cl (ref. 7) and nonheme-iron protein: cytochrome c1 (ref. 8) remain constant.1'

Transcription

1 ON THE MECHANISM OF ELECTRON TRANSFER IN COMPLEX III OF 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 unit with a diameter of Al and a molecular weight of about 300,000.2 Preparations of the complex contain per cent by weight of lipid and (in the presence of bile salts) are water soluble.3 Under appropriate conditions (involving dilution of the bile salts), the units aggregate into vesicular membranes.4 The complex exhibits reduced coenzyme Q-cytochrome c reductase activity with high specific activity.3 The following accumulated evidence strongly supports the contention that the complex represents the subunit of the inner mitochondrial membrane which catalyzes the corresponding span (reduced coenzyme Q-cytochrome c reductase) of the electron transfer chain: (i) The complex has been prepared from mitochondria by three different procedures. All preparations have the identical composition in terms of their content of cytochromes and nonheme-iron protein (ii) At all stages in the purification of the complex, the ratios cytochrome b: cytochrome cl (ref. 7) and nonheme-iron protein: cytochrome c1 (ref. 8) remain constant.1' 6 (iii) All preparations of the complex exhibit, on ultracentrifugal analysis, a single peak with constant sedimentation characteristics,2 consistent with the minimum particle weight as assessed by cytochrome cl content. (iv) The dimensions of the particle as seen by electron microscopy are consistent with this molecular weight, and are similar to the dimensions of the "base-piece" repeating units of the mitochondrial inner membrane. (v) The complex represents the purest form in which the component species have been isolated without loss of native characteristics relating to spectra, oxidation-reduction potential, and solubility. (vt) Specific inhibitors of reduced coenzyme Q-cytochrome c reductase (such as antimycin A) exhibit much the same inhibitory behavior with the isolated complex as with intact mitochondria.3 Cleavage of the complex results in loss of both enzymic activity 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 examples of such a multiprotein enzyme. This is not to say that it might not be possible to demonstrate partial reactions both with the intact complex and with isolated fragments. 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 In addition, certain significant observations relating to spectral changes in the active complex have also been reported.13 It is the purpose of this communication to integrate these findings into a tentative model of the mechanism of electron transfer within the complex, and to relate this model to the problem of the primary process of energy transduction in the mitochondrion. The following are the salient features of the model which will be developed (cf. 798

2 VOL. 57, 1967 BIOCHEMISTRY: BAUM ET AL. 799 Fig. 1): Electron flow through the complex is considered to be a discontinuous process. Electrons from reduced coenzyme Q are considered to be fed into the complex at two separate sites: at one of the cytochromes b, and at a second site which might involve a new functional group, X. Initially, the orientation of the complex is treated (for the purposes of description only) as an extended V. In this model, cytochromes b and cl are out of contact with each other at the ends of the arms of the V (possibly in a lipid milieu); X is at the base angle. When cytochrome b is first reduced, there is thus no flow of electrons. However, reduction of X by a second electron results in a conformational change so that the angle at X becomes more acute. In this conformation cytochromes b and cl are in functional contact 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 now reoxidize this reduced form of the complex, allowing it to return to its original orientation. (e) (Fe) (I (~±±-X) OH, QH+HW STATE A j~~~~~~~s~ ~ H ( IN 1wo ST EPVS) A (CN);T (of-x) STATE B FIG. 1.-Tentative model of the mechanism of electron transfer within complex III of the mitochondrial electron transfer chain. The model is described and discussed in the text; the diagram is purely illustrative and is not (Fe-) (FX (Fe intended to reflect actual spatial relationships of the components within the complex. Cytochromes b, cl, and c are represented by the corresponding letters only. QH2, QH,. and I X~A-+H -" Q represent, respectively, the reduced, ( XH) (j+( XH) semiquinone, and oxidized forms of coenzyme STATE E STATE D \ STATE C Q. (Fe) represents the nonheme-iron protein of the complex; no attempt has been made antimycin A to define its oxidation state in any of the arbitrarily designated states of the complex. (Fe) WC) (Fe) I ~ ~~~~~~~~~I ~ (tg * Q+2H- OH, (2 x H- ( H) STATE H STATE G STATE F The evidence upon which this model is based will now be summarized and the model and its implications will then be developed in more detail. Experimental Evidence. -The relevant findings upon which this model is based may 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 two atoms 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 electrophoresis in polyacrylamide gel) is a basic protein of low molecular weight.' This last component, although not yet characterized, is purified together with cytochrome cl in most fractionation procedures and is soluble in acidified methanol.

3 800 BIOCHEMISTRY: BAUM ET AL. PROC. N. A. S. The complex is thus, in terms of composition, a remarkably elaborate apparatus for the transfer of electrons from reduced coenzyme Q to cytochrome c. Indeed, it is this very complexity which compels speculation that within the structural organization of the complex lies the capacity for the integration and transduction of 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 the enzymic digestion of the complex,' have led to three tentative assignments pertaining to the organization of components within the complex: the nonheme-iron protein is on the "outside"'6 of the complex, possibly associated with phospholipid and with bound coenzyme Qlo; cytochrome c1 is on the "outside" of the complex, and intimately associated with the site of action of antimycin A; and "core protein" is on the "inside" of the complex, probably associated with other components by virtue of interactions involving free sulfhydryl groups (sulfhydryl group reagents such as mersalyl are required to cleave "core protein" from the complex).1' There are eight readily titratable sulfhydryl groups on the "outside" of the complex; these are not essential for enzyme activity. In concentrated detergents or in the presence of guanidine hydrochloride, a total of 23 sulfhydryl groups are titratable in the (denatured) complex. (3) Reduction of the complex (or treatment with antimycin A) imparts to it a markedly increased conformational stability. Thus, although reduction (or treatment with antimycin A) does not affect the titration of the sulfhydryl groups on the "outside" of the complex, it markedly decreases the number of other sulfhydryl groups which can be titrated in the presence of denaturing agents. Similarly, reduction of the complex (or treatment with antimycin A) prevents the cleavage of a segment of the complex containing cytochrome b from a segment containing cytochrome cl under conditions where the oxidized complex undergoes rapid cleavage.9' 10 (These conditions are rather specific and involve treatment either with guanidinium salts or with detergent plus ammonium sulfate.) The cleavage of the complex is inhibited to the extent of about 50 per cent in the presence of a redox system with a potential of about mv, a potential between the mid-point potentials of cytochrome b and of cytochrome cl.10 (4) Reduction of the complex (or treatment with antimycin A) does not inhibit the c(leavage from the complex of the nonheme-iron protein.9' 10 Following cleavage of the noonheme-iroin protein from the reduced (or antimycini A-treated) complex, cleavage of the remainder of the complex is still inhibited. Thus the component whose reduction (at a potential of mv) inhibits this latter cleavage cannot be the nonheme-iroin protein. However, in contrast to the effects of reducing agents on the cleavage, reduction of the complex with dithionite or with ascorbate (the latter of 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. Available evidence suggests that the site in the complex most sensitive to tryptic digestion is the nonheme-iron protein.' (5) Under selected conditions, ill which electrons are fed very slowly into the active complex, cytochrome b is partially reduced without concomitant reduction of cytochrome ci. Subsequently, at what appears to be a critical point,, cytochrome cl is reduced relatively rapidly with concomitant oxidation of cytochrome b."

4 VOL. 57, 1967 BIOCHEMISTIY: BAUM ET AL. $01 In experiments using glycerol or D20 as solvents, cytochrome b is rapidly reduced by reduced coenzyme Q, but a crossover point is observed between cytochrome b and cytochrome cl. One interpretation of these findings is that a proton is involved in the electron transfer process between cytochrome b and cytochrome c1.'3 Such a proton requirement might relate to the event associated with the critical point in the discontinuous reduction of the complex mentioned above. (6) Antimycin A mimics reducing agents in having a stabilizing effect on the conformation of the complex." 10 Treatment with antimycin A decreases the number of sulfhydryl groups titratable under denaturing conditions and inhibits the cleavage of the enzyme by guanidinium salts or detergent plus salt; antimycin A also retards the mersalyl-induced release of "core protein" from the complex."' 12 Indeed, the release of "core protein" requires the destruction of the antimycinbinding site of the complex. Antimycin A completely inhibits the enzymic activity of the complex when added in amounts stoichiometric with the cytochrome c,.9 1lIoreover, when the reduced complex is treated with antimycin A and is subsequently treated with a small amount of ferricyanide (in the presence of an excess of reduced coenzyme Q), cytochrome cl immediately becomes oxidized whereas cytochrome b becomes almost completely reduced.'3 (7) The cytochrome b of the complex is readily autoxidizable. Treatment with antimycin A increases this autoxidizability, an effect which is much more pronounced in the presence of high levels of cyanide (about 0.1 M), or when the complex is damaged by treatment with organic solvents. (Cyanide, in the absence of antimycin, appears to cause a partial inhibition of electron transfer between cytochromes b and cl.) Complex III may be subjected to controlled tryptic digestion to a point where enzymic activity is destroyed but where the guanidine-induced cleavage of the complex is still inhibited by antimycin A. As in the case of cyanide treatment, such tryptic digestion appears, as judged from spectral data, to inhibit electron transfer between cytochromes b and cl.' It is, therefore, of interest that treatment of such trypsin-inactivated preparations of the complex with antimycin A also renders the cytochrome b exceptionally autoxidizable. Interpretation.-There appear to be three recognizable functional segments of the complex: the cytochrome b segment, the cytochrome cl segment, and the nonheme-iron protein segment. The structural linkage between the two heme segments is susceptible to cleavage by guanidinium salts or by ammonium sulfate in the presence of high concentrations of detergent. This linkage is modified, or in some way protected from the cleaving agents by the reduction of some grouping (with an oxidation-reduction potential between that of cytochrome b and that of cytochrome c,). Anitimycin A also protects this linkage from cleavage (even when the complex is oxidized). "Core protein" can only be released from the complex when this linkage is broken. The nonheme-iron protein apparently has a role in the transfer of electrons between the two heme segments. Cyanide (at high concentrations) and tryptic digestion may well each inhibit the catalytic activity of the complex 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 cytochrome cl 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-

5 802 BIOCHEMISTRY: BAUM ET AL. PROC. N. A. S. idizability of cytochrome b. It is noteworthy that the extent of this autoxidizability can be correlated with the extent of isolation of cytochrome b from other oxidationreduction components of the complex. (It is assumed, in drawing this conclusion, that tryptic digestion or treatment with cyanide prevents interactions involving the nonheme-iron protein, and that antimycin A effectively prevents interactions between 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 inicrease in the conformational stability of the complex when an unidentified group (with a potential between and mv) is reduced. Whatever the precise nature of this effect, it is likely that anl actual conformationial change occurs, involving the spatial interrelationships of the cytochrome b and cytochrome c1 segments of the complex. The studies on the tryptic digestion of the complex (see section 4 above) suggest that the nonheme-iron protein also undergoes a conformational change upon reduction, to a form which is more susceptible to proteolytic digestion. An alternative interpretation is that a conformational change of the whole complex upon reduction renders the nonheme-iron protein more accessible to trypsin. In the light of the above considerations we now propose the following model for electron transfer within the complex, in the presence of an excess of reduced coenzyme Q (cf. Fig. 1): cytochrome b is not in direct contact with cytochrome c1; thus, although cytochrome cl has a more positive oxidation-reduction potential than cytochrome b, the substrate (reduced coenzyme Q) may reduce cytochrome b without reducing cytochrome cl. Subsequent to the reduction of cytochrome b by one electron, a second electron (possibly from the semiquinone form of coenzyme Q) is fed into the system to reduce another component. This other component could be either the second cytochrome b or else X, a functional group on either core protein or the uncharacterized fifth species of the complex. This second reduction might involve a proton (see section 5 above), and the functional group involved might indeed be associated with the labile linkage between the two cytochromecontaining segments of the complex. Upon this second reduction there is a conformational change of the whole complex whereby functional contact is made between cytochrome b and cytochrome c1. An electron is now transferred from cytochrome b to cytochrome cl, possibly through the mediation of the nonheme-iron protein. 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 this state (state D in Fig. 1) with cytochrome b in redox equilibrium with cytochrome c1. In the presence of cytochrome c, however, XH is oxidized, and the components of 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 (+ 100 mv?) would be more favored than the reduction of cytochrome c by cytochrome cl (+ 220 mv). Subsequently, however, cytochrome c1 is presumed to be reoxidized by a second molecule of cytochrome c so that the system completes an entire electron transfer 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 be explained ill terms of this model (see states F-H in Fig. 1). Antimycin can react

6 VOL. 57, 1967 BIOCHEMISTRY: BAUM ET AL. with the reduced complex (as demonstrated by a shift in the wavelength of the band of reduced cytochrome b)'7 without affecting the states of reduction already established between the cytochromes, but rendering cytochrome b more autoxidizable. The transient oxidation of the whole complex by the ferricyanide momentarily causes a conformational change making cytochrome b more readily accessible to electrons transferred from the substrate. Once fully reduced, however, the cytochrome b can no longer come into functional contact with the cytochrome cl segment of the complex, and so is not reoxidized. This would be because antimycin A blocks the flow of electrons to cytochrome cl following the conformational change attendant 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 linkage between the cytochrome b and cl segments of the complex. It is also likely that such a group would be associated with the antimycin-sensitive site. The specificity of the conditions necessary for the cleavage of the complex give some clue as to the nature of the labile linkage. Thus, it is likely that the residue involved would be one which is stabilized by reduction, would have the necessary oxidation-reduction potential (about mv), and would require a proton for the reductive step. Moreover, it would be sensitive to hydrolytic cleavage in the presence of guanidine or guanidine derivatives.'0 One 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 isolated complex, or within the complex as it exists natively in the inner membrane of the mitochondrion. The tentative localization of some of the components within the complex' is of little help in deciding upon the spatial arrangement of prosthetic groups. The only real clue to the solution of this latter problem is the nature of the electron donor itself. In the intact mitochondrion the native, and preferred, substrate is reduced coenzyme Qio.'8 This is a lipid-soluble mobile component which is thought to transfer electrons from reduced complex I or II to complex III by moving through the lipid phase of the inner mitochondrial membrane.19 In the case of the isolated complex, the preferred substrate is reduced coenzyme Q2'20 In this case it appears that the extreme water-insolubility of higher homologues renders very difficult their interaction with an aqueous suspension of the bile-salt dispersed complex. In either case, however, it would appear likely that the initial interaction between the lipid-soluble substrate and the heme of cytochrome b takes place within a lipid milieu at one surface of complex III. (The nonheme-iron protein and cytochrome cl, both of which have been tentatively assigned to the "outside" of the complex, may also abut this same hydrophobic region.) In all probability, the second electron would also have to be fed into the complex in a lipid phase, but since the identity of the postulated functional group, X, remains to be established, further speculation as to the site of the reaction involved is not possible. However, it is interesting to speculate that once X has been reduced and cytochromes b and cl have been brought into contact by virtue of the resultant cotnformational change, the transfer of electrons between the cytochromes might take place in a polar region within the complex. Such a new orientation might effectively shield cytochrome b from further reacting with reduced coenzyme Q. Hence some of the discontinuities in electron flow which we have discussed might 803

7 804 BIOCHEMISTRY: BAUM ET AL. Pitoc N. A. S. reflect changes not only in spatial interrelationships but also in the actual phases within which the reactive groupings are localized. In principle, the proposed discontinuous nature of the electron transfer process within complex III could be demonstrated unambiguously by stop-flow techniques or by single-turnover titrations. Such studies have, in fact, been initiated. If the proposed model is shown to be valid, serious consideration will have to be given to the 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 have been suggested in connection with the mechanism of action of cytochrome oxidase.2' Moreover, complex IV of the mitochondrial electron transfer chain (cytochrome oxidase) has been titrated with phenazine methosulfate-dpnh, the reduction of one of the copper atoms being monitored by electron paramagnetic resonance spetroscopy.22 This titration provided evidence of a discontinuity of electron flow within the complex. (This interpretation is only valid if results obtained at room temperature are comparable with those obtained at very low temperatures.) In addition, there is evidence that a proton may be required in electron flow between cytochromes a and a3 in complex IV.13 correct If the model which we propose for electron transfer within complex III is in principle, then it is tempting to relate it to the primary energy-conserving process in mitochondria. The cyclic wave of conformational changes which we suggest would represent a satisfactory solution to the problem of the integration and transduction of the potential energy changes involved in the over-all electron transfer. The actual transduction process could be the formation of a covalent bond between groups 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 or lactone) and a thiol group; or it could be the movement, say, of a proton from one site in the complex to another. In any event, it is clear that any description of this primary transduction process must ultimately rest upon a detailed description of the process of electron transfer within the complexes of the respiratory chain of the mitochondrion. * This work was supported in part by program-project grant GM-12,847 from the National Institutes 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: Medicine, Columbus, Ohio. Department of Physiological Chemistry, Ohio State University School of 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

8 VOL,. 57, 1967 BIOCHEMISTRY: BAUM ET AL. 805 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 "cytochrome b" was a contaminant, possibly of microsomal cytochrome b5 or myoglobin, since a ratio of the cytochromes obtained with washed mitochondria corresponded closely to the value obtained 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 topographical 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 Beinert, H., in Biochemistry of Copper, ed. J. Peisach, P. Aisen, and W. E. Blumberg (New York: Academic Press, 1966), p. 213.