ON THE MECHANISM OF ELECTRON TRANSFER IN COMPLEX IIIOF THE ELECTRON TRANSFER CHAIN*

BY H. BAUM, t .J. S. RIESKE,t H. I. SILMAN, § AND S. H. LIPTON

INSTITUTE FOR ENZYME RESEARCH, UNIVERSITY OF WISCONSIN, MADISON

Communicated by David E. Green, January 11, 1967

Complex III of the mitochondrial electron transfer chain is an asymmetric unitwith a diameter of 80-100 Al and a molecular weight of about 300,000.2 Prepara-tions of the complex contain 20-30 per cent by weight of lipid and (in the presenceof bile salts) are water soluble.3 Under appropriate conditions (involving dilutionof the bile salts), the units aggregate into vesicular membranes.4 The complexexhibits reduced coenzyme Q-cytochrome c reductase activity with high specificactivity.3 The following accumulated evidence strongly supports the contentionthat the complex represents the subunit of the inner mitochondrial membranewhich catalyzes the corresponding span (reduced coenzyme Q-cytochrome c reduc-tase) of the electron transfer chain: (i) The complex has been prepared frommitochondria by three different procedures. All preparations have the identicalcomposition in terms of their content of cytochromes and nonheme-iron protein.3 5 6(ii) At all stages in the purification of the complex, the ratios cytochrome b: cyto-chrome cl (ref. 7) and nonheme-iron protein: cytochrome c1 (ref. 8) remain con-stant.1' 6 (iii) All preparations of the complex exhibit, on ultracentrifugal analysis,a single peak with constant sedimentation characteristics,2 consistent with theminimum particle weight as assessed by cytochrome cl content. (iv) The dimen-sions of the particle as seen by electron microscopy are consistent with this molec-ular weight, and are similar to the dimensions of the "base-piece" repeating unitsof the mitochondrial inner membrane. (v) The complex represents the purest formin which the component species have been isolated without loss of native char-acteristics relating to spectra, oxidation-reduction potential, and solubility. (vt)Specific inhibitors of reduced coenzyme Q-cytochrome c reductase (such as anti-mycin A) exhibit much the same inhibitory behavior with the isolated complex aswith intact mitochondria.3 Cleavage of the complex results in loss of both enzymicactivity and the capacity to bind antimycin A.9-1'By all the above criteria the complex must be considered as a single, integrated

enzymic unit. Indeed it probably represents one of the best-documented exam-ples of such a multiprotein enzyme. This is not to say that it might not be possibleto demonstrate partial reactions both with the intact complex and with isolatedfragments. All available evidence, however, suggests that such reactions would be,to some extent, artifactual.An intensive study has recently been made to extend the available knowledge of

the composition and structural organization of the complex.1 10-12 In addition,certain significant observations relating to spectral changes in the active complexhave also been reported.13 It is the purpose of this communication to integratethese findings into a tentative model of the mechanism of electron transfer withinthe complex, and to relate this model to the problem of the primary process ofenergy transduction in the mitochondrion.The following are the salient features of the model which will be developed (cf.

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Fig. 1): Electron flow through the complex is considered to be a discontinuousprocess. Electrons from reduced coenzyme Q are considered to be fed into thecomplex at two separate sites: at one of the cytochromes b, and at a second sitewhich might involve a new functional group, X. Initially, the orientation of thecomplex is treated (for the purposes of description only) as an extended V. In thismodel, cytochromes b and cl are out of contact with each other at the ends of thearms of the V (possibly in a lipid milieu); X is at the base angle. When cyto-chrome b is first reduced, there is thus no flow of electrons. However, reductionof X by a second electron results in a conformational change so that the angle atX becomes more acute. In this conformation cytochromes b and cl are in functionalcontact with one another, although cytochrome b is now not accessible to substrate(possibly by virtue of having moved out of a lipid milieu). Cytochrome c Caln nowreoxidize this reduced form of the complex, allowing it to return to its originalorientation.

(e)

(~±±-X) OH, QH+HWSTATE A

(Fe)(I

(of-X)STATE B

j~~~~~~~s~ ~ H

(Fe-) (FX (Fe

I X~A-+H -" ( . XH) (j+( XH)STATE E STATE D \ STATE C

antimycin A

(Fe) WC) (Fe)I ~ ~~~~~~~~~~I

( IN 1wo STEPVS) A (CN);T

(tg * Q+2H- OH, (2 x H- ( H)STATE H STATE G STATE F

FIG. 1.-Tentative model of the mechanismof electron transfer within complex III of themitochondrial electron transfer chain. Themodel is described and discussed in the text;the diagram is purely illustrative and is notintended to reflect actual spatial relationshipsof the components within the complex.Cytochromes b, cl, and c are represented bythe corresponding letters only. QH2, QH,and Q represent, respectively, the reduced,semiquinone, and oxidized forms of coenzymeQ. (Fe) represents the nonheme-iron proteinof the complex; no attempt has been madeto define its oxidation state in any of thearbitrarily designated states of the complex.

The evidence upon which this model is based will now be summarized and themodel and its implications will then be developed in more detail.

Experimental Evidence. -The relevant findings upon which this model is basedmay be summarized as follows:

(1) The complex has been shown to contain the following protein components:one molecule of cytochrome cl (mol wt 40,000)6; two molecules of cytochrome b(mol wt 25,000-30,000)14; one molecule of nonheme-iron protein containing twoatoms of iron (mol wt 30,000)'$; 3-4 molecules of "core protein" (mol wt 40,000-50,000)12; and a fifth component which (judged by its behavior upon electro-phoresis in polyacrylamide gel) is a basic protein of low molecular weight.' Thislast component, although not yet characterized, is purified together with cytochromecl in most fractionation procedures and is soluble in acidified methanol.

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The complex is thus, in terms of composition, a remarkably elaborate apparatusfor the transfer of electrons from reduced coenzyme Q to cytochrome c. Indeed, itis this very complexity which compels speculation that within the structuralorganization of the complex lies the capacity for the integration and transductionof the energy released during the flow of electrons from a two-electron donor,reduced coenzyme Q, to a one-electron acceptor, cytochrome c.

(2) Fractionation studies, titrations of sulfhydryl groups, and studies on theenzymic digestion of the complex,' have led to three tentative assignments per-taining to the organization of components within the complex: the nonheme-ironprotein is on the "outside"'6 of the complex, possibly associated with phospholipidand with bound coenzyme Qlo; cytochrome c1 is on the "outside" of the complex, andintimately associated with the site of action of antimycin A; and "core protein" ison the "inside" of the complex, probably associated with other components byvirtue of interactions involving free sulfhydryl groups (sulfhydryl group reagentssuch as mersalyl are required to cleave "core protein" from the complex).1' Thereare eight readily titratable sulfhydryl groups on the "outside" of the complex; theseare not essential for enzyme activity. In concentrated detergents or in the pres-ence of guanidine hydrochloride, a total of 23 sulfhydryl groups are titratable inthe (denatured) complex.

(3) Reduction of the complex (or treatment with antimycin A) imparts to ita markedly increased conformational stability. Thus, although reduction (ortreatment with antimycin A) does not affect the titration of the sulfhydryl groupson the "outside" of the complex, it markedly decreases the number of other sulfhy-dryl groups which can be titrated in the presence of denaturing agents. Similarly,reduction of the complex (or treatment with antimycin A) prevents the cleavageof a segment of the complex containing cytochrome b from a segment containingcytochrome cl under conditions where the oxidized complex undergoes rapid cleav-age.9' 10 (These conditions are rather specific and involve treatment either withguanidinium salts or with detergent plus ammonium sulfate.) The cleavage of thecomplex is inhibited to the extent of about 50 per cent in the presence of a redoxsystem with a potential of about + 100 mv, a potential between the mid-pointpotentials of cytochrome b and of cytochrome cl.10

(4) Reduction of the complex (or treatment with antimycin A) does not inhibitthe c(leavage from the complex of the nonheme-iron protein.9' 10 Following cleavageof the noonheme-iroin protein from the reduced (or antimycini A-treated) complex,cleavage of the remainder of the complex is still inhibited. Thus the componentwhose reduction (at a potential of + 100 mv) inhibits this latter cleavage cannot bethe nonheme-iroIn protein. However, in contrast to the effects of reducing agents onthe cleavage, reduction of the complex with dithionite or with ascorbate (the latterof which reduces the nonheme-iron protein and cytochrome cl but not cytochrome b)increases the rate at which the enzyme is inactivated by tryptic digestion. Avail-able evidence suggests that the site in the complex most sensitive to tryptic diges-tion is the nonheme-iron protein.'

(5) Under selected conditions, ill which electrons are fed very slowly into theactive complex, cytochrome b is partially reduced without concomitant reduction ofcytochrome ci. Subsequently, at what appears to be a critical point,, cytochromecl is reduced relatively rapidly with concomitant oxidation of cytochrome b."

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In experiments using glycerol or D20 as solvents, cytochrome b is rapidly reducedby reduced coenzyme Q, but a crossover point is observed between cytochrome band cytochrome cl. One interpretation of these findings is that a proton is involvedin the electron transfer process between cytochrome b and cytochrome c1.'3 Sucha proton requirement might relate to the event associated with the critical point inthe discontinuous reduction of the complex mentioned above.

(6) Antimycin A mimics reducing agents in having a stabilizing effect on theconformation of the complex." 10 Treatment with antimycin A decreases thenumber of sulfhydryl groups titratable under denaturing conditions and inhibitsthe cleavage of the enzyme by guanidinium salts or detergent plus salt; antimycinA also retards the mersalyl-induced release of "core protein" from the complex."' 12Indeed, the release of "core protein" requires the destruction of the antimycin-binding site of the complex. Antimycin A completely inhibits the enzymic activityof the complex when added in amounts stoichiometric with the cytochrome c,.91lIoreover, when the reduced complex is treated with antimycin A and is sub-sequently treated with a small amount of ferricyanide (in the presence of an excessof reduced coenzyme Q), cytochrome cl immediately becomes oxidized whereascytochrome b becomes almost completely reduced.'3

(7) The cytochrome b of the complex is readily autoxidizable. Treatmentwith antimycin A increases this autoxidizability, an effect which is much morepronounced in the presence of high levels of cyanide (about 0.1 M), or when thecomplex is damaged by treatment with organic solvents. (Cyanide, in the absenceof antimycin, appears to cause a partial inhibition of electron transfer betweencytochromes b and cl.) Complex III may be subjected to controlled tryptic diges-tion to a point where enzymic activity is destroyed but where the guanidine-inducedcleavage of the complex is still inhibited by antimycin A. As in the case of cyanidetreatment, such tryptic digestion appears, as judged from spectral data, to inhibitelectron transfer between cytochromes b and cl.' It is, therefore, of interest thattreatment of such trypsin-inactivated preparations of the complex with antimycinA also renders the cytochrome b exceptionally autoxidizable.

Interpretation.-There appear to be three recognizable functional segments ofthe complex: the cytochrome b segment, the cytochrome cl segment, and thenonheme-iron protein segment. The structural linkage between the two hemesegments is susceptible to cleavage by guanidinium salts or by ammonium sulfatein the presence of high concentrations of detergent. This linkage is modified, or insome way protected from the cleaving agents by the reduction of some grouping(with an oxidation-reduction potential between that of cytochrome b and that ofcytochrome c,). Anitimycin A also protects this linkage from cleavage (even whenthe complex is oxidized). "Core protein" can only be released from the complexwhen this linkage is broken. The nonheme-iron protein apparently has a role inthe transfer of electrons between the two heme segments. Cyanide (at high con-

centrations) and tryptic digestion may well each inhibit the catalytic activity of thecomplex by affecting this function of the nonheme-iron protein.Two lines of evidence suggest that in the oxidized complex, the cytochrome b

which is first reduced is not in oxidation-reduction equilibrium with the cytochromecl segment of the complex. The first of these is the observation (section 5, above)of a discontinuous flow of electrons through the complex. The second is the autox-

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idizability of cytochrome b. It is noteworthy that the extent of this autoxidizabilitycan be correlated with the extent of isolation of cytochrome b from other oxidation-reduction components of the complex. (It is assumed, in drawing this conclusion,that tryptic digestion or treatment with cyanide prevents interactions involvingthe nonheme-iron protein, and that antimycin A effectively prevents interactionsbetween the cytochrome b and c1 of the complex, as indicated in section 3 above.)

Available evidence (see section 3 above) favors the notion that there is an ini-crease in the conformational stability of the complex when an unidentified group(with a potential between + 100 and + 220 mv) is reduced. Whatever the precisenature of this effect, it is likely that anl actual conformationial change occurs, in-volving the spatial interrelationships of the cytochrome b and cytochrome c1segments of the complex. The studies on the tryptic digestion of the complex(see section 4 above) suggest that the nonheme-iron protein also undergoes aconformational change upon reduction, to a form which is more susceptible toproteolytic digestion. An alternative interpretation is that a conformational changeof the whole complex upon reduction renders the nonheme-iron protein more ac-cessible to trypsin.

In the light of the above considerations we now propose the following model forelectron transfer within the complex, in the presence of an excess of reduced co-enzyme Q (cf. Fig. 1): cytochrome b is not in direct contact with cytochrome c1;thus, although cytochrome cl has a more positive oxidation-reduction potentialthan cytochrome b, the substrate (reduced coenzyme Q) may reduce cytochrome bwithout reducing cytochrome cl. Subsequent to the reduction of cytochrome b byone electron, a second electron (possibly from the semiquinone form of coenzyme Q)is fed into the system to reduce another component. This other component couldbe either the second cytochrome b or else X, a functional group on either core pro-tein or the uncharacterized fifth species of the complex. This second reductionmight involve a proton (see section 5 above), and the functional group involvedmight indeed be associated with the labile linkage between the two cytochrome-containing segments of the complex. Upon this second reduction there is a con-formational change of the whole complex whereby functional contact is made be-tween cytochrome b and cytochrome c1. An electron is now transferred from cyto-chrome b to cytochrome cl, possibly through the mediation of the nonheme-ironprotein. A further conformational change, this time in the nonheme-iron protein,might be involved as part of the mechanism of this second electron transfer process.Unless a further oxidant is introduced into the system, it remains poised in thisstate (state D in Fig. 1) with cytochrome b in redox equilibrium with cytochromec1. In the presence of cytochrome c, however, XH is oxidized, and the componentsof the cytochrome b segment of the complex revert to their original conformation(state E in Fig. 1). The reduction of cytochrome c (+ 250 mv) by XH (+ 100mv?) would be more favored than the reduction of cytochrome c by cytochrome cl(+ 220 mv). Subsequently, however, cytochrome c1 is presumed to be reoxidizedby a second molecule of cytochrome c so that the system completes an entire electrontransfer cycle and returns to state A.The anomalous effect of the addition of ferricyanide to the reduced complex in

the presence of antimycin A and reduced substrate (see section 6 above) can beexplained ill terms of this model (see states F-H in Fig. 1). Antimycin can react

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with the reduced complex (as demonstrated by a shift in the wavelength of theband of reduced cytochrome b)'7 without affecting the states of reduction alreadyestablished between the cytochromes, but rendering cytochrome b more autoxidiz-able. The transient oxidation of the whole complex by the ferricyanide momen-tarily causes a conformational change making cytochrome b more readily accessibleto electrons transferred from the substrate. Once fully reduced, however, thecytochrome b can no longer come into functional contact with the cytochrome clsegment of the complex, and so is not reoxidized. This would be because antimycinA blocks the flow of electrons to cytochrome cl following the conformational changeattendant upon the reduction of X.

If indeed electron transfer in the complex does involve a new functional group,X, it appears likely that this group is intimately associated with the labile linkagebetween the cytochrome b and cl segments of the complex. It is also likely thatsuch a group would be associated with the antimycin-sensitive site. The specificityof the conditions necessary for the cleavage of the complex give some clue as to thenature of the labile linkage. Thus, it is likely that the residue involved would beone which is stabilized by reduction, would have the necessary oxidation-reductionpotential (about + 100 mv), and would require a proton for the reductive step.Moreover, it would be sensitive to hydrolytic cleavage in the presence of guanidineor guanidine derivatives.'0One point which has not been considered in discussing the model as outlined in

Figure 1 is the actual localization of the various reactive groupings within the iso-lated complex, or within the complex as it exists natively in the inner membraneof the mitochondrion. The tentative localization of some of the components withinthe complex' is of little help in deciding upon the spatial arrangement of prostheticgroups. The only real clue to the solution of this latter problem is the nature of theelectron donor itself. In the intact mitochondrion the native, and preferred, sub-strate is reduced coenzyme Qio.'8 This is a lipid-soluble mobile component whichis thought to transfer electrons from reduced complex I or II to complex III bymoving through the lipid phase of the inner mitochondrial membrane.19 In thecase of the isolated complex, the preferred substrate is reduced coenzyme Q2'20In this case it appears that the extreme water-insolubility of higher homologuesrenders very difficult their interaction with an aqueous suspension of the bile-saltdispersed complex. In either case, however, it would appear likely that the initialinteraction between the lipid-soluble substrate and the heme of cytochrome btakes place within a lipid milieu at one surface of complex III. (The nonheme-ironprotein and cytochrome cl, both of which have been tentatively assigned to the"outside" of the complex, may also abut this same hydrophobic region.) In allprobability, the second electron would also have to be fed into the complex in alipid phase, but since the identity of the postulated functional group, X, remains tobe established, further speculation as to the site of the reaction involved is notpossible. However, it is interesting to speculate that once X has been reduced andcytochromes b and cl have been brought into contact by virtue of the resultantcotnformational change, the transfer of electrons between the cytochromes mighttake place in a polar region within the complex. Such a new orientation mighteffectively shield cytochrome b from further reacting with reduced coenzyme Q.Hence some of the discontinuities in electron flow which we have discussed might

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reflect changes not only in spatial interrelationships but also in the actual phaseswithin which the reactive groupings are localized.

In principle, the proposed discontinuous nature of the electron transfer processwithin complex III could be demonstrated unambiguously by stop-flow techniquesor by single-turnover titrations. Such studies have, in fact, been initiated. If theproposed model is shown to be valid, serious consideration will have to be given tothe possibility that the suggested mechanism may prove to be a general phenomenon.Indeed models with some features in common with the one which we propose havebeen suggested in connection with the mechanism of action of cytochrome oxidase.2'Moreover, complex IV of the mitochondrial electron transfer chain (cytochromeoxidase) has been titrated with phenazine methosulfate-DPNH, the reduction ofone of the copper atoms being monitored by electron paramagnetic resonancespetroscopy.22 This titration provided evidence of a discontinuity of electron flowwithin the complex. (This interpretation is only valid if results obtained at roomtemperature are comparable with those obtained at very low temperatures.) Inaddition, there is evidence that a proton may be required in electron flow betweencytochromes a and a3 in complex IV.13

If the model which we propose for electron transfer within complex III is correctin principle, then it is tempting to relate it to the primary energy-conserving processin mitochondria. The cyclic wave of conformational changes which we suggestwould represent a satisfactory solution to the problem of the integration and trans-duction of the potential energy changes involved in the over-all electron transfer.The actual transduction process could be the formation of a covalent bond betweengroups brought into contact with one another by the conformational change, e.g.,between coenzyme Q and X, or between an aldehyde (if X were a reducible acid orlactone) and a thiol group; or it could be the movement, say, of a proton from onesite in the complex to another. In any event, it is clear that any description of thisprimary transduction process must ultimately rest upon a detailed description ofthe process of electron transfer within the complexes of the respiratory chain of themitochondrion.

* This work was supported in part by program-project grant GM-12,847 from the NationalInstitutes of General Medical Sciences (USPHS).

t On leave of absence from the Royal Free Hospital School of Medicine, London, W.C. 1,England.

Present address: Department of Physiological Chemistry, Ohio State University School ofMedicine, Columbus, Ohio.

§ On leave of absence from the Department of Biophysics, The Weizmann Institute of Science,Rehovoth, Israel. Recipient of a U.S. government grant under the Fullbright-Hays program.

lBaum, H., H. I. Silman, J. S. Rieske, and S. H. Lipton, in preparation.2Tzagoloff, A., P. C. Yang, D. C. Wharton, and J. S. Rieske, Biochim. Biophys. Acta, 96, 1

(1965).31Hatefi, Y., A. G. Haavik, and D. E. Griffiths, J. Biol. Chem., 237, 1681 (1962).4Green, D. E., D. W. Allmann, E. Bachmann, H. Baum, K. Kopaczyk, E. F. Korman, S. H.

Lipton, D. H. MacLennan, D. G. McConnell, J. F. Perdue, J. S. Rieske, and A. Tzagoloff, Arch.Biochem. Biophys., in press.

5 Rieske, J. S., R. E. Hansen, and W. S. Zaugg, J. Biol. Chem., 239, 3017 (1964).6Rieske, J. S., W. S. Zaugg, and R. E. Hansen, J. Biol. Chem., 239, 3023 (1964).7Reported ratios of cytochrome b : cytochrome cl in whole mitochondria have been greater

than those reported in purified complex III. This extra "cytochrome b" apparently remains in

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the soluble protein fractions during fractionation of mitochondria. The data of a recent report(Vanneste, W. H., Biochim. Biophys. Acta, 113, 175 (1966)) may indicate that this extra "cyto-chrome b" was a contaminant, possibly of microsomal cytochrome b5 or myoglobin, since a ratioof the cytochromes obtained with washed mitochondria corresponded closely to the value ob-tained with purified complex III.

8 Measured by electron paramagnetic resonance spectroscopy.9 Rieske, J. S., and W. S. Zaugg, Biochem. Biophys. Res. Commun., 8, 421 (1962).10 Rieske, J. S., H. Baum, C. D. Stoner, and S. H. Lipton, in preparation.

Rieske, J. S., S. H. Lipton, H. Baum, and H. I. Silman, in preparation."2Silman, H. I., J. S. Rieske, H. Baum, and S. H. Lipton, in preparation.13 Baum, H., and J. S. Rieske, Biochem. Biophys. Res. Commun., 24, 1 (1966).14Zaugg, W. S., and J. S. Rieske, Biochem. Biophys. Res. Commun., 9, 213 (1962).15 Rieske, J. S., D. H. MacLennan, and R. Coleman, Biochem. Biophys. Res.-Commun., 15, 338

(1964).16 The terms "outside" and "inside" are used more to imply functional accessibility than topo-

graphical location.'7 A. M. Pumphrey, J. Biol. Chem., 237, 2384 (1962).8 L. Szarkowska, Arch. Biochem. Biophys., 113, 519 (1966).19 Green, D. E., Radiation Res., Suppl., 2, 504 (1960).20Lipton, S. H., and J. S. Rieske, unpublished results.21 King, T. E., H. S. Mason, and M. Morrison, ed., Oxidases and Related Redox Systems (New

York: John Wiley and Sons, 1965), vol. 2.22Beinert, H., in Biochemistry of Copper, ed. J. Peisach, P. Aisen, and W. E. Blumberg (New

York: Academic Press, 1966), p. 213.

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