Cytochrome c binding to enzymes and membranes

Biochimica et Biophysica Acta, 346 (1974) 261-310 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 86019

C Y T O C H R O M E c B I N D I N G TO E N Z Y M E S A N D M E M B R A N E S

PETER NICHOLLS

Institute of Biochemistry, University of Odense, Niels Bohrs alle, DK-5000 Odense (Denmark)

(Received August 5th, 1974)

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

II, Cytochrome c and small ligands . . . . . . . . . . . . . . . . . . . . . . . . . 263

Ill. Cytochrome c and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

IV. Cytochrome c and non-specific membranes . . . . . . . . . . . . . . . . . . . . 276

V. Cytochrome c and the inner mitochondrial membrane . . . . . . . . . . . . . . . 282

VI. Properties of bound cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . 296

VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

I. INTRODUCTION

The in te rpre ta t ion o f the curve relat ing cy tochrome c concent ra t ion to catalyt ic

activity, first ob ta ined by Kei l in [I ] for the oxida t ion of cysteine by submi tochondr ia l

part icles (Fig. 1), has been a mat te r o f cont roversy f rom the outset. Kei l in himself,

influenced perhaps by a pape r o f Ar rhen ius [2] tha t had been b rough t to his a t ten t ion

by George Nut ta l l , in terpre ted the result in terms o f Schutz 's law, which predicts

tha t the velocity o f an enzyme react ion is p ro po r t i ona l to the square roo t o f the

p roduc t o f enzyme concent ra t ion , substra te concent ra t ion and t ime [3]:

v = ~ v ~ e ~ - ( 1 )

This law is in fact a much bet ter app rox ima t ion to the kinetics o f some cyto-

ch rome c systems than any simple rec iprocal law of the Michael is type (Eqn 2):

v = k es/(Km ÷ s) (2)

Nevertheless , the idea was not exper imenta l ly fruitful and the concept tha t a

Michael is complex exists between cy tochrome c and its oxidase was p roposed by

Abbreviations: ANS, 8-anilino-l-naphthalene sulfonate; TMPD, tetramethyl-p-phenylenediamine.

262

2512

2O0

E E 0

E E

~oo

D

5O

2 3 4 5 6 7 8 9 110 111 12 Cytochrorne concent r(~t ion

Fig. 1. Effect of cytochrome c concentration on the rate of oxidation of cysteine by cytochrome c oxidase particles. From Fig. 4 of Keilin [1 ].

Stotz et al. [4] in 1938. Chance [5] extended the idea in 1951 by proposing ternary complexes of cytochrome c, H202 and peroxidases, although ironically the enzyme which does seem to form a complex with cytochrome c, cytochrome c peroxidase of yeast [6], was the one which showed no sign of a monomolecular reaction step in Chance's system.

The first serious challenge to the idea of a functional cytochrome c-cytochrome oxidase complex came from Smith and Conrad [7] in 1956, who showed that the kinetic time course of cytochrome c oxidation could be explained only by postulating the formation of inhibitory complexes between ferric cytochrome c and the enzyme. The possibility then existed that the complex with ferrocytochrome c was also inhibitory rather than catalytically active, because the first order character of cytochrome c oxidation at very high [c] concentrations showed that the binding constants for the ferric and ferrous cytochrome must be almost identical. The idea that an enzyme might be incapable of distinguishing between its substrate and product came as a surprise to the present author, then a student at Cambridge, and, he remembers, to his teachers. Such kinetic patterns had, however, first been described by Henri [8] for the case of amylase.

The interpretation of this finding remained uncertain for some time. Smith and Conrad themselves emphasized the idea of inhibition by all forms of cytochrome c, while Slater [9] preferred an explanation in terms of a redox equilibrium between cytochrome c and the oxidase. The most complete kinetic analysis was that produced by the late Koen Minnaert [10] who devoted his short research life to a solution of this oxidase problem and to posing a question about the redox behaviour of the system [11 ] that has stimulated some of today's most interesting studies (he is also the only colleague to have urged the present author to publish his work [12] because otherwise his own studies would preempt it).

263

The relevance of these results and ideas to cytochrome c in situ in the mitochondrial membrane was not clear. Tsou [13] had shown that "endogenous" cytochrome c of Keilin-Hartree submitochondrial particles was catalytically and chemically different from "exogenous" cytochrome c with which such particles also interact. The idea was put forward that cytochrome c in situ was shielded by and complexed to mitochondrial lipid and a number of early attempts were made to isolate or prepare such a "native" lipocytochrome c [14,15].

Better knowledge of both cytochrome c and membrane structure has revised these ideas and forced the posing of more precise questions. In consulting the rest of this article, it is suggested that the reader refer continuously to Fig. 2, the "left" (and likely binding) side of cytochrome c as determined by Takano et al. [16], asking the question "how will it fit?". Meanwhile the author will address the following current problems:

(a) Is anion interaction (binding) with cytochrome c or cation interaction (binding) with enzyme or membrane the major cause of the salt dissociability shown by cytochrome c complexes as ionic strength is increased?

(b) If enzymes (and the mitochondrial membrane) show approximately equal affinities for ferrous and ferric cytochrome c, how can the measured E'o for cytochrome c be lower in the bound state?

(c) How many functional binding sites are there for cytochrome c on a "molecule" of cytochrome aa3?

(d) Are the mitochondrial cytochrome c binding sites lipid or protein in nature? (e) Is all the mitochondrial cytochrome c membrane-bound, and if so, are

all the binding sites identical?

II. CYTOCHROME c AND SMALL LIGANDS

Cytochrome c in the ferrous and ferric state binds small anions and cations. Evidence for this binding has been obtained by electrophoresis [17], X-ray crystallo- graphy, redox potential effects [ 19 ], and ascorbate reducibility [20,21 ]. In crystals [ 18 ] of some mammalian cytochromes c (Fe 3÷) one anion is located approximately 5 /~ from the haem plane close to isoleucine 81, and another in the region of asparagine 52. Both regions contain lysines or arginines that could be involved in the anion binding. On reduction, some of these ligands are displaced [16] (the accessible pocket in front of the haem is blocked by phenylalanine 82)*.

Electrophoretic studies with ferricytochrome c [17] indicated a binding order (at pH 6.9):

phosphate > chloride > iodide ~ sulphate ~ cacodylate.

* Margoliash (personal communication) indicates that this interpretation may not be valid (cf. legend to Fig. 2).

264

l 9

B ~ ' 5 ¸

Fig. 2. Molecular structure ofcytochrome c: computer generated stereoscopic drawings of cytochrome c viewed from the left side. Numbered circles are alpha-carbon positions. Unnumbered circles show the haem group (FE indicates iron atom) and certain key side-chains. ' N ' is marked on nitrogen atoms of the pyrroles and of histidine 18 (co-ordinated to Fe), and 'S ' on the iron co-ordinated sulphur atom of rnethionine 80. A: oxidized (ferric) cytochrome c from horse. B: reduced (ferrous) cytochrome c from tuna. Facing the viewer are the positively charged lysines 55, 60, 72, 73, 86, 87 & 88 as well as arginine 91. Towards the back ( 'r ight ' side) are lysines 5, 7, 8, 13, 22, 25, 27, 39, 53, 79, 99 & 100 as well as arginine 38. Acidic residues are found at positions 61, 62, 66, 69, 92 & 93 in the upper part of the molecule. Note the apparent movement upon oxidoreduction of residues lysine 13, tryptophan 59, tyrosine 67, tyrosine 74 and phenylalanine 82 (although the last mentioned shift according to Margoliash (personal communication) may be exaggerated in these reconstructions); a loop in the 20's also moves and the haem ring 'slumps' in its pocket on reduction. Amino-acid sequences are as follows for the two cytochromes : (on page 265)

265

1 5 10 15 20 25 horse aG D V E K G K K I F V Q K C A Q C H T V E K G G K tuna aG V D A K G K K T F V Q K C A Q C H T V E N G G K

30 31 35 40 45 50 horse H K T G P N L H G L F G R K T G Q A P G F T Y T D tuna H K V G P N L W G L F G R K T G Q A E G Y S Y T D

55 60 61 65 70 75 horse A N K N K G 1 T W K E E T L M E Y L E N P K K Y I tuna A N K S K G I V W N N T D L M E Y L E N P K K Y I

80 85 90 91 95 100 horse P G T K M 1 F A G I K K K T E R E D L I A Y L K K tuna P G T K M I F A G I K K K G E R Q D L V A Y L K S

104 horse A T N E tuna A T S - -

From Fig. 13A and B of Takano et al. [16]. One-letter notation conforms with Tentative Rules of IUPAC-IUB Commission on Biochemical Nomenclature (Biochim. Biophys. Acta (1968) 168, 6-10).

TABLE 1

ANION BINDING BY CYTOCHROME c

Anion Complex with: Kd (app) Ref. (Fe 3+ c) (~tM)

Fe a+ c Fe 2+ c

HzPO4- yes yes 25-100" [22,24] C1- yes no? 50-70* [22,24] I - yes ? ~j Ka(C1-) [17] SO42- yes? ? :> Ka(I-) [17] Fumarate yes yes? - - [22] Succinate no ? - - [22 ] Citrate yes yes - - [22] ADP yes ? 250 [22,24]

* Other methods (ascorbate reduction kinetics, etc. refs 20,21) give higher values (Kd 1.0-2.0 raM). pH values from 6-7, 25 °C, horse heart cytochrome c.

T h a t is, p h o s p h a t e h a s t he g r e a t e s t a n d c a c o d y l a t e t h e l eas t effect o n c a t i o n i c

m o b i l i t y . S o m e o f t h e i ons we re t i gh t l y b o u n d a n d o n l y r e m o v e d b y e l ec t rod ia lys i s .

T h e r e d u c e d p r o t e i n , h o w e v e r , b i n d s a n i o n s less f i rmly a n d c a t i o n s m o r e f i rmly [22].

A s s h o w n in T a b l e I, s o m e a n i o n s ( i n c l u d i n g c h l o r i d e ) d i s t i n g u i s h b e t w e e n t h e f e r r o u s

a n d fe r r i c f o r m s , wh i l e o t h e r s ( i n c l u d i n g p o s s i b l y p h o s p h a t e ) b i n d to b o t h . I n t h i s

r e s p e c t t h e s m a l l a n i o n s r e s e m b l e t h e l a r g e r l i gands .

B e t w e e n o n e a n d t w o a n i o n s c a n be b o u n d q u i t e t i g h t l y (Kd ~ 0.1 m M ) [22,23 ].

M a r g a l i t a n d S c h e j t e r [19,23] h a v e m e a s u r e d r e d o x p o t e n t i a l s in t h e p r e s e n c e a n d

a b s e n c e o f s u c h a n i o n s . E q u a t i o n s 3 a n d 4 s u m m a r i z e t h e o b s e r v a t i o n s a t p H 7

a n d 25 °C:

266

Em ~ 275 mV

Em ~ 255 mV

A _ ¢3+ ~.

Kd ~ 0.1 mM

m_ c 2+

Ka ~ 0.9 mM

A _

C 3+

K e = x

m-- C 2+

~.~

Kd ~'~ 0.3x

c a+ A-

I x_ c2+ A-

~- c 3+ A-

.~. c 2+ A-

Em ~-~ 225 (3)

mV

I - + 0

Em ~ 285 (4) mV

I --> high

According to these workers, when the ionic strength becomes very low, the redox potential of the free cytochrome c is greater than that of the anion-complexed c, implying that the anion (usually CI-) binds more tightly to c 3+ than c 2+. On the other hand, when the ionic strength increases, the situation changes in such a way that the free c now has a lower potential than the bound c. Therefore, at high ionic strength ( > 0.01) the anions are bound more tightly to the reduced form. Or put in yet a third way, the action of ionic strength is to decrease the potential of free c and increase that of anion-bound c. Although more detailed work is needed and some of the authors ' assumptions [23] may be questionable (e.g. the assumption that the potential is proportional to ~/I/(1 4- 6 ~/I) outside the range of I values actually employed), the reviewer believes that these observations may help to answer question (b) above.

Direct determinations of anion binding by gel filtration [24] confirmed the predictions based on the redox potentials at low ionic strengths, as well as the electrophoretic observations [17,22]. Table I lists the binding constants obtained. No spectroscopic effects of such binding have been seen (unlike the cases of the iron- binding ligands cyanide, azide and imidazole [25]). The presence of such ligands does however diminish the rate of reduction of cytochrome c by ascorbate [20,21 ]. Ka values calculated from the inhibition of the ascorbate or hydroquinone reactions [26] are about ten times higher than those obtained by redox and direct binding studies (Table I). Possibly a different anion-binding site is being monitored. Recent studies [27] of the Cr3+-cytochrome c 2+ complex produced by the reduction of c 3÷ by Cr 2+ indicate that the Cr a tom is located between asparagine 52 and tyrosine 67; thus giving a clue as to the route of ascorbate reduction.

Not all redox reactions are blocked by this anion. The reaction with ascorbate

267

free radical (about l0 a times faster than the reaction with ascorbate itself in 0.1 M phosphate) is almost unaffected by phosphate concentration. Ascorbate itself appears

o / to reduce the liganded c at a rate some 2/o that of the uncomplexed c [20,28]. Prob- ably the reducing agent itself forms a complex with ferricytochrome c. Evidence has been obtained by NMR for a ferrocyanide-ferricytochrome c complex [29], and the Cr 2+ reduction is promoted by bridging anions [30].

Mochan and Nicholls [28] suggest that the reaction with ascorbate is affected similarly by enzyme binding and by phosphate binding, but that ascorbate and other non-reducing anions do not interact at the same sites. In view of the multiple binding sites now found [24] this conclusion needs tempering. The confusing character of the observations on ligand binding by cytochrome c itself lies in the uncertainty as to which particular site is being monitored by a given technique. We may hope that the availability of detailed structural data (Fig. 2) will encourage investigators to re-examine the various ion effects and to define more closely the interaction areas on the molecule.

III. CYTOCHROME c AND PROTEINS

Cytochrome c forms complexes both with proteins capable of specific electron transfer reactions and with proteins incapable of such electron transfer. The former include: cytochrome c peroxidase of yeast, cytochrome b2 (L-lactate cytochrome c reductase) of yeast, and possibly cytochrome aa3 (mammalian cytochrome c oxidase). Phosvitin and antibodies are among the proteins which bind cytochrome c but cannot accept or donate electrons.

IliA. The phosvitin-eytochrome c complex [31] Phosvitin is a highly charged (120 phosphoserine residues /217 total residues)

protein of mol. wt 35000 obtained from eggs [32]. Because of its charge at neutral pH it appears to have a random coil configuration. Under these conditions at least twenty cytochrome c molecules can be bound along its length (maximum 780 /~), each occupying a "site" of about six negative charges. The complex sedimented as such in the ultracentrifuge, the S°2o,w value (1.8 for cytochrome e and 2.2 for phosvitin) increasing to 10.5 with 20 moles e bound/mole phosvitin. Within the complex cytochrome c was less readily reducible than in free solution, but the ferric protein retained the 695 nm band characteristic of the native state with unperturbed sulphur-iron linkage [33,34]. Neither the redox potential nor the affinity of the ferrous cytochrome have been examined in this useful model system.

IIIB. The Fab fragment-cytochrome c complexes [35] Fab fragments of Ig antibodies formed in rabbits against various cytochromes c

will form complexes with the antigenic cytochrome e [35]. Such complexes appear to involve the action of fragments recognizing both charges [36,37] and hydrophobic

268

[35,36] groups on cytochrome c. The former ("anti-oxidase") fragment type reacts with k ,~ 0.8 • l06 M -1 " S - 1 , the latter ("anti-isoleucine 58") type with k ~-~ 0.45 ' 106

M -1 • s -L Both these and other Fab fragments bind to a total of four sites on each c molecule with Kd ~ 0.01 ~zM in 0.05 M phosphate-0.05 M borate pH 8.

Smith et al. [37] have used the inhibitory action of Fab fragments to probe for reductase- and oxidase-specific binding sites on the native cytochrome c molecule. The "anti-oxidase" Fab fragment blocks interaction between c and aaa but not between c and succinate-c reductase, while the "anti-isoleucine 58" Fab fragment does not produce any inhibition. Salemme et al. [38] have pointed out that explanations of these results in terms of indirect effects are also possible.

11IC. The yeast peroxidase-cytochrome c complex The first functional complex between cytochrome c and a soluble enzyme protein

was the yeast peroxidase cytochrome c complex obtained by Mochan [39] working in Buffalo. Beetlestone [6], who demonstrated competitive inhibition by ferric cytochrome c in the reaction of Eqn (5), had put earlier investigators on the trail of this functional (ferrocytochrome c) complex. Although the result [6] was confirmed kinetically and the analogy with cytochrome c oxidase extended [40], the poor quality of the original yeast enzyme discouraged more physicochemical studies.

2 cytochrome c F e 2+ -~- H 2 0 2 cytochrome cperoxidas 2 ecytochromecFe 3+ + 2 0 H-(5)

The preparation of Yonetani and Ray [41] put an end to these difficulties. However, their initial kinetic results suggested that not only was their enzyme much purer and more active than that originally isolated [42] but that it also utilised a different catalytic mechanism [43]. In Cleland's terminology [44], it was a question of distinguishing an initially ordered "bi-uni-uni-uni-ping-pong" mechanism from an initially random similar mechanism (Eqn 6), that also included product inhibition.

(Yonetani and Ray [43]) (ordered) bi-uni-uni-uni-ping-pong

A B P B P

EA , EAB , F --- FB --- E 6(a)

(Nicholls and Mochan [45])

E

A B P B

- E A ~ EAB --- EXP --- F --- FB T\/I B EB A (random)

-- EP ~ E 6(b)

The latter mechanism (6b) contains stable cytochrome c peroxidase-cytochrome

0.08

O.OE

0,04

0 .02

0 . 0 0 r_ 4(~D

(a)

I 4,.~0 J 5~)0 J 5,~0 I J

A(n rn )

269

Fig. 3. Spectrum of the cytochrome c-cytochrome c peroxidase complex. (a) oxidized form (both species ferric). (b) reduced form (both species ferrous). From Fig. 4 of Mochan and Nicbolls [46].

C 2+ and cytochrome c peroxidase-cytochrome C 3+ complexes. We tried to show that Eqn 6 (b) was more consistent with the observations of product inhibition and could account for the kinetics of both the old (low-turnover) and new (high-turnover) enzymes [45]. The predicted complexes were detected both by the ultracentrifuge and by column chromatography [46], with dissociation constants not far from those predicted kinetically. Fig. 3 shows the spectra of the complexes isolated chromato- graphically. Although the overall reaction (Eqn 5) involves two molecules of cytochrome c, the complex contains only one such molecule, as shown both by the chro- matographic results (Fig. 4a) and from a titration using the decline in ascorbate reducibility of c in the complex [28] (Fig. 4b).

The optical spectra of both components are unaffected in the complex (Fig. 3), but more powerful direct methods have since confirmed the 1 : 1 cytochrome c peroxidase: cytochrome c complex formation; complexation modifies the NMR spectrum [47,48] and quenches the fluorescence of 8-anilino-l-naphthalene sulphonate (ANS)-apocytochrome c peroxidase or protoporphyrin-apocytochrome c peroxidase [49]. The latter results, together with those demonstrating apocytochrome c peroxidase-cytochrome c complexes by Sephadex chromatography [28], showed that the peroxidase haem group is not needed for complex formation.

Correspondingly, cytochrome c peroxidase containing dimethyl protohaem ester instead of protohaem, which is much less catalytically active (in terms of turnover) than native cytochrome c peroxidase, binds cytochrome c as tightly as native cytochrome c peroxidase both under catalytic conditions and during gel chromatography [50].

Horse radish peroxidase, whose kinetic behaviour led to the prediction [40] that no c-enzyme complex occurs, does not form a Sephadex-detectable complex [46].

270

I

2£

%6

0.8

0.4

B 0"I L

2 6

1.2

II 0

1 14 18 22 26 30 Fract ion no.

4 A

8,0

ZO E

6.0

5.0 E o

4~3 o ?.

3.0 .~_

2,0 EL

1.0

100

u3 - 6(

20 • & / / ' e -

l i i i i i i 1.1 .d i

4 8 12 16 30 /~M cy tochrome c perox idase

4 B

Fig. 4. Evidence for stoichiometric binding of cytochrome c to cytochrome c peroxidase. A. Elution profile from Sephadex G-75 gel. © ©, absorbance at 408 nm (total haem); © . . . . . C~, calculated c/enzyme ratio; Peak I, complex; Peak lI, free cytochrome c. From Fig. 3 of Mochan and Nicholls [46]. B. Suppression of ascorbate reducibility of cytochrome c by cytochrome c peroxidase addition. Rate of reduction of 1.5 #M cytochrome c by ascorbate in 5 mM phosphate pH 7 at varying cytochrome c peroxidase concentrations. From Fig. 4 of Mochan and Nicholls [28].

Serum albumin, with considerable negative charge at pH 6.25, gave evidence of only slight interaction [46]. The binding to cytochrome c peroxidase is thus rather specific (cf. Table II). Like the reaction with cytochrome c oxidase (below) it is sensitive to ionic strength (or cation concentrat ion) and blocked by polycations [50]. Guanidi- nated [51] cy tochrome c (retaining the charges) is catalytically active and binds to the enzyme, while acetylated [52] cytochrome c (which has lost the positive groups) is inactive and a poor ligand [50].

In a significant analogy with cy tochrome c oxidase [6,40], it was found that cy tochrome c peroxidase interacts almost equally well with the ferrous and ferric

271

TABLE II

RATE AND DISSOCIATION CONSTANTS FOR THE REACTION OF CYTOCHROME c WITH ENZYMES AND PROTEINS

Data in this table include information obtained from both static (Kd) and dynamic studies (Kra). However, the Kd values given for cytochrome aaa and for cytochrome c peroxidase are true dissociation constants and have been corrected for kinetic effects (e.g. of enzyme and reductant concentration).

Protein (enzyme) Ref. ko, kofr Kd* (M -1 's -l) (s -1) (~M)

(a) Fab fragment 0.8 )< 106 ? ~ 0.01 [36] (anti-oxidase)

(b) Cytochrome c 3 × 108+ ~ 2000? + 6.0-8.0 [43,45,49] peroxidase (yeast)

(c) Cytochrome b2 10a? (> 200?) + 0.01 ++ [54,55] (yeast)

(d) Cytochrome aa3 ~ 108+ ~ 800 + 8.0-15.0"* [40,47,61] (beef heart) [63,156]

* In 50 mM salt, pH 6.5-8.0; most systems show a direct proportionality between Kd and buffer concentration; 25-30 °C.

** Lipid dependent. + From turnover rates.

++ For static system (not equal to korf/kon).

forms o f cy toch rom e c. Both types o f complex are dissociated by increasing phosphate

buffer concentrat ion [45]. Fig. 5 shows that the Km (for cytochrome c Fe E+) and Ki (for cy tochrome c Fe 3÷) is almost directly proport ional to potassium phosphate concen-

tration f rom 5 mM to 300 raM, with a minimum value o f about 2.0 ~M (pH 7, 25 °C).

This compares with a minimum value of 0.5 ~M for the complex under non-catalytic conditions obtained by ascorbate reactivity changes [28]. Values obtained by the fuorescence method [49] agree closely with those obtained kinetically [45] (see

Table II). Nicholls and Mochan [53] suggested that the cytochrome c peroxidase - cyto-

chrome c complex, if crystallized, would prove the most convenient X-rayobject to study the cy tochrome c oxidation mechanism. So far the co-crystallization has not been achieved. The first such crystallized complex has turned out to be the one between

cy tochrome b 2 (also a yeast enzyme, L-lactate cytochrome c reductase) and cyto chrome c obtained by Baudras and his coworkers [54,55].

IIID. The cytochrome b2-cytochrome c complex

A very tight complex is formed between the haemoflavoprotein cy tochrome b2 and cy tochrome c, not only in solution but also in the crystalline state [54]. The ratio o f c to b 2 haem in the complex is 0.25 (one c per tetrahaem unit). The complex also appears in sucrose gradient ultracentrifigation, and is dissociated with decreasing pH or increasing ionic strength. Lysozyme does not compete

272

0 02 0.4 0.6 0.8 1D 1.2 [Cytochrome c2+]'l(#M "~)

o

~.5 }.o ~.5 T.o T.5 log [Buffer]

Fig. 5. Effect of buffer concentration on the kinetics of complex formation between cytochrome c and cytochrome ¢ peroxidase under catalytic conditions. (a) Lineweaver-Burk plots of oxidation rates in: © ©,0.01M;D [~,0.03 M; • •,0.1 M; O-- O,0.3 M; • . .•, 0.5 M phosphate buffer; 27 ~M H202 at 25 °C. (b) Secondary plots of: • - m , --log Km (pKm) for cytochrome c; • - - O , log V; • • , log V/Km against log [phosphate]. From Fig. 7 of Nicholls and Mochan [45].

for the binding site, and the deflavoenzyme still binds cytochrome c. A binding constant of 0.01 ~M was reported, which, if correct for 0.02 M buffer as stated, represents an affinity 200 times greater than that shown by cytochrome c peroxidase or normal aaa (below). As with these enzymes, however, both ferric and

ferrous c were bound. The initial cytochrome c reaction occurs with a rate of 10 a M -1 s -1 (implying

an off constant of ~-~ 1 s -1 in the absence of allosteric effects) [55]. The bound molecule can only accept one of the twelve electrons in the tetrahaeme-tetraflavin molecule of b2. Baudras et al. [55] postulate a form of negative co-operativity between subunits to account for the tight binding of only the one molecule of cytochrome c.

IIIE. The cytochrome aaa-cytochrome c complex Whether this complex really involves the protein or the lipid components of

the oxidase is uncertain. The original students of the complex, the reviewer in T. E. King's laboratory in Oregon [12,40], and Orii and Sekuzu in Okunuki 's laboratory in Osaka [56], preferred the idea of protein-protein interaction.

As with cytochrome c peroxidase, the original evidence for complex formation was kinetic; product inhibition by ferricytochrome c. Nicholls [40] presented a number of indirect arguments for the catalytic activity of the complex, but direct evidence for electron transfer within the isolated complex was slow to appear. The original c-aaa complex, prepared by ultracentrifugation [40] or by chromatography [56], had a relatively low turnover. Such complexes were prepared simply by mixing cytochromes c and aaa at low ionic strengths, and the postulated catalytic mechanism [10] required rapid dissociability of the product ferricytochrome c.

Subsequently, Kuboyama et al. [57] obtained a more stable complex by soni- cating the cytochrome c-oxidase mixtures. This stable complex appeared to have a

273

o~ ,~

,/i, ~ a~

0.0 4()0 L I 450 50O 55O 600 640

Wavelength (nm)

Fig. 6. Spectrum of the cytochrome c-cytochrome aa3 complex ( 'Kuboyama ' type). - . . . . I, oxidized form; - 2, dithionite reduced form. From Fig. 2 of Kuboyama et al. [57].

relatively low catalytic activity, yet sufficient for an effective reconstitution of a succinate oxidase system on addition to succinate-c reductase [58]. The spectrum of this, the most stable form of the c-aaa complex, is shown in Fig. 6. The complex could be rechromatographed in 25 mM salt without dissociation, and several hundred millimolar NaCI were required for partial splitting. Cholate, however, induced im- mediate fission. The cytochrome c environment is in some way abnormal in the sonicated complex because an appreciable proportion of the bound c reacts with carbon monoxide [59].

The complex obtained by mixing is different. Its formation is readily reversed by 50-100 mM salt [40]; electron transfer takes place between ferrocytochrome c and cytochrome a in the complex [40,60]; and the stoichiometry of formation appears to be consistently lower than in the sonicated complex, one molecule of c being bound per aaa molecule instead of two (i.e. 0.5 mole c/mole haem a rather than one) [28]. This complex is almost certainly catalytically active [61]. Kinetic observations indicate that both ferro- and ferricytochrome c react with isolated cytochrome aa3 The Ko is about 30 ~tM in 100 mM phosphate pH 7.4, declining to 1 EzM or less as the ionic strength tends to zero [61,62].

From a comparison of the kinetics of the Smith-Conrad assay and the ascorbate-tetramethyl-p-phenylenediamine (TMPD) assay systems, the reviewer [61,63] has calculated that the association and dissociation rate constants cannot be less than 108 M -1 's -1 and 500 s -1 respectively. Although the labile Orii-type c oxidase complex may therefore be kinetically competent, in Chance's terminology, as a catalytic intermediate, the reviewer admits that in some ways it is less interesting than the probably incompetent Kuboyama c-oxidase complex. This is because no striking new properties appeared in the former complex as isolated. Only more recently with spin labelling and fluorescence techniques are such new properties

274

observed. But the Kuboyama complex has a number of new properties: It is (a) more effective than the constituent cytochromes in restoring succinate oxidase activity [58]; (b) capable of an extra reaction with carbon monoxide [59]; (c) stable against gel chromatography and dilute salt solutions [57,59]; (d) possessed of a unique infrared spectrum [59]; and (e) responsive to uncouplers and possiblyADP in certain media [64].

From these properties the reviewer concludes that the Kuboyama complex must be vesicular or micellar in nature. Even in the absence of coupling behaviour the evidence indicates that cytochrome c in this preparation, compared to the usual kinetic complex, is relatively inaccessible. If the coupling characteristics are confirmed this would inevitable place this complex among the other reconstituted systems described below. Perhaps small micelles with the cytochrome c binding sites on aa3

facing inwards represent the simplest hypothesis. Complexes between cytochrome c and cytochrome c oxidase played a special

part in the theory of Yakushiji and Okunuki who supposed that cytochrome c was needed for the reaction with 02 as well as for the reduction of a~ +. Although this hypothesis was later proved incorrect, the idea led to some valuable work intended to identify the c-oxidase interaction chemically. Both modified c and modified oxidases were used. Acetylated and succinylated cytochromes c were inactive, while guanidinated c retained full activity [65], indicating the requirement for positively charged groups rather than lysine residues per se. Hettinger and Harbury [51 ] showed that in some systems the guanidinated protein had an even higher affinity for the oxidase than the native cytochrome.

Although catalytically inactive, the acetylated cytochrome c derivatives could still catalyse the reduction of cytochrome a by hydroquinone [65] and bind to the aa3 molecule land hence inhibit its activity [66] towards native cytochrome c). Similar activity was retained in trinitrophenylated cytochrome c although succinylated protein was completely inert [65]. Observations of this kind encouraged the idea that one of the lysines was also implicated in electron transfer [67], perhaps by interaction with the formyl group of haem a.

At least six lysines must be acetylated before catalytic activity is lost; acetylation of the first few lysines has little effect on spectrum or activity [52,68]. Lysine-22 of beef heart is apparently the most rapidly acetylated, and a positive charge here is not essential for activity. Introduction of a single trinitrophenyl group at lysine-13, which left the molecules as a whole cationic in character, increased the apparent Km at least three-fold while leaving turnover almost unaffected [69].

Position 13, unlike position 22, is occupied by a positive residue (lysine or arginine) in all species examined. However, doubly acetylated cytochrome c (acetyl groups on both lysine 13 and lysine 22) was more active than 1-trinitrophenyl- cytochrome c [52,68,69].

When adventitious inhibitors have been removed by electrodialysis, there are no significant differences in the Km values or turnovers for any of the animal cytochromes c reacting with beef cytochrome oxidase [70]. However, yeast cytochrome c may have a somewhat lower affinity for the mammalian oxidase [71 ]. This leaves the

275

invariant or conservative lysines-13,-27, -53 (-54 in yeast), -72, - 73 , -79 , -86 , -87 (-87, -88 in rattlesnake) and arginines-38 and-91 as possibly needed charges. Margoliash and others [71a] have recently reported that a large part of the methionine 80 side of the cytochrome c molecule is required only for the mitochondrial cytochrome c reductase activity and not for reactivity with the oxidase. Modification of Tyr 74 (iodination), Trp 59 (formylation), Tyr 67 (nitration) and even Met 80 itself(carbox- ymethylation) left appreciable reaction rates with the oxidase. Derivitization of lysine 13, on the other hand, sharply reduced activity with the oxidase without affecting the capacity to accept electrons. Binding constants for these modified cytochromes ought to be examined in more detail.

The binding site on the oxidase protein, if indeed it is on the protein, is un- known. The measured affinity for cytochrome c is normally unaffected by the redox state of the enzyme and by complex formation with terminal inhibitors [61]. Van Buuren and his colleagues have suggested otherwise for the inhibitions by cholate [72] and fluoride [73]. Both agents appeared to decrease the affinity for cytochrome c as well as the turnover (increasing Km by ~ 5 0 ~ in the case of cholate and ,~250 ~/o in the case of fluoride). However, as these inhibitors are capable of multiple interactions with the oxidase, it is not necessary to postulate site-site interactionto explain these data.

If complex formation involves protein-protein interaction [40,67], aspartate or glutamate residues on one of the aa3 peptides must interact with c. King [74] has found that his complex can be formed with "lipid-free" ( < 2 ~ lipid) oxidase (see also ref. 75), yet such preparations usually contain at least 2 moles of tightly bound cardiolipin per mole aa3 [76]*. The catalytic affinity of the enzyme for cytochrome c can also be modified by addition of different lipids to depleted systems. Zahler and Fleischer [77] found an increase in Km on addition of lipid (together with an increase in turnover) but Chuang and Crane[75], rather more reasonably, obtained decreases in Km together with increases in turnover. Thus, excess phospholipids decreased Km from 12 ~tM to 2.5 ~zM while increasing V five-fold (in 16 mM phosphate 10 mM citrate buffer pH 6.5) [75]. Emasol-1130 also decreased the Km, indicating that added lipid was not contributing the combining site, but making that site more accessible (see Table V). The site itself could still be either protein or the tightly bound cardiolipin.

Table II summarizes some of the rate constants and binding constants obtained for cytochrome c and four of the proteins discussed. Because these values are very sensitive to ionic strength and pH, comparisons should be made cautiously.

Cytochrome c can also engage in electron-transfer reactions without detectable stable intermediate complexes. Thus, electron transfer occurs at a rate of 1.5" 104 M - 1 " s -1 between horse cytochrome c and Pseudomonas c- 551 in a reaction independent of ionic strength, under conditions where no complex formation could be detected by ultracentrifugation [78]. Cytochrome c~ reacts with cytochrome c in a bimolecular reaction [79] with a rate of between 3 and 4 • l0 T M -1 - s -1 at low ionic strength**.

* King (personal communication) reports -< 0.1 ~ lipid in his enzyme, i.e. no tightly bound cardi lipin.

** The original paper [79]gives a rate ten times smaller than this, apparently due to a misprint.

276

Although no kinetic evidence for complex formation in this reaction was found, the marked dependence on ionic strength suggests that such a complex may be involved as an intermediate. Kaminsky et al. [79a] have recently obtained evidence for a cytochrome cl-c complex by fluorescence probe techniques. Azurin, the copper protein from Pseudomonasfluorescens, also reacts with cytochrome c in an apparently bimolecular fashion [80]. A reaction between oxymyoglobin and ferricytochrome c was also found to take place without detectable complex formation [80a].

IV. CYTOCHROME c AND NON-SPECIFIC MEMBRANES

It is difficult to identify the mitochondrial binding site because cytochrome c can bind to negatively charged phospholipids as well as to proteins; either cardiolipin or cytochrome oxidase peptide could therefore be involved.

IVA. Formation of isooctane-soluble complexes ("lipo-cytochrome c") Certain mixed mitochondrial phospholipid-cytochrome c complexes, soluble

in isooctane, are active in reconstituting succinate and NADH oxidase activity in preparations treated with bile salts [ 14, 15]. Of the major mitochondrial lipids, cardiolipin and phosphatidylinositol by themselves formed complexes (10 moles lipid P/mole c) that were isooctane insoluble, while lecithin formed no complex [8l]. Only phosphatidylethanolamine among the pure phospholipids formed an isooctane soluble complex similar to that formed by the mixed lipids (despite the absence of effective charge?) [81,82]. Both phosphatidylcholine and phosphatidylethanolamine increased the isooctane solubility of the acidic lipid-c complexes, giving mixed complexes with 20-25 btmoles lipid P/~tmole cytochrome c, of which 10 ~tmoles P remained acidic [81] (see Table III).

If cytochrome oxidase is depleted of lipid, lipid-containing cytochrome c preparations, but not soluble cytochrome c preparations, will restore the activity [83 ]. Alternatively, one can activate the enzyme with phospholipids and then use free cytochrome c. Km values are consistently lower (~-~1.5 ~M compared to 5.0 ~M) and maximal turnover about the same (600-800 s -1 at pH 6, 37 °C) when free c and normal (or lipid-repleted) enzyme are used [83] (see Table V below). Although Chuang et al. [84] suggest that lipid is needed for both the binding of c and the turnover of the enzyme, the reviewer finds the "dual" role unproven. Ivanetich et al. [85,86] have reported that a complex formed with 4 ~moles cardiolipin, 15 ~moles phosphatidylethanolamine and 22 ~moles phosphatidylcholine per ~mole cytochrome c is en- zymically active in the ferric form with N A D H - e reductase and in the ferrous form with cytochrome c oxidase, even at pH 7 where the rate of exchange between complex and free c was very low. In contrast to most other findings, at pH values below 6 the ferrous complex was more stable to exchange than was the ferric complex.

The major problem in studies of "lipo-cytochrome c" at high ratios of protein to lipid is the uncertain structure of the complex. X-ray scattering data [87] indicate

277

_ _ _ Q . • •

O

-~ 2 ~

1 \

\, 0 ~ I I I ~ I

10 -5 10 -4 10 -3 10 -2 10 -1 1 Ionic s t rength ( M ) - - - ~

Fig. 7. Effect of ionic strength on cytochrome c binding to phospbatidylinositol monolayers and bilayers. Bound cytochrome c monitored at 410 n m . • 0, bilayer binding against varying [KCI]; . . . . . monolayer binding against varying [KCI]. From Fig. 8 of Steinemann and L~iuger [90].

that the phosphatidylcholine/phosphatidylserine/cytochrome c complex in isooctane has a 'molecular weight' of about 1.3" l06, with a phospholipid: protein ratio of 29 : 1. This is a large aggregate but smaller than a sonicated liposome (see below). Studies with cytochrome c and preformed membrane systems have helped to clarify the problem of structure.

IVB. Interaction with phospholipid monolayers and bilayers Cytochrome c in the subphase both binds electrostatically and penetrates

hydrophobically into a monolayer of phospholipid spread in a Langmuir trough [88,89]. Expulsion of bound c from phosphatidylethanolamine films required com- pression to 40 dynes/cm plus the presence of up to 1 M NaC1 in the subphase [88]. An initial electrostatic binding was postulated, followed by a less readily reversible hydrophobic bonding. When negative charges were present (cardiolipin or phosphatidic acid) binding at high pressures ( ~ 4 0 dynes/cm) was facilitated. Above a certain pressure pure electrostatic binding occurred without appreciable penetration [89].

Using monolayers spread on glass plates, as well as bimolecular (black) membranes, Steinemann and L~iuger [90] found that cytochrome e molecules could be bound to phosphatidylinositol at a concentration approximating hexagonal close packing, without appreciable spectral changes in either ferrous or ferric form. Bound e Fe 3+ was ascorbate-reducible, although the product c Fe z+ appeared to be more autoxidizable than ferrocytochrome c in solution. As shown in Fig. 7, binding to a bilayer appeared to be more sharply salt-sensitive than binding to the monolayer.

Gulik-Krzywicki et al. [91] and Letellier and Schechter [92] have reported

278

Fig. 8. Letellier-Shechter model of cytochrome c binding to cardiolipin lamellae. Densely hatched heart shaped areas represent protein molecules. (e) electrostatic lamella~ phase (proposed for ferricytochrome c); (1) lamellar phase without protein; (h) hydrophobic lamellar phase (proposed for ferrocytochrome e), note decrease in thickness oflamella. From Fig. 2 of Letellier and Shechter [92].

structural changes in both protein and membrane when lamellar complexes of cytochrome c and phosphatidylinositol [91] or cardiolipin [92] are examined by X-ray diffraction or by fluorescence and circular dichroism. Under their conditions, ferricytochrome c comes to resemble one of Theorell's acid forms, in which the haem crevice is opened. Under other conditions (below) the native structure is retained, and the reviewer wonders whether the effects seen are not a secondary consequence of the very acid environment of the phospholipid head groups at the high negative lipid concentrations employed. Such differences are not seen with ferrocytochrome c, and it is proposed [91,92] that ferrocytochrome binding is hydrophobic while that of the ferri-form is electrostatic. The hydrophobic ferrous complex, like that of Ivanetich et al. [86] under acid conditions, is not readily dissociated by salt.

Fig. 8 shows the picture of cytochrome c binding to the lamellae in the ferric and ferrous states, as developed by the French workers [92]. The idea that cytochrome c penetrates the membrane, even in the ferric form, as well as binding electrostatically, is supported by the discovery of Kimelberg and Papahadjopoulos [93,94] that the promotion of ionic permeability in phospholipid vesicles [93] by cationic proteins is correlated with their ability to "penetrate" monolayers [94].

IVC. Interaction with liposomes The introduction of the aqueous phospholipid dispersion [95], liposomes, as a

means of trapping and binding proteins and other ions in solution altered the outlook of those studying interactions between cytochrome c and lipids [96]. Cytochrome c can be readily trapped within the multilamellar liposomes formed by gently dis-

279

Fig. 9. Electron micrograph of20~ cardiolipin-80~ egg lecithin liposomes in cross section, in the absence of cytochrome c. Uranyl acetate/lead citrate staining, x 173 000. From Fig. 5 of Kimel- berg et al. [97].

persing negatively charged phospholipids in aqueous medium containing cytochrome c [97]. Fig. 9 shows an electron micrograph cross section of the pure phospholipid liposome, and Fig. 10 the corresponding electron micrograph of the liposome containing cytochrome c. The electrostatic binding of the protein has increased the interlamellar spacing; possibly other changes have taken place at the molecular level similar to those envisaged in Fig. 8.

Both ferrous and ferric cytochrome c can be bound to negatively charged liposomes, either internally or on the external surface. The binding is vulnerable to increases in ionic strength [98]. Externally bound c, but not internally trapped c, can be reduced by dithionite or ascorbate [98,99]. The latter reagent does not react as fast as with cytochrome c in solution [100]. Both internally [98] and externally [100], ferric cytochrome c is bound more tightly than the ferrous protein. The ferrous protein is also less effective in reducing the electrophoretic mobility of nega-

280

Fig. 10. Electron micrograph of 2 0 ~ cardiolipin-80~ egg lecithin liposomes in cross section, prepared in the presence ofcytochrome c (0.14 ~mole cytochrome c~ 10 mg lipid). As Fig. 9, x 173 000. From Fig. 7 of Kimelberg et al. [97].

281

tively charged liposomes (Miller, N. and Nicholls, P., unpublished), and in modifying the fluorescence of phospholipid-bound 8-anilino-l-naphthalene sulphonic acid and 12(9-anthroyl) stearic acid [101,102]. Kimelberg and Papahadjopoulos [93] do not seem to have examined the two reduction states for differential effects on cation permeability; the dramatic effects they observed were all obtained with the ferric

protein. When located externally, phosphatidic acid seems to be the most effective

binding site for cytochrome c, only 4--5 molecules being needed to complex one c molecule [100]. With other negatively charged lipids (cardiolipin, phosphatidylserine etc.) about 10 charges are required to trap one molecule of c internally or bind it externally [93,97,99]. This number is similar to that found for the isooctane-soluble lipid-cytochrome c complexes [81] (see Table III). The latter probably represent "micellar" forms while the liposome complexes retain their normal lamellar con- formation.

Cytochrome c binding to cardiolipin-containing liposomes can be monitored by the fluorescence quenching of 12 (9-anthroyl) stearic acid retained amphipathically in the membrane [102]. Quenching is reversed by KC1 or alkaline pH, and the rate of quenching is markedly slowed in presence of Mg 2+. The binding appears to have a fast phase (k >/ 10 v M -a "s -a) and a slow phase (k ~ 0.05 s-l) ; single-site binding followed by aggregation, perhaps [100]. 5 ~ 12 (9-anthroyl) stearic acid in cardiolipin vesicles could be quenched by 1 mole e/80-100 moles cardiolipin. Full quenching may require only about 10)/o occupation of the available e binding sites.

In addition to promoting permeability changes [93] in the vesicles, cytochrome c has been reported to induce significant phase changes in the lipid hydrocarbon interior [91] and to modify the transition temperature of the membrane to which it is complexed [103,104]. The latter may be a local effect, as binding of cytochrome c to an 11 ~ phosphatidic acid system ( ~ 1 ~ molar binding of c) did not affect the transition temperature of the major fraction of lipid [100].

The effect of cytochrome c on cation permeability is inhibited by the addition of cholesterol to the membrane [104]. Concomitantly, in accord with the theory of Kimelberg and Papahadjopoulos, cholesterol blocks the monolayer expansion induced by cytochrome c [104].

IVD. Interaction with a photosynthetic reaction centre The only natural membrane system, other than the mitochondrion, which

reacts rapidly with mammalian cytochrome c, seems to be the photosynthetic reaction centre of Rhodopseudomonas spheroides [105]. Ke et al. found that mammalian cytochrome c was an effective electron donor to photooxidized P870. In order to function in this way, an electrostatic complex was formed between a ferrocytochrome e molecule and a reaction centre complex containing P870. Although such complexes could bind ~ 2 4 molecules of cytochrome e, only one (or at the most two) of these molecules could act as a rapid electron donor. The cytochrome c-reaction centre complex was dissociated by salts and polycations were strongly inhibitory. The

282

TABLE III

CYTOCHROME c BINDING BY LIPIDS AND ARTIFICIAL MEMBRANE PREPARATIONS

Lipid or vesicle composition Conditions Cytochrome c stoichiometry Ref. nmoles/nmole nmoles/nmole " lipid negative charge

1. 20 ~ cardiolipin 15 mM KCI 0.03 0.15 [97,99 ] 80~ lecithin* (pH ~ 8.0) (K~ ~ 200 ~zM)

2. 25 ~ phosphatidic acid 4 mM KC1 0.05 0.2 [100] 75 ~ lecithin** (pH 6.8) (Kd ~ 1 ~tM)

3. Mixed mitochondrial 30~/o ethanol 0.04 0.1 [81] lipids (40% acidic)*** 33 mM Tris-HCl

(pH 7.0) 4. Cardiolipin*** (pH 7.0) 0.2 0.1 [81] 5. Phosphatidylinositol*** (pH 7.0) 0. l 0.1 [81 ] 6. 10~o cardiolipin

55 % lecithin 35 % pH 5 0.025 0.25 [85,86] phosphatidylethanolamine +

7. 67% lecithin 10 mM phosphate 0.03 0.1 [87] 33 % phosphatidylserine*** (pH 7.0)

8. Phosphatidylserine** 10 mM KC1 0.12 0.12 [93] (pH 7.4) (Kd ~ 25 EzM)

* Binding (internal and external) to liposomes as formed. ** Binding (external) to preformed vesicles.

*** Complex between cytochrome c and lipids extractable into isooctane. + Binding to preformed vesicles followed by precipitation and deoxycholate solubilization.

e lect ron t ransfer rate was ~ 27 000 s -~, the highest such rate measured in a cyto-

ch rome c complex (see Section VID). The second-order rate of complex fo rmat ion

was found to be 3.8 • l0 s M -1 • s -1 by Prince et al. [106]. The la t ter workers also

found tha t the co r respond ing react ion with Rhodopseudomonas spheroides cy tochrome

c2, an acidic protein, was s t imula ted by polylysine over the same concent ra t ion range

tha t b locked the reac t ion with m a m m a l i a n c.

Table I I I summar izes the observat ions repor ted on cy tochrome c b inding by

artificial membranes and phospho l ip id systems.

V. CYTOCHROME c AND THE INNER MITOCHONDRIAL MEMBRANE

Binding o f cy tochrome c to mi tochondr i a or submi tochondr i a l part icles can be

fo l lowed in two ways: (i) by moni to r ing b inding directly, and (ii) by moni to r ing

increases in resp i ra t ion rate. The first me thod fails to dis t inguish cata lyt ic and

non-ca ta ly t ic sites; the second me thod in t roduces kinetic compl ica t ions tha t have no

s imple and universal ly accepted mode o f resolut ion. Some workers have ignored these

p rob lems ; bu t they lie a t the hear t of the d isagreements in the field. Fig. 11 compares

results ob ta ined with non-phosphory la t i ng submi tochondr ia l part icles by the two

methods [107]. Both methods indicate a heterogenei ty in b inding sites,with the first

283

320C

280

2401

200C

1600 .,E

~ 1200

8OO

40o, |

-20 -10 0 I I I I I I I / I I

lO 20 30 40 50 60 7o 8o 90 lOO

lO-,/[c? l l A

U / / / /

&O / / / / /

,o. / . . ' i / ~ o

O r 0 lm(IOWC)1'0 ,20~ 310 4() Kin(high c)

[c],UM

l l B

Fig. 11. Cytochrome c binding to submitochondrial particles (Keilin-Hartree type). A. Effect of cytochrome c concentration on spectrophotometric assay of cytochrome c oxidase activity. Line- weaver-Burk plot for reaction in pH 7.4, 0.02 M phosphate. From Fig. 4 of Nicholls [107]. B. Direct- binding profiles for cytochrome c and submitochondrial particles. Ratio of [c]/[haem a] in complex plotted against [c] concentration in pH 7.4, 0.02 M phosphate. • . . . . . • , normal Keilin-Hartree particles; • O, normal Keilin-Hartree particles subtracting endogenous c concentration; O ©, cytochrome c deficient (Tsou) particles. From Fig. 5 of Nicholls [107].

group o f sites binding quite tightly (Kd ~< 2 EzM at 20 m M phosphate) and the second group binding much more loose ly (Kd ~> 20 ~ M in 20 m M phosphate) . In addition, the results with the catalytic system indicate that the turnover is higher with the loose binding sites. Hence the Lineweaver-Burk plots tend to be concave downwards .

284

Neither type of heterogeneity has so far been reported with isolated cytochrome oxidase preparations (see Section IIIE).

VA. Topology of cytochrome c binding The original submitochondrial particles [108] contained cytochrome c in a

form that was not readily dissociable; apparently some kind of lipid-linked species. This was the more unexpected because cytochrome c in the initial minced muscle could be extracted by salt solutions [13]. Development of the ideas (i) that most submitochondrial particles have an inverted configuration with respect to the original mitochondria (see e.g. refs 109, 110), and (ii) that cytochrome c trapped inside liposomes shows similar features [98], allowed an interpretation of the observations in terms of a normal localisation of cytochrome c on the outside of the inner mitochondrial membrane. A group of papers presented at the 1969 Johnson Foundation symposium gave almost identical pictures of the relative position of cytochrome c with respect to the other cytochromes in the mitochondrion (cf. Fig. 1 of Racker et al. [111 ], Fig. 5 of Lee [112], and Scheme 2 of Nicholls et al. [113]).

The assigned location of cytochrome c was supported by a number of experimental observations, including the following:

(a) Cytochrome c is synthesized outside the mitochondrion [114] and the inner membrane is cytochrome c impermeable; unfortunately for this simple argu- ment, the outer membrane of the mitochondrion is also cytochrome c impermeable [115].

(b) Antibodies against cytochrome c were effective inhibitors of mitochondrial respiration but not of submitochondrial particles unless trapped internally Llll]; unfortunately the analogous and very effective competitive inhibitors, the polylysines, gave more ambiguous results [113,11 l, 116 ].

(c) Cytochrome c-depleted mitochondria show Km values for cytochrome c in the restoration of respiration that are sensitive to external ion concentration and are unaffected by valinomycin (expected to modify internal cation concentration) [113,117].

(d) Reduction by ascorbate (± phenazine methosulphate) and by succinate indicated external binding in mitochondria and internal binding in submitochondrial particles [112].

(e) Cytochrome c in yeast is accessible not only to cytochrome oxidase but also to cytochrome c peroxidase [118], which appears to be a loosely bound enzyme probably located in the intermembrane space [119].

(f) Cytochrome c oxidation by mitochondria free of outer membrane is rapid and unaffected by the non-ionic detergent Lubrol while submitochondrial vesicles prepared by mechanical fragmentation and showing the 90 A particles (ATPase) externally, oxidize cytochrome c only in the presence of Lubrol [120].

However, the surprising unanimity that appeared to prevail in 1969 was due not only to the empirical evidence but to theoretical predilections resulting from the growing ascendancy of the chemiosmotic theory of respiratory chain phosphory-

285

2.O " 0 n

I

m 0 t t 2.0 ~ ~" %)

~E :::t

A

!.C ' ~ ~ •

, ,

1.0

0 , . ' / . .O

\ + 6 0 m M KCI

0 .5 # g va l i nomyc in

t t t / / _ L - _

A D P 5 7 4 # M

. - :~

~+ 60raM KCI

I I I ,, /.A..-- 2.0 67

I Z M C 3÷

Fig. 12. Effect of valinomycin and KCI on cytochrome c binding to cytochrome c-deficient mito chondria. Respiration rate with succinate plus glutamate plotted against added cytochrome c concentration in the presence of valinomycin (upper traces), O O, mannitol-suerose-Tris-phosphate medium; • 0, mannitol-sucrose-Tris-phosphate medium + 60 mM KCI; and in the presence of ADP (lower traces); • ~, mannitol-sucrose-Tris-phosphate medium; • A, mannitol-sucrose-Tris-phosphate medium + 60 mM KCI. (a ÷ aa) indicates amount of mitochondrial haem a present (2 × [aa3] concentration). From Fig. 2 of Nicholls et al. [117].

iation. Within a year of the Johnson Foundation meeting, an attempt was made [118 ] to construct a compromise proposal with separate respiratory chains containing all

the cytochromes, including c, on both sides of the inner membrane. The ambiguity of the topological data often permitted interpretation in terms of heterogeneous

populations. On the other hand, the reviewer feels, and felt, that the results [117] with c-depleted mitochondria in presence of permeant and impermeant cation systems (Fig. 12) were difficult to explain away. Although the suggestion has been made,

both by the author and by others, that cytochrome c may be able to react and be oxidized on both sides of the inner mitochondrial membrane, satisfactory evidence for differences in rates or binding constants has not been obtained which can be reliably linked to such topological differences. In the ensuing discussion I shall ignore these possibilities and treat cytochrome c binding as analogous in all types of sub-mitochondrial particle.

The definitive experiment linking cytochrome c location with the postulates of the chemiosmotictheory was carried out by Hinkle et al. [121], who showed that it was possible to incorporate cytochrome aa3 (mammalian cytochrome c oxidase)

286

into an artificial membrane system that was uncoupler- and valinomycin-sensitive provided that cytochrome c and reductant were added on one side of the liposome membrane only. These observations, confirmed by Jasaitis et al. [122] and by Hunter and Capaldi [123] (albeit with slightly modified interpretations in the latter case) leave little doubt concerning the physiological importance of binding c to one side of the membrane. Although reconstituted vesicles can apparently oxidize cytochrome c added to either side, when c is present on both sides such vesicles (unlike mitochondria and submitochondrial particles) are uncoupled [124]. A similar asymmetry across a black membrane can be induced by fusing cytochrome oxidase vesicles to one side and then adding cytochrome c to that side [125].

VB. Specificity of cytochrome c binding One site in mitochondria or submitochondrial particles is quite specific for

cytochrome c. Keilin and Hartree [108] found that the trapped (endogenous) c of the submitochondrial particle is much more active in the oxidation of succinate than added (exogenous) e. Tsou [13] showed that exogenous c nevertheless became as active as endogenous c when pre-added so that it was incorporated during the preparation of the submitochondrial particles. Estabrook [126,127] found that the apparent "inactivity" of exogenous c was largely due to a very marked effect of buffer-cation concentration on the Km for added c in the succinate oxidase system. At zero ionic strength exogenous c had the same activity as endogenous c; the effect of cations appeared to be exerted at the cytochrome c-cytochrome c oxidase step.

The specific binding site involved is present at a concentration equal to or slightly greater than the amount of cytochrome a3 in submitochondrial particles [107,128]. As with the binding by proteins (Section llI) and non-specific membranes (Section IV) this binding is largely electrostatic in nature. Guanidinated cytochrome c proved as good a ligand and catalyst as normal cytochrome c, both in a rat liver succinate oxidase system and a beef heart ascorbate-aa3 system (Fig. 13) [51]. Fig. 13 shows that in the former case the modified c binds even more effectively than the normal e. As with the isolated cytochrome aa3 system, there is no species specificity in cytochrome c binding [70]. In Keilin-Hartree submitochondrial particles there is some evidence that the specific binding sites can be titrated preferentially by non-specific inhibitors such as the high molecular weight polylysines [116].

The specific cytochrome c-binding site is located internally in submitochondrial particles, although such particles are often leaky and slowly lose their cytochrome c to salt solution [129]. But there are also binding sites elsewhere on such particles, including the outside [112,t13]. This less-specifically bound c may also engage in reaction with the particulate oxidase system, although apparently not with the cytochrome c reductase complexes (See comments in Section VA).

Direct binding studies (cf. Fig. 11 B) have identified both types of site in intact mitochondria, either from rat liver [130] or from pigeon heart [102,131]. Williams and Thorp [130] found 3-5 nmoles of available c binding sites/mg protein, with an apparent Kd of 0.4 ~xM in a 0.25 M salt-free sucrose medium. The sites seemed to

287

7

~5

~4

c &2

'~IC

~6

0 2

0

Cytochrome c (#M) Cytochrome c (#M) Ca) (b)

Fig. 13. Binding of guanidinated cytochrome c by rat liver mitochondria and beef heart submitochondrial particles. (a) Effect of cytochrome c on succinate (50 mM) oxidation by cytochrome c-deficient rat liver mince in 0.033 M phosphate pH 7.4. (b) Effect of cytochrome c on ascorbate (33 mM) oxidation by Keilin-Hartree particles in 0.05 M phosphate pH 7.0, 37 °C. © ©, normal cytochrome c; ×, 57 % guanidinated cytochrome c; • • , 100% guanidinated cytochrome c. From Fig. 1 of Hettinger and Harbury [51].

involve both protein and phospholipid. Cytochrome c binding was unaffected by the respiratory or energy state of the mitochondria. These authors did not remove endogenous cytochrome c (0.34).4 nmoles/mg) nor the outer membrane. Swelling, or phospholipase D treatment, which presumably increased inner membrane accessibility, increased the number of binding sites to about 10 nmoles/mg protein without markedly affecting K~ (phospholipase A dramatically reduced the number of binding sites). Vanderkooi et al. [102,131] have identified 3 nmoles c binding sites/mg protein in cytochrome c-depleted pigeon heart mitochondria (initial cytochrome c concentration 0.5-0.6 nmoles/mg). The majority of these sites (low affinity) have a Kd of 0.3 ~M for oxidized horse heart c but 2.0 ~tM for the reduced cytochrome. The sites do not distinguish between oxidized and reduced yeast cytochrome c (Kd ,~ 0.4 ~zM). Of the 3 nmoles sites/mg, approx. 0.5 nmoles constitute tight binding sites (Kd ~ 0.03 ~M in 0.225 M mannitol, 0.05 M sucrose, 0.04 M MOPS buffer pH 7.2), presumably the specific c-binding sites. Whether these latter sites distinguish between ferrous and ferric c is not discussed by the authors (but see below).

Similar results have been obtained with beef heart mitochondria and submitochondrial particles [112], as well as with non-phosphorylating beef heart particles (Fig. 11) [107,132] and with a reconstituted NADH oxidase system [133]. Before our present ideas of mitochondrial topology were elaborated, Machinist et al. [134] put forward the idea of two populations of mitochondrial c-bonding sites, later developed by the reviewer (see below) to explain the kinetics of succinate oxidation [107]. Table IV lists the stoichiometries (nmoles c/mg protein) and apparent Ko values (dissociation constants) for the two types of binding site in the various mitochondrial and submitochondrial preparations.

288

TABLE IV

BINDING OF FERRICYTOCHROME c BY THE M1TOCHONDRIAL MEMBRANE

Preparation Apparent Kd (~tM) Stoichiometry Ref. (nmoles/mg protein)

(a) Whole rat liver mitochondria* 0.4 1.5-5.0 [130] (b) Cytochrome c-depleted ~ 0.03 0.5

pigeon heart 0.3 2.5 ( [131 ] mitochondria** (2.5 for Fe 2+ c) (~ 3.0 total) )

(c) Beef heart 2.0 0.4 ) [107] submitochondrial ~ 2 0 > 3.0 particles*** 25 17 [l 32]

(d) Cytochrome c-depleted 2.0 0.8 ) beef heart submitochondrial particles*** ~ 2 0 ~-2.0 t [107]

(e) N A D H oxidase - - 11 [133] system (submitochondrial particles) +

* 0.2l M sucrose. ** 0.22 M mannitol, 0.05 M sucrose, 0.04 M morpholinopropane sulphonate buffer, pH 7.2.

*** 0.02 M potassium phosphate pH 7.4. + 0.66 M sucrose, 0.05 M Tris pH 8.0.

VC. Kinetic aspects of cytochrome c binding The cytochrome c oxidase system. T h e first k ine t i c p a r a d o x o f c y t o c h r o m e c

b i n d i n g was t he o n e p o s e d by S m i t h a n d C o n r a d [7] w h o c o n t r a s t e d t he f irst o r d e r

b e h a v i o u r o b s e r v e d w i th t i m e (Fig . 14) a n d t he ze ro o r d e r b e h a v i o u r o b s e r v e d w i t h

1.0

O,'

t,l

oO.O"

. c

E

0 , 0 ~ I I I I I I I I I I L I I I

T i m e - 1 div, = 11,5 se¢

Fig. 14. Smith-Conrad kinetics of cytochrome c oxidation by submitochondrial particles. Semi- logarithmic plots of ferrocytochrome c concentration against time after addition of Keilin-Hartree particles to the indicated cytochrome c solutions in 67 mM phosphate buffer pH 7.4 at 25 °C. Spectro- photometry at 415 nm (A), 550 nm (B, C, D, E) and 520 nm (F.). From Fig. 2 of Smith and Conrad [7].

289

cytochrome c concentration above a certain level (Fig. 1). This paradox was essentially resolved in the "mechanism IV" of Minnaert [10], given in Eqn 7:

C2 + + aa +Km c2 + a3 + (7a)

c2+aa kac3 ÷ a2 ÷ 0 2 c3 +a3 + (7b)

fast

c3 + a3 + K~ e3 + + a3 + (7c)

The apparently conflicting ideas of inhibitory product complexes (c 3+ a 3+) and active complexes with bound c playing an analogous role to that of endogenous c [135] were adequately reconciled [62].

However, a second kinetic paradox was promptly posed. Eqn 7 gives rise to the kinetic expression:

k3[c 2+ ] [e] v = (8)

K m + [c 2+ ] + (Km/Kp) [c 3+]

The denominator is independent of the reduction state of e, and the expression therefore always first order in c with time, when K m = Kp. That is, time courses such as those in Fig. 14 can only be obtained when the affinity of the binding site for the two redox forms of cytochrome c is the same. Time courses of this kind are in fact obtained under almost all conditions: (a) varying the buffer composition of the medium [70]; (b) varying the cation concentration and type [136,137]; (c) varying the type of enzyme preparation used [138]. Only by moving to alkaline conditions were Yonetani and Ray [137] able to obtain time courses that curved up in logarithmic form (Kp < Km). This could be attributed to a tighter binding of the alkaline form of cytochrome e.

However, direct binding studies [102,131] gave Kd(C 2+) ~ Kd(C3+), an interpretation supported by redox potential measurements which gave Em (bound c) < E m (free c) [131,139]. Thermodynamic balance requires that if the ferric form is bound more tightly, then the redox potential must be less positive, and vice versa [102,129]. Binding at the specific sites (0.5 nmole/mg, Table IV)maynot therefore distinguish the two redox forms, although binding at the non-specific sites (2.5-10 nmoles/mg, Table IV) undoubtedly does. Alternatively, mechanism IV (Eqn 7) may be incorrect. But so far no satisfactory alternative has been proposed [61].

The idea that only non-specific sites discriminate cannot account for the redox observations on endogenous cytochrome ¢ of normal mitochondria and submitochondrial particles [139]. This problem is discussed below (Section V|).

What are the actual values of Km (and/or Kp) obtained from the mechanism of Eqn 8? Table V lists the values obtained by various workers; Table II gives estimates of rate constants for the on and off steps. These kinetic constants can be compared with binding constants obtained with the isolated oxidase and other

290

TABLE V

Km AND K~ VALUES FOR CYTOCHROME c BINDING TO AND OXIDATION BY VARIOUS CYTOCHROME c OXIDASE (aa3) PREPARATIONS

All data in this table are kinetic in origin, but have been checked and corrected where necessary for the effects of enzyme concentration etc. (cf. Table II). If the kinetics are of the Minnaert [10] variety, then both Km and K~ values represent true binding (dissociation) constants.

Conditions Km (c Fe 2+) Ki (c Fe 3+) Preparation p H - - Tempe--rature Buffer TN* (~tM) (~M)

(°C) (mM) (s -~)

Ref.

SMP a 7.4 25 67 - - ~ 20 ~ 20 [7] SMP 7.4 25 65 (200) 12 12 [10] aa3(SS) ~ 7.4 25 67 - - 8 8 [7] aa3(Y) a 7.0 25 45 150 8.3 7.9 [137] aa3(F) a 6.0 25 65 500 ~ 8 ~ 8 [73 ] aa3(F) 7.4 25 65 b 240 30 - - [62] aa3(Y) 7.0 25 50 g 180 2.6 - - [63 ] aaa(F) 6.0 37 11 c 800 ~ 1.5 - - [83] aas(FLD) a 6.0 37 l l e 80 2.5 - - [83] aaa(FLD) 6.0 37 I 1 ~ 800 5.0 - - [83 ] aaa(F) 6.5 37 16 e 725 2.5 - - [75] aa3(FLD) 6.5 37 16 e 100 10.0 - - [75] aaa(FLD) 6.5 37 16 t 550 4.0 - - [75]

a S M P = Keilin-Hartree type submitochondrial particles; SS - Smith-Stotz preparation [7]; Y = Yonetani preparation [137]; F = Fowler preparation [62]; F L D - Lipid- depleted preparation [83].

b ascorbate assay, in presence of 30 mM ascorbate. c ascorbate-TMPD assay, in presence of 6 mM citrate, 8.33 mM ascorbate, 1.1 mM TMPD. d plus added mitochondrial phospholipids and 'lipo-cytochrome c'. e ascorbate-TMPD assay, in presence of 10 mM citrate, 13 mM ascorbate, 1.1 mM TMPD. f plus Emasol-ll30 (5 mg/mg protein). g ascorbate-TMPD assay, in presence of 10 mM ascorbate, 1.0 mM TMPD. * TN = turnover in electrons/second/cytochrome aaa.

pro te ins (Tab le II) a n d w i t h the m i t o c h o n d r i a l m e m b r a n e i tse l f (Tab le IV). T h e

v a r i a t i o n in Km wi th ion ic s t r eng th is c o m p a r e d wi th the re la ted v a r i a t i o n in Kso fo r

r e c o n s t i t u t i o n o f succ ina te ox idase ac t iv i ty in Fig. 16, of. the ac t ion o f buffer con-

c e n t r a t i o n on the b i n d i n g o f c to c y t o c h r o m e c pe rox ida se (Fig. 5 above) . T h e

m a j o r conc lu s ions a re :

(i) Km and Kp are l inear ly p r o p o r t i o n a l to ion ic s t r eng th in p o t a s s i u m p h o s p h a t e

buffers , and p r o b a b l y af fec ted by ca t i on c o n c e n t r a t i o n ;

(ii) Km a n d Kp fall to less t h a n 1 ~ M as ion ic s t r eng th falls b e l o w 5 m M salt .

The succinate and N A D H oxidase systems. R e s t o r a t i o n o f succ ina te o r N A D H

ox idase ac t iv i ty to c y t o c h r o m e c -dep le ted sys tems is cha rac t e r i s ed by a r e q u i r e m e n t

fo r a ce r t a in a m o u n t o f c y t o c h r o m e c. T h e c o n c e n t r a t i o n o f a d d e d c g iv ing 50 ~o

r e s t o r a t i o n (Kso) is ex t r eme ly var iab le , un l ike the c o r r e s p o n d i n g c o n c e n t r a t i o n (Kin)

r e q u i r e d to ac t iva te the c y t o c h r o m e c ox idase s tep a lone [107]. Fig. 15 i l lus t ra tes

the cu rve re la t ing c y t o c h r o m e c c o n c e n t r a t i o n to ac t iv i ty fo r s u b m i t o c h o n d r i a l

par t ic les ox id i z ing succ ina te in 67 m M phospha t e . U n d e r these cond i t i ons the Km

291

10C ,l lr01-- '~ " / ~ J"""~'l ~ • 9C

8c B ~ ' , ' . , ' i , . 4 _ 70 A - ~ " / /

>, 60

• g 50

~ 3 0

2O

10

i i 0 8 7 6 5 4

- log [¢yt.c]

Fig. 15. Cytochrome c binding pattern in the reconstitution of succinate oxidase activity to cytochrome c-deficient submitochondrial particles. Percentage increase in manometric activity towards succinate plotted against the negative logarithm of added cytochrome c concentration. Tsou-type cytochrome c-deficient beef heart particles, 30 °C, 17 mM succinate, 0.067 M phosphate pH 7.4. O, experimental points. , curve A, theoretical sigmoid curve for simple non-cooperative binding (n = 1), Kd = 1 FM; . . . . . , curve B, theoretical curve for activity dependent on one site bound to cytochrome c in every group of ten; . . . . . curve C, theoretical curve for activity dependent on 10% of sites being bound at random (Kd~ 20 ~tM). After Fig. 1 of Nicholls [107] and unpublished data.

for cy tochrome c oxidation itself is about 15 FM (Table V). Even the "high affinity" sites (Fig. 11) are not 50% saturated at 0.5 I~M, as is the succinate oxidase system of

Fig. 15. It can be shown that full activity can be restored to such systems by binding

not more than 20 % of the normally active sites [117]. Conversely, c-depleted preparations with as little as 10% of their original complement of c are still fully active towards N A D H and succinate [107,140,102].

The theoretical curves in Fig. 15 are derived for three possible cases: (i) that the cy tochrome c dependence of succinate oxidase activity follows the usual saturation curve (A); (ii) that any 10 % of the sites need be filled to reconstitute full activity (C);

and (iii) that at least one site in each neighbouring group of ten sites must be filled to reconstitute activity (B). Evidently the data are insufficiently accurate to distinguish the three possibilities, a l though other evidence would support either (ii) or (iii).

Addit ion o f a terminal inhibitor such as azide (cf. Table VI), rendering the

oxidase reaction rate-limiting, increases the Kso for cytochrome c five or ten-fold, to a value equal to the high affinity Ks obtained by direct measurements o f cytochrome c

oxidase activity [107]. This enables us to reconcile the finding of Estabrook [126,127] that Kso for succinate oxidation increases almost in propor t ion to [K+] 2 with the direct proport ional i ty o f Km with ionic strength for cytochrome c oxidase; increasing buffer concentrat ion both tends to dissociate cytochrome c and to make the terminal oxidation rate limiting [127]. Fig. 16 illustrates the behaviour o f Ks o in the succinate oxidase [107,127], p-phenylenediamine oxidase [135], and cytochrome c [41] (or ascorbate [62]) oxidase activities with increasing phosphate buffer concentrat ion.

292

E

I

® a + PPDA ~o

uccinate

• high[c] ~ . ~

cyt.¢. or ascorbate ~o

;.5 ;.o o'.s - log [Phosphate]

Fig. 16. Effect of ionic strength on real and apparent Km values for cytochrome c binding to submitochondrial particles and to cytochrome aa3. -log Km (apparent) plotted against -log [phosphate] for oxidase activity ; ©-- ©, succinate (c-deficient submitochondrial particles) [107 ]; [], succinate plus azide [107 ]; A -- ~ , succinate (c-deficient submitochondrial particles [127]; II. II, p- phenylenediamine (NichoUs, P, unpublished); • • , cytochrome c oxidase [40,107] ('low c' and 'high c' points from Fig. 11A) A, cytochrome c oxidase [137]. N.B. ascorbate oxidation [62,135] points lie close to the 'high c' values for cytocbrome c oxidation. Modified after Fig. 6 of Nicholls [107].

At each ionic strength used we have: (i) K5 o (succinate ÷ azide) - Kso (PPDA) -= Km (high affinity); (ii) Kso (succinate alone) ~ 0.2 Km (high affinity); (iii) Km (high- affinity site) ~ 0.1 Km (low-affinity site).

Somewhere between the f lavoproteins and cy tochrome c lies a region of branching (interchain) electron transfer. The more effective the branching the less cy tochrome e is needed to reconsti tute full activity, up to the limit imposed by the turnover of the terminal oxidase. Thus, certain submitochondr ia l particles depleted of coenzyme Q and cy tochrome e can show full restorat ion of activity on the addit ion of either c o m p o n e n t (Redfearn, personal communicat ion) . As cy tochrome c is added to deficient particles, its steady state reduct ion in presence of succinate declines f rom a m a x i m u m of ~ 60 ~ to 20 ~ or less (Fig. 17). With increasing ionic strength there is a decrease in the apparen t affinity for c accompanied by an increase in the steady-state reduction at each level of cy tochrome e added [141 ]. Maximal activity in 10 m M potass ium phosphate is obtained at a cy tochrome e concentra t ion (0.08 ~tM) five times smaller than the aa3 concentrat ion. Table VI summarises the appar - ent Km values obtained with submitochondr ia l particles [141].

Phosphorylating e-deficient systems. Jacobs and Sanadi [128] showed that res torat ion of activity in State 3 to e-depleted mi tochondr ia by addit ion of cy tochrome e gave rise to a t i trat ion curve with an end-point corresponding to the amoun t of endogenous c removed. This contras ted with uncoupled submitochondr ia l particles

293

~o

"5 40

N 20

~ o

1.5

7

1.C

::t ,

0.5,

I I I / . t I

/

t (a+ oa)

I / I //I 0 1.0 2.0 3.0 5.4

~/~ C 3÷

Fig. 17. Effect of ionic strength on respiration rates and steady state reduction of cytochrome c added to cytochrome c-deficient submitochondrial particles. Oxidation of 8.3 mM succinate in potassium phosphate pH 7.2 buffers at 25 °C by Tsou-type Keilin-Hartree particles [13] (0.8 ~.M haem a). • • , 0.01 M (phosphate); O O, 0.05 M; A A, 0.1 M. Upper curves, steady state reduction; Lower curves, respiratory rate. From Fig. 44 of Kimelberg [141].

(preceding section), and might have implied an absence of significant branching reactions in the State 3 system. Fig. 18 illustrates the kind of behaviour shown by c-depleted mitochondria [63,141]. Apparent titrations are exhibited not only in

State 3, but also in state 3u and State 4, where [ C ] ( t i t r a t i o n ) ~--- [C](origlnal endog . . . . . ) ~

2[aa3]. The steady-state behaviour of cytochrome c is quite different under the three conditions. The ADP cross-over point [142], localized on the substrate side of c in the c-depleted system, shifts to the oxygen side as c is bound. Addition of uncoupler causes a much greater oxidation of cytochrome c than addition of ADP (V in State 3u > V in State 3).

As Fig. 15 indicated, binding data can be plotted either as titration curves [128] or as Michaelis-Menten plots [141]. Table VI summarises the apparent Km values obtained for intact c-depleted mitochondria when the data are analysed in the latter way. In contrast to the results with submitochondrial particles, V with mitochondria is unaffected by external ionic strength, whereas the /(so (apparent Km) is more sensitive in the mitochondria than in the particles.

At the apparent titration point the responses to ADP and to uncoupler indicate

294

TABLE VI

APPARENT Km VALUES FOR RESTORATION OF CATALYTIC ACTIVITY TO CYTO- CHROME c - DEPLETED MITOCHONDRIA AND SUBMITOCHONDRIAL PARTICLES Unlike the data in Table II, IV and V, the data in this table do not represent true dissociation constants. See text for interpretations.

System Substrate

1. Submitochondrial 10 mM particles** succinate

10 mM succinate

2. Rat liver Succinate mitochondria***

Succinate

3. Rat liver Succinate mitochondria

Succinate

Conditions Km app TN ma~* Ref. (~tM) !s- ')

0.02 M phosphate pH 7.4 0.15 10-20 [107]

plus 4 mM NaN3 2.0 4-8 [107] State 3 (20 mM phosphate + ADP) 0.08 30-50 [63]

plus 0.25 mM NaN3 0.20 10-20 [63 ] State 3u (plus CCCP +) 0.36 45-80 [63 ]

plus 0.67 mM Na N3 1.82 20--40 [63]

4. Submitochondrial 10 mM particles ascorbate ÷

TMPD Rat liver 10 mM ascorbate mitochondria -- TMPD

0.01 M phosphate pH 7.4 0.9 ~ 150 [63,141 ] 0.01 M phosphate pH 7.4 0.7 ~ 260 [63,141 ]

* Electrons/second/cytochrome aa3.

** Prepared according to Tsou [13]. *** Prepared according to Jacobs and Sanadi [128].

+ CCCP" carbonyl cyanide m--ehlorophenyl hydrazone.

tha t a l though full act ivi ty has been restored, the oxidase react ion is more rate-

l imit ing than in the presence of excess c. In accord with this idea, azide decreases the

V for succinate ox ida t ion and increases Km ( a p p a r e n t ) (Table VI) [63]. In the un-

inhibi ted mi tochondr ia , only 25 ~o of the oxidase is b o u n d by cy tochrome c at the

t i t ra t ion point . When excess c-deficient mi tochondr i a are added to such a system

the free cy tochrome c can be taken up and the freshly added mi tochondr i a also

become funct ional [141]. The equi l ibr ium charac te r of cy tochrome c b inding to

deple ted mi tochondr i a in the presence o f varying salt concent ra t ions permi t ted the

va l inomycin exper iment (Fig. 12) tha t helped demons t ra te the external loca t ion of

cy tochrome c on the inner membrane [117].

The heterogenei ty o f cy tochrome c-binding sites seen in d i rec t -b inding studies

(Table IV) [131] is also seen in cata lyt ic studies with intact mi tochondr ia . Fig. 19

i l lustrates the L ineweave r -Burk plots ob ta ined with c-deficient ra t liver mi tochondr i a

oxidiz ing succinate and ascorba te plus te t ramethyl -p-phenylenediamine (cf. Fig. 11).

There is a high-affinity site where Km and V are s imilar for the two substra tes ; a

second pa thway exists for the ascorba te system, however, with a ten-fold higher K~

and a three-fold greater /I.

The results suggest tha t the b ind ing site for cy tochrome c in mi tochondr i a is

[ 0 I.,3 6

8

~4c

1.2

"7 o.8

::t

0.4

295

I I

"~/,<e I .51.-

/

(a4-a3) I I i /z.-

0 0 .5 1.0 1.5 3.4 #M C 3.

Fig. 18. Effect of mitochondrial energy state on respiration rates and steady state reduction of cytochrome c added to cytochrome c-deficient mitochondria. Oxidation of 10 mM succinate + 2.5 mM glutamate (in presence of rotenone) in mannitol-sucrose-Tris medium pH 7.4 at 25 °C by cytochrome c-deficient rat liver mitochondria [128] (0.18 ~zM haem a). A A, state 4; O O, state 3 ( + 215 FM ADP); • • , state 3u (+0.83 ~tM CCCP). Upper curves, steady state reduction; Lower curves, respiratory rate. From Fig. 1 of Nicholls and Kimelberg [63].

I//~MO, sec -I

,]

I

0 2 0 4 0 6 0 8 0 100 I / # M C 3"

Fig. 19. Are there two populations of catalytically active cytochrome c binding sites in mitochondria? Lineweaver-Burk plots of respiratory rates against cytochrome c concentration for cytochrome c-deficient rat liver mitochondria respiring in mannitol-sucrose-Tris medium pH 7.1 at 25 °C. Other conditions as in Fig. 18. • A, succinate oxidation, state 3; • • , succinate oxidation, state 3u; A A, ascorbate-TMPD oxidation, state 3; O O, ascorbate- TMPD oxidation, state 3u. Succinate activity measured in presence of 4 ~tM rotenone, ascorbate- TMPD in presence of 0.27 ~tM antimycin. From Fig. 43 of Kimelberg [141 ].

296

exposed to the external medium, whereas in Keilin-Hartree submitochondrial particles it is partially hidden, although the greatest affinity is always shown at the lowest cation concentrations. When maximal turnovers are compared, on the other hand, the Keilin-Hartree oxidase is phosphate-sensitive while the mitochondrial enzyme is phosphate-insensitive.

Does cytochrome c have any direct effect on mitochondrial energization? Conversely, does energization modify cytochrome c binding? Jacobs and Sanadi [143] and Morrison et al. [144] found that excess cytochrome c diminished P :O ratios in ascorbate oxidation. MacLennan et al. [145,146] found that P : O for succinate with c-depleted mitochondria increased with increasing cytochrome c levels. The former observation may have been due to the presence of low affinity sites in uncoupled fragments; the latter phenomenon was satisfactorily explained as a common feature of all systems with reduced flux. Thus malonate addition mimicked e depletion. At low fluxes alternative energy-dissipating pathways can compete effectively with the ATP synthetase. Energy-linked processes that do not involve electron transfer are unaffected by removal of cytochrome e [145]. Coupled c-deficient submitochondrial particles [146] could not interact with cytochrome c. Their energy-linked functions (reversed electron transfer and transhydrogenation) were fully active in the presence of ATP.

If mitochondrial energization involves the generation of a membrane potential, would one not expect some effect on the binding of cytochrome c? The reviewer does not know the answer to this rhetorical question. However, the results shown in Fig. 18 and summarized in Table VI indicate that energization has little effect on binding of this polycation. State 4, State 3, and uncoupled mitochondria have similar affÉnities [63,102].

v[. PROPERT[ES OF BOUND CYTOCHROME c

How much do the properties of cytochrome c change on binding to proteins or membranes? Originally it was thought that the 552 nm band in situ indicated a shift due to binding. Keilin and Hartree [147] showed that this was due to the presence of cytochrome el, and that the a-peak of bound c alone is identical with that of e in solution. Since that time, subtle rather than gross changes have usually been expected for bound forms of cytochrome c, although dramatic changes in CO binding have been reported for the (intramicellar?) oxidase-e complex [59] and in ferricytochrome e for the phosphoinositide-e complex [91].

VIA. The redox potential of bound eytochrome c As discussed briefly above (Section VC), membrane-bound cytochrome c

usually shows a redox potential below that of the free cytochrome (see Table I of ref. 139). A decline from a value between + 270 and ÷ 285 mV to a value between 225 and 235 mV occurs on binding inside artificial phospholipid vesicles [98] or

297

TABLE Vll

REDOX POTENTIALS FOR HORSE HEART CYTOCHROME c OBTAINED IN SOLUTION AND IN COMPLEXES

State of cytochrome c Conditions Em Ref. (mV)

1. Free (a) low (ionic strength) -c 275 [19,23] (b) high + 255

2. Phosphate (a) low + 225 [19,23] complex (b) high + 285

3. Free ~ 280 [131,139] 4. Liposome-bound ÷ 240 [131]

(externally) 5. SMP-bound ~ 227 [139]

(a) endogenous mannitol, sucrose, MOPS* (b) trapped excess ~ 235 [139]

6. Mitochondrial ÷ 233 [139] (a) endogenous (b) bound excess + 230 [131]

7. Liposome-bound KCl-succinate** ~ 225 [98] internally

8. CcP complex ( -~- 285 [148] 9. aa3 complex _sucr°se-MOPS*** ÷ 255 [148]

* 0.225 M mannitol. 0.075 sucrose, 20 mM morpholino propane sulphonate buffer, pH 7.0. ** 0.15 M KC1, 0.01 M succinate buffer pH 7.5.

*** 0.20 M sucrose, 20 mM morpholino propane sulphonate buffer pH 7.0. SMP, submitochondrial particles; mitochondria from pigeon heart; aa3, beef heart cytochrome c oxidase; CcP, yeast cytochrome c peroxidase.

submi tochondr i a l part icles and to intact p igeon hear t mi tochondr i a [139]. Baker ' s

yeast cy tochrome c shows a smaller Em shift on b inding than horse or beef hear t

cy tochrome c, bo th with l iposomes and with mi tochondr i a [131]. N o difference is

seen between cy tochrome ¢ b o u n d at the specific site (if there is such a site) and

extra cy tochrome c b o u n d at the low affinity sites.

Binding to isolated enzymes has less effect on the Em of cy tochrome ¢. N o

change is seen in the presence of yeast cy tochrome c peroxidase or cy tochrome bs,

and the Em in complexes with isolated cy tochrome c oxidase and succinate cyto-

ch rome c reductase lies between the value in solut ion and tha t for the membrane -

b o u n d cy tochrome [148]. These results are summar ized in Table VII . Fig. 20

i l lustrates the kinds of redox t i t ra t ions achieved with the membranes and with the isolated proteins.

Two kinds o f exp lana t ion may be offered as a way out of the conflict be tween

these results and the kinetic da ta (Section VC):

(i) Tha t b inding to proteins , including specific enzyme-b ind ing sites, does no t

d iscr iminate between Fe 2+ and Fe 3+ forms, but b inding to l ipids, including non-

specific l ipopro te in -b ind ing sites, does discr iminate . Wi th a pure p ro te in (yeast

peroxidase) , the kinetic [45], b inding [46,49], and redox [148] observat ions are in

298

295-

245

195

~" 'r45 E

u~ 345

295

24~

19~

145

Baker's Yeast

o ~ ~ 2 3 - - ' ~ 5

t~

Horse Heart ~ . ~ ' ~ i ~

o:, , Io - - Ox/Red

20 A

O.35O

0.300,

0.25C

0.20C

0.15C

Em zo=O'290 n~¢,." O n=l

EmT.o'O.260V ( ~ , ~ " ~ '

Ern Z0=0.250 V

I t 1tO t 0.O1 0,1 1.O 10Q Ox/Red.

2O B

agreement. The 'partial' shift in Eo' seen with cytoclarome c oxidase [148] can then be attributed to two populations of binding sites (specific, protein, non-discriminating, and non-specific, negative lipid, discriminating).

(ii) That the redox potentials measured for the bound forms represent the potential of anion-liganded cytochrome c at zero ionic strength (Eqn 3) [19,23]. In any kinetically active system, where the binding site is readily accessible to the medium, the Eo' will rise until it is approximately the same as that for unliganded c

299

0 . 4 0 0

0 , 3 0 0

0 .20C

O J O 0

0

. , 1

• .,...,,,.~ v -- E m 7..0 = 0 . 2 8 5 V

I ;o ©x/Red

20 C

Fig. 20. Redox titrations of cytochrome c bound to mitochondria and to mitochondrial enzymes. A. Comparison of yeast and horse heart cytochrome c potentials free and bound to cytochrome c-depleted pigeon heart mitochondria. Mannitol-sucrose-MOPS medium, pH 7. Titration of 30 I~M cytochrome c (+ dye mediators). Q, © O, free; i , [ ] [~, plus 1.4 mg mitochondrial protein/m]; • , A A, plus 2.8 mg mitochondrial protein/ml. Open symbols, oxidation. Closed symbols, reduction. From Fig. 2 of Vanderkooi et al. []3] ]. B. Effect of cytochrome aa3 on potential of horse heart cytochrome c. Sucrose-MOPS medium pH 7, 550-540 nm. Titration of 4 ~tM cytochrome c (-F dye mediators). IS], () oxidative and reductive titrations of free c; ©, A plus 4 ~.M aa3; Q, • plus 12 I~M aa3. From Fig. 1 of Vanderkooi and Erecinska [148]. C. Effect of cytochrome c peroxidase on potential of horse heart cytochrome c. Sucrose-MOPS medium pH 7. 550-540 nm. Titration of 4 ~.M cytochrome c (÷ dye mediators). O, • , oxidative and reductive titrations of free c; (3, A, plus 20 [tM cytochrome c peroxidase of yeast. From Fig. 2 of Vanderkooi and Erecinska [148].

(on the Eo' o f which, ionic strength has the opposite effect [23]). In the closed systems (including the cristal-enfolded spaces o f intact mi tochondr ia ) the effective

ionic strength may be zero (?) because ions are excluded. It should also be remembered that there is more than one cationic binding site on

cytochrome c (Fig. 2) and that cy tochrome c-induced aggregations, both o f artificial membranes and of mitochondria, must involve a double binding reaction, while the

catalytic reactions involve only a single binding step.

VIB. Reactivity with cyanide and ascorbate The clever observation of Tsou [13], that Keilin-Hartree particles retain a

substrate-reducible c cytochrome in the presence o f sufficient cyanide to complex exogenous c, was flawed [129]. What he saw was almost certainly cy tochrome c l ; bound cy tochrome c itself continues to react with added cyanide, in submitochondrial particles [127], in liposomes [98], and in the cytochrome oxidase-cytochrome c complex [28]. Nevertheless, Tsou 's observation is still regularly quoted [149,150] as an example o f the all©topic modification o f bound cytochrome c. Old observations never die . . . . .

300

Both in the liposomes [98] and in the oxidase-c complex [28] the rate of the cyanide ~ c reaction is slightly reduced compared to that of free cytochrome c. But the liposomal reaction rate was only clearly affected relative to that with free c when the external medium was made alkaline; as suggested by Kimelberg and Lee [98], this is due to liposomal impermeability to ions. In the presence of valinomycin and uncoupler (permitting H + equilibration across the membrane) the CN - reactivity of liposomal c was increased to the free value [100].

Reduction of cyanferricytochrome c by dithionite gives cyanferrocytochrome c [151], a thermodynamically unstable species that undergoes crevice closure to form ferrocytochrome c. The rate of crevice closure is almost unaffected by cytochrome c binding to submitochondrial particles [129] or to the oxidase [28].

Catalytic activity of submitochondrial particles towards ascorbate [135] or cysteine [1 ] requires the addition of soluble cytochrome e, while the oxidation of p-phenylenediamine or ascorbate + tetramethyl-p-phenylenediamine can be cata- lysed via the endogenous cytochrome c of such particles. Slater [135] found that ascorbate was a poor reductant for endogenous cytochrome c. However, it is capable of reducing the bound cytochrome and the major reason for the low catalytic activity seems to be kinetic [129] (the reduction of c becomes the rate-limiting step). The decline in the rate of c reduction by ascorbate [28] on complex formation with cytochrome c peroxidase or cytochrome aas is possibly analogous to the decline seen when small anions bind to cytochrome c. A similar decrease occurs on binding externally to negatively charged liposomes [100] (this phenomenon should be distin- guished from the lowered ascorbate accessibility seen when cytochrome c is bound inside liposomes [98,99] or submitochondrial particles [112]). Whether a phenomenon that can be mimicked by small anions in solution should be termed allotopic seems doubtful.

Table VIII summarises the changes in reaction rates with ascorbate on binding of small anions, proteins and membranes, to cytochrome c.

At least one type of redox catalysis increases on binding either cytochrome c or the normally inhibitory high molecular weight polylysines to the cytochrome c binding site in mitochondria and submitochondrial particles. This is the tetrachloro- hydroquinone oxidase activity [152]. The reviewer does not pretend to understand how polylysine can substitute for cytochrome c in a redox system of this kind.

VIC. Spectroscopy of bound cytochrome c Although the visible spectra of both ferric and ferrous cytochrome c remain

unchanged in the bound state [147], spin labelling of the cytochrome c gives an EPR signal that is modified on binding both to proteins and to membranes. Barratt et al. [153] found that a nitroxymaleimide derivative labelled a number of cytochrome c lysines. The motion of the labels was hindered on complex formation with lipid, although the ORD spectrum of the cytochrome c was unchanged. Baker's yeast cytochrome c specifically labelled at the cysteine-102 sulphydryl [154] also showed line broadening and partial spin immobilization on binding to cytochrome c peroxi-

301

TABLE VIII

RATES OF REACTION WITH ASCORBATE FOR CYTOCHROME c 1N THE FREE AND BOUND STATES

Cytochrome c Conditions k(ascorbate) Ref. (M-1 . s-l)

I. 'Free' (a) 25 °C, pH 7.0-7.5 ~ 500 [21,28] (Tris cacodylate)

(b) ÷ 5 mM phosphate 150 2. H2PO-4 complex 100 mM phosphate 10-15 [21]

(Ka ~ 2mM) pH 6.83 3. Cytochrome c peroxidase complex 5 mM phosphate 10 [28]

pH 7.0 4. aa3 complex 5 mM phosphate 8 [28]

pH 7.4, 2 mM ascorbate

5. Liposome-bound 4 mM phosphate -~10 [100] cytochrome c pH 6.8

6. Submitochondrial 50 mM phosphate 15 [129] particles pH 7.4 plus

8.3 mM cyanide

dase and mitochondrial membranes. The former binding showed only slight broadening of the resonance peaks, but the membrane binding gave a more marked effect.

This facilitated an elegant proof of the KC1 accessibility of externally loaded but not

internally loaded cytochrome ¢ in submitochondrial particles [154]. Azzi et al. [155] specifically labelled methionine-65 of the horse heart cyto-

chrome. Label in this position was also immobilized on binding to the membrane. When bound, the labelled Met-65 became viscosity-insensitive, suggesting that it

faces or interacts with the binding site. The immobilization was much more marked than with the Cys-102 label [131], indicating that the latter residue may face out-

wards when bound. Fig. 21 shows the effect of membrane binding on the two types of spin label [131].

N M R spectroscopy may offer an even more powerful tool for monitoring cytochrome e binding [47]. The hyperfine-shifted low-field porphyrin ring methyl

resonances of the cytochrome c haem are broadened and brought closer together on binding to cytochrome c peroxidase [48]. The two haems (of c and cytochrome c peroxidase) are far apart ( > 25 A). Binding of cytochrome c to cytochrome aa3

can also be monitored by fluorescence quenching of 4-C1-7 NO2 benzo-2-oxa-l,3 diazole-modified cytochrome c [156]. This method confirms both the c: aa3 ratio obtained by measurements of ascorbate-e reactivity [28] and the binding constant determined kinetically. The intrinsic fluorescence of ferrocytochrome c due to tryptophan-59 is also modified on binding to cardiolipin vesicles, suggesting that H-bonds involving this residue may be disrupted on complex formation [157].

The 695 nm band of ferricytochrome c is a fairly sensitive intrinsic monitor of the intactness of the methionine-80-haem linkage and hence the closed character

302

Boker'$ yeost cyt.ochPome c

~ * Mitocho.ndria c)

Horse heQrt CytOchr'ome c

~ndr'iG

b i t )

Fig. 21. Effect of binding on EPR spin label signals of horse and yeast cytochromes c. Yeast cytochrome c spin labelled at cysteine-102, and horse heart cytochrome c spin labelled at metbionine -65 (by incubation with 3-(2 iodoacetamide-2, 2, 5, 5 tetramethyl)-l-pyrrotidinyloxyl). 10 ~M samples incubated alone (upper traces) with aerobic pigeon heart mitochondria (middle traces) and with reduced pigeon heart mitochondria (lower traces) in presence of succinate and KCN. From Fig. 7 of Vanderkooi et al. [131].

of the haem crevice [158]. This bond is present in normal mitochondrial (bound) c [159] as well as in liposomal-trapped c [100]. It is also retained in the phosvitin- cytochrome c complex [31]. Binding to either proteins [31] or lipids [85] increases the stability of the cytochrome c towards thermal denaturation (Fig. 22A); it also seems to shift the apparent pK for the transition to the inactive alkaline form towards a more basic value (Fig. 22B) [85]. But complex formation destabilizes cytochrome e against the action of denaturants [86] which, in the free cytochrome, displace the equilibrium of ferricytochrome c towards the inactive species that lack the 695 nm band [160,161].

VID. Electron transfer rates involving bound eytoehrorne c

Electrons can be transferred to or from bound cytochrome e in redox-active systems such as enzymes or the mitochondrial membrane. The question has been raised whether such electron transfer involves a slow step associated with the bound cytochrome c molecule itself, for example crevice opening and/or closing. Creutz and Sutin [162] have estimated a limiting rate of 30 s -1 (25 °C, pH 6.5, I = 1.0 M) for the reduction mechanism of cytochrome c by dithionite that proceeds via a

303

o

E tO O~

O :E

J

1 0 2'0 3 0 4 0 ' ' 5 0 6 0 Temperature (*C)

22 A

X

I I

p l - t

22 B

Fig. 22. Effects of temperature and pH on the 695 nm band of ferricytochrome c in the free and bound states. A. Variation with temperature. © ©,freecytochromec. • • , phosv i t in - bound cytochrome c; 0.65 mM cytochrome c ± 0.04 mM phosvitin in 50 mM Tris-Cl pH 7.5 (at 10 °C). From Fig. 7 of Taborsky [31]. B. Variation with pH. , free cytochrome c; • - - 0 , cytochrome c-phospholipid complex. Complex with mixed mitochondrial lipids formed in 25 mM deoxycholate at 25 °C. From Fig. 2 of Ivanetich et al. [85].

304

crevice opening step (the "adjacent attack" pathway). Stellwagen and Shulman [29] obtained (by N M R line broadening measurements) maximal velocities of 200 s - ' for the reduction of cytochrome c by ferrocyanide and 2 • l04 s -1 for the oxidation by ferricyanide (pH 7, 100 mM ferri- or ferrocyanide, 24 °C).

Mitochondrial cytochrome c is oxidized in situ with a rate constant [63] between 300 and 400 s -1. Maximal reduction rates are rarely seen with physiological substrates, but can be estimated as being greater than 100 s -1. Reduction by isolated cytochrome cl in dilute (5 ~M) solution occurs at 180 s 1 and the maximum rate (assuming only limited complex formation, Ka ~ 20 ~tM) may be at least five-fold greater than this C> 900 s -1) [79].

The maximum rate of cytochrome c oxidation by the isolated oxidase [61] occurs at pH values between 6.0 and 6.5, and is about 750 s ~ at 25 °C; the rate of oxidation by cytochrome c peroxidase is 1300 s -1 for horse cytochrome c and 7000 s for yeast cytochrome c (25 °C, 50 mM acetate pH 6.0) [43,45]. Remarkably enough, the highest measured unimolecular rate of cytochrome c oxidation is the photo- oxidation in the unphysiological complex with a bacterial photosynthetic centre. A rate equal to 27000 s -1 is reported by Ke et al. [105].

There is thus no evidence for a rate-limiting step associated with cytochrome c itself in any of the complexes. The transfer rates are apparently normally governed by the other member of the complex. In nearly every case the rate constant exceeds the value of 30-60 s --~ characteristic of crevice opening and closure [25,162]. Ox- idation and reduction of cytochrome c within the complexes thus seems to take place via a "remote attack" pathway without kinetically detectable conformational inter- mediates.

vii. CONCLUSIONS

What are my tentative answers to the five questions posed on p. 263. (a) Although the redox potential of cytochrome c is affected both by anion

binding and by ionic strength, and although general ionic strength effects must modify all electrostatic binding constants, the evidence still suggests that the major cause of cytochrome c dissociation from enzymes and from the mitochondrial membrane by salts is the increasing cation concentration. Simple competition between monovalent cations and cytochrome c for the binding site can account for most of the observations, and the catalytic sites can bind c equally well in media containing anions that affect cytochrome c and in "nonbinding" media (Tables II, I l l and IV, Figs 5, 7, 16 and 17 and the accompanying discussion in Sections I I IC and E, IVC and VC).

(b) Measured Eo' values for bound cytochrome c that differ from the Eo' for cytochrome c in solution are found either with the cytochrome at non-specific binding sites or with the cytochrome at catalytic binding sites shielded from the medium. The reviewer predicts that in all cases where a functional binding site fully accessible from the medium is occupied, the Eo' at that site will be identical with the Eo' in

305

solution (as in the case of cytochrome c peroxidase). See Table VII, Fig. 20 and the accompanying discussion in Sections II, IIIE, IVC, VC and VIA.

(c) Each "molecule" of cytochrome aaa (or each pair of haem a equivalents) is associated with one tight catalytically functional binding site for cytochrome c. At least one and possibly more secondary sites exist with higher dissociation constants. Cytochrome c may not need to dissociate into free solution in order to move from a secondary to a primary site or vice versa. Electron transfer from the secondary site(s) may also occur. See Tables IV, V and VI, Figs 6, II, 17, 18 and 19 and the discussion in Sections IIIE, VD and E and VID.

(d) The specific mitochondrial inner membrane binding sites ( ~ 0 . 5 - 1 . 0 Ezequiv./g protein) are associated with the protein of or with the cardiolipin specifically associated with cytochrome c oxidase. Secondary mitochondrial binding sites (2.5-10 ~equiv./g protein) are probably due to the general cardiolipin of the inner membrane or phosphatidyl inositol of the outer membrane. The specific binding sites are all located on the outside of the inner membrane but the secondary sites can be found on either side of that membrane. Cytochrome c may not need to dissociate in order to move from one site to another and electron transfer may be possible from some of the secondary sites. (See Table IV, Figs 1 l, 12, 16 and 18 and the discussion in Sections VA, B and D and VID.)

(e) Mitochondria as isolated contain between 0.2 and 2.0 moles cytochrome c/mole cytochrome aa3 (i.e. per 2 equivalents of haem a). When the quantity of mitochondrial cytochrome c is less than that of aaa, it is probably all bound to the proposed external inner membrane sites. Under such conditions (Section VD) 20 of the full complement of c is sufficient for full respiratory activity with flavoprotein- linked substrates. When the quantity of mitochondrial cytochrome c exceeds that of aaa, secondary sites must come into play, either: ( i )ou te r membrane sites (cytochrome bs); (ii) sites on intermembrane proteins (e.g. cytochrome c peroxidase of yeast); (iii) secondary sites on cytochrome aa3 ; (iv) secondary sites on other inner membrane cytochrome complexes; or (v) non-specific cardiolipin/ phosphatidyl inositol sites. (See Sections VD and E and also Wojtczak and Zaluska [115].)

Attempts to demonstrate availability of cytochrome c in free solution in the intermembrane space have failed. Fig. 23 .qlustrates the picture developed by 1969 [117]. The postulated pattern is plausible, but to the reviewer's knowledge not a single one of thecytochromec interactions suggested in thefigure has been proved in situ.

A better idea must surely come from studying cytochrome c interactions with the Hinkle-Racker-Jasaitis cytochrome oxidase liposomes [121,122] which contain both specific and non-specific binding sites in definable and variable amounts. Perhaps this review has been written too soon.

ACKNOWLEDGMENTS

I must thank all those who have sent me reprints and preprints for this review, and engaged in correspondence on my five questions, including A. Azzi, A. Baudras,

306

Outer membrQne

IntrocrFsto{ spoce

NADH s u b s t ~ t e s

NADH ---m-

XH 2

ll..-

e- ~'~ . . . . . . 1 i e-T'"

Inner mernbrone

MQtrix

A.-~ __ .rBH 2

0~ "2 2e Ii,

Fig. 23. Can cytochrome c play a role as electron carrier in the intracristal space? Scheme from Fig. 3 of Nicholls et al. [117].

F. L. Crane, M. Erecifiska, G. P. Hess, B. Ke, H. K. Kimelberg, T. E. King, R.

L~iuger, G. Lenaz, A. N. Malviya, L. Smith, A. Ste inemann, K. J. H. van Buuren,

J. Vanderkooi , B. F. van Gelder, and L. Wojtczak.

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Cytochrome c binding to enzymes and membranes

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