerning i t s properties. P u r i f i c a t i o n of cyto-chrome d may be e s s e n t i a l to eliminate interference and to de-termine which components are intimately associated with i t s mechanism of action. Once p u r i f i e d i t may be possible to de-termine i f other components are required as a source of elec-trons for the reduction of dioxygen to water. 120 PART I I . THE ORGANIZATION OF THE CYTOCHROMES IN THE RESPIRA-TORY CHAIN OF E. COLI The a b s o r p t i o n band at 560 nm i n membrane p a r t i -c l e s of E. c o l i which has been a t t r i b u t e d to cytochrome b-^  has been r e s o l v e d by low temperature (77 0 >K) d i f f e r e n c e spec-troscopy i n t o m u l t i p l e components (9). No work other than that of Haddock and Sch a i r e r (81) has taken i n t o considera-t i o n these a d d i t i o n a l components i n proposing a scheme f o r the o r g a n i z a t i o n of the cytochromes of the r e s p i r a t o r y chain of E. c o l i . My i n t e n t i o n i n t h i s p a r t of the t h e s i s i s to describe the r e s u l t s concerning the r e s o l u t i o n and p r o p e r t i e s of the cytochromes absorbing i n the b-region and t h e i r r e l a -t i o n s h i p to the other cytochromes i n the NADH and succinate oxidase systems. RESULTS Low temperature (77^K) d i f f e r e n c e spectra of p a r t i c l e s pre-pared from c e l l s grown to the exponential and s t a t i o n a r y phaw-ses of growth. The room temperature d i f f e r e n c e s p e c t r a of p a r t i -c l e s prepared from E. c o l i c e l l s grown to the exponential and s t a t i o n a r y phases of growth are shown i n F i g . 2 of Part I. Fig u r e 33 shows the d i t h i o n i t e , NADH, and succinate reduced versus o x i d i z e d d i f f e r e n c e spectra of the same type of p a r t i -c l e s taken at 77°K. Spectra taken at 77°K tend to show much 121 FIGURE 33. Low temperature (77°K) reduced versus o x i d i z e d d i f -ference s p e c t r a of p a r t i c l e s prepared from c e l l s grown to the exponential (L) and s t a t i o n a r y (S) phases of growth. The par-t i c l e s were prepared from c e l l s grown on succinate to the r e -quir e d phase of growth and suspended i n 0.05 M phosphate buf-f e r , pH 7.0, c o n t a i n i n g 1 M sucrose to a concent r a t i o n of 7.2 mg protein/ml and 5.4 mg pr o t e i n / m l , r e s p e c t i v e l y . The cyt o -chromes were reduced w i t h d i t h i o n i t e (D), NADH (N), or s u c c i -nate (S) and the spectr a were scanned at 60 nm/min, using a 1 nm bandwidth, aga i n s t an o x i d i z e d reference sample. 121a W a v e l e n g t h <nm 122 sharper a b s o r p t i o n bands and higher e x t i n c t i o n c o e f f i c i e n t s than spectra taken at room temperature. These f a c t o r s a l l o w f o r the r e s o l u t i o n of m u l t i p l e absorption bands i n the Soret r e g i o n and the cytochrome b r e g i o n i n p a r t i c u l a r . The Soret r e g i o n showed peaks at 427 nm and 437 nm due to the b cyto-chromes and cytochrome d, r e s p e c t i v e l y . The substrate r e -duced d i f f e r e n c e spectra showed that cytochrome d was reduced greater than 90% by both NADH and succinate i n the anaerobic steady s t a t e . The r e d u c t i o n of the cytochrome b components and a l s o cytochrome a-^  by the'!.® substrates was not as com-p l e t e . The v a r i a t i o n i n the l e v e l of the cytochromes i n exponential and s t a t i o n a r y c e l l s has already been discussed but without reference to the r e s o l v e d components of the cyto-chrome b peak. Low temperature d i f f e r e n c e spectra were taken of the alpha-band re g i o n of the b-cytochromes using a narrow-er bandwidth, an expanded s c a l e , and slower scan speed to ob-t a i n maximum r e s o l u t i o n of these components. E. c o l i was grown on glucose to the exponential phase of growth, and on succinate to the exponential or s t a t i o n a r y phases of growth, and on the complex medium to the s t a t i o n a r y phase of growth. Low temperature d i f f e r e n c e spectra of the a-band r e g i o n of cytochrome b-^  i n membrane p a r t i c l e s prepared from these c e l l s showed two major peaks at 556 and 558 nm, and w i t h minor com-ponents present as shoulders on the main peaks at 548-550 nm and at 562 nm. Cytochrome b ^ g was the major component i n 123 a e r o b i c a l l y grown exponential phase c e l l s w i t h cytochrome ^558 a P P e a r i - n g a s a shoulder ( F i g . 34). Cytochrome b ^ g i n -creased i n amount r e l a t i v e to b ^ g i n the t r a n s i t i o n from the exponential to the s t a t i o n a r y phase of growth. This was most d r a m a t i c a l l y demonstrated when the c e l l s were grown on a complex medium. In our preparations cytochrome b ^ ^ w a s d i f f i c u l t to d i s t i n g u i s h even i n e a r l y exponential phase c e l l s grown w i t h a h i g h a e r a t i o n r a t e , where i t has been r e -ported to be at i t s highest c o n c e n t r a t i o n (51). In membrane p a r t i c l e s from c e l l s grown on complex media to the s t a t i o n a r y phase both NADH and succinate d i d not reduce the e n t i r e pool of b-type cytochromes ( F i g . 35). NADH and suc c i n a t e , r e s p e c t i v e l y , reduced 72% and 58% of both cy-tochrome b ^ g and cytochrome b ^ g . The incomplete r e d u c t i o n of the b-type cytochromes was most c l e a r l y demonstrated i n the d i t h i o n i t e - r e d u c e d minus substrate-reduced d i f f e r e n c e spectra. Here a c-type cytochrome appeared as an even more prominent shoulder at 548-552 nm on the peak of the b cyto-chromes i n d i c a t i n g that i t was l e s s r e d u c i b l e than cytoshro chromes b^^^ and b ^ g by both NADH and succinate. The shoul-der due to cytochrome b ^ ^ w a s a b s e n t showing that t h i s cyto-chrome had been completely reduced by NADH and by succinate. The same behaviour was observed w i t h p a r t i c l e s from c e l l s grown to the exponential phase of growth (shown l a t e r i n F i g . 49). The l e v e l of r e d u c t i o n of cytochrome b ^ g was s l i g h t l y greater than that of cytochrome b ^ ^ i n the substrate reduced -124 FIGURE 34. Low temperature d i t h i o n i t e reduced versus o x i d i z e d d i f f e r e n c e s p e c t r a of the cytochrome b reg i o n of p a r t i c l e s grown under d i f f e r e n t c o n d i t i o n s . A l l p a r t i c l e preparations were suspended i n 0.05 M phosphate b u f f e r , pH 7.0, c o n t a i n i n g 1 M sucrose. The spect r a were measured at 77°K at a scan speed of 10 nm/min and a band width of 0.5 nm. Gluc-E, p a r t i -c l e s (11.0 mg protein/ml) prepared from c e l l s grown on glucose to the exponential phase; Succ-E, p a r t i c l e s (5.6 mg p r o t e i n / ml) prepared from c e l l s grown on succinate to the exponential phase; Succ-S, p a r t i c l e s (4.1 mg protein/ml) prepared from c e l l s grown on succinate to the s t a t i o n a r y phase; complex-S, p a r t i c l e s (5.2 mg protein/ml) prepared from c e l l s grown on com-pl e x media to the s t a t i o n a r y phase. 124a 3X10"2 COMPLEX-s u c c - s S U C C - E G L U C - E 5 2 0 5 5 0 W a v e l e n g t h - n m 5 8 0 125 FIGURE 35. Reduction of cytochrome b by succinate and NADH. P a r t i c l e s were prepared from c e l l s grown on complex media to the s t a t i o n a r y phase of growth. Spectra were run at 77°K i n the same manner as F i g . 34. P a r t i c l e s were suspended to a con c e n t r a t i o n of 4.5 mg protein/ml. D, d i t h i o n i t e - r e d u c e d minus a i r - o x i d i z e d d i f f e r e n c e spectrum; N, NADH_-reduced minus a i r - o x i d i z e d d i f f e r e n c e spectrum; S, succinate-reduced minus a i r - o x i d i z e d d i f f e r e n c e spectrum; D-S, d i t h i o n i t e - r e d u c e d min-us succinate-reduced d i f f e r e n c e spectrum; D-N, d i t h i o n i t e - r e -duced minus NADH-reduced d i f f e r e n c e spectrum. 125a I 1 - J 540 560 580 Wavelength nm 126 anaerobic steady s t a t e i n most p a r t i c l e p reparations. This was most c l e a r l y demonstrated at 77°K i n d i f f e r e n c e spectra reduced w i t h ascorbate plus PMS (FiLg. 36). The sharp de-crease i n absorption on the red s i d e of the absorption band of the cytochrome b r e g i o n i n the d i t h i o n i t e imunus ascorbate w i t h PMS d i f f e r e n c e spectrum w i t h p a r t i c l e s from both expo-n e n t i a l and s t a t i o n a r y c e l l s i n d i c a t e d that cytochrome b ^ ^ was a l s o more reduced by ascorbate plus PMS. This suggested that cytochromes and b^^ 2 have higher oxidation=reduc-t i o n p o t e n t i a l s than cytochrome b ^ g . P o t e n t i o m e t r i c t i t r a t i o n s of the cytochromes In order to determine the thermodynamic r e l a t i o n -ship of the cytochromes of the r e s p i r a t o r y chain of E. c o l i i t i s necessary to know the o x i d a t i o n - r e d u c t i o n p o t e n t i a l s of these cytochromes. The p o t e n t i o m e t r i c data f o r cytochrome d, presented i n Part I ( F i g . 14), showed i t had a midpoint ox-i d a t i o n - r e d u c t i o n p o t e n t i a l of +260 mV. F i g u r e 37 shows an o x i d a t i o n - r e d u c t i o n t i t r a t i o n of cytochrome a^ i n membrane p a r t i c l e s from s t a t i o n a r y phase c e l l s . The spectrummat v a r i -ous p o t e n t i a l s was scanned against an o x i d i z e d sample and the height above the b a s e l i n e of the peak f o r reduced cyto-chrome a-^  at 594 nm was p l o t t e d versus the p o t e n t i a l . The r e s u l t i n g sigmoidal curve was r e s o l v e d by the Nernst equation to give a midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l of +147 mV 127 FIGURE 36. Reduction of cytochrome b i n p a r t i c l e s from expo-n e n t i a l (L) and s t a t i o n a r y (S) c e l l s by ascorbate i n the pre-sence of PMS. The p a r t i c l e s were obtained from c e l l s grown on glucose to the exponential phase of growth and on complex med-ium to the s t a t i o n a r y phase of growth and were suspended to a con c e n t r a t i o n of 11.0 mg protein / m l and 5.2 mg p r o t e i n / m l , r e s -p e c t i v e l y . The cytochromes were reduced w i t h d i t h i o n i t e (D) or ascorbate (A) plus PMS and the spectra scanned i n a s i m i l a r manner to those i n F i g . 34. 1 1 I 1 I 1 I I I 5 4 0 5 6 0 5 8 0 5 4 0 5 6 0 5 8 0 Wav e l e n g t h ,nm 128 FIGURE 37. Pot e n t i o m e t r i c t i t r a t i o n of cytochrome a^. P a r t i -c l e s were prepared from c e l l s grown on succinate to the s t a t i o -nary phase of growth and suspended to a conce n t r a t i o n of 8.3 mg p r o t e i n / m l i n 0.1 M phosphate b u f f e r , pH 7.0. The mediators used were 50 yM PMS, 50 yM PES, 50 yM duroquinone, 5 yM DCIP, and 25 yM f e r r i c y a n i d e . The redox s t a t e of cytochrome a-^  was determined from the height of the absorbance peak at 594 nm. Both o x i d a t i v e and r e d u c t i v e t i t r a t i o n s were performed using NADH (0.2 M) to decrease the p o t e n t i a l and f e r r i c y a n i d e (0.2 M) to increase the p o t e n t i a l . I n the lower f i g u r e the concentra-t i o n s of o x i d i z e d and reduced cytochrome a-^  were determined from the upper f i g u r e . The t h e o r e t i c a l l i n e f o r n=l i s drawn through the p o i n t s . 128a 129 f o r cytochrome a-^ . The r e a c t i o n i n v o l v e d the t r a n s f e r of one e l e c t r o n . P o t e n t i o m e t r i c t i t r a t i o n s of the cytochrome b r e -gion i n p a r t i c l e s from c e l l s grown to the exponential phase of growth gave a two step r e d u c t i o n curve ( F i g . 38). The s i g -moidal curve i n the p l o t of p o t e n t i a l versus l o g ( o x i d i z e d form/reduced form) i n d i c a t e d that the cytochrome b r e g i o n was made up of more than one component w i t h d i f f e r e n t midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l s . The o r i g i n a l curve of absor-bance versus p o t e n t i a l was r e s o l v e d a r i t h m e t i c a l l y i n t o two components and r e p l o t t e d . This time the r e s o l v e d curve y i e l d -ed two s t r a i g h t l i n e s , each w i t h a slope of n = 1 which could be assigned to a hi g h p o t e n t i a l cytochrome b w i t h a midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l of 4-165 mV and to a low poten-t i a l cytochrome b w i t h a midpoint p o t e n t i a l of 4-36 mV. To r e l a t e the measured p o t e n t i a l s to the d i f f e r e n t cytochromes observed i n the d i f f e r e n c e spectrum of the cyto-chrome b r e g i o n , the d i f f e r e n c e spectrum of t h i s r e g i o n was measured at 22 °C at two p o t e n t i a l s (-48 mV and 4-140 mV) which represented each of the p l a t e a u regions of the t i t r a t i o n ci*.\ curve ( F i g . 39). The spectrum at 4-140 mV i n d i c a t e d that the hig h p o t e n t i a l component had an absorption peak, at 560 nm. Su b t r a c t i n g t h i s spectrum from th a t measured at -48 mV y i e l d -ed the spectrum of the low p o t e n t i a l component w i t h a peak at 557.5 nm. Other components such as the c-type cytochromes ab-130 FIGURE 38. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome b. P a r t i -c l e s were prepared from c e l l s grown on succinate to the expo-n e n t i a l phase of growth and suspended i n 0.1 M phosphate buf-f e r , pH 7.0, to aoeoncentration of 8.0 mg pr o t e i n / m l . The med-i a t o r s used were 50 yM PMS, 50 yM PES, 50 yM duroquinone, 7 pM DCIP, and 25 yM f e r r i c y a n i d e . The redox t i t r a t i o n was c a r r i e d out using the same procedure as i n F i g . 37. The redox s t a t e of the cytochrome b components was determined from the height: of the absorbance peak at 560 nm. The data i n the middle f i g - . ' ure was r e s o l v e d f o r the c o n t r i b u t i o n of two components and p l o t t e d on the r i g h t s i d e . The t h r o r e t i c a l l i n e s f o r n=l are drawn through these p o i n t s . 130a 131 FIGURE 39. S p e c t r a l a n a l y s i s of the cytochrome b components reduced at d i f f e r e n t p o t e n t i a l s . The d i f f e r e n c e spectra were scanned at +140 mV and -48 mV against a reference sample o x i -d i z e d w i t h f e r r i c y a n i d e using c o n d i t i o n s s i m i l a r to those i n F i g . 38. Scan speed, 120 nm/min; band width, 1 nm. P a r t i c l e s were suspended to a co n c e n t r a t i o n o f 6.6 mg pr o t e i n / m l . The broken l i n e represents the d i f f e r e n c e between the spectra mea-sured at -48 mV and at +140 mV. 131a 540 560 580 Wavelength -nm 132 sorbing at 548-552 run and cytochrome b ^ ^ * which could be de-te c t e d i n spectra measured at low temperatures, could not be adequately r e s o l v e d by redox t i t r a t i o n i n t h i s manner e i t h e r because they occurred i n too small q u a n t i t i e s or t h e i r redox p o t e n t i a l s too c l o s e l y overlapped w i t h those of the two major components present. However, the asymmetry of the curve mea-sured at +140 mV i n d i c a t e d that a minor component having a peak at about 565 nm was present. In some cases the r e s o l v e d t i t r a t i o n curves deviated from exact l i n e a r i t y . This might be due to the c o n t r i b u t i o n of a component w i t h a s l i g h t l y d i f -f e r e n t redox p o t e n t i a l . I t i s l i k e l y t hat the low p o t e n t i a l cytochrome b w i t h a peak at 558 nm at room temperature i s cy-tochrome b^^g (at 77°K) and the high p o t e n t i a l cytochrome b w i t h a peak at 560 nm i s cytochrome b ^ g (at 77°K) . The min-or component w i t h a peak at about 565 nm detected at +140 mV i s probably cytochrome b^^ 2 ( a t 77°K). Besides v a r y i n g i n amount, the midpoint o x i d a t i o n -r e d u c t i o n p o t e n t i a l s of the two major components comprising the cytochrome b peak appeared to vary s l i g h t l y w i t h the growth phase. The low p o t e n t i a l b cytochrome of very e a r l y exponential phase c e l l s had a midpoint p o t e n t i a l of +15 mV wh i l e the high p o t e n t i a l cytochrome b had approximately the same value as i n mid or l a t e exponential c e l l s ( F i g . 40). In s t a t i o n a r y phase c e l l s the midpoint p o t e n t i a l of the h i g h p o t e n t i a l cytochrome was as h i g h as +205 mV w i t h the low p o t e n t i a l cytochrome having approximately the same value as i n mid or l a t e exponential c e l l s . These small changes i n 133 FIGURE 40. P o t e n t i o m e t r i c t i t r a t i o n of cytochrome b i n p a r t i -c l e s prepared from e a r l y exponential c e l l s . The p a r t i c l e s were prepared from c e l l s grown on succinate to the e a r l y expo-n e n t i a l phase of growth and suspended i n 0.1 M phosphate b u f f e r , pH 7.0, to a con c e n t r a t i o n of 4.2 mg pr o t e i n / m l . The experiment was c a r r i e d out i n a s i m i l a r manner to F i g . 38. 133a 134 JIGURE 41. Po t e n t i o m e t r i c t i t r a t i o n of cytochrome b i n p a r t i -c l e s prepared from s t a t i o n a r y c e l l s . The p a r t i c l e s were pre-pared from c e l l s grown on succinate to the s t a t i o n a r y phase of growth and suspended i n 0.1 M phosphate b u f f e r , pH 7.0,l;'t6 a concentration of 7.8 mg pro t e i n / m l . The experiment was c a r r i e d out i n a s i m i l a r manner to F i g . 38. 134a 0.06 56 0 200 100 0 -100 E n , mV T 0 1 1 0 1 L O G ^ 1 3 5 redox p o t e n t i a l s could be due to environmental changes w i t h i n the membrane r a t h e r than being due to the presence of a new cytochrome since i t i s known that the midpoint o x i d a t i o n r e -d u c t i o n p o t e n t i a l s of the cytochromes are very s e n s i t i v e to en-vironmental f a c t o r s (62). These data are summarized i n Table 3 The amount of the high p o t e n t i a l cytochrome was de-creased i n amount r e l a t i v e to the low p o t e n t i a l component i n e a r l y exponential phase c e l l s when compared to mid or l a t e ex-p o n e n t i a l phase c e l l s . However, i n s t a t i o n a r y phase c e l l s the amount of the h i g h p o t e n t i a l cytochrome was increased r e l a t i v e to the amount of the low p o t e n t i a l component ( F i g . 41). The l e v e l s of cytochromes b ^ g and b ^ g , r e s o l v a b l e at 77 °K, cor-r e l a t e d w i t h the l e v e l s of the h i g h and low p o t e n t i a l cyto-chromes, r e s p e c t i v e l y , i n a l l these types of c e l l s . The redox p o t e n t i a l of cytochrome b-^  i n mitochondria has been shown to increase by over 200 mV upon a d d i t i o n of ATP (162). A d d i t i o n of pulses of ATP, to give a f i n a l concentra-t i o n of 0.5 mM, to the system i n which the redox p o t e n t i a l of cytochrome b was being measured showed no apparent e f f e c t on :bhe midpoint p o t e n t i a l of e i t h e r of the major b cytochromes of E. c o l i . Cyanide, which i n t e r a c t s w i t h t e r m i n a l oxidases, could p o s s i b l y a f f e c t the midpoint p o t e n t i a l of a t e r m i n a l ox-idase. In an attempt to i d e n t i f y a component absorbing i n the cytochrome b-region w i t h cytochrome o, the redox t i t r a t i o n of cytochrome b was c a r r i e d out i n the presence of 14 mM KCN. 136 TABLE 3 MIDPOINT OXIDATION-REDUCTION POTENTIALS AND AMOUNT OF THE MAJOR B CYTOCHROMES OF RESPIRATORY PARTICLES PREPARED FROM CELLS AT DIFFERENT PHASES OF GROWTH3 Growth phase Cytochrome Midpoint % t o t a l p o t e n t i a l (mV) cytochrome b E a r l y exponential b556 + 15 73 b558 +169 27 Mid exponential + 34 60 "B556 +165 40 S t a t i o n a r y $558 +334 40 b558 +196 60 C e l l s were grown on glucose, succinate or complex media. Midpoint p o t e n t i a l s are the averages of se v e r a l determinations. The cytochromes are i d e n t i f i e d by the p o s i t i o n of the absorp-t i o n maximum of the reduced form at 77°K. 137 There was no e f f e c t of cyanide on the measured redox poten-t i a l s . Hendler et al«(70) have examined the redox poten-t i a l s of the b cytochromes i n p a r t i c l e s of E. c o l i W6. They found three b-type cytochromes w i t h midpoint o x i d a t i o n - r e d u c -t i o n p o t e n t i a l s of -50 mV, +110 mV, and +220 mV. Since these r e s u l t s are d i f f e r e n t from those given above, we have compared the redox t i t r a t i o n curves obtained using the mixture of me--/ d i a t o r s employed by Hendler et al.^with that used i n our stud-i e s ( F i g . 42). The same p a r t i c l e p r e p a r a t i o n was used. A l -though both t i t r a t i o n curves suggested the presence of two components, the curves were not i d e n t i c a l . Whereas the reduc-t i o n of the low p o t e n t i a l component was 957<> complete at -50 mV w i t h our mixture of mediators, t h i s cytochrome was only 657> reduced w i t h the mixture of mediators used by Hendler et a l . A n a l y s i s by the Nernst equation of the data ob-= ta i n e d w i t h t h e i r mediator mixture showed that the low poten-t i a l component d i d not y i e l d a l i n e a r p l o t w i t h a slope of 59 mV as found w i t h our mediators, r a t h e r the slope was greater than t h i s value and could not be analyzed simply. With both mixtures of mediators the high p o t e n t i a l component gave a mid-p o i n t o x i d a t i o n - r e d u c t i o n p o t e n t i a l of +165 mV. In none of our t i t r a t i o n experiments could we detect the presence of the three b-type cytochromes detected by Hendler et a l . I t may be that the l a c k of e q u i l i b r a t i o n of the mediators w i t h the redox systems was r e s p o n s i b l e f o r the d i f f e r e n c e s between these r e -138 FIGURE 42. E f f e c t of d i f f e r e n t mixtures of mediators on the p o t e n t i o m e t r i c t i t r a t i o n curve of the cytochrome b components. For both p l o t s A and B p a r t i c l e s were prepared from c e l l s grown to the exponential phase of growth on succinate and suspended i n 0.1 M phosphate b u f f e r , pH 7.0, to a concentra-t i o n of 5.5 mg p r o t e i n / m l . The mediators used f o r p l o t A were 50 uM PMS, 50 uM PES, 50 uM duroquinone, and 50uuM quinhydrone. The mediators used f o r p l o t B were 50 uM 2-hyd-roxy-1,4-naphthoquinone, 25 uM pyocyanine, 50 pM PMS, and 50 u M quinhydrone. The t i t r a t i o n s were c a r r i e d out i n s i m i l a r manner to F i g . 38. 138a E n , m v 139 s u i t s and suggests that i n t e r p r e t a t i o n of t i t r a t i o n data must be made w i t h c a u t i o n (163). A f u r t h e r reason f o r the d i f f e r e n c e s between our r e s u l t s and those of Hendler et al.may be that f r e s h l y pre-pared p a r t i c l e s were used i n our experiments w h i l e frozen and thawed m a t e r i a l was used by Hendler et a l . Reduction k i n e t i c s of cytochrome b and the e f f e c t of HOQNO The i n t e r a c t i o n of the hi g h and low p o t e n t i a l cyto-chromes w i t h the r e s p i r a t o r y chain was now examined. The r e -dox s t a t e of cytochrome b was followed w i t h time using NADH and succinate as substrates ( F i g . 43). A f t e r a d d i t i o n of NADH, cytochrome b assumed i t s aerobic steady s t a t e l e v e l of red u c t i o n u n t i l the oxygen i n the s o l u t i o n was depleted. The i n i t i a l r a p i d phase of re d u c t i o n on anaerobiosis was followed by a slower phase of re d u c t i o n i n d i c a t i n g a non-homogeneous pool of cytochrome b. Reduction of cytochrome b w i t h s u c c i -nate followed the same p a t t e r n except that s i n c e the o x i d a t i o n r a t e of succinate was slower, a longer time was r e q u i r e d to reach anaerobiosis. The pool of r a p i d l y reduced cytochrome was l a r g e r w i t h succinate than w i t h NADH. However, the r a t e of r e d u c t i o n i n t h i s i n i t i a l r a p i d phase was slower w i t h suc-c i n a t e than w i t h NADH. As p r e v i o u s l y observed ( F i g . 35), both substrates d i d not reduce the t o t a l cytochrome b pool as demon-s t r a t e d by the increased l e v e l of re d u c t i o n obtained f o l l o w i n g the a d d i t i o n of d i t h i o n i t e . 140 FIGURE 43. Reduction, of cytochrome b by NADH and succinate. The p a r t i c l e s were prepared from c e l l s grown on suc c i n a t e to the s t a t i o n a r y phase and suspended i n 0.1 M phosphate b u f f e r , pH 7.0. The redox s t a t e of cytochrome b was followed at the wavelength p a i r of 558-540 nm using a dual wavelength spectro-photometer. NADH (2 mM) or succinate (1.67 mM) was added at the p o i n t i n d i c a t e d (zero time) i n the presence or absence of HOQNO (73 yM and 44 yM w i t h NADH and succi n a t e , r e s p e c t i v e l y ) and the progress of the s p e c t r a l change i s followed from r r i g h t to l e f t . The t o t a l volume i n the cuvettes was 1.5 ml. A few c r y s t a l s of sodium d i t h i o n i t e were added at D. The par-t i c l e s were suspended to a concent r a t i o n of 2.7 and 5.4 mg protein/m l f o r the experiments i n v o l v i n g NADH and succi n a t e , r e s p e c t i v e l y . D 0 . 0 1 S u b s t r a t e HOQNO +HOQNO 3 mm NADH 0^ o S u c c 141 P r i o r a d d i t i o n of HOQNO to the membrane p a r t i c l e s slowed the o x i d a t i o n r a t e of both substrates. The aerobic steady s t a t e was lower i n the presence of HOQNO. Upon anaer-o b i o s i s the r a p i d phase of re d u c t i o n w i t h NADH was more mark-edl y i n h i b i t e d than t h a t w i t h succinate. When succinate was used HOQNO als o decreased the s i z e of the pool of cytochrome b which underwent r a p i d r e d u c t i o n . The r a t e of the subse-quent slow phases of re d u c t i o n w i t h both substrates was also i n h i b i t e d by HOQNO. The t r a n s i t i o n to anaerobiosis was not as sharp i n the presence of HOQNO. E s s e n t i a l l y the same r e -s u l t s were found f o r the k i n e t i c s of cytochrome b r e d u c t i o n i n p a r t i c l e s prepared from c e l l s grown to the exponential phase of growth ( F i g . 44). The m u l t i p h a s i c nature of the k i n e t i c s of cyto-chrome b re d u c t i o n i n p a r t i c l e s prepared from s t a t i o n a r y c e l l s u s ing NADH, w i t h and without HOQNO, was more c l e a r l y demonsSr t r a t e d i n F i g . 45. The logarithm of r e s i d u a l o x i d i z e d cyto-chrome b was p l o t t e d against time a f t e r anaerobiosis. The i n -h i b i t o r HOQNO slowed the r a t e i n both the f a s t and slow phases of r e d u c t i o n . The m u l t i p h a s i c nature of red u c t i o n w i t h s u c c i -nate was not as c l e a r i n the dual wavelength t r a c e s shown i n F i g . 43. The m u l t i p h a s i c nature of the re d u c t i o n of cyto -chrome b by succinate i n the presence and absence of HOQNO was shown by a s i m i l a r method ( F i g . 46). The e f f e c t of the concent r a t i o n of HOQNO on the NADH oxidase and succinate oxidase a c t i v i t i e s and the aerobic 142 FIGURE 44. Reduction of cytochrome b by succinate and NADH i n p a r t i c l e s prepared from c e l l s grown on succinate to the expon-e n t i a l phase of growth. The p a r t i c l e s were suspended i n 0.1 M phosphate,buffer, pH 7.0, to a concentration of 4.9 mg p r o t e i n / ml. The experiments were c a r r i e d out i n a s i m i l a r manner to those i n F i g . 43. NADH (2 MM) or succinate (1.67 mM) was add-ed at the p o i n t ( s ) i n d i c a t e d (S) i n the presence of"absence of HOQNO (37 uM and 30 uM, r e s p e c t i v e l y ) . Progress of s p e c t r a l change i n followed from r i g h t to l e f t . N A D H + H O O N O [ _ 0 . 0 1 A 3 0 s + H O Q N O S u c c i n a t e 143 FIGURE 45. Reduction of cytochrome b by NADH i n the presence and absence of HOQNO. The redox s t a t e of cytochrome b f o l l o w -i n g anaerobiosis was measured as described i n F i g . 43. Log AA was determined as described i n the t e x t . HOQNO (50 -pM) , when present, was added p r i o r to the a d d i t i o n of NADH (2mM). The p a r t i c l e s were suspended at a concentr a t i o n of 6.8 mg p r o t e i n / ml. 143a 144 FIGURE 46. Reduction of cytochrome b by succinate i n , t h e pre-sence and absence of HOONO. The redox s t a t e of cytochrome b f o l l o w i n g anaerobiosis was measured as described i n F i g . 43. Log AA was determined as described i n the t e x t . HOQNO (50 uM), when present, was added p r i o r to the a d d i t i o n of succinate (1.67 mM). The p a r t i c l e s were suspended at a concentration of 6.8 mg prote i n / m l . 144a 145 steady s t a t e l e v e l of reduction, of cytochrome b i s shown i n F i g . 47. There was a decrease i n oxidase a c t i v i t y to a p l a t -eau l e v e l w i t h i n c r e a s i n g concentrations of HOQNO. The p l a t -eau l e v e l of i n h i b i t i o n was 70% and 30% f o r NADH and s u c c i -nate oxidases, r e s p e c t i v e l y . The same s i t u a t i o n was found f o r c e l l s grown to the exponential and to the s t a t i o n a r y phases of growth. The aerobic steady s t a t e l e v e l s of r e d u c t i o n of cy-tochrome b i n p a r t i c l e s from c e l l s grown to the exponential phase was higher w i t h NADH than w i t h succinate which i s i n agreement w i t h the higher a c t i v i t y of NADH oxidase compared w i t h succinate oxidase. However, the aerobic steady s t a t e l e v e l of r e d u c t i o n w i t h NADH decreased w i t h i n c r e a s i n g concen-t r a t i o n of HOQNO w h i l e i t remained almost unchanged w i t h suc-c i n a t e ( F i g . 47). The same p a t t e r n was seen w i t h p a r t i c l e s from c e l l s grown to the s t a t i o n a r y phase although i n t h i s case the aerobic steady s t a t e l e v e l s were lower. This i s i n l i n e w i t h the lower NADH and succinate oxidase a c t i v i t i e s of s t a t i o n a r y phase c e l l s . The extent of i n h i b i t i o n of oxidase a c t i v i t y d i d not p a r a l l e l the changes i n the aerobic steady s t a t e l e v e l of red u c t i o n . Thus, at a conc e n t r a t i o n of HOQNO where the NADH and succinate oxidase a c t i v i t i e s were maximally i n h i b i t e d , the aerobic steady s t a t e l e v e l of r e d u c t i o n was l i t t l e a f f e c t e d . 146 FIGURE 47. E f f e c t of con c e n t r a t i o n of HOQNO on NADH and suc-c i n a t e oxidase a c t i v i t i e s , and on the aerobic steady- s t a t e l e -v e l s of r e d u c t i o n of cytochrome b. Data f o r p a r t i c l e s pre-pared from c e l l s grown to the exponential..and s t a t i o n a r y phas-es of growth are shown i n .1, 2 and 3, 4, r e s p e c t i v e l y . The p a r t i c l e s were suspended to a con c e n t r a t i o n of 4.9 mg p r o t e i n / ml f o r exponential phase c e l l s and 5.4 mg protein/ml f o r s t a -t i o n a r y phase c e l l s . The .data were obtained from experiments s i m i l a r to those described i n F i g . 43 and F i g . 44. Oxidase a c t i v i t y , \ g i v e n as a percentage of the c o n t r o l l e v e l s w i t h suc-c i n a t e (S; 1.67 mM) ,and NADH(IdN; 2 mM) i n the absence of i n h i -b i t o r , was determined by c a l c u l a t i n g the i n v e r s e of the time r e q u i r e d f o r the system to become anaerobic a f t e r a d d i t i o n of the su b s t r a t e . Anaerobiosis was i n d i c a t e d by the i n i t i a l up-ward d e f l e c t i o n i n the t r a c e of cytochrome b re d u c t i o n . HOQNO was added p r i o r to the a d d i t i o n of substrate. The aerobic steady s t a t e l e v e l of r e d u c t i o n of cytochrome, b was determined immediately a f t e r the a d d i t i o n of substrate and c a l c u l a t e d as a percentage of the d i t z K i o n i t e - r e d u c i b l e cytochrome b. 147 Low temperature d i f f e r e n c e spectra of the d i f f e r e n t redox  sta t e s of Cytochrome b. Immediately a f t e r the a d d i t i o n of the su b s t r a t e s , NADH or suc c i n a t e , the samples were plunged i n t o l i q u i d N 2 to trap the cytochromes i n t h e i r aerobic steady s t a t e . The d i f -ference spectra were then run at 77°K. 'Ks can be seen from the spectra i n F i g . 48, both cytochromes b ^ g and b ^ g were reduced i n the aerobic steady s t a t e by NADH and by succinate; The percent r e d u c t i o n r e l a t i v e to the d i t h i o n i t e - r e d u c e d l e -v e l was higher f o r cytochrome b ^ g than f o r cytochrome b ^ g , 2.8% and 2.3%, r e s p e c t i v e l y , w i t h NADH, and 1.5% and 1.3%, r e s p e c t i v e l y , w i t h succinate. In the presence of HOQNO the re d u c t i o n of cytochrome b ^ g r e l a t i v e to cytochrome b ^ g was f u r t h e r enhanced. These spect r a were taken w i t h p a r t i c l e s from c e l l s grown to the s t a t i o n a r y phase. In t h i s case the apparent aerobic steady s t a t e l e v e l of re d u c t i o n of cytochrome b w i t h NADH i n the presence of HOQNO was s l i g h t l y increased i n cont r a s t to the data presented i n F i g . 47. I have no explana-t i o n f o r t h i s d i f f e r e n c e but i t was observed c o n s i s t e n t l y w i t h low temperature spectra of s t a t i o n a r y phase c e l l s o x i d i z i n g NADH i n the presence of HOQNO. Cytochrome b ^ g was s l i g h t l y more reduced than cytochrome b ^ g when measured r e l a t i v e to the d i t h i o n i t e l e v e l of re d u c t i o n i n the low temperature d i f -ference spectra of the anaerobic steady s t a t e w i t h both NADH and succinate as substra t e s . The same p a t t e r n was seen when the cytochromes were trapped i n t h e i r redox s t a t e s by r a p i d l y 148 FIGURE 48. E f f e c t of HOQNO on the steady s t a t e l e v e l s of r e -duction of cytochrome b. The d i f f e r e n c e spectra at 77°»K were obtained as i n F i g . 34. The cytochromes reduced i n the aero-b i c and anaerobic steady s t a t e s are shown on the l e f t and r i g h t sides of the f i g u r e , r e s p e c t i v e l y . The aerobic steady s t a t e spectra were obtained by immersing the cuvette i n t o l i -q u id N 2 immediately a f t e r the a d d i t i o n of NADH (2 mM) or s u c c i -nate (1.67 mM) to the p a r t i c l e suspension. HOQNO, 110 uM when present, was added p r i o r to the a d d i t i o n of the substrates. The p a r t i c l e s were prepared from c e l l s grown on succi n a t e to the s t a t i o n a r y phase and suspended i n the b u f f e r used i n F i g . 33 to a concentr a t i o n of 4.1 mg prote i n / m l . 148a 149 f r e e z i n g at 77 °K immediately a f t e r the i n i t i a l r a p i d phase of r e d u c t i o n by NADH o c c u r r i n g at anaerobiosis. In the anaerobic steady s t a t e the presence of HOQNO f u r t h e r enhanced the r e d u c t i o n of cytochrome b ^ g r e l a -t i v e to that of cytochrome b ^ g . This was more marked w i t h NADH than w i t h succinate as the substrate. In agreement w i t h the r e s u l t s shown i n F i g . 4jf7, the extent of r e d u c t i o n of the to.ta.1 cytochrome b pool was lower i n the anaerobic steady s t a t e i n the presence of HOQNO. There may als o have been s l i g h t s h i f t i n the wave-le n g t h of maximum absorption of cytochrome b ^ g and b ^ g i n the presence of HOQNO. The absorption peak of cytochrome ^556 w a s s b i f t e d by about 0.5 nm towards lower wavelengths w h i l e that of cytochrome b ^ g was s h i f t e d by about the same amount towards higher wavelengths r e s u l t i n g i n sharper d e f i n -i t i o n of the two peaks i n the low temperature d i f f e r e n c e spectrum. This suggested that HOQNO may i n t e r a c t w i t h the b cytochromes d i r e c t l y . P o t e n t i o m e t r i c t i t r a t i o n s of the b cy-tochromes i n the presence of HOQNO were complicated by the i n t e r f e r e n c e of the i n h i b i t o r w i t h the e q u i l i b r a t i o n of the mediators w i t h the cytochromes, but no d e f i n i t e e f f e c t on the redox p o t e n t i a l s was detected. The d i f f e r e n c e s p e c t r a measured at 77°K of the ae-r o b i c and anaerobic steady s t a t e s of r e d u c t i o n of the b cyto-chromes i n p a r t i c l e s from exponential c e l l s are shown i n F i g . 49. There was a smaller amount of cytochrome b ^ g than cyto-150 FIGURE 49. E f f e c t of HOQNO on the steady s t a t e l e v e l s of r e -duc t i o n of cytochrome b in. p a r t i c l e s from exponential c e l l s . The d i f f e r e n c e spectra at 77°K were obtained as i n F i g . 34. The cytochromes reduced i n the. aerobic and anaerobic steady s t a t e s are shown on the l e f t and ri g h t , sides of the f i g u r e , r e s p e c t i v e l y . HOQNO, 110 uM, when present, was added p r i o r to the a d d i t i o n of the substra t e s . The p a r t i c l e s were prepared from c e l l s grown on succinate to the exponential phase of growth and suspended i n the b u f f e r used i n F i g . 34 to a con-c e n t r a t i o n of 10.6 mg prote i n / m l . 150a I I I I I I 5 4 0 5 7 0 5 4 0 5 7 0 W a v e l e n g t h , n m 1 5 1 chrome b ^ g i n these p a r t i c l e s compared w i t h p a r t i c l e s from s t a t i o n a r y c e l l s as shown e a r l i e r . The aerobic steady s t a t e l e v e l of re d u c t i o n of the b cytochromes i n p a r t i c l e s o x i d i z -i n g NADH and suc c i n a t e , trapped at 77°K, was higher than i n p a r t i c l e s from s t a t i o n a r y c e l l s . The data was i n general ag-reement w i t h that presented i n F i g . 48 f o r p a r t i c l e s from s t a -t i o n a r y c e l l s except that the aerobic steady s t a t e l e v e l of red u c t i o n of the b cytochromes decreased s l i g h t l y i n exponen-t i a l p a r t i c l e s o x i d i z i n g NADH i n the presence of HOQNO. Examination of the compartmentalization of cytochrome b. With both NADH and suc c i n a t e , r e d u c t i o n of cyto-chrome b d i d not occur i n a s i n g l e phase. This suggested that there were separate pools of cytochrome b w i t h each pool con-t a i n i n g both cytochrome b ^ g and b ^ g . To examine the p o s s i b -l e overlap of these cytochrome b pools werlooked at the k i n e -t i c s of r e d u c t i o n i n the presence of both substrates ( F i g . 50). When NADH was added i n the aerobic phase to a pr e p a r a t i o n a l -ready o x i d i z i n g succinate there was a stepwise i n c r e a s e i n the aerobic steady s t a t e l e v e l of red u c t i o n . When both NADH and succinate were present the aerobic steady s t a t e l e v e l of r e -duction was the sum of the l e v e l s , observed w i t h each substrate alone. More complete r e d u c t i o n of the cytochromes a f t e r anae-r o b i o s i s occurred i n the presence of both substrates than w i t h each separately. A l s o , there was a l a r g e r amount of cytochrome 152 FIGURE 50. Reduction of cytochrome b i n the presence of both NADH and succinate. The redox s t a t e of cytochrome b was f o l -lowed as described in. F i g . 43. The. p a r t i c l e s were prepared as described i n F i g . 43.and suspended to a concent r a t i o n of 6.8 mg pro t e i n / m l . NADH (2 mM) (N), succinate (1.67 mM) (S), and a few c r y s t a l s of sodium d i t h i o n i t e (D), were added at the poi n t s i n d i c a t e d . Progress of s p e c t r a l change i s followed from r i g h t to l e f t . 1 5 3 b reduced i n the i n i t i a l r a p i d phase under these c o n d i t i o n s . I f succinate was added a f t e r the r a p i d phase of cytochrome b re d u c t i o n had occurred i n the presence of NADH, there was a f u r t h e r r a p i d phase of r e d u c t i o n of t h i s cytochrome. Rever-si n g the order of a d d i t i o n of the substrates gave a s i m i l a r r e s u l t . This suggested that the cytochrome b was p a r t i a l l y compartmentalized i n t o d i f f e r e n t pools. When the k i n e t i c s of r e d u c t i o n of cytochrome d were followed using NADH as substrate a b i p h a s i e r e d u c t i o n curve was a l s o observed ( F i g . 51). In con t r a s t to cytochrome b, most of the cytochrome d was r a p i d l y reduced. With succinate as substrate the re d u c t i o n k i n e t i c s of cytochrome d looked more homogeneous w i t h only a small amount being s l o w l y r e -duced. • The combination of succinate and NADH r a p i d l y reduced almost the e n t i r e amount of cytochrome d. Thus, compartment-a l i z a t i o n of the pool of cytochrome d i s not as marked as w i t h cytochrome b. 154 FIGURE 51. Reduction of cytochrome d i n the presence of both NADH and succinate. The. same p r e p a r a t i o n was used as i n F i g . 50. The redox s t a t e of cytochrome d was followed using the wavelength p a i r 628-605 nm. NADH (2 mM) (N), "succinate (1.67 mM) (S), and d i t h i o n i t e (D) were added at the p o i n t s i n d i c a t e d Progress of s p e c t r a l change i s fo l l o w e d from r i g h t to l e f t . 155 DISCUSSION The d i f f e r e n c e s p e c t r a of E. c o l i membrane p a r t i -c l e s taken at room temperature demonstrate the presence of cytochromes d, a-^ , and b-^  and a carbon monoxide bi n d i n g p i g -ment, cytochrome o. However, spectra taken at 77°K r e v e a l the presence of a d d i t i o n a l components w i t h overlapping a-ab-s o r p t i o n bands i n the cytochrome b-^  region. The r e l a t i v e a-mounts of the components comprising the cytochrome b^aabsorp-t i o n peak change w i t h the t r a n s i t i o n from exponential to s t a -t i o n a r y growth. Cytochrome b ^ g i s the major component i n c e l l s grown to the exponential phase of growth w h i l e cyto-chrome b ^ g becomes prominent i n the s t a t i o n a r y phase of growth (9, 81). Other components absorbing at approximately 550 nm and 562 nm appear as minor shoulders on the major ab-s o r p t i o n bands. Shipp (9), using f o u r t h order d i f f e r e n t i a l a n a l y s i s of the low temperature d i f f e r e n c e spectra of the cy-tochrome b-^  a-absorption band re g i o n reported components w i t h absorption peaks at 548-549, 552-553, 556-557.5, 559-562, and 564-566 nm. V a r i a t i o n s occurred i n the l e v e l s of these com-ponents as a f u n c t i o n of growth phase, carbon source and as a r e s u l t of mutation. The f u n c t i o n of these components or t . t h e i r arrangement i n the r e s p i r a t o r y chain was not determined However, according to Haddock and Sc h a i r e r (81) cytochrome ^556 a n d cytochrome o compose the primary r e s p i r a t o r y pathway i n exponential c e l l s w h i l e cytochrome b c c o and cytochrome d 156 compose an a l t e r n a t e pathway formed i n the t r a n s i t i o n to s t a -t i o n a r y growth. In the f i r s t p a r t of t h i s t h e s i s I showed that both cytochrome d and cytochrome o p a r t i c i p a t e d i n the NADH and succinate oxidase systems. The c o n t r i b u t i o n to these sys-tems was. d i r e c t l y p r o p o r t i o n a l to the r e l a t i v e amounts of these t e r m i n a l oxidases i n the membrane. In t h i s p a r t of the t h e s i s I have examined the p a r t i c i p a t i o n of the other c y t o -chromes i n the NADH and succinate oxidase systems. Substrate reduced minus o x i d i z e d d i f f e r e n c e spect-r a showed th a t succinate and NADH were able to reduce cyto-chrome i3cjcjg and cytochrome b ^ g i n the anaerobic steady s t a t e but could not reduce the e n t i r e pool of e i t h e r cytochrome i n c e l l s grown e i t h e r to the exponential or to the s t a t i o n a r y phase of growth.wiWith the exception of cytochrome b^g2> t n e other components, cytochrome c,and cytochrome , al s o appear not to be t o t a l l y r e d u c i b l e by these substrates. Ascorbate i n the presence of PMS reduced a s l i g h t l y l a r g e r f r a c t i o n of the cytochrome b ^ g pool than of the cytochrome b ^ g pool suggesting that cytochrome b^^g was the higher p o t e n t i a l cyto-chrome of the two. Examination of the d i t h i o n i t e reduced mi-nus ascorbate plus PMS reduced d i f f e r e n c e spectrum measured at 77°K i n d i c a t e d that cytochrome b ^ ^ w a s almost e n t i r e l y r e -du c i b l e by ascorbate plus PMS suggesting that i t was a high p o t e n t i a l component. The thermodynamic r e l a t i o n s h i p s of the cytochromes were determined i n order to understand t h e i r arrangement i n 157 the r e s p i r a t o r y system of E. c o l i . Cytochrome d, as shown i n the f i r s t p a r t of t h i s t h e s i s , i s a one e l e c t r o n c a r r i e r and has a midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l of +260 mV. The h i g h p o t e n t i a l of cytochrome d i s i n l i n e w i t h i t s r o l e as a t e r m i n a l oxidase. Cytochrome a-^  i s a l s o a one e l e c t r o n c a r -r i e r w i t h a midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l of +147 mV. Cytochrome a^ has been proposed to be a t e r m i n a l oxidase i n E_. c o l i (11) although no evidence f o r t h i s f u n c t i o n has been found. The measured p o t e n t i a l f o r t h i s component seems to be too low f o r i t to serve as a t e r m i n a l oxidase wheSe compared w i t h the value of cytochrome d or that found f o r cytochrome a^ (+380 mV) (164). The cytochrome b^ r e g i o n can be r e s o l v e d i n t o two major components absorbing at 560 nm and 557.5 nm at 22°C (corresponding to cytochrome b ^ g and b^^g, r e s p e c t i v e l y , at 77KK) w i t h midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l s of +165 and +36 mV, r e s p e c t i v e l y , i n exponential c e l l s . The l e v e l of the h i g h p o t e n t i a l component, as determined by p o t e n t i o m e t r i c t i t r a t i o n , i ncreases i n the t r a n s i t i o n to s t a t i o n a r y growth p r o v i d i n g a d d i t i o n a l evidence that the h i g h p o t e n t i a l compo-nent i s cytochrome b ^ g . The midpoint p o t e n t i a l s of these two b-cytochromes v a r i e d s l i g h t l y w i t h the growth phase. This v a r i a t i o n i s probably due to environmental i n f l u e n c e s and not due to the development of new components. The midpoint poten-t i a l s of the other components (c-type cytochromes and cyto-chrome b^g 2) abosrbing i n the cytochrome b r e g i o n could not be determined s i n c e t h e i r absorption peaks were too small. Fur-1 5 8 thermore, t h e i r redox p o t e n t i a l s may overlap w i t h those of the two major b-cytochromes. However, i t i s apparent from the substrate reduced spectra described e a r l i e r that cyto-chrome b^g2 i s a h i g h p o t e n t i a l b cytochrome. Hendler et al.(70) reported the presence of three b-cytochromes w i t h p o t e n t i a l s of -50 mV, +110 mV, and +220 mV i n E. c o l i . In none o f my preparations d i d I observe these cytochromes. This d i f f e r e n c e could p o s s i b l y be a t t r i b u t e d to use of d i f f e r e n t s t r a i n s and growth c o n d i t i o n s or the use by Hendler et a l * of c e l l s which were stored frozen. My work was c a r r i e d out w i t h f r e s h l y grown c e l l s . Furthermore, as d i s -cussed i n " t h e Results s e c t i o n , the mediator mixture used by Hendler et a l . d i d not appear to e q u i l i b r a t e s a t i s f a c t o r i l y w i t h the cytochromes. Cytochromes b ^ g and b ^ g do not appear to have p r o p e r t i e s analogous to cytochromes b^ and b^ i n m i t o c h o n d r i a l systems. In mitochondria cytochrome b^ i s reported to have a midpoint p o t e n t i a l of +30 mV while b^ has a p o t e n t i a l of -30 mV which changes to +245 mV upon the a d d i t i o n of ATP (162). This change has been suggested to be i n v o l v e d i n energy t r a n s -duction,? The r e d u c t i o n k i n e t i c s and the midpoint o x i d a t i o n -r e d u c t i o n p o t e n t i a l s of the E. colijcytochromes were unaffec-ted by ATP. Hendler et al.(70) a l s o found that the midpoint p o t e n t i a l s of the E. c o l i cytochromes were unaffected by ATP. In c o n t r a s t , ATP increased the. amount of cytochrome b reduc-i b l e by succinate at anaerobiosis i n Mycobacterium p h i e l (165). 159 Cyanide which i s an i n h i b i t o r of t e r m i n a l oxidases could p o s s i b l y a f f e c t the midpoint p o t e n t i a l of the c y t o -chrome oxidase. In an attempt to i d e n t i f y cytochrome o w i t h a component absorbing i n the cytochrome b^ region the poten-t i o m e t r i c t i t r a t i o n of cytochrome b-^  was c a r r i e d out i n the presence of cyanide. No e f f e c t was seen. The behaviour of the cytochromes of E. c o l i i n d i f f e r e n t steady s t a t e s of r e d u c t i o n were examined to deter-mine t h e i r o r g a n i z a t i o n i n the r e s p i r a t o r y chain. The aero-b i c steady s t a t e l e v e l of r e d u c t i o n of a cytochrome i s deter-mined by the r e l a t i v e r a t e of input of e l e c t r o n s from the sub-s t r a t e through the dehydrogenase, and the r a t e of output of e l e c t r o n s through the t e r m i n a l oxidase to oxygen. The c l o s e r a component i s to oxygen i n a sequence the lower should be i t s aerobic steady s t a t e l e v e l of r e d u c t i o n . Other f a c t o r s can a f f e c t t h i s s t r i c t l y k i n e t i c s i t u a t i o n such as the r e l a -t i v e midpoint p o t e n t i a l s of the cytochromes and the a c c e s s i b i -l i t y of cytochromes to e l e c t r o n s from the substrate. Whenbeibectron" f l o w away from the cytochrome i s slowed then the higher p o t e n t i a l component tends to becomee more reduced. The aerobic steady s t a t e l e v e l of r e d u c t i o n of cytochrome b^ was g e n e r a l l y l e s s than 10%. I t was s l i g h t l y higher i n exponential c e l l s probably because there was a g greater e l e c t r o n f l u x through a smaller cytochrome pool than i n the s t a t i o n a r y c e l l s . E l e c t r o n s from NADH f l u x through 160 the r e s p i r a t o r y chain. 10-20 times f a s t e r than e l e c t r o n s from succinate because of the r e l a t i v e a c t i v i t i e s of t h e i r dehydro-genases , yet the steady s t a t e l e v e l of re d u c t i o n of cytochrome b-^  w i t h NADH i s only 1.5 to 2.0 times as great as that w i t h succinate. When both substrates are present together, how=' ever, the aerobic steady s t a t e l e v e l of r e d u c t i o n i s equiva-l e n t to the sum of the i n d i v i d u a l aerobic steady s t a t e s even though the e l e c t r o n f l u x has been increased by only 10%. This suggests that t h e r e must be some compartmentalization of the pathways of e l e c t r o n flow from NADH and succinate to oxy-gen. Both cytochromes b ^ g and b ^ g were reduced i n the ae-r o b i c steady s t a t e by both substrates i n exponential and s t a -t i o n a r y c e l l s . In a l l cases there was a s l i g h t l y greater r e -duction of cytochrome b ^ g than of cytochrome b ^ g i n the aerobic steady s t a t e i n agreement w i t h t h e i r midpoint o x i d a -t i o n - r e d u c t i o n p o t e n t i a l s . These r e s u l t s i n d i c a t e that each compartmentalized pool of b-cytochromes must c o n t a i n both cytochromes b ^ g and b ^ g . The k i n e t i c s of re d u c t i o n of the cytochrome b pool supports the idea of compartmentalization of, but i n t e r a c t i o n between, the cytochromes of the NADH and succinate oxidase systems. A f r a c t i o n (307o) of the cytochrome b-^  pool i s r e -duced r a p i d l y by NADH upon anaerobiosis f o l l o w e d by a slower phase of re d u c t i o n . Both cytochrome b ^ g and b ^ g are r e -duced i n both phases. With succinate the s i z e of the pool which i s r a p i d l y reduced upon anaerobiosis i s l a r g e r (45%) but the f i n a l l e v e l of r e d u c t i o n (70%) i s l e s s than w i t h NADH 161 (80%). When the substrates are used i n combination more of the cytochromes are reduced r a p i d l y upon anaerobiosis i n d i c a -t i n g t h a t there i s some compartmentalization of the b-cyto-chromes between the NADH and succinate oxidase systems. How-ever, the extent of r e d u c t i o n of the b-cytochromes i n the r a -p i d phase when both substrates are used together i s not equ i -v a l e n t to the sum of the i n d i v i d u a l pools of r a p i d l y r e d u c i b l e cytochrome when NADH and succinate are used alone. This i n d i -cates that some of the components' are common to both the NADH and succinate oxidase systems. A c e r t a i n f r a c t i o n (10-15%) of both b-cytochromes comprising the cytochrome b-^  f r a c t i o n i s not reduced by NADH or suc c i n a t e , alone or together. V i r t u a l -l y complete r e d u c t i o n of the b-cytochromes would be expected i n the presence of NADH which has a redox p o t e n t i a l of -340 mV. Since the r e d u c t i o n l e v e l .expected w i t h NADH or s u c c i -nate, on the b a s i s of midpoint p o t e n t i a l s , was not obtained there may be an a c c e s s i b i l i t y b a r r i e r preventing the r e d u c t i o n of some of the cytochrome b pool . This b a r r i e r appears to be lower f o r succinate because the r e d u c t i o n of cytochrome b by succinate approaches more c l o s e l y the expected l e v e l . The b a r r i e r may be due to denatured cytochrome or to separate cy-tochrome pools not d i r e c t l y l i n k e d to the substra t e s . The b i p h a s i c nature of r e d u c t i o n of cytochrome b has a l s o been observed i n m i t o c h o n d r i a l preparations. The f i r s t order r a t e constant f o r the f i r s t phase was s i g n i f i c a n t -l y higher than the r a t e constant f o r the second phase (166). 162 Eisenbach and Gutman (166) have a t t r i b u t e d the f a s t and slow phases of r e d u c t i o n to a c t i v e and s l u g g i s h forms of cytochrome b and that t h e i r i n t e r c o n v e r s i o n i s a dynamic c o n t r o l mechan-ism f o r the r e g u l a t i o n of e l e c t r o n f l u x i n the system. As w i t h E. c o l i , on the b a s i s of midpoint p o t e n t i a l s more com-p l e t e r e d u c t i o n of the b-cytochromes by NADH or succinate would be expected. An a c c e s s i b i l i t y b a r r i e r was suggested to e x p l a i n these r e s u l t s (167.) . The b a r r i e r between succinate and cytochrome b, appeared to be much lower than that between NADH dehydrogenase and cytochrome b (168). This i s s i m i l a r to the s i t u a t i o n observed w i t h E. c o l i . This " b a r r i e r " f o r redox e q u i l i b r a t i o n ( a c c e s s i b i l i t y b a r r i e r ) i n mitochondria can be lowered by the a d d i t i o n of ATP (169, 170), antimycin (171), or by i n c r e a s i n g the pH from 7.0 to 9.0 (173). The mechanism of a c t i o n of these various agents i n causing greater r e d u c i b i l i t y of the b-cytochromes i n mitochondria can only be speculated (172). Mediators such as PMS al s o appear to remove the acces-s i b i l i t y b a r r i e r i n E. c o l i as seen by the more r a p i d and com-p l e t e r e d u c t i o n of the b-cytochromes i n the p o t e n t i o m e t r i c t i -t r a t i o n s where these mediators are present. PMS because of i t s a b i l i t y to penetrate membranes (174) may shunt e l e c t r o n s around b a r r i e r s w i t h i n the membrane. Further support f o r the p a r t i a l compartmentaliza-t i o n of the NADH and succinate oxidase chains i s given by the observation that NADH oxidase a c t i v i t y i s i n h i b i t e d by 707o i n the presence of HOQNO wh i l e succinate oxidase a c t i v i t y i s i n -.163 h i b i t e d only by 30%. Moreover, HOQNO has a greater e f f e c t on the r a t e of r e d u c t i o n by NADH than by succinate of the r a p i d l y -r e d u c i b l e cytochrome b pool. This i n d i c a t e s that the main suc-c i n a t e oxidase pathway i s l e s s s e n s i t i v e e t o i n h i b i t i o n by HOQNO than the main NADH oxidase pathway. The part of the succinate oxidase pathway which overlaps w i t h itehe main NADH oxidase path-way i s i n h i b i t e d , however. D i f f e r e n t i a l s e n s i t i v i t y to i n h i b i t i o n of the suc-c i n a t e and NADH oxidase a c t i v i t i e s has been observed i n other organisms. In A,_ v i n e l a n d i i . s u c c i n a t e oxidase a c t i v i t y was l e s s i n h i b i t e d than NADH oxidase by HOQNO (25). In Rhodopseu-domonas p a l u s t r i s , where the succinate oxidase system i s l e s s s e n s i t i v e than the NADH oxidase system to i n h i b i t i o n by antimy-c i n , an i n h i b i t o r which i n mitochondria acts at the same s i t e as HOQNO (175), a branched system which shunts e l e c t r o n s around the s i t e of i n h i b i t i o n has been proposed. This type of com-p a r t m e n t a l i z a t i o n appears analogous to the s i t u a t i o n i n E. c ^ 1  c o l i . At a con c e n t r a t i o n of HOQNO which i n h i b i t e d the NADH and succinate oxidase a c t i v i t y maximally there i s l i t t l e e f f e c t on the aerobic steady s t a t e l e v e l of r e d u c t i o n of the b cytochromes. This suggests that HOQNO e f f e c t i v e l y blocks e l e c -t r o n flow both before and a f t e r these cytochromes. This con-curs w i t h e a r l i e r r e p o r t s f o r the s i t e s of i n h i b i t i o n by HOQNO i n the NADH oxidase chain of E. c o l i (86, 97, 176) and i n Kleb- s i e l l a aerogenes (177). The lower r a t e of r e d u c t i o n i n the r a -164 p i d phase upon anaerobiosis supports the idea of a block be-for e the b cytochromes, wh i l e the enhanced r e d u c t i o n of cy t o -chrome b ^ g r e l a t i v e to cytochrome b ^ g i n the presence of HOQNO i n the aerobic steady s t a t e i s c o n s i s t e n t w i t h a block a f t e r both of the b cytochromes. There need not be two s i t e s o f HOQNO bi n d i n g to e x p l a i n t h i s phenomenon. M i t c h e l l (178) has proposed t h a t ubiquinone i n mitochondria goes through an ox i d a t i o n - r e d u c t i o n c y c l e y i e l d i n g intermediates which can both o x i d i z e and reduce the cytochrome b pool. HOQNO could block the normal f u n c t i o n i n g of t h i s c y c l e and th e r e f o r e would e f f e c t i v e l y b l ock e l e c t r o n flow i n t o and out of the cytochrome b p o o l . Antimycin and HOQNO both act as e l e c t r o n t r a n s p o r t i n h i b i t o r s i n mitochondria, a c t i n g near cytochrome b. Brandon et al.(175) have reported that both antimycin and HOQNO caused increased r e d u c t i o n i n mitochondria of one of the b-cytoshro chromes (b^gg) but only antimycin caused a red s h i f t (2-3 nm) i n the other b-cytochrome (b^g-^) . Antimycin i s not an e f f e c -t i v e i n h i b i t o r i n E. c o l i perhaps because of i t s l a r g e r s i z e than HOQNO. HOQNO causes only a small e f f e c t on the spectrum of the b-cytochromes i n E. c o l i . There appears to be a shar-pening of the absorption bands of cytochrome b ^ g and b ^ g i n the reduced plus HOQNO minus o x i d i z e d d i f f e r e n c e spectra run at 77°K which i s p o s s i b l y a t t r i b u t a b l e to a s l i g h t s h i f t i n the absorption peaks away from each other (0.5 nm i n each d i r -165 e c t i o n ) . This does not n e c e s s a r i l y mean d i r e c t i n t e r a c t i o n of the i n h i b i t o r w i t h the cytochromes but may be due to environ-mental i n f l u e n c e s . The t r a n s i t i o n to anaerobiosis i s normally i n dual wavelength experiments i n d i c a t e d by the sharp r i s e i n the ab-so r p t i o n at 558 nm due to the r a p i d r e d u c t i o n of tcytochrome b-^ . However, i n the presence of HOQNO t h i s t r a n s i t i o n i s not as sharp. White (123) has shown i n H. parai n f l u e n z a e that i n h i b i -t i n g e l e c t r o n flow w i t h HOQNO r a i s e s the K f o r oxygen of the te r m i n a l oxidases, cytochromes d and o. P o s s i b l y because of t h i s e f f e c t the r e s i d u a l oxygen i s u t i l i z e d more slowly and the t r a n s i t i o n to anaerobiosis i s not as marked. The k i n e t i c s of re d u c t i o n of cytochrome d are much more homogeneous than those of cytochrome b. In the aerobic steady s t a t e the l e v e l of r e d u c t i o n of cytochrome d i s e s s e n t i -a l l y 6% which i s i n l i n e w i t h i t s r o l e as a ter m i n a l oxidase. Both NADH and succinate reduce the major part of the cyto-chrome d pool r a p i d l y at anae r o b i o s i s . This would suggest that e l e c t r o n flow from both succinate and NADH converge at the l e v e l of the t e r m i n a l oxidase. However there i s s t i l l s;.r. some compartmentalization as i n d i c a t e d by the slower r e d u c t i o n of a small p a r t of the cytochrome d po o l . A working model f o r the r e s p i r a t o r y chain i n E. c o l i i s presented i n F i g . 52. The cytochrome components are arranged according to t h e i r redox p o t e n t i a l s as determined i n t h i s t h e s i s . Cytochrome o, f o r which no absorption peak has 166 FIGURE 52. Model proposed f o r the .arrangement of the cyto-chromes of the r e s p i r a t o r y chain o f E. c o l i . The components are arranged according to t h e i r midpoint o x i d a t i o n - r e d u c t i o n p o t e n t i a l s . The s o l i d l i n e s i n d i c a t e primary pathways of e l e c -t r o n flow w h i l e the dashed l i n e s i n d i c a t e secondary pathways. The e f f e c t i v e s i t e s of HOQNO i n h i b i t i o n are represented by the s o l i d bars. 16.6 a 167 been i d e n t i f i e d i n E. c o l i , has been a r b i t r a r i l y placed at the l e v e l of cytochrome b ^ g , although i t s midpoint o x i d a t i o n - r e -d u c t i o n p o t e n t i a l has not been determined. NADH and succinate feed e l e c t r o n s i n t o separate chains which i n t e r a c t and overlap to some degree, but converge at the l e v e l of the te r m i n a l o x i -dases. In exponential c e l l s the primary r e s p i r a t o r y pathway i s cytochrome b ^ g and cytochrome o. In s t a t i o n a r y c e l l s the cytochrome b ^ g and cytochrome d pathway develops and i s pos-s i b l y l i n k e d through cytochrome b^,^ to the dehydrogenases. The primary NADH oxidase pathway i s more s e n s i t i v e to i n h i b i -t i o n by HOQNO than the primary succinate pathway. The dashed l i n e s represent the pathways of NADH and succinate o x i d a t i o n which are HOQNO-insensitive and HOQNO-sensitive, r e s p e c t i v e l y . The slow phase of cytochrome b r e d u c t i o n may be due to a cyt o -chrome pool which i s not d i r e c t l y on the pathway. Cytochrome , the f u n c t i o n of which i s not known, i s placed o f f the r e s -p i r a t o r y chain but i n e q u i l i b r i u m w i t h i t . The c-type cyto-chrome^) and cytochrome b^^2» which are not in c l u d e d i n t h i s scheme, but which are present i n the membrane p a r t i c l e s of E. c o l i , may not be e s s e n t i a l f o r normal r e s p i r a t o r y a c t i v i t y . This was suggested by the absence of cytochrome b ^ ^ a n d cyto-chrome C i j i j Q i n a f u n c t i o n a l r e s p i r a t o r y system i n a 5-aminolev-u l i n i c a c i d - r e q u i r i n g mutant of E. c o l i grown i n the presence of haematin to r e c o n s t i t u t e the cytochrome system C81). Compartmentalized e l e c t r o n t r a n s p o r t systems have been suggested f o r other b a c t e r i a l species such as. Haemophilus  papainfluenzae (103), Azotobacter v i n e l a n d i i (25), Halobacter-168 ium cutirubrum (132)., and Rhodops eudbirionas' p'aiustris (133). The scheme presented f o r the o r g a n i z a t i o n of the cytochromes of the r e s p i r a t o r y chain may be an o v e r s i m p l i f i c a -t i o n of the t r u e s i t u a t i o n . . We have n e i t h e r considered the i n -t e g r a t i o n of the other oxidase systems nor have we considered the overlap of these cytochrome systems w i t h the n i t r a t e reduc-tase system of E. c o l i . Another f a c e t of great importance i s the i n t e r a c t i o n of the energy transducing components w i t h the r e s p i r a t o r y components which r e q u i r e s f u r t h e r a t t e n t i o n i f the problems of o x i d a t i v e phosphorylation are going to be solved. 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