The interaction of c-type cytochromes withRhodospirillum rubrum reaction centers

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Authors Rickle, Gregory Kent, 1951-

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THE INTERACTION OF C-TYPE CYTOCHROMES WITH RHODOSPIRILLUM RUBRUM REACTION' CENTERS

byGregory Kent Rickie

A Thesis Submitted to the Faculty of the•DEPARTMENT OF c h e m i s t r y

In Partial Fulfillment of the Requirements For the Degree ofMASTER OF SCIENCE

In the Graduate CollegeTHE UNIVERSITY OF ARIZONA

1 9 7 7

STATEMENT BY AUTHOR

This thesis has been submitted in partial ful­fillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate ac­knowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manu­script in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: K ■

APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below:

MICH&EIXA.^CUSANOVICH Associate Professor

of ChemistryDate

ACKNOWLEDGMENTS

I would•like to gratefully acknowledge Dr= Michael A. Cusanoyich for his guidance in my graduate studies and Dro Frank Rizzuto for his aid in construction of the flash photolysis apparatus. In addition, I. would like to ac­knowledge my wife, Nina, whose sacrifice, patience, and understanding made this work possible.

TABLE OF CONTENTS

PageLIST OF TABLES o © o. o © © © © © © © © © © © © © © © ©. VILIST OF ILLUSTRATIONS © © © © © © © © © ©, = . • © „ © . » viiABSTRACT © © © © © © © © © © © © © © © © © © © © © © © 5CINTRODUCTION © © © © © © © © © © © © © © © o © © © © © IMATERIALS AND METHODS.. © . . . . . « . © . . . © . © . 23

Preparation of Chromatophores © . © © . © . . . © , 25Extraction of Reaction Centers © © © © © © © © © © 26

RESULTS © o o © © o o o o o © o o e o o o o o o o © o o 3ICharacterization of Reaction Centers © © © © © © © 31Effects of Detergent and Organic Solvents . on Reaction Centers © © .. . © © © © . . © © © © © 35Effect of Detergent on the Oxidation-

Reduction Properties of Cytochrome c . © © = . . 38General Kinetic Properties © . . . © © . © . . . . 40The Effect of Ionic Strength on the Reduction

of Oxidized Reaction Centers by Cytochrome c © © 42Horse . Heart Cytochrome c » . © © © . . . © . © 42R. rubrum Cytochrome c2 © . . . . . . . « © . © 48R© capsulata Cytochrome £ 2 .. = © . © © « © © © 51S \3 © © © © © © © © © © © o © © © © © © © © 5 4

Effect of pH on the Reduction of OxidizedReaction Centers by Cytochrome c © . „ . © . © . 58

Horse Heart Cytochrome c . = © . . « . © . © . 58pH Effects with R. rubrum Cytochrome £ 2 . . . © 62pH Effects with R. capsulata Cytochrome £ 2 © © 66Summary © © © © © © © © © © © © © © © © © © © © 66

The Effect of Detergent on the Reduction ofOxidized Reaction Center by Cytochrome £ © © © . 70

Interaction with Mon-Physiological Reductants © © © 70DISCUSSION © © © © ©. © © © © © © © © © © © © © © © © © 79

Co nc1us rons © © © © © © © © © © © © © © © © © © © © 85iv

VTABLE OF CONTENTS (Continued)

PageAPPENDIX As DERIVATION OF RATE LAWS , . . = « = ........ 86APPENDIX B; FLASH TECHNIQUE . . . . . . . . . . . . 91LITERATURE CITED . . . . . . . . . . . . . . . . . . . 94

LIST OF TABLES

Table1.2.

3 o4 o

5.

6;*.,;

7.

8.

9*

10 o

11.

12.

PageProperties of c-type cytochromes . . . . . . . . 12Amino acid residues in the vicinity of theheme ^ Ige . . * . 6 . . . . . . . . . . . . . . . 20Reaction center properties and/or components . „ 34Effect of LDAO on the oxidation-reduction potential and auto-oxidation of horse heart

c . . . . . . o . o . o . . . . . . . 39The photo-oxidation of horse heart cytochrome c as a function of ionic strength . . . . . . . . 45The photo-oxidation of R.. rubrum cytochrome c^as a function of ionic strength „ „ . . . . . . . 49The photo-oxidation of R. capsulata cytochrome c^ as a function of ionicstrength . . . . . . . . . . . . . . . . . . . o 53Rate constants and apparent charges atinfinite dilution . . . . . . . . . . . . . . . . 56The photo-oxidation of horse heart Cytochrome c as a function of pH . . . . . . . . . . . . . . 60The photo-oxidation of R. rubrum cytochrome c^as a function of pH . . . . . . . . . . . . . . . 64The photo-oxidation of R. capsulatacytochrome as a function Of pH . . . . . . . . 68The photo-oxidation of horse heart cytochrome c as a function of LDAO concentration . . . . . . 74

vi

LIST OF ILLUSTRATIONS

Figurelo

2°

. 3 o

4

So

6 o

7.

8.

9 o

10.11.

12.

PageThe absorption spectra of R. rubrumreaction centers . . „ , = . . . » . . . . . . . 3Light minus dark difference spectra of R. rubrum reaction centers, in 50 mMphosphate buffer, pH'7.0 . . . . . . o . . . . . 5Structures of Triton X-100 and Laurylamine-N-oxide (LDAO), two de­tergents commonly used in reactioncenter preparations . . . . . . . . . . . . . . 10Tertiary structure of horse heartcytochrome c . ...... . . . = . . . . . . . . . . 14:Tertiary structure of R. rubrumcytochrome c . . . . . . . . . . . . . . . . . 15Absorption spectra of cytochrome c . . . . . . . 16Schematic diagram of power supply and associated circuitry used in the flash spectrophotometer . . . . . . c . . . . . . . . 24Plot of logarithm of molecular weight versus mobility of standard proteinsrelative to the tracking dye . . . . . . . . . . 32Oxidized minus reduced spectra of reaction centers in the a-band regionof c-type cytochromes . . . . . . . . . . . . . 33The effect of detergent on the reactioncenter spectra . . . . . o . . . . . . . . . . . 36The effect of acetone-methanol (7/2)on reaction center spectra . . . . . . . . . . . . 37The reduction of photo-oxidized re­action center by horse heart cytochrome c as a function of ionic strength „ . .. . . 44

vii

• viiiLIST OF ILLUSTRATIONS (Continued)

Figure Page13 o Debye-Huckel plots, at pH 7.0 and 9,0

for reduction of reaction center byhorse heart cytochrome c_ . = = «, « « «, = « , » , 46

14.' The reduction of photo-oxidized re- .action center by R. rubrum cytochrome c-as a function of ionic strength . . . . . . . . . 47

15» : Debye-Huckel plots at pH 7.0 and 9.0 forreduction of reaction center by R. rubrumcytochrome c^ . . . . . , . . . . . . . . . 50

16. The reduction of photo-oxidized reaction center by R. capsulata cytochrome c- asa function of ionic strength . . . . . . . . . . 52

17. Debye-Hucke1 plots.at pH 7.0 and 9.0 forreduction of reaction center by R.capsulata cytochrome c^ . . . . . . . . . . . . « 55

18. The reduction of photo-oxidized re­action center by horse heart cytochromec as a function of pH . . . . . . . . . .. . . 59

19. The dependence of k]_2 on pH for horseheart cytochrome c at 25°C . . . . . . . . . . . 61

20. The reduction of photo-oxidized re­action centers by R. rubrum cytochromec_ 2 as a function of pH . . . . . . . « » .... 63

21. The dependence of k%2 on pH for R.rubrum cytochrome c^ at 25°C . . . . . . . . . . 65

22. The reduction of photo-oxidized reaction centers by R. capsulatacytochrome ^ as a function of pH . . . . . . . 67

23. The dependence of k]_2 and k 23 on pH forR. capsulata cytochrome ^ at 25°C . . . . . . . 69

24. The effect of LDAO concentration on re­duction of reaction center by horseheart cytochrome c . . . . . . . . . . . . . . . 71

IXLIST OF. ILLUSTRATIONS (Continued)

Page25 o The effect of low detergent concentra­

tions on reduction of photo-oxidized reaction center by horse heart cyto­chrome c at high ionic strength i « » . . . . . 72

26o Reduction of photo-oxidized reaction center by horse heart cytochrome c

0 *6 LDAO b o O' b o o b o o o o ' O o b ' o b o o o 7 227= Plot of absorbance vs. time for biphasic

auto-reduction of reaction center at pH 7o 0 in 50 mM phosphate = « = » =. = » = = = . . = 75

28 o The effect of pH on of auto­reduction of reaction centers . = = = = * = . = 77

29. The effect of detergent on the k ^ g ofauto-reduction of reaction.; centers o = = ..... . 78

30 i A typical trace obtained from theflash spectrophotometer = = . = = = = = » . . . 93

ABSTRACT

The reduction.of photo-oxidized reaction center from RhQdos.prilli.um rubrum by c-type cytochromes obtained from horse heart, R. rubrum, and R. capsulata has been investigated. These studies were designed to obtain in­formation on a physiological mechanism of electron trans­port for O’-type cytochromes and compare these results with available studies with non-physiological reactants.

The reaction between c-type cytochromes and re­action centers was found to be ionic strength and pH dependent, with electron transfer preceded by complex formation between the reactants« From Debye-Huckel analysis the charge on the reaction center was found to be -4.6 to -4.9 at the site of electron transfer. First-order rate constants, i.e., complex disassociation and electron transfer, were found to be pH and ionic strength independent for R. rubrum cytochrome Cg and horse heart cytochrome c but not R. capsulata .cytochrome £ 2 °

It is concluded that electron transfer takes place at the exposed heme edge. The available data suggests that amino acid side chains distant from the site of electron transfer might be involved in binding the reactants.

x

Finally it appears that complex, formation involves modes of binding other than electrostatic imparting a high de­gree of specificity to the reaction center for c-type

INTRODUCTION

The purpose of this work is to gain insight as to mode of interaction between the photochemically active reaction center from Rhodosprillium rubrum and its physio­logical reductant cytochrome c^ as well as c-type cytochromes from other sources0 Reaction centers are the smallest known entities capable of producing the initial reactions of photosynthesis, namely charge separation. Reaction centers were first isolated by Reed and Clayton.(1968) from chromato- phores of a carotenoidless mutant of the bacteria Rhodo- psendomonas sphaeroides using the detergent Triton X-100 to solublize the chromatophore membrane, Since Reed and Clayton's first preparation reaction centers from many photosynthetic bacteria have been isolated (Neith and Drews, 1974? Lin and Thornber, 1975? Noel, Van der Rest and Gingras, 1972), however the best characterized reaction centers are those from R„ sphaeroides and R= rubrum using the detergents Triton X-100 and lauryldimethylamine-N-oxide (LDAO) as solublizing agents.

Pure reaction center preparations contain protein, bacteriochlorophyll a, bacteriopheophytin a, iron, ubiqui­none, and caro.tenoid, which are absent in the carotenoidless

1

mutants (Feher and Okamura,. 1977), The protein component consists of three polypeptides of which two are necessary for photochemical activity (Okamura, Steiner and Feher?1974)o The polypeptides possess a high degree of hydro- phobic residues which is not unexpected since in vivo the reaction center is an integral part of the membrane (Steiner et al., 1974)= The polypeptides have acidic isoelectric points and possess molecular weights of between 21,000-33?000 with some dispute in the actual weights due to questions concerning the validity of using SDS gel electrophoresis to size membrane proteins (Banker and Cotman, 1972) •„ The exact role of the proteins in reaction centers is unknown but at least part of their role is structural (Okamura et al,? 1974). The long wavelength absorptions in the reaction center spectra (shown in Figure 1) are due to bacteriochlorophyll and bacterio- pheophytin. The 865 nm and 802 nm peaks are due to bacterio chlorophyll while the 755 ran peak is attributed to bacteriopheophytin. The ratio of bacteriochlorophyll to bacteriopheophytin has been found by acetone-methano1 extraction to be two bacteriochlorophylls to One bacterio­pheophytin (Reed and Peters? 1972)= However? optical spectra are more consistent with a ratio of three bacterio­chlorophylls to one bacteriopheophytin (Feher? 1971),Peaks at 598 and 362 nm are due to bacteriochlorophyll?

1.0 .

0.8

y 0.6. <S0 04- <

0.2

00400 6&0

WAVELENGTH (nm)3(!)0 800500

WAVELENGTH

Figure 1. The absorption spectra of R. rubrum reaction centers.The dashed line represents bleaching observed under strong illumination of the sample obtained by using the IR-2 mode of a Cary 14 spectrophotometer.

w

450^535 ran carotenoids, and 278 ran protein, When the de­tergent concentration .is high, pheophytinization of bacterio- chlorophyll occurs.(Noel et al.,, 1972), an observation which may explain why a discrepancy exists in the ratio. Illumi­nation causes extensive changes in the long^wayelength absorption maximum as can be seen in the light minus dark differences spectra in Figure 2. Host notable is the com­plete disappearance of the maxima at 865 ran, and a blue shift in the 802 ran maxima t o .796 ran. The nature of the interactions between pigment molecules is not clearly known but it is believed that the 865 hm. maxima arises from bacteriochlorophyll dimer; formation and is the primary electron donor (Norris et al., 1971) while the bacterio- pheophytin serves as the p r i m a r y electron acceptor in bacterial photosynthesis (Fajer et al., 1975).

Recent studies have shown that the secondary electron acceptor in the reaction center is likely an iron-quinone complex (Feher and Okamura, 1977), although others believe it to be only ubiquinone (Morrison, Runquist and Loach, 1977)« The secondary acceptor is capable of reducing the oxidized bacteriochlorophyll in a back reaction if the bacterio­chlorophyll has not already been reduced but the reaction is slow compared to cytochrome reduction (Prince, Cogdell and Crofts, 1974). The purpose of the carotenoid is unknown but it is believed by some to serve, like the protein, in

5

AA

♦ .04

♦02

♦01 1

.00 650 750600 800

- jO I

WAVELENGTH (nm)

Figure 2. Light minus dark difference spectra of R. rubrum reaction centers, in 50 mM phosphate buffer, pH 7.0.

a structural role as reaction centers from carotenoidless mutants are less stable than their wild-type counterparts (Van Der Rest and Gringras, 1974)»

Several kinetic studies using flash photolysis have been made using reaction centers and c-type cytochromes (Ke, Chaney and Reed, 1970; Prince et al«, 1974). Ke et al. (1970) using the reaction center from R-26 (the carotenoidless mutant of Rhodopseudomonas sphaeroides), explored the effects of pH, ionic strength and inhibitors upon the kinetics of the reduction of the photo-oxidized reaction center by horse heart cytochrome c. They found that the reaction was collision independent, i,e., a complex was formed and that the reaction center could bind up to 24 cytochromes, The progress of the reaction was monitored by following absorbance changes at 550 nm (cyto­chrome c oxidation) or 865 nm (reaction center reduction) and was found to be first-order with a rate constant of 2.8 x 10^ sec at pH 7.5 and .Oil M ionic strength. Ke et al. (1970) investigated only the portion of the reaction that occurred within 1 ms after flashing which was sub­stantially less than 100% complete at low cytochrome c- reaction center ratios and did not elaborate on how the remainder of the reaction centers was reduced after being photo-oxidized. Increasing the ionic strength decreased the rate of reaction indicating a positive-negative charge

interactiono However, at. pH 9.3, where the rate was maximum, the reaction was found to be ionic strength independent suggesting no charge interactions were taking place * The: reduction of oxidized phototraps was inhibited by poly- lysine, suggesting along with the ionic strength data that interaction, between the reaction center and horse heart cytochrome c is electrostatic in nature except at pH 9.3 and that weakly ionizable groups on the cytochrome and/or reaction center are responsible for the complex formation. The electrostatic interaction was not unexpected since the reaction center has a pi of, ~5 and horse heart cytochrome c has a pi of 10 W .

Prince et al. (1974) obtained somewhat different results than Ke et al. (1970). Using the same reaction center, but prepared by a different method Prince et al. (1974) studied the kinetics of reduction of photo-oxidized reaction centers with horse heart cytochrome c and bac­terial cytochrome c^ from R. sphaeroides, the physiological reductant. Cytochrome oxidation was found to be collision dependent, i.e., second-order, over the concentration range investigated with a rate constant of 3.8 x 10®S "*"M at pH 7.5 and an ionic strength of 10 mM for horse heart cytochrome c and 8.2 x 10^M ^ for R. sphaeroides cyto­chrome c_ 2 under the same conditions. The reaction was ionic strength dependent with second-order rate constants at

infinite dilution of 1.1 x 10^M and 1.9 xfor horse heart cytochrome c and R. sphaeroides cytochrome c^f respectively at pH 7.5. . The rate constants at infinite, dilution suggest a diffusion controlled reaction and as the rates become slower at higher ionic strength for both cyto­chromes , a positive-negative electrostatic interaction is also suggested. The latter point is interesting Since both the reaction center and the bacterial cytochrome possess a net negative charge at pH 7.5'. Thus it is suggested that at the site of electron transfer the charges responsible for. interaction are other than the net charge on at least one. of the reactants. The .reaction was found to be pH independent between pH 4.5-pH 11 for cytochrome..c9 and cytochrome c (pH 6-11). Unlike the work of Ke et al.(1970), the rate was found to be ionic strength dependent throughout the pH range 4-11, suggesting weakly ionizable groups were not involved in electron transfer and that this transfer was collision dependent mediated by electrostatic interactions.

The main difference between the experiments of Ke et al. (1970), and that of Prince et al. (1974), was the type and amount of detergent used in the preparation of the reaction centers. Ke et al. (1970) used a preparation with Triton X-100 as the solubilizing detergent ultimately yielding a reaction center with no detectable amount of

detergent present (Reed and Clayton, 1968)= However, it was found later that this preparation was contaminated with endogenous bacterial5 cytochrome c^ (Reed) 1969)i The preparation used by Prince et al. (1974.) Utilizes lauryl- dimethylamine-N-oxide (LDAO) as the solubilizing detergent and all experiments contained 2% LPAOo However, the effects of this high detergent concentration on the cytochromes was not reportedo Both Triton X-100 and LDAO are non-ionic at neutral pH as their structures in Figure 3 indicate, however, LDAO is a zwitterion and is cationic below pH 6.

Cytochromes of the c-class contain a. heme group which is bound to the protein -through covalent thioether linkages to cysteine amino acid side chains„ Cytochromes of c-type are generally low molecular weight containing from 80 fo 130 residues depending upon their source. The function of cytochrome £ in both bacteria and higher life forms is that of an electron carrier. This function is carried out by the iron atom in the prosthetic heme group which alternates between a +2 and +3 oxidation state.

Cytochrome c from mammalian and cytochrome Cg from bacterial sources differ substantially in a number of physical properties. The midpoint potentials are generally 320-370 mV for cytochrome while those for cytochrome c are lower, approximately 260 mV. In general the poly­peptide chain is slightly longer in the cytochrome Cg

LAURYLAM INE-N-O XIDE

9 h3CH_(CH_) —N-O"

3 CH3

TRITON X -100

CH. CH

c h3c c h 2c c h 3 c h 3

CH2CH2 ° 1 o8

Figure 3, Structures of Triton X-100 and Laurylamine-N-oxide (LDAO), two detergents commonly used in reaction center preparations.

11(up to 20 more amino acids) and the N-terminus is free unlike mammalian cytochrome c which is acylated. Cyto­chrome c_ has an isoelectric point of approximately 10 while cytochrome Cg has isoelectric points ranging between 5 and 10 depending on the source« The physical-chemical properties of the cytochromes used in the experiments de­scribed here are presented in Table 1. Despite the dif­ferences in physical-chemical properties, there is re­markable structural similarity between c-type cytochromes regardless of the source. Of some 67 eukaryote, and 8 photosynthetic bacterial cytochromes c that have been sequenced, 12 seguencei, positions, haye^ been found to be totally invariant (Dickerson and Timkovich, 1974) „ Most of these amino acid side chains, are associated with the heme or sharp bends in the polypeptide chain. In addition to the 12 invariant amino acid residues, there are several general structural features present. One such feature is that of positive charge consisting mainly of lysine residues that encircle the heme edge on the front of the molecule. Acidic residues are absent from the area around the heme crevice but instead are clustered around the upper rear of the molecule (Dickerson and Timkovich, 1974). The sig­nificance of these clusters of positive and negative charges may be in binding the cytochrome to specific ions or the membrane although definitive data is not available.

Table 1, Properties of c-type cytochromes«

Horse Heart Cytochrome c

R. rubrum Cytochrome Cg

R. capsulata Cytochrome

Molecular Weight 12,380 12,480 13,330

Midpoint Potential pH 7.0 (mV)

260 320 368

Isoelectric Point 10.0 6.2 7 .1

13From the x-ray crystallo'graphic structures of three

eucaryotic and three bacterial c-type cytochromes (Dickerson and Timkoyich, 1974; Salemme et alo f ■1973; Almassy and Dickerson, 1977; Dickerson et al., 1971; Takano et al.,1973; Ashida. et al., 1973) r there emerges a similar tertiary structure for all c-type cytochromes„ These similarities include a heme surrounded by hydrophobic residues with one edge exposed and ringed with positive charges, a region of di-helix near the beginning of the polypeptide chain located on the top of the molecule, and a cluster of negative charges on the rear of the molecule as shown in Figures 4 and - 5 & Ths' absorption sppotra1' (Figure 6 ) o f , all c-type cytochromes is characterized by maxima at approximately 550 nm, 521 nm, 415 nm, 316 run, and 272 nm for the reduced form and maxima at approximately 525 nm, 408' nm, and 357 nm for the oxidized form.

The horse heart cytochrome C is typical of mammalian cytochrome c and because of its ready availability it has been the subject of extensive research. The biological role of cytochrome c is to transfer electrons between cytochrome b-c^ complex and cytochrome a-a^ in the inter­membrane of the mitochondria. The molecule consists of a single polypeptide chain of 104 residues (MW = 12,400 daltons) in the shape of a prolate spheroid with dimensions of 30 x 34 x 34 2. as determined by x-ray crystallography in

14

Tii n

Figure 4. Tertiary structure of horse heart cyto­chrome c.

chrome

A BS

OR

BA

NC

E

16

1.50 -

1.0 0 -

.5 0 -

4doWAVELENGTH(nm)

3 bo 600500

Figure 6. Absorption spectra of cytochrome c. , reduced form; — , oxidized form.

17the oxidized state (Dickerson et al„, 1971)» Of the 104 residues, 19 are lysine, 2 are arginine, and 12 are acidic givingv the protein. at isoelectric vppint., of . It), 4 o The polypeptide chain can be divided into two regions, residues 1-47 and 48-91, on either side, of the heme„ The larger region (1—47) is to the right of the heme crevice and con­tains the thioether linkages to the heme via cysteine residues 14 and 17, The porphyrin ring forms four ligands to the iron atom while the nitrogen from histidine 18 and sulfur from methionine 80 comprise the fifth and sixth ligands. The heme crevice is surrounded by hydrophobic residues leaving only one edge accessible.to the solvent- creating a largely hydrophobic environment in the interior and leaving the surface predominately charged. Propionic acid chains on the heme are located at the bottom of the crevice and hydrogen bonded to residues tyrosine-48 and tryptophan-59 and to the polypeptide backbond chain at threonine 40. Openings appear on both sides of the heme due to the folding of the polypeptide chain and have been termed channels (Dickerson et al., 1971).' In the left

Ichannel tryptophan 59 and tyrosine 74 lie parallel to. one another and are 6.% apart, and both are close to tyrosine- 67 which is nearer to the heme. In the right channel phenylalanine 10 and tyrosine 97 are parallel while phenylal­anine 46 and tyrosine 48 are parallel in the front of the

18moleculeo The proximity to one another of aromatic residues and the fact that the residues are parallel to each other has caused some, to speculate that electron transfer might, occur through overlapping ir molecular orbitals of the aro­matic residues (Dickerson et ai., 1971) but this model, is now discounted (Dickerson and Timkovich,, 1974) «

Bacterial cytochrome c^ from the purple non-sulfur bacteria Rhodospirillium rubrum functions as the primary electron donor to photo-oxidized bacteriochlorophyll (Taniguchi and Kamen, 1965). The cytochrome was first isolated by Vernon-and' Kamen (1953) and it is the only cytochrome eg whose tertiary structure has; been determined. , by x-ray crystallography (Salemme et al.f 1973). The molecule consists of a single polypeptide chain with 112 amino acid residues (MW = 12,480) and is, in the oxidized state, ellipsoidal with dimensions 25 x 33 x 40 5L The heme is surrounded by hydrophobic residues leaving only one edge exposed. The heme is attached covalently by thioether linkages to cysteines 14 and 17. As in the mammalian cytochrome c, nitrogen from histidine and sulfur from methione constitute the fifth and sixth ligands to the heme iron. Around the heme crevice are located 11 lysine residues which form a ring of positive charge on the front side of the molecule. There are four areas of a-heliCal structure, residues 2-10, 64-71, 75-80, and 96-110

19similar to horse heart cytochrome c* The methionine sulfur-iron bond seems to be somewhat distorted by inter- action with the nearby tyro s ine*-7 0 hydroxy l oxygen which may help stabilize the oxidized form by dispersal of charge away from the iron (Salemme e f . a l e , 1973),

The absorption spectra of cytochrome c^ is inde­pendent of pH between 6-11 in both oxidized and reduced forms (Horio and Kamen t 1960) but the oxidation-reduction potential decreases from 380 mV at pH 5,0 to 220-240 mV at pH 10o0 (Wood and CusanQvich, 1975),

Cytochrome c0 from R= capsulata is similar in amino acid sequence to that of R. rubrum (Table 2) however> its tertiary structure is unknown, but believed to be like that of R» rubrum cytochrome c^ due to sequence homology. Differences Occur in rates of reduction by potassium ferro- cyanide for R, rubrum cytochrome c^ and R, capsulata cyto- chrome c^ which has been ascribed to differences in the distribution of charge around the exposed heme edge (Wood, Post and Cusanovich, 1977),

As a model system for electron transport in vivo, oxidation and reduction of cytochrome c with iron hexa- cyanides has been studied extensively (Stellwagen and Schulman, 1973; Wood and Cusanovich, 1975; Wood et al,,1977)o The iron-hexacyanides were chosen due to their stability and the fact that they are one electron transfer

20Table 2o Amino acid residues in the vicinity of the heme

edge,,K, lysine? V, valine? T, threonine? Y, tyrosine? F , phenylalanine? A> alanine? S, serine? D, aspartic acid? Gy glycine;

Cytochrome c Amino Acid PositionSource 27 28 46 47 48 49 50 51 52 88 89 90

R. rubrum K V Y A Y S D S Y K S K

Ro capsulata K ‘ T F K Y K D S I - K T G

Horse Heart K T F T Y T D A N _a T K

a »' A dash indicates sequence deletion at indicated position.

21agents as is cytochrome c„ Kinetic studies reveal that intermediate complexes are formed for both the oxidation and reduction, with second-order rate constants (complex forma­tion) of 5 x 106M for reduction and 1,3 x 10^M ^for oxidation at pH 7,0 and infinite dilution (Wood andGusanovich, 1975) for R« rubrum cytochrome c^. Furtherthe second-order rate constants are pH and ionic strength dependent, with Debye-Hucke1 plots yielding a net effective charge for R, rubrum cytochrome c^ of +1,3 and +.7 for the oxidized, and reduced states, respectively. The second-1 order rate constant for the oxidation was maximum at approxi­mately ;pH 9,0, while that for the reduction gradually de­creased between pH 4-10, First-order rate constants (electron transfer following complex formation) were found to be independent of pH and ionic strength for oxidation,however, they were ionic strength but not pH dependent

-1for reduction and had values of 450 sec . for oxidation“" Xand 250 sec for reduction of R, rubrum cytochrome c^

at pH 7,0 and an ionic strength of ,035 M,From the kinetic data.a mechanism for electron

transport is postulated, at least in the case of ferro- ferricyanide as given by equation 1 involving a commonpathway for both oxidation and reduction,

i

22"3+ o ^ 9 ^'34 94-cyto C (Fe ) + F — - FH • • •• •• - OR * 0 + cyto C (Fe )

k21 k32 k43 (1)

where F represents ferrocyanide ion? FH, complex of ferri- cytochrome c and ferrocyanide ion? OR, complex of ferro-cytochrome c and ferricyanide ? 0, ferricyanide» Despiteits simplicity, the iron hexacyanide model system has one important drawback, it is a nonphysiological reactant for cytochrome c„ The in vivo reactants of cytochrome c are cytochrome complex b-c^, and a-a^, in respiratory organisms and cytochrome b and the reaction center for cytochrome c^. Ail: - of- the* physiological:: reactants are : membrane bound which makes isolation and purification in the absence of solubilizing agents difficult. Of all the physiological reactants the one which is most easily isolated and best characterized is reaction centers from photosynthetic bacteria. It is the objective of this work to characterize the interaction of reaction centers from R. rubrum with c-type cytochromes and to, compare and contrast these results with those obtained with non^physiological reactants. From these studies it is anticipated that information bearing on the mechanism of biological electron transfer will be obtained.

MATERIALS AND METHODS

The flash photolysis apparatus used for the studies described here is similar to one constructed by Ke,Trehane and McKibben (1963)? except that it is a single beam instrument. Two 4 microfarad capacitors which can be . charged from 0-6 kilovolts are discharged through a single EP-5-lOOc Xenon flash tube giving a maximum flash energy of 144 joules, with a duration of 30-60 microseconds. The capacitors are charged by means of a 6 KV power supply and discharged using a Xenon-G trigger module- and a Maxwell,. 80200 high voltage spark-gap, A detailed schematic of the power supply and flash apparatus is given in Figure 7, The monitoring beam is produced by a tungsten halogen lamp.The light is collimated by use of a lens and filtered by narrow band Baird interference filters before passing through the sample cell. The absorption cell was 5 cm long and requires a volume of 7,5 ml, shorter path length cells can be used provided the concentration of the transient species is higher, A Schoffel GM 100-2 monochronometer with a range of 0-900 nM was attached to the photomulti­plier tube housing which contained a RCA IP28 photomul­tiplier tube. The signal from the multiplier tube is fed into a Tektronix 5130N storage oscilloscope and the

23

trigger

flashlamp

1 -27kilohm

2-10 megaohm

3-2 megaohm

4- 8 ufd. 10 kv5-500 pfd 15 kv

\

Figure 7. Schematic diagram of power supply and associated circuitry used in the flash spectrophotometer.See Appendix B.

display photographed with a Honeywell Pentax Spotmatic 35 mM camera.

The kinetics of oxidation and reduction were monitor'ed at 550 nm (Cytochrome oxidation) and 605 nm (reaction center reduction), From the absorbance, changes, a reduced minus oxidized extinction coefficient of 19,500 for horse heart cytochrome c (Watt and Stuftevant, 1969) and assuming one.to one stoichiometry a reduced-minus oxidized extinction coefficient for the reaction center was determined at 605 mru

Preparation of ChromatophoresRhodospirillium rubrum whole cells which had been

grown on Hunter medium, harvested and frozen were used for the preparation of chromatophores (Loach et al«, 1963) the whole cells were suspended in glyclyglycine buffer (.1 M, pH 7 = 0) and 3 grams, of EDTA added for every 100 grams of cellso The pH was adjusted to pH 7=0 and the suspension sonicated with an Ultrasonic W350 sonifer at full output and 50% pulsed cycle for at least 12 minutes at 0°-10o C= The crude cell extract was then centrifuged at 40,000 xg for 30 minutes in a Spinco-Model L ultracentrifuge in order to remove large particles. The resultant supernatent con­taining the chromatophores was then spun for 60 minutes at 140,000 xg. The pellet containing the chromatophores was

26resuspended in the glyclyglycine and again spun for 60 minutes at 140,000 xg. The chromatophores were then re­suspended •in a small volume of glyclyglycine buffer to make a stock solution of chromatophores (Agg^ = 100-180) which were stored at 2°C in the dark until used =

Extraction of Reaction CentersThe reaction centers were prepared by the method

of Noel et alo (1972). The stock solution of chromato-phores was diluted with enough potassium phosphate buffer(50 mM, pH 7,0) to give an absorbance at 880 nm of 38.The nonionic detergent lauryl dimethylamine-N-oxide isadded to bring, the final detergent concentration to .3%

V( /V) with a 30% stock solution„ The mixture was stirred for 10 minutes in the dark at room temperature and 15 ml was placed into a 25 ml centrifuge tube and enough 0.6 M sucrose was then introduced, at the bottom, to fill the tube. Centrifugation at 100,000 xg for 90 minutes gave centrifuge tubes containing a top layer rich in reaction centers. This top layer is carefully removed and enough ammonium sulfate added to give a 33% saturated solution.The mixture was then spun at 10,000 xg for 30 minutes and the pellet containing the reaction centers was resuspended in sodium carbonate buffer (50 mM, pH 9.30). This solution was dialyzed against a 100 fold volume of potassium phos-

ophate buffer (50 mM, pH 7.0) twice at 2 G. The resultant

27solution was centrifuged 10,000 xg for 30 minutes to remove any suspended material and stored as.a reaction center stock solution at 2°C, The stock solution concentration ranges between 5-20 mM and is generally diluted ten fold before use. It is necessary to keep the reaction centers in the dark at all times in order to avoid loss of activity» This solution may be stored for weeks at 10°C in the dark but it may have to be recentrifuged in order to remove suspended material that forms upon storage.

Horse heart cytochrome c was obtained from Sigma Chemical Company, Before use it was reduced with sodium dithionite and desalted by passing the sample through a Sephadex G-.25 molecular sieve column. The sample was then concentrated on an Amicon pressure dialysis cell to a con­centration of 300-700 uM in 10 mM phosphate buffer, pH 7.0.

R. rubrum cytochrome c^ was absorbed on a DEAE cellulose column from the high speed supernatent left from the chromatophore preparation described earlier. The cyto­chrome £ 2 was eluted with salt solution (,2 M NaCl) and then depending on the protein (280 nm) to Soret peak ratios it was further purified. If the ratio was less than 0,4 the sample was reapplied to a DEAE column buffered with 20 mm Tris-Cl, pH 7.3 and eluted with NaCl (10 mM), if the ratio was between .4 and 1,2, the sample was fractionated on a Sephadex G-75 molecular sieve column, if the ratio is

28greater than 1.2 the sample is purified by ammonium sulfate fractionation with cytochrome c^ coming down in the 95 and 100% . cuts. After use in kinetic, experiment s., the cytochromes were routinely repurified by ammonium sulfate fractionation« The resultant cytochrome c^ was desalted and concentrated to SOO-TOO ]iM in 10 mM Tris^chloride pH 7,0 before use.R 0 capsulata c^ was prepared in a similar manner.

Lauryldimethylamine-N-oxide was a gift from the Onyx Chemical Company and was used without further puri­fication as a 30% W/v solution. 1-4 napthaquinone from Kodak was recrystallized with ethanol before use. All other chemicals were reagent grade.

The number and molecular weights of the poly­peptides in the reaction center was determined by SDS gel electrophoresis. The reaction center (Ag^ — «6 ) was ex­tracted with 10 volumes of acetone, the mixture was centri­fuged and the pellet washed with 10 volumes of water. The pellet was then dissolved in one half volume of buffer (50 mM Tris-Cl , = 5 SDS, and 1% dithiothretol, pH 8.0).The solution was heated to .65—80 C for 30 minutes and .1 ml of the denatured protein solution was mixed with 1 drop glycerol and 1 drop of .05 bromophenol blue, .01 ml of this solution was then layered onto the gel. Marker proteins were denatured and used in a similar fashion to give 7 ug protein per gel. The gels consisted of 10% (W/v) acylamide

that had been recrystallized from ethanol with .29% (W/v) methylene bisaryl amide and were polymerized after degassing by.,.adding> enough; .ammhEi m.■■.pe■a?S3 6at:e- ah4 .:N:/.J..,H■, -r tetramethylethylenediamine (TEMED) to give a final con- • centration of 1.1 mg/ml and .05% (v/v) respectively. The gels were pre-run. at 60 V for.3 hours, and then were electrophoresed for 6 hours at 50 V and 25 me. The electrode and gel buffers were the same, 50 mM tris-chloride and .1% SDS at pH 8.0. The gels were fixed and stained overnight in a solution of .2% Coomasie blue, 7=5% acetic acid and 5=0% methanol; the excess stain was removed by electrophoresis.

Carotenoids were determined by extraction of the reaction center inbo acetone and using an extinction coef­ficient of 94 mM 1cm ■*" at 485 nM (Van ber Rest and Gingras,

• 1974) .Kinetic studies were done on sample solutions con­

taining reaction centers (.5-2.0 uM), cytochrome c (5-100 yM), ascorbic acid (.5 mM), 1-4 napthaquinone (100 uM) was used to increase the size of the secondary electron ac­ceptor pool allowing repeated flashes of a single sample, and the appropriate buffer. The detergent LDAO was .03% unless otherwise stated.

For ionic strength studies the buffer was 10-20 mM phosphate at pH 7.0 or 20 mM glycine at pH 9.0. The

30appropriate ionic strength was obtained by adding sodium chloride as not. to change the volume. The effects of pH were.studied,at.constant ionic strength with the buffers. imidazole pH 5^7 6 Tris-chloride 7-8, and glycine 9-11,

To study the. effects of detergent upon the kinetics, the detergent concentration was raised or lowered by dialysis against a solution of the desired detergent con­centration, In order to determine the midpoint potentials of cytochromes in the presence of detergent the solutions were made anaerobic by bubbling with argon for at least 45 minutes before adding protein and then.titrating the cyto­chrome with potassium ferrocyanide as described by Margalit and Schejter (1970), The autooxidation was followed by mixing reduced cytochrome with detergent and monitoring the loss of absorbance at 550 nm (cytochrome oxidation) in a Cary Model 118 spectrophotometer,

RESULTS

Characterization of Reaction Centers Although reaction centers have been previously pre­

pared from Ro rubrum (Noel et al0, 1972) it was necessary to fully characterise the reaction centers used in this work and establish their basic physical-chemical properties. SDS gel electrophoresis of the purified reaction centers yielded three bands after destaining,. Figure 8 is the plot of the Rp value versus the log of the molecular weights and yield values of 33,000, 24,000, and 17,000 daltons. The purified reaction centers were found to be free of cyto­chromes as evidenced by the lack of detectable heme absorp­tion bands in the reduced minus oxidized difference spectra (Figure 9)„, The reaction centers were found to contain one caroteneid, determined as spirillOxanthin, based on the absorbance at 485 nm (Van Der Rest and Gingras, 1974). Finally ratios of the absorption band in the reaction center were in good agreement with previously published results (Noel et al., 1972? Van Der Rest and Gingras,1974), The properties of reaction centers used in this work and those previously reported are compared in Table 3.

XThe differential extinction coefficient at 605 nm was found

— 1 —1to be 37 ± 3 mM cm” from the average of eight trials31

3210

1.0M O B ILITY

Figure 8 . Plot of logarithm of'molecular weight versus mobility of standard proteins relative to the tracking dye.Protein markers used in determining the line are indicated by o and were bovine serum albumin (6 8 ,0 0 0 ), alcohol de­hydrogenase from yeast (37,500), a-chymotrypsin (21,600), trypsin (15,100, and horse heart cytochrome c (11,700).Rf values for the three polytides obtained from the re­action center are denoted by [ and yield values of 33000, 24000, and 17000 daltons respectively.

33

— 1_---------- <----------- r♦.02'

WAVELENGTH (nm)

Figure 9. Oxidized minus reduced spectra of reac­tion centers in the a-band region of c-type cytochromes.The base line is represented by curve a with curve b ob­tained by the addition of sodium dithionite to the reference cuvet and potassium ferricyanide to the sample. Cytochromes were found to absent as evidenced by a lack of absorption in curve b in the 550 nm region.

34Table 3„ Reaction center properties and/or components,

Property and/or Component

; literature Values

Values Obtained in this Study

Peptides . ' 3. 3Carotenoids 1 1

Chlorophyll 3-4 3—4Bacteriopheophytin 1-2 1-2

Ubiquinone 1 NDMidpoint Potential

(my)..440 ND

Isoelectric Point 4-6 acidic

As causes 2.3 2. 3

A3 6 5 ^ 2 7 5 1 1

Cytochromes 0 0

Peptide Molecular 31,50-0 33,000Weights 24,000 24,000

2 1* 0:00; 17,000

35based on the ratio of AAg^g/AA^^Q and a difference extinc-

— 1 — 1tion coefficient of 19=5 mM cm for horse heart cytochromeCo From a ratio of A & g q g / 5g 3 *3. detatznined*by 1 ight-induced difference spectra (Figure 1) a differential ex-

_1tinction coefficient of 122 mM cm was calculated for the. absorbance change at 868 nm (P8 6 8 ) and is in excellent agreement with previously published values (Van Der Rest and Gingras, 1974)»

Effects of Detergent and Organic Solvents On Reaction Centers

Reaction centers are stable in low concentrations of LDAO (c .3) but at.higher concentrations changes begin to appear in the optical spectra accompanied by loss of activityo These changes are depicted in Figure 10 at wave­lengths between 650 and 1000 nm. As the LDAO concentration is increased absorption bands at 802 nm and 862 nm de­crease while the 755 nm band increases suggesting phe— opbytinization is occurring. When enough HC1 was added to cause complete pheophytinization a new band occurred at 680 nm and the 802 and 868 nm bands disappeared com­pletely. When a mixture of acetone and methanol (7:2 V/V) was added in increasing proportions to reaction centers, a similar effect was observed as shown in Figure 11. In the past, ratios of bacteriopheophytin to bacteriochlorophyll were determined by extraction with acetone-methano1

36

.7

.5 •

< -4

Q .3

iobo800WAVELENGTH (nm)

Figure 10. The effect of detergent on the reaction center spectra.Curve a, .03% LDAO; b, 1.4% LDA0; c, 1.4% LDAO + 1 M HC1. Buffer was 50 mM phosphate at pH 7.0 initially.

37

0.6 -

0.7

0.5--

Z 0 4 - -

^ 0.3-.

< 0.2-

0.1 ■ -

800 900700WAVELENGTH

Figure 11. The effect of acetone-methanol (7/2) on reaction center spectra.Curve a, 0% acetone-methanol; b, 21% acetone-methanol; c , 43% acetone-methanol; d, 60% acetone-methanol. Buffer was 50 mM phosphate, pH 7.0.

• 38mixtures, which might lead to erroneous results based on work presented here.

Effect of Detergent on the Oxidation-Reduction Properties of Cytochrome c

The kinetic studies to be reported here were con­ducted in the presence of detergent making it necessary to obtain data on the redox properties of cytochrome c in the presence of detergent. The oxidation-reduction midpoint potential of horse heart cytochrome c was determined in 50 mM phosphate buffer, pH 7=0, with 0, .03, and 2% LDAO.As shown in Table 4, .03% LDAO results in a 30 mV decrease in midpoint potential of,cytochrome:cj in 2% LDAO the cyto­chrome is clearly denatured evidenced by a decrease in the midpoint potential of 134 mV. The rate of auto-oxidation was also found to be a function of LDAO concentration. In the presence of 2% LDAO the rate of auto-oxidation was found to 30 times that of. a .03% solution, which in turn was greatly increased over the 0% LDAO which did not auto- oxidize at all in the time range investigated (30 min).The rate of auto-oxidation did not follow pseudo first- order kinetics so for comparative purposes only the initial rates are reported in Table 4. Although the midpoint po­tential and rate of auto^oxidation were effected by .03% LDAO, it is believed that changes in cytochrome structure were minimal as evidenced by the kinetics of oxidation of

Table 4 0 Effect of LDAO on the oxidation-reductioh potential and auto­oxidation of horse heart cytochrome c»

% LDAO (V/V)aEm (Gytochrone c) (mV)

Em (Ferri- Ferrocyanide) (mV)

Rate of Auto- Oxidation CuM/sec)

ooo 278 ± 5 421 negligible

0 = 03 247 ± 8 422 0.029

2.00 144 ± 10 382 0.680

a. Buffer 0.05 M potassium phosphate, pH 7=0.

40cytochrome c by reaction centers in the presence and ab­sence of LDAO which are presented in later sections.

General Kinetic Properties The reduction of photo-oxidized reaetibn- centers«

by three different c-type cytochromes was investigated as a function of pH and ionic strengtho' Further, the effect of detergent concentration and the interaction of oxidized reaction centers with non-physiological reductants was studied. The following discussion will outline the analysis of kinetic data used to obtain the appropriate rate constants. The reaction of c-type cytochromes with oxidized reaction centers is complex and can be best de­scribed by the mechanism given in equation 1 (p. 2 2 ). Application of the steady state approximation yields equation 2 (see Appendix A for the derivation).

^ k12k23[R] ,2)obs k 1 2 R J +k 2 3+k 21

where kQ^s is the observed pseudo first-order rate con­stant, R is the initial concentration of reduced cytochrome c arid the individual rate constants are defined in equa­tion 1 .

At low concentrations of R, complex formation (controlled by k^ 2 and k2j) becomes rate limiting as a

41linear relation between R and k ^ g is found (see subsequent sections), hence

kobs - ki2 [ R ] + k 21 (3>

At high concentrations of R> ko^s becomes independent of the cytochrome concentrationr hence ko^s = kgg.

Equation 2 can be rearranged to give equation 4 which will yield k23 and ki2// k23+k21^ 0

k23"i*k21^ o b s - + '''23 12 • (4>

Thus a plot of k^ks as. [R] atvlow. cytochrome, cqn - centration yields k.2^ (the intercept) and k^ 2 (the slope) . Further, taking k2^ from the intercept of a plot of ko^s vs. [R], the slope of the inverse plot (equation 4) can be used to calculate k^ 2 as a check to determine if the re­sults are consistent.

In all cases to be reported (unless otherwise noted) plots of In(AA) vs. time were linear for at least three half-lives when the oxidized reaction center was reduced by cytochrome, provided the cytochrome was present in at least a two fold excess. Values of k ^ and k^3 (equation 1 ) were not obtainable as the reaction is not measurably reversible.

42The Effect of Ionic Strength on the Reduction of

Oxidized Reaction Centers by Cytochrome cThe effects of ionic strength upon the reduction of

oxidized reaction center by cytochrome c was studied over an ionic strength range of% = 03 M t o „ 17 M in 0„ 02 M po-: tassium phosphate, pH 7.0 by the addition of the appropriate amount of NaCl <, The rate of reaction and ionic strength of a solution can be related by equation 5 (Frost and Pearson, 1961), known as the Debye-Hucke1 equation«

log: k: = log kOT + 2ZaZbayu (5)

Where n is the ionic strength of the solution? zaZb the product of charges on the interacting molecules? 2a.is a proportionality constant equal to approximately one in aqueous media and k^ is the rate constant at infinite dilu­tion <> A plot of log k versus V]I is known" as a Debye- Huckel plot and yields from the slope, Z^Z^ and the intercept, kroo Although the absolute values of k^ andZ Z, are somewhat questionable due to uncertainties in the a. dapplication of the Debye-Huckel relation to polyvalent ions in solution (Cummins and Gray, 1977) the relative values are useful»

Horse Heart Cytochrome cThe kinetics of reduction of oxidized reaction

center by horse heart cytochrome c follow the rate law

43expressed in equation 2 at any given ionic strength as illustrated in Figure 12A where k vs. [cytochrome c] is plotted and Figure 12B where l/k^bs as [cytochrome c]^^ is plotted. The effect of ionic strength at pH 7.0 upon the rate constants given in equation. 2 are presented in Table 5. The second-order rate constant, k ^ , was found to vary with ionic strength while the first-order rate constants ^ 2 3 an< ^21 were independent of ionic strength as shown in Figure 12A, the second-order plot and Figure 12B, the reciprocal plot. Figure 13 presents the Debye- Huckel plot for horse heart cytochrome c at pH 7.0. The

is negative indicating a interaction at the site of electron transfer. Taking a value of +1.3 for the charge at the site of electron transfer on horse heart cytochrome c at pH 7.0 (Morton, Overnell and Harbury, 1970) a value of -4.9 is calculated for the charge on the reaction center at pH 7.0. The value of kj^ at infinite dilution is 1.3 x lO^M ^ which suggests the reaction is approaching the diffusion con­trolled limit, (~1 0^ M ■*") „ The effects of ionic strengthon at pH 9.0 were investigated and summarized in Figure14. Values of kj^ at pH 9.0 were single point determina­tions calculated by subtracting from k ^ ^ and dividingby the cytochrome £ concentration. The product of charges is diminished (-6.3 to -5.9) but still indicates a

Figure 120 The reduction of photo-oxidized re­action center by horse heart cytochrome c as a function of ionic strength*A« plots of kgbg versus horse heart cytochrome c con­centration at 25°C, pH 7.0, and different ionic strengths; 0, -04S M? A, .089 M; Q , .089 M? O, =138 M? ©, .168 M. Reaction center concentration 1.0 M.B. inverse plots (see equation 5) for the reduction of photo-oxidized reaction center with horse heart cytochrome c at 250C, pH 7.0 and different ionic strengths? q, .048 M A* .078 M? D, .089 M? Of .138 M? o, =168 M=, Reaction center concentration 1.0 viM.

1000■24

8 0 0 20

■166 0 0

■12

4 0 0

200

20 40CYTOCHRO M E X 10'

60 80 20 401 /CYTOCHROME X 10'

60 8 0

Figure 12. The reduction of photo-oxidized re­action center by horse heart cytochrome c as a function of ionic strength.

45Table 5. The photo-oxidation of horse heart cytochrome c

as a function of ionic strength.

ua:'

^ 2(M"1S _1 ) x l b - 7 "

k l2(M"“1S- 1 )x lO “ 7

k 23(s” 1 )

k 21

(s " 1 )

.048 5.80 6.90 6250 10

.078 2.10 2.40 6250 10

.089 0.99 0.92 6250 10

.638 0.56 0.59 6250 10

.168 0.37 0.41 6250 10

a.appropriate

Buffer . 010 potassium* phosphate,. pHi 7 .,0, amount of NaCl.

plus

vs.b. Determined

[cytochrome c].from initial slopes of plots of kobs

c. Determined from inverse plots (see text) o

\

46

10 •

10:

id]— ,--,— j—0 .10 .20 .30 40 .50 60

(IO N IC STRENGH)1

■10'.10 .20 .30 40 .50 .60 t/2

Figure 13. Debye-Huckel plots at pH 7.0 and 9.0 for reduction of reaction center by horse heart cytochrome c.A. Debye-Huckel plot at pH 7.0 for horse heart cytochromeB. Debye-Huckel plot at pH 9.0 for horse heart cytochrome

ulul

Figure 14. The reduction of photo-oxidized reactioncenter by R. rubrum cytochrome c~ as a function of ionic .strength. “ — - 2

A. Plots of k0bs vs. R. rubrum cytochrome eg concentration at 25'q G, pH 7.0, and dTfferent ionic strengths? o, .03 M?Q, .06 M?. A, .12 M; .15 M. Reaction center concentra­tion 1.0 ]iM.B. Inverse plots (see equation 4) for the reduction of photo-oxidized reaction center with R. rubrum cytochrome£ 2 at 25°C, pH 7.0, and different ionic strengthsO, .03 M? Q, o06 M? A, .12 M? .15 M. Reaction center concentra­tion 1.0 pH. '

47

1000

80 0

6 00

4 0 0

200

20 60 100 1/CYTOCHROME X 10"

10 20 30 6 40CYTOCHROME X 10

Figure 14. The reduction of photo-oxidized reaction center by R. rubrum cytochrome c_ as a function of ionic strength.

obs

48positive-negative interaction at the site of electron transfer. It is impossible to determine the charge at the site of electron transfer on either reactant as both are unknown at pH 9,0, Although the product of charges is diminished, at infinite dilution is approximatelythree fold larger which is a reflection of the pH dependence upon the reaction (see following sections),

R. rubrum Cytochrome CgThe reduction of the oxidized reaction center with

R. rubrum cytochrome c^ was found to be ionic strength dependent. Figure 14A presents second-order plots used in determining k ^ and k^^ at different ionic strengths and Figure 14B is the corresponding reciprocal plots. The various rate constants at different ionic strengths are summarized in Table 6 , It can be seen that k ^ is ionic strength dependent while kgj and kg^ are not. The de­pendency of k^2 upon ionic strength follows the Debye- Huckel equation as is shown in Figure 15, The slope is negative meaning the interaction charge at the site of electron transfer is positive-negative, assuming a charge of +.7 on ferrocytochrome Cg (Wood and Cusanovich, 1975) at the site of electron transfer a value of -4,6 is cal­culated for the site of electron transfer on the reaction center. This value is in excellent agreement with the

49Table 6 . The photoroxidation of R. rubrum cytochrome

as a function of ionic strength.

Pa:/ CM"1s'1 lxlO" 7kC :12

(M"1S™1 )xlO" 7k23(s-1)

k 21(s-1)

0 O w 2,80 3.10 7100 24,06 1.40 1.62 7100 24.09 0 . 80 0.82 7100 24,12 0.79 0.88 7100 24.15 0.62 0.75 7100 24

a. Buffer, .010vM: potassiuwphqsphate,. pH 7,0, plus appropriate amount of NaCl, „

bo Determined from initial slopes of plots of ko^s vs. [cytochrome £2 3.

c. Determined from inverse plots (see text).

50

10 - |

10 ..

10(IONIC STR ENG TH )172

Figure 15. Debye-Huckel plots at pH 7.0 and 9.0for reduction of reaction center by R. rubrum cytochromeA. Debye-Huckel plot at pH 7.0 for R. rubrum cytochromeB. Debye-Huckel plot at pH 9.0 for R. rubrum cytochrome

<N fN

CMol

ulol

51previously calculated charge for the reaction center site of electron transfer from the horse heart cytochrome c studieso The value of infinite dilution was foundto be 1 x 10^M "*"S which is substantially less than that found for horse heart cytochrome c» The effect of ionic strength o n .k^2 for R. rubrum cytochrome Og at pH 9.0 is shown in Figure 15« The values of ah pH 9.0 are singlepoint determinations. Again the slope was diminished com­pared to pH 7.0 (-3.20 to -2.70) but still negative, k ^ at infinite dilution was larger at pH 9.0 than pH 7=0 (1 x 1 0 8m"1S" 1 vs". 1.7 x 108M-1S-1) .

R. capsulata Cytochrome CgThe effect of ionic strength at pH 7.0 on the re­

duction of oxidized reaction center by R. capsulata cyto­chrome Cg yielded somewhat different results from horse heart cytochrome c and R. rubrum cytochrome c^. The second order plots are shown in Figure 16 and demonstrate that varies as a function of the ionic strength. At high ionic strength (.09 - .15) reciprocal plots were not linear and kg^ was estimated from layover of second-order plots, at lower ionic strengths (<.09) k^^ was determined from re­ciprocal plots. In this manner it was found that kg^ was dependent upon ionic strength in marked contrast to the other cytochromes Studied. Table 7 summarizes the effect of

Figure 16 =, Tlie1 reduction of photo-oxidized reaction center by R= capsulata cytochrome c, as a function of ionic strength* -A* Plots of k0bs vs. R. capsulata cytochrome eg concentra­tion at 25°Cr pH 7.0, and different ionic strengths;O, o03 M ; .06 M? Q, .09 M$ A, .12 M? ©, • .15 M. Reaction center concentration is 1.0 uM.B. Inverse plots (see equation 4) for the reduction of photo-oxidized reaction center with R. rubrum cytochrome £ 2 'at 25°G, pH 7.0, and different ionic strengths; o, .03 M;

.06 M; D, .09 M; A, .12 M; ©, .15 M. Reaction center concentration is 1.0 uM=

52

1200

20001000

800- 1500

60 0

4 0 0

5 0 0200-

20 4 0 6 0 ICYTOCHROME X10(

40 80 1201/CYTOCHROME X lO

Figure 16. The reduction of photo-oxidized reaction center by R. capsulata cytochrome as a function of ionic strength.

Table 7 o Ttie photo-oxidation of R, capsulata cytochrome c„ as a function of ionic strength.0

Ma '

kb12

(Ms;Ls f 1 JxlO r‘7

k c12 k23

ts "1 )

k 21te"1)

kd23

( s '1 )

Oo 03 3.60 3.70 12500 10 noturnover

0,06 1.24 1.47 2500 10 noturnover

0.09 0.74 0.89 714 3 200

0.12 0.48 0.55 625 8 200

0 .15 0.35 0.43 556 8 170

a„ Buffer: .010 M potassium phosphate plus approp riate amount of NaCl.

b., Determined from initial slopes of k0^s vs. [cytochrome o^]»

Co Determined from inverse plots (see text)»do Estimated from layover of second-order plots.

' 54ionic strength on the various rate constants at pH 7.0.The Debye-Huckel plot for reduction by R= capsulata cyto­chrome £2 is given in Figure 17 and has,a negative slope

8yielding a value of at infinite dilution of 2 x 10M "*"S ^. Using a value of -4.7 as the. charge at the site of electron transfer on the reaction center a value of +1.0 is calculated for the charge at the corresponding site on R. capsulata cytochrome c g. From studies on the reduction of R. capsulata cytochrome Cg by potassium ferrocyanide an apparent charge of +.7 has been determined (Wood et al.f 1977) and is in reasonable agreement with that estimated; from studies^. wit%: the . reaction center re­ported here. At pH 9.0 the Debye-Huckel plot for k ^ , from single point determinations> yields the same charge product as pH 7.0. The value of k ^ at infinite dilution at pH 9.0 was three fold greater than at pH 7.0

SummaryTable 8 summarizes the values of k ^ at infinite

dilution and the apparent charges for the interaction of R. rubrum reaction centers and the various c-type cytochromes studied. The value of kg^ appears to be independent of ionic strength in all cases with ionic strength depen­dent only in the case of R. capsulata cytochrome £ 2 ° The values of k ^ vary with different cytochromes, likely due

55

10i

10 .

10.

10 .10 .20 .30 .40 .5 0 .60

(IONIC STRENGTH)172.10 20 .30 4 0 .50

Figure 17. Debye-Huckel plots at pH 7.0 and 9.0 for reduction of reaction center by R. capsulata cytochrome c^.A. Debye-Huckel plot at pH 7.0 for R. capsulata cytochrome Cg. B. Debye-Huckel plot at pH 9.0 for R. capsulata cytochrome c^.

Table 8„ Rate constants and apparent charges at infinite dilution.

Cytochromeka12

(M-1S_1)k 21

(s-1)k23

(s-1)Product, of Charges

Charge on Cytochrome

Charge onReactionCenter0

Horse Heart Cytochrome c

pH 7.0 pH 9.0

1,3 x 10 q 4.5 x 10*

6250ND

—6 .33 -5.90

+ 1 .3° ND

-4.9 ND

R. rubrumCytochrome ^

pH 7.0 pH 9.0

R. capsulata Cytochrome c^

pH 7.0 pH 9.0

1.0 x 10 1.7 x 10

24ND

7100ND

•3.20 • 2.6 6

2.0 x 106.0 x 10

10ND

NDND

•4.72■4.71

+ 0.7 ND

+1.0'ND

- 4 . 6 ND

-4.7gND

a. Determined from y-intercept of Debye-Huckel plot,b. Determined from slope of Debye-Huckel plot.c. Morton.et al., 1970.d. Not determined.

Table 80 (Continued)

e. Wood and Gasanovich, 1975,f= Calculated using a value of -4,7 for the charge on the reaction

center. .g. Determined by dividing product of charges by cytochrome charge.

58to structural differences at or near tile srte of electron transfer. Most notable is the fact that the apparent charge at the site of electron transfer on the reaction center appears to be independent of the cytochrome used (making reasonable estimates of the charge on the cytochromes), and falls in the range -4,5 to -5,0, This result indicates that the cytochromes are all seeing the same region of the reaction center hence reacting in a mechanistically identical fashion. Finally at pH 9,0 the value of k ^ is substantially increased with the three cytochromes studied relative to pH 7,0 and the product of apparent charges is decreased for horse heart cytochrome £. and R, rubrum cytochrome c^ but remains constant for R, capsulata cytochrome c,..

Effect of pH on the Reduction of Oxidized Reaction Centers by Cytochrome c

Horse Heart Cytochrome cThe reduction of oxidized reaction center by horse

heart cytochrome c was found to be pH dependent. Figure 18A presents second-order plots at various pH values and Figure 18B gives the reciprocal plots as a function of pH,It can be seen that k ^ i-s PH dependent but k^^ and k ^ are not, The.values of the various rate constants as a function of pH are summarized in Table 9, Figure 19

Figure 18= The reduction of photo-oxidized reaction center by horse heart cytochrome c as a function of pH=:A= Plots of k0bs vs= horse heart cytochrome c concentration at 25°Cj, =08 M ionic strength, and different pH values?A, pH 6 = 0? O., pH 7 = 0?-O, pH 8 = 0? o, pH 9 = 0? o, pH 10 = 0 = Reaction center concentratlen is 1.0 uM=B= Inverse plots (see equation 4) for the reduction of photo^pkidized reaction center with horse heart cytochrome c at 25°C, =08 M ionic strength, and different pH values?2T, pH 6 = 0? O, pH 7 = 0? O, pH 8 = 0? o, pH 9 = 0? ©, pH 10 = 0 = Reaction center concentration is 1=0 yM=

obs

59

,10002 4 0 0

2000 - ■800

16006 0 0

1200

400

8 0 0

2004 0 0

40 80 120 .1/CYTOCHROME X 10'

10 20 30 4 0CYTOCHROME X1C£

Figure 18. The reduction of photo-oxidized reaction center by horse heart cytochrome c as a function of pH.

60Table 9, The photo-oxidation of horse heart cytochrome c

as a function of pH.

PH

7ak12xl0_

k21 (S-1) k23 (s-1)

-7^k^2xl0in h r 1)

6.0 1.2 10 6250 1.47 .0 1.6 10 6250 1.8

7.5° 2.4 — — — —8.0 3.8 10 6250 4.29.0 6.1 10 6250 7.69*5? 5.2 — —

1 0 .0 2 . 6 10 6250 ; 2.8

1 1 .0° .4

a. Determined from initial vs. cytochrome c concentration.

slope of plots of kobs

b. Determined from slope of inverse plot (see text)c. Determined from single point (see text) 0

[Cytochrome c] is 10.0 uM.

61

6.0-

40 -

20 -

10-

1210pH

Figure 19. The dependence of k,_ on pH for horse heart cytochrome c at 25°C.o, .08 M and , .16 M ionic strengths.

62graphically represents the pH dependence of = A maxima is reached at about pH 9,0 and pK values of 8.5 and 9.5 are estimated, suggesting at least two weakly ionizable.groups are involved in complex formation. The effect of ionic strength upon kj^ at various pH values are also presented in Figure 19, and demonstrate that k ^ is lowered with in­creased ionic strength regardless of pH.

pH Effects with R. rubrum Cytochrome Cg

A pH dependence was found for the reduction of oxi­dized reaction center by R. rubrum cytochrome c^. Values for the rate constant s:weres determined^from second-order, plots shown in Figure 20A and the correspohding reciprocal plots in Figure 20B„ The results are tabulated in Table 10 and show that k ^ is pH dependent while and k^^ arepH independent. The dependency of k12 upon pH is illus­trated in Figure 21 and yields approximate pK values of8.5 and 9.5 as found for horse heart cytochrome c. In­creasing the ionic strength lowered k^ 2 throughout the pH range investigated and is also shown in Figure 21. ThepH effects implicate at least two groups with pK values of8 .5 and 9.5 that are involved in binding the reaction center and cytochrome through electrostatic interactions„

Figure 20„ The reduction of photo-oxidized reaction centers by R„ rubrum cytochrome c^ as a function of pH.Ao Plots of k0bs vs<, R, rubrum cytochrome eg concentration at 25°C and .08 M ionic strength and different pH values?Of pH 7e0; 0, pH 10.0; A, pH 8.0; © r pH 9.0. Reaction center concentration is 1.0 # 1.B. Inverse plots (see equation 4) for the reduction of photo—oxidized reaction center by R. rubrum cytochrome c. at 25°C and .08 M ionic strength and different pH values;Of pH 7.0; Q, pH 10.0; A, pH 8.0; ©, pH 9.0. Reaction center concentration is 1.0 uM.

63

centers

1200

1000

BOO

6 0 0 -

4 0 0 -

200

10 20 30 4 0CYTOCHROME X 106 1/CYTOCHROM E X 10

Figure 20. The reduction of photo-oxidized reaction by R. rubrum cytochrome c^ as a function of pH.

64Table 10, The photo-oxidation of R, rubrum cytochrome c2

as a function of pH,

pH

-7a12x

k21(s-b k 2 3 (s " 1 )k12:t10( H ' V 1)

7,0 1,3 20 7100 1.57,5° 1,6 — —8,0 3,0 20 7100 3.49,0 4.8 20 7100 5.59, 5C 2.1 ■ ——

10,0 1.8 20 7100 2.0

1 1 ,0? 0 . 2

a. Determined from plots kobs VSo [cytochrome c^].b. Determined from inverse plots (see text)c. Determined from single point (see text),

[Cytochrome c^] is 10 uM,

65

4.0 •

3.0

• O

2.0

1.0

H—10

pH

Figure 21. The dependence of k12 on pH for R. rubrum cytochrome Cg at 25°C.o, .08 M and D, .16 M ionic strengths.

66pH Effects with R 0 capsuiata .Cytochrome £2

The reduction of reaction center by R, capsuiata cytochrome c2 . was found- to be: pHv,dependentc Figure 22A presents second-order plot and shows that is pH de­pendent - Reciprocal plots were not linear (Figure 22B) and ^ 2 2 was estimated from layover of the second-order plotso Table 11 summarizes the effect of pH upon the rate constants which is illustrated in Figure 23. At least two pK values at 8.5 and 9.5 are suggested for both k^ 2 and at high and low ionic strength.

Summary .The reaction between oxidized reaction center and

cytochrome c was found to be similar for each cytochrome investigated. The second-order rate constant was pH de­pendent for each cytochrome„ The first-order rate constants were pH independent for horse heart cytochrome c and R, rubrum cytochrome c2 but pH dependent for R. capsuiata cytochrome c2. Ionic strength and pH studies implicate weakly iohizable groups with pK 8,5 and 9,5 that form electrostatic interactions responsible for complex forma­tion prior to electron transfer consistent with the mech­anism in equation 1 for all three cytochromes investigated.

Figure 220 The reduction of photo-oxidized reaction Centers by R 0 capsulata cytochrome Cg as a function of pH0

. Ac Plots of kobs vs. R. capsulata cytochrome eg concentra­tion at 25°C and .08 M ionic Strength and different pH values? ©r’ pH 10.0? □, pH 7.0? A, pH 8=0? o, pH 9.0. Re­action center concentration is 1.0 yM.B. Inverse plots (see equation 4) for the reduction of . photo-oxidized reaction center by R. rubrum cytochrome c„ at 25°Cy .08 M ionic strength and different pH values?©> pH 10.0? D, pH 7=0? A, pH 8.0? o, pH 9.0. Reaction center concentration is 1.0 yM.

67

10001200

10008 0 0

• 8 0 0

6 0 0

4 0 0

400

200-

200

4 0 801/CYTOCHROME X10

120I 20 3 0 4 0CYTOCHRO M E X 10

10

Figure 22. The reduction of photo-oxidized re­action centers by R. capsulata cytochrome c2 as a function of pH.

68Table 11. The photo-oxidation of R. capsulata cytochrome

c_ 2 as a function of pH.

pH

_7ak^^xio"(M~1S“1) k 2 1 (S"1) k23 )

-7bk12x10 (M~ 1S ~1): ,

7.0 1.1 5 600° 0.97. 5d 1.3 — — ——8.0 2.0 5 1200 2.39.0 3.2 5 1200 3.39.5d 2.5 — — ——

1 0 . 0 O'. 9 5 400° 0.71 1 . od 0.3 ——

kobs vsa. Determined . [cytochrome c^bo Determined

from the initial Slope ].from slope of inverse

of plots of

plot (seetext).

c. Estimated from layover of plots of vs. [cytochromes

d. Determined from single point (see text. [Cytochrome c^l is 10.0 pH.

69

4.0"

^9x

2.0 -

pHFigure 23. The dependence of k-^ and k23 on pH for

R. capsulata cytochrome £2 at 25°C.e, .08 M and A, .16 M ionic strength for kj_2 ; at .08 M (O) ionic strength for k2 3 « For k23 abcissa is x 10- .

70The Effect of Detergent on the Reduction of Oxidized Reaction Center by Cytochrome c

Reduction of oxidized reaction center by horse heart cytochrome c was found to be dependent upon LDAO concen­tration. Figure 24 shows the dependency of upon theLDAO concentration. As the LDAO increased from 0 to .005% (Figure 24A) the rate was cut nearly in half but leveled off until a concentration of .1 % was reached and then the rate decreased in a linear fashion (Figure 24B). At high ionic strength was independent of LDAO concentrationas illustrated in Figure 25. From the second-order plot at 0% LDAO presented in Figure 26A and the corresponding reciprocal plot in Figure 25B it was found that and^21 <° not change from the values determined in .03% LDAO but k.^2 bad increased. Table 12 compares the rate constants at 0% LDAO and .03% LDAO. The data is consistent with the role of LDAO as an inhibitor at lower concentrations of LDAO (0-.l%). At higher concentrations of LDAO ionic effects due to impurities in the LDAO and denaturation of both the reaction center and the cytochrome probably become important in reducing the rate.

Interaction With Non-Physiological ReductantsIn the absence of cytochrome c the reduction of re­

action center by recombination with the primary acceptor was biphasic in nature (Figure 27) with apparent first-order

Figure 24«, The effect, of LDAO concentration onreduction of reaction center by horse heart cytochrome o„Ao The effect of low LDAO concentration on reduction of photo-oxidized reaction center by horse heart cytochrome c in 50 mM phosphate buffer, pH 7o0. Horse heart cytochrome concentration is 10=4 yM (©) and 5=2 yM (o)=Bo The effect of high detergent concentration on reduction of photo-oxidized reaction center by horse heart cytochrome c (10o2 yM) in 50 mM phosphatef pH 7*0 and 250C=

71

400

200-

100

.01 .02 .03 .04 .20 4 0 .60 .80LDAO (•/<>)

Figure 24. The effect of LDAO concentration on reduction of reaction center by horse heart cytochrome c.

72

7 0

60.

50.

40 -

30

2 0 -

OS0401 02 03LDAO (%>)

Figure 25. The effect of low detergent concentra­tions on reduction of photo-oxidized reaction center by horse heart cytochrome c at high ionic strength.10.2 at pH 7.0 and .167 M ionic strength and 25°C.

Figure 26= Reduction of photo-oxidized reactioncenter by horse heart cytochrome c at 0% LDA0oA. Riot of hobs vs-, horse heart cytochrome c concentration at 0% LDAO in 50 mM phosphate buffer? pH 7.Of at 25°Co

■ .. ' ' Bo The inverse plot (see equation 4) for reduction of photo-oxidized reaction center by horse heart cytochrome c at 0% LDAO in 50 mM phosphate buffer? pH 7*0? at 25°C=

73

240Q -500

.400

1600300

n

n12 00-

200

800-

.100400-

20 40 80CYTOCHROME X 10*

40 80 120 1/CYTOCHROME X 10'

Figure 26. Reduction of photo-oxidized reaction center by horse heart cytochrome c at 0% LDAO.

74Table 12. The photo-oxidation of horse heart cytochrome c

as a function of LDAO concentration.Buffer 50 mM phosphate, pH 7e0„

k^2xl0 ~ 7 _7a3cl2xlO% LDAO (m""1s” 1 ) kjlts"1) k 23(S'1)

0 CMCM 10 6520 2-4

. -03 1,0 10 6520 o .

a.. Determined from inverse plot (see text) .

75.10

.01

O01 3.53.02.0 2.51.5TIME (SEC.)

Figure 27. Plot of absorbance vs. time for biphasic auto-reduction of reaction center at pH 7.0 in 50 mM phos­phate.The fast phase, c , can be separated from the biphasic curve, a, by subtraction of the slow phase, b.

76-1 -irate constants of 6.0 S and 0.5 S . In the presence of

ascorbate the biphasic nature or rate constants did not change at pH 7.0 but did at higher pH. Figure 28 shows the percent fast reaction and kQ,g as a function of pH. At pH 11.0 the reaction was monophasic but it is impossible to determine whether the change is due to the reaction center or to the ascorbate because ascorbate has a pK at 11.5. The reaction center has a large negative charge at the site of electron transfer (-4.6 to -4.9) as determined earlier. The charge on ascorbate is also negative and becomes increasingly' so as the. pH is raised which would make any reaction between.ascorbate and the reaction center slower rather than faster at higher pH. Therefore it seems unlikely that the pH effect is not due to ascorbate reduc­tion but rather recombination with the primary acceptor.The LDAO had little or no effect on reduction of reaction center at pH 7.0 in the presence of ascorbate as illustrated in Figure 29. Ferrocyanide ion did not effect the kinetics of reduction of photon-oxidized reaction centers over the concentration range investigated (0-100 mM) at pH 7.0.

77

24

20.

16-

U1AA

1 2 -

8

4 -

6

100

— 4-7

-75

-50

-25

8 10 11 12PH

Figure 28. The effect of pH on k , of auto­reduction of reaction centers.In the presence of .5 mM ascorbate (o) and % fast reaction (□) and .08 ionic strength.

fast

re

actio

n

78

8 -

6 -

n

4-

.06.01 .04 .05.02 .03L D A O (%)

Figure 29. The effect of detergent on the ^Q-s of auto-reduction of reaction centers.At pH 7.0 for the fast phase (o) and slow phase (□) at 25°C in 50 mM phosphate buffer.

DISCUSSION

The reaction center used in the kinetic studies was pure based on optical spectra and component analysis as compared to other studies (Noel et aley 1972? Van Der Rest and Gingras? 1974). The effects of LBAO and organic solvents upon the spectra indicate that some of the bacteriochlorophyll is easily phenophytinized and that phenophytinization might have taken, place in previous ex­periments when determining the bacteriochlorophyll content of reaction.centers- (Van.Der Rest and Gingras,: 1974 ?t Reed and Peters, 1972?'Steiner et al., 1974} resulting in high values for bacteriophedphytin„ The long wavelength absorp­tion bands of bacteriochlorophyll and bacteriopheophytin vary a great deal in position depending upon the solvent (Vernon and Seely, 1966) „ Different environments created by detergent micelles and/or organic solvents mixed with aqueous media may cause shifts and overlaps of peaks in region 600-1000 nm making it difficult to identify and determine concentrations of the reaction center pigments in this region of the spectrum. An explanation for the absorption band appearing in Figure 3 at 680 nm may be that two populations of bacteriopheophytin existing in different

79

80environments cause two absorption bands one at 680 nm and 755 nm for bacteriopheophytin»

The detergent LDAO was found to have a pronounced effect upon horse heart cytochrome c_ at a concentration of 2%. The rapid rate of auto-oxidation and the drastic change in midpoint potential indicate that protein de- . saturation is occurring. The exact changes in the cyto­chrome structure are unknown but due to the rate of auto- oxidation (Table 4) it can be suggested that the heme crevice is opened with the iron more accessible to oxida­tion 6 At the low concentrations used in this work (,03%) the effects of- LDAO were minimal as evidenced by similar values for the rate constants in the basence of LDAO which appears to inhibit complex formation at low ionic strength but not electron transfer. Based on the results presented here 2% LDAO clearly denatures cytochrome c making the re­sults Of previous kinetic studies using high detergent concentrations (Prince et al«, 1974) difficult to in­terpret o

The effect of ionic strength on the interaction of oxidized reaction centers and c-type cytochromes was similar in all cases investigated in regards to k ^ of the mechanism given in equation 1. Thus electron transfer was preceded by the formation of a complex between the cytochrome and reaction center and complex formation is

81largely due to electrostatic interactions„ The studies reported suggest a charge of -406 to -4.9 on the reaction center using available estimates of the charge on the cyto- 'chromes. It is interesting to note that the charge upon the cytochrome is always positive regardless of its iso­electric point indicating a specific site upon the cyto­chrome is responsible for binding the reaction center.This finding agrees well with previous work of Prince et alo (1974) and with the iron hexacyanide model system (Wood and Cusanovich, 1975). Ke et al. (1970) found that at the pH of maximal rate (9.3) the reaction was independent of ionic strength, suggesting no ionic interact^oms-: were: taking place. In this study? the finding of Ke? Chaney and Reed (1970) were not borne out in that the reaction was ionic strength dependent over the pH range 6-11. In work by Prince et al. (1974) the reduction of oxidized reaction center by cytochrome c was found to be purely second-order, i.e., no complex formation unlike the work presented here. There are two possible reasons for the discrepancies: (1 ) high detergent concentration could haveprohibited complex, formation by raising the ionic strength or denaturing the reactants"? and (2 ) the reaction was investigated over a narrow range of cytochrome concentra­tions (0-40 ym) so that layover in the second-order plots indicative of complex formation was not observed. The

82first-order rate constants (for electron transfer and break­down of complex) were found to be ionic strength independent for horse heart cytochrome c_ and R» rubrum cytochrome c^ r however, for R, capsulata cytochrome c^ ^ 2 3 was found to be ionic strength dependent.

In terms of k^g the reduction of phqto-oxidized reactions centers by e-type cytochromes was pH dependent in all cases investigated. The pH profiles of the reaction yield maxima at approximately pH 9.0 in all cases and sug­gest approximate pK values of 8.5 and 9.5. These results correspond well with those reported by Ke et al. (1970) who found a maxima at 9. 3 and rpK. values . of, &> 2 : and. 9.7, but are in complete disagreement with the work of Prince et al. (1974) who found no pH dependence in the pH range 5-11. It is difficult to separate the relative contribu­tions of the reaction center from the cytochrome- in terms of ionization as no structural information is presently available concerning reaction centers. From studies on the oxidation of R. rubrum ferrocytochrome c^ by potassium ferricyanide pK values of relative rates.of the various species could be accommodated by the results obtained with reaction centers. The oxidation of ferrocytochrome c by potassium ferricyanide is pH independent from pH 7.0 to 9.4 (Brandt et al., 1966). This observation could be interpreted in terms of the ionizations leading to altered

83values of k.^2 resulting from ionizations of side chains associated with the reaction center only. The almost identical pH profiles observed with the three different cytochromes would provide further support for the dominant role of the reaction center. However, the very similar structure of various cytochromes in the region of the heme edge (Table 2) with lysine residues the dominant ionizable side chains may be responsible for the similar pH profiles.

Only in the case of R. capsulata cytochrome c^ was ^23 found to be pH dependent. This can be interpreted as that once a complex is formed for horSe heart cytochrome c and R. rubrum cytochrome c^ it is.inert to changes in solvent conditions or changes in the conformation of the molecule important for electron transfer. For R. capsulata the complex is not inert to the solvent and further changes in the molecule may occur due to ionizable side chains. Electron transfer could be effected changing the conforma^ tion of the molecule at the site of electron transfer driven by pH or ion binding at sites distant from the electron transfer site.

The reaction center was found to have an apparent charge of 4«5-5.6 at the site of electron transfer regard­less of the cytochrome used, indicating all the cytochromes are reacting at the same site on the reaction center. The apparent charge on the cytochrome at the site of electron

84transfer is always positive and is consistent with the iron hexacyanide model system. The most prominent area on the c type cytochromes that possess a positive charge is the front of the molecule around the exposed heme edge. This ring of positive charge around the heme edge is highly conserved among c type cytochromes strongly implicating the exposed heme edge as the site of electron transfer.

The mode for electron transport involving the reac­tion center and c type cytochromes is consistent with the iron hexacyanide model system. In the case of the reaction center the complex formed with cytochrome is.very tightwith association constants of 1.3 x 1 0 8m"1, 4-:«, l. x; loSd""1,

7 - 1and 1 x 10 M for horse heart cytochrome o, R. rubrum cytochrome £ 2 , and R. cpasulata cytochrome £ 2 respectively, indicating a high degree of specificity. These association constants are 2-3 orders of magnitude larger than those found for iron hexacyanides (Wood and Cusanovich,1975) where the binding is mainly electrostatic, suggesting modes of binding other than electrostatic, i.e., hydrogen or hydrophobic, are important in the reaction center- cytochrome complex accounting for its large association constant. The value of for oxidation of horse heartcytochrome £ is larger than R« rubrum cytochrome £g which also agrees with the iron hexacyanide model system (Wood and Cusanovich, 1975, Morton et al=, 1970).

Conclusionslo The reduction of photo-oxidized reaction

center,, .bx, e-type cytochromes follows,- a complex mechanism, involving electrostatic interactions of weakly ionizable groups»

2e. The reaction occurs at the same site on the reaction center and cytochrome regardless of the source of cytochrome indicating a general mechanism for electron transfero With c-type cytochrome electron transfer pre­sumably taking place on the front of the molecule at the exposed heme edge. The mechanism of electron transport involving the reactioncenter and c-type cytochromes is totally consistent with the iron hexacyanide model system for electron transport,

3, LDAO appears to open-up the cytochrome c molecule and expose the heme group making it more auto- oxidizable, especially at LDAO concentrations greater than ,06%, This fact could explain some of the anomalies between this work and the work of Prince et al, (1974) who routinely used a LDAO concentration of 2%,

APPENDIX A

DERIVATION OF RATE LAWS

Rewriting the complex mechanism given by equation 1 of the text in letter notation equation i is obtained:

^1 2 ^23 ^34A + B — AB ~ AB' D + E (i)^21 k32 k43

Where AB and AB * are meant to represent the oxidized re­action. Genter-ferrocytochrome complex and the reduced reaction center-ferricytochrome complex, respectively.In: the kinetic analysis only the rate limiting first order steps will be monitored, hence equation ii is a reasonable representation of the observed kinetics,

k12 k23A + B - • C --- D + E (ii)k21 k43

For the analysis to be described kgg has been taken as thelimiting rate constant to the right as this is the stepaccounting for the observed spectral change, The reactionof oxidized reaction centers with c-type cytochromes isessentially irreversible, hence k^^[D][E] and/or k^2 (AB)'\ . . " are negligible for the conditions used here as a result of

86

87the large oxidation^-reduction potential differences between the reaction center (~440 mV) and the cytochromes (260-370 mV) <,

Application of the steady state approximation for the production of C yields equation iii.

making the substitution (A) - A-)-(C)-(D) where A^ is the initial concentration of A and Co = = 0 equation v isobtainedo

dt k1 2 [A][B] - (k12 + k2 3 )[C] 0 (iii)

or on rearrangement

k^2 [B] —. (k^2 t k2 3 ) [C] (iv)

(v)kiaCE] + k2i + k23

since

(Vi)

substituting [C] from equation v into equation vi results in equation vii

k 12k 23^Ao ^ B = 0kl2[Bi + k2i + k23^

as long as [B]» [.A [B0] is unchanged with ■ time andk^^[E] is small, equation vii becomes equation viii

+ a[D] - g = 0

wnere

a = kl2k23l-Bo-1k12[Bo 3 + k21 + k23

g = k12k23 Ao3 Bo3k12^Bo 3 + k21 + k23

(vii)

since

(viii)

(ix)

Integration of equation viii yields

ln[D] - Ing/a = at

Since 3/a = [Aq]

(x)

89The observed rate constant from the slope of ln[D] vs, t plot will be a or kQ^s, Inversion of equation ix gives equation xii

1 1 + k21 + k23 1kbbs k23 ki2k23 [B ]o

For the situation when ^q2^Bo^<<k23 equation, i becomes

kioA + B -r-' - C (xiii)21

Thus with [Bo]»[Aq]

since [C] =•" [A ] - [A]

= k12[B03[a] - k 2 1[Ao-A] (XV)

ordAdt = CA](k1 2 [Bo] + k21 - k2 1 EAGJ (xvi)

Integration of equation xiv yields equation xv only ifk1 2 [B][A] > k21[Cj

lnLAoJ ~ k12*-Bo^ + k21^t " (xv)

90Thus the plots of vs0 [B^] should be linear with aslope of k^2 and y-intercept of provided the conditionused in obtaining equation xv is applicable.

APPENDIX B

FLASH TECHNIQUE

The flash photolysis apparatus outlined in Figure 7 is of classical construction. The spark-gap is triggered after charging the capacitors to 2-5 kilovolts„ Simul­taneously with the firing of the spark-gap, a trigger pulse is applied to windings on the flash tubes to insure ionization and immediate flash lamp breakdown. The halogen lamp (monitoring lamp) was fitted with an independent cooling fan to minimize vibrations, and was operated on direct current by a Sorenson QSB6-30 variable power supply.

As a photomultiplier tube is essentially a constant current device, switched load resistors were used to vary the output voltage. The load resistance could be varied from 1 megaohm to 10 kilohm by switching the desired load resistor across the input signal.

After a trace was run and the reaction complete the oscilloscope was triggered again to obtain a base line. The trace was then photographed and later enlarged on graph paper. Voltages at various times were converted to ab­sorbance and a least squares fit was used to determine kobs» The reactions were first-order with ln[AA] vs. time

91

92plots linear over at least three half-lives« A typical trace is shown in Figure 30=

In order to charge the low inductance capacitors a power supply was constructed and is shown in Figure 7= Alternating current was fed into a neon light transformer, (primary voltage 1 2 0 , secondary 1 2 ,0 0 0 ) from a varic and then half rectified by two diodes (Varo VF' 25*40) to give a 6 kilovolt output, In order to keep the capacitors from discharging back through the power supply and creating a potential hazard when the power was cut, a 2 megaohm re­sistor was placed in the line (Victoreen HPB 15% tolerance). With this resistor the time required to charge the capaci­tors was 30-60 seconds. AS a safety measure a means of discharging the capacitors without firing the flash lamp was required. This was accomplished by using a vacuum relay switch (Jennings Radio Cbrporation, Model RP100X4963D21B20) that discharged the capacitors to ground.

i-------- 1—5 10 •H -----------------1-----------------1----------------- 1-----------------1-----------------1 -15 20 25 30 35 40TIME(ms)

Figure 30. A typical trace obtained from the flash spectophotometer.

LITERATURE CITED

Almassy, R,, and Dickerson, R, E. (1977) California Institute of Technology, Work in progress.

Ashida, To, Tanaka, No, Yamane, To-, Tsukihara, T., and KakudOf (1973) J. Blocliem, 73, 463«

Banker, G», and Cotman, C. (1972) j . Biol. Chem. 247,5856.

Brandt, K., Parks, P., Czerlinski, G., and Hess, G.(1966) J. Biol. Chem. 241, 4180.

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