- Email: [email protected]
The question of localized or delocalized proton circuits
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Discussion Forum Is transmembrane A/Xn+essential to mitochondrial energy coupling? Bayard T. Storey and Chuan-pu Lee Transmembrane A/2w is not obligatory
Bayard 1". Storey. Ph. D., is m:~ociate research profes:sor o f obstetrics and gynecology, physiology, and physical biochemistry at the University o f Pennsylvania School o f Medicine. He has worked on the mechanisms o f electron transport in plant mitochondria and more recently has been studying the mechanism o f energy coupling in skeletal muscle mitochondria, using submitochondrial particles, and its relation to O~ and CO= handling in this tissue. His interests also include the energy metabolism o f mammalian spermatozoa and the reactions leading to fertilization in rnammaL~.
c. P. Lee, Ph.D., D.Phil., is professor o f biochemist O' at the Wayne State University School o f Medicine. She has long been interested in the process o f mitochondrial energy transduction: her work with Professor Lars Ernster in the 60~ establL*hed submitochondrial particles as a powerful tool for the study o f this process. Her present research deals with the chemical mechanism o f the mimchondrial energy coupling proce~w and its implications in neuromuscular d&eases.
The inner mitochondrial membrane acts as a fuel cell. The multi-enzyme assembly of the respiratory chain mediates the isothermal combustion of hydrogen from N A D H and succinate generated in the mitochondrial matrix and from L-3-glyeerol phosphate (L-3-GP) in the cytosol. This isothermal combustion is accomplished in a series of electron transport steps, in which electrons removed from the substrate are transported to oxygen by the various electron transport carriers of the respiratory chain. The actual reduction of oxygen to water is carried out by the enzyme complex, eytochrome oxidase. The combustion of substrate hydrogen occurs with a large positive free energy change which is conserved as A T P by coupling between electron transport and the A T P synthetase in the inner mitochondrial membrane. The question which has exercised mitochondrial biochemists for many years is: what is the mechanism by which this free energy change of electron transport is conserved in the inner mitochondrial membrane and then utilized to make ATP? For convenience, this process is referred to as 'energy coupling'. Two main hypotheses have
M~trten Wikstr6m - The question of localized or delocalized proton circuits ~ldrten Wikstrom. ~I.D., Dr.Med.Sci., born in Helsinki, Finland in 1945, is docent in medical biochemistry at the University o f Helsinki since 1973 and presently carries out his second five-year term as senior lecturer at the Department o f Medical Chemistry o f this University. He was a post-doctoral fellow at the University o f Amsterdam in 1971 72 and visiting aswociate professor a~ the University o f Pennsylvania in 1975-76. His particular research interests are in bioenergeties with emphasis on the mechanZsms o f electron transfer and energy conservation by cytochrome comph,xes. In 1977 he was awarded the ["EELS Anniversa O, Prize for achievements in the field o f cytochrome hioenergetics.
While it is widely agreed that an electrochemical gradient of protons(A/2w) serves as an obligatory intermediate thermodynamic force that links mitochondrial (as well as photosynthetic and bacterial) electron transfer to phosphorylation, the more precise nature of this force is still uncertain. A coupling mechanism of oxidative phosphorylation involving localized proton circuits was proposed by Williams ~ at about the same time as MitchelP proposed the chemiosmotic theory. In the latter, the coupling between respiration (or electron transfer, more generally) and phosphorylation is achieved through A/2w which is delocalized as a gradient between the bulk aqueous phases on each side of the inner mitochondrial (bacterial or photosynthetic) membrane. In contrast, 'localized proton' theories (several variations on this theme have been proposed since Williams' proposal) postulate that the coupling occurs through a non-osmotic A/2H- that is strictly localized to the membrane (to proteins within the membrane or to the membrane/water interphases). Thus, the proton circuitry that connects electron transfer complexes, which generate A~w, with (
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been proposed to account for both the energy conservation and the energy utilization in this process. The H + ion is central to these hypotheses. Considerable evidence has accumulated to indicate that some form of H + transfer is a key component of the primary reaction of energy couplingL This H + transfer can be formulated as a transmembrane reaction, generating an H + electrochemical potential, A/2w, across the inner mitochondrial membrane, as required by the chemiosmotic hypothesis 2. It can also be formulated as an intramembrane H + fluxa: more specifically, a direct H + transfer between membrane proteins occurring in a 'limited energy transfer domain' in the membrane 4. These two hypotheses are here designated the transmembrane and intramembrar~e hypotheses for convenience. A schematic view of them is shown in Fig. 1. It appears to be easy in principle to differentiate between these two hypotheses. In practice, it has proved very difficult experimentally. A suspension of intact mitochondria will release H + into the suspending medium on 'energization' of the inner mitochondrial membrane, leading to Ap.w as predicted by the chemiosmotic hypothesis. Energization may be accomplished by the addition of oxidizable substrate to mitochondria in the presence of 02 or by addition of 02 to mitochondria which have become anaerobic in the presence of substrate. The rate and extent of H + release, and the stoichiometry of H + released per 0 atom consumed, are all critically dependent on the exact reaction conditions employed for energizationL Submitochondrial particles, prepared by sonication of intact mitochondria, have also been extensively studied with regard to energy coupling, because these are a purified preparation of fragments of the inner mitochondrial membrane. These particles have the properties of sealed vesicles of 'inverted' configuration: the side of the membrane facing the matrix space in the intact mitochondrion becomes the outer surface of the vesicle, while the side originally facing the cytosol becomes the inner face 5. The chemiosmotic hypothesis predicts that H + transfer in submitochondrial particles leading to At2w should be opposite to that observed in intact mitochondria. This is what is observed: H + uptake occurs on energization of these submitochondrial particles. The rate, extent, and stoichiometry of this process are again critically dependent upon the experimental conditions. which has resulted in much controversyL In addition, these par-
the ATP synthase that consumes it, will be delocalized through the aqueous phases in the chemiosmotic theory but localized to the membrane in the 'localized proton' theory. The difference between the two ideas is not merely an academic one, but is reflected in rather different structural and catalytic requirements of the system, e.g. in terms of protein/ protein interactions in the membrane, possible requirement of H+-conducting catalysts, etc. It has also bearings on the other unsolved aspects of oxidative phosphorylation, viz. the mechanisms by which At~LH is generated by electron transfer complexes and is used by ATP synthase. The consensus on a kind of At2w as the coupling parameter is an important achievement, but clearly only the beginning to our understanding of the molecular mechanism. Against this background the question addressed by Storey and Lee is a fundamental one. But before discussing their thesis I should like to give a very brief experimental background to the problem. In my mind the qocalized proton' idea has a weaker position at present. As demonstrated quite frequently today, A/2H* is indeed generated between the bulk aqueous phases as a result of electron transfer. While proponents of the 'localized proton' idea usually regard this as a consequence of side reactions with little or no direct significance for the phosphorylation of ADP, the application of an artificial bulk phase A#H* has been shown to lead to ATP synthesisa. The problem therefore becomes a kinetic one, but as Thayer and Hinkle 4 have shown, the artificial bulk phase Ap,H*induces ATP production at least as fast, if not faster, than does respiration. On the other hand, Sorgato and her collaborators 5 (and see cited Refs for other related work) have shown that under steady state conditions the rate of ATP synthesis seems to be quite insensitive to the measured bulk phase At2w. This could mean that the main proton current is of a localized character in the steady state. But alternatively the rate of ATP synthesis may be a complicated function of (bulk phase) Ai2w. For instance, there may be a threshold A/2w (due to the opening of the H + gate in the ATP synthase or to the removal of its natural inhibito&?) above which the rate of ATP synthesis may be extremely sensitive to small variations in the absolute magnitude of A/2w that may be difficult to detect experimentally. The reported dependence of the rate of ATP synthesis on an artificial A0,w is not incompatible with this possibility4. There is also another group of experimental findings (see review by Ernster 7) that have been interpreted to indicate that the transfer of energy (protons) occurs in discrete domains of the membrane rather than in a delocalized osmotic fashion. 1 will briefly comment on these experiments later because they are closely related to the data 8 discussed by Storey and Lee. The interpretation by Storey and Lee is subject to general criticism that also applies to other related data. The chemiosmotic notion relates to coupling between respiration a n d p h o s p h o r y lation by means of a delocalized Ao,w. Therefore, this notion cannot be disproved by demonstrations of (i) respiratory control. (ii) the related uncoupler-induced oxidation of cytochrome b in the aerobic steady state, or (iii), 'energy-linked' changes in fluorescence of extrinsic probes, even though the submitocbondrial particles could be shown unequivocally not to sustain At2w between the aqueous phases due to excessive leakiness of the membrane. It is self-evident that energy conservation occurs primarily (in terms of separation of charge with associated protolytic effects) at the local level of the molecular A#w generators, i.e. electron transfer complexes. The question
168
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ticles are permeable to sucrose, unlike the intact mitochondria6; this question of differing permeability is, unfortunately, us0ally ignored. While these results are all consistent with the transmembrane hypothesis, they do not differentiate between the two: the observed H + movements to and from the suspending medium could well be reactions secondary to the primary reaction of energy coupling. There are several experimental findings obtained with beef heart submitochondrial particles which favor the intramembrane hypothesis, however. These are: (a) oligomycin-induced respiratory control is unaffected by partially inhibitory concentrations of the membrane-bound inhibitors of electron transport, rotenone and antimycinT; (b) the titration profile of oligomycin inhibition of ATPsupported NAD + reduction by succinate is unaffected by partially inhibitory concentrations of either rotenone or malonateL (c) the ATP-driven NADH/NADP + transhydrogenase is inhibited by the mitochondrial ATPase inhibitor protein with titration in parallel with ATPase activity even when the mitochondrial ATPase activity is not rate-limiting to the transhydrogenase reaction g. A similar result is observed with the energy-linked enhancement of 8-anilino-napthalene sul-
addressed here is whether delocalization of A/Zw is necessary to achieve coupling to ATP synthesis (chemiosmotic), or whether the main contact to the ATP synthase is established more locally in the membrane. By analogy, bacteriorhodopsin undergoes primary light-induced chemical/structural changes that are much faster than the subsequent light-insensitive translocation of protonsL If, as claimed by Storey and Lee, the membranes in their preparation are indeed fully permeable to H + (but see below), the rate-limiting step of electron transfer may be delocalization of H + from the primary A/2H+generators under their experimental conditions. This would explain the findings i-iii without disproving the idea that ATP synthesis may still require the delocalization of A/2w. Also other findings (reviewed in Ref. 7) indicating 'energization' effects in localized domains near the ATP synthase or respiratory chain complexes are really not unexpected, and do not in my opinion provide any evidence for a localized mode of coupling between respiration and phosphorylation. Baum et al. lo studied the combined inhibitory effects of the ATPase inhibitor oligomycin and the respiratory chain inhibitor rotenone on the rate of the ATP-linked reduction of NAD + by succinate. The results were suggested to imply localized domains of energy transfer between ATPase and respirat-
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fonate (ANS) supported by A T P when 7-chloro-4nitrobenzofurazan (Nbf-Cl) is the ATPase inhibitoP °. These results are compatible with the intramembrane hypothesis but not with the transmembrane hypothesis. A n o t h e r approach to distinguishing between the hypotheses was sought using a different submitochondrial membrane preparation: 'open' fragments of inner membrane of skeletal muscle mitochondria which are incapable of sustaining a transmembrane A/2w. Skeletal muscle mitochondria have an active L-3-GP oxidase activity; the L-3-GP dehydrogenase is located on the outside of the inner mitochondrial membrane which is impermeable to the substrate L-3-GP n. Both the succinate and N A D H dehydrogenases are located on the inside of the inner mitochondrial membrane which is impermeable to the substrate N A D H . The particular set of permeability properties and dehydrogenase locations possessed by the inner membrane of skeletal muscle mitochondria allows one to differentiate between 'right-side-to', "inside-out', and 'open' submitochondrial particles (Fig. 2). The 'right-side-to' particles should oxidize L-3-GP but not N A D H , while the 'inside-out' particles should oxidize N A D H but not L-3-GP, if the permeability barriers native to the inner membrane of these mitochondria remain intact. If those barriers are broken down such that N A D H and L-3-GP are freely permeant, then we have the 'open' particle, able to oxidize both substrates. Note that, for the "open' particle, geometry is unimportant. Such a particle may have the shape of a vesicle but be infinitely 'leaky'; it may have the shape of a m e m b r a n e strip, as shown in Fig. 2; it may be a M6bius strip, or its three-dimensional equivalent, a Kline bottle. The important point is that there exists no functional 'inside' or 'outside', and so no transmembrane gradient can be generated or sustained. Preparations of submitochondrial particles prepared from rabbit skeletal muscle mitochondria by high pressure disruption (rather than by sonication) have the properties of "open' fragments of the inner mitochondrial membrane, yet retain the capacity for energy coupling TM. (a) They show respiratory control and uncoupler-induced cytochrome b oxidation with N A D H , L-3-GP, and succinate as oxidative substrates. (B) The uncoupled rate of N A D H oxidation by the particles is the same as that observed with mitochondria treated with II.4% cholate and is itself not affected by cholate. Intact mitochondria do not oxidize N A D H . The uncoupled rate of succinate and L-3-GP oxidation by the particles is the same as that by uncoupled intact mitochondria, and is not further increased by 0.4% cholate. (c) Cytochromes b and c in the particles are reduced to the same degree by each of the three oxidizable substrates in anaerobiosis. No further reduction of cytochrome is observed when one substrate is added to particles whose cytochrome is already reduced with another. (d) Energization of the particles by adding 02 pulses to a particle suspension, rendered anaerobic with either succinate of L-3-GP, results in H + efflux to the medium; this efflux is little affected by the presence of K + plus either the permeant anion, SCN-, or the ionophore, valinomycin. (e) An energy-linked decrease in fluorescence emission of the fluorescent probe quinacrine, enhanced by the anion SCN=, is observed. This reaction is essentially the same as that observed with beef heart sonic submitochondrial par-
ory chain complexes. However, there are alternative explanations consistent with delocalized coupling TM. Most of these could be tested if Ap,,+ could be measured simultaneously in this type of experiment. Ernster et al. (see Ref. 7) showed that while the ATP-linked nicotinamide nucleotide transhydrogenase reaction is much less sensitive to oligomycin and uncoupling agents than is the ATP-linked reduction of N A D + by succinate (the latter is indeed expected to require a much higher A/~w than the former), the two activities are inhibited in parallel by the natural ATPase inhibitor of Pullman and Monroy ~. While these results show no evidence of localized coupling between the respiratory chain and the ATPase, they certainly strongly suggest such an interaction between the latter and the transhydrogenase. Now moving back to the results of Storey and Lee, a verification of the proposed 'openness' of the m e m b r a n e s in their preparation seem necessary. If it consists of membrane sheets, this should be seen by electron microscopy. But i f E M shows closed vesicles the suggested infinite membrane 'leakiness' should be verified biochemically by probing for the presence of a waterpermeable but sucrose-impermeable space. Light scattering measurements (see Ref. 6 of Storey and Lee) are indirect and do not seem sufficient for this purpose due to the unspecificity of this technique in determining the size of submitochondrial particles or mitochondria. If a solute-impermeable space is found by direct biochemical measurements, the membranes" permeability to H + could be measured directly. These controls are essential in my opinion because the observed unusual substrate accessibility need not necessarily reflect a 'generalized' permeability of the membrane. Instead, it may represent a more specific effect on the organization of the enzymes in the membrane. Assuming now that these checks reveal open membrane sheets or the equivalent, the reported experiments would still not prove the proposed thesis, as 1 already concluded above. Only a demonstration of oxidative phosplaorylation in such a preparation might provide such proof. But even if A T P were produced the notion of localized coupling would not be proven unless the P/O ratio were comparable to that in intact mitochondria, or at least of a considerable magnitude. As indicated in the beginning, the 'localized H +' hypothesis must allow for the finding that some A T P may be produced by the delocalized AtiH'. By the same token the chemiosmotic theory would allow for the possibility of a small fraction of H + current being carried more locally, e.g. over short distances at the membrane/water interphases. From this it is clear that a compromise is possible between the two notions. 50% of the current may be localized and 50% carried through the aqueous phases. It is, of course, also possible that this proportion may vary depending on the conditions (transient and steady states, for instance; cf. above). However, at the present time, these speculations are ahead of experimental reality. While I think that the data of Storey and Lee (as well as the other data cited) provide interesting directions for future experimental work, they are not presently sufficient to motivate a departure from the chemiosmotic interpretation of A/2w. This interpretation is supported by the available data, and there are no absolute contradictions. In contrast, the indications of more localized proton circuits are, at least so far. more ambiguous. ~,,,+,,,~,,,~ot,,r,~l,,~,,,,,,
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ticles, in which the reaction has b e e n d e m o n s t r a t e d to be a m o n i t o r of i n t r a m e m b r a n e H + transfer to the probe: an a p p a r e n t i n t r a m e m b r a n e 'acidification'. The inner m i t o c h o n d r i a l m e m b r a n e is i m p e r m e a b l e to the substrates N A D H and L - 3 - G P **. The ability of these subm i t o c h o n d r i a l particles to oxidize N A D H at the same rate as m i t o c h o n d r i a t r e a t e d with cholate (which destroys the m e m brane's p e r m e a b i l i t y barrier), shows that the N A D H dehydrogenase of the particles is fully accessible to this substrate, in contrast to intact mitochondria. Yet the L - 3 - G P d e h y d r o g e n ase is also equally accessible. The particles c a n n o t be a m i x e d p o p u l a t i o n of finside-out' and 'right-side-to' particles: the c y t o c h r o m e s are c o m p l e t e l y reduced by e i t h e r N A D H or L-3-GP; in a mixed population, each substrate could achieve only partial reduction (Fig. 2). The conclusion s e e m s clear: open f r a g m e n t s of the inner m i t o c h o n d r i a l m e m b r a n e oxidize substrates with e n e r g y coupling, yet totally lack the g e o m e t r y for sustaining a t r a n s m e m brane A~w. They d e m o n s t r a t e i n t r a m e m b r a n e H + transfer to the f u o r e s c e n t p r o b e quinacrine in an e n e r g y - l i n k e d reaction which is identical to that observed in sonic particles, which are 'inside-out' vesicles. The direction of e n e r g y - l i n k e d H + movement with respect to the s u s p e n d i n g m e d i u m is opposite in the two particles: e n e r g y q i n k e d H + efflux is o b s e r v e d with ' o p e n ' skeletal muscle particles while e n e r g y - l i n k e d H + u p t a k e is o b s e r v e d with 'inside-out' b e e f heart particles. This H + u p t a k e by the 'inside-out' particles is m a r k e d l y e n h a n c e d in the presence of K ÷ and v a l i n o m y c i n or S C N - - conditions which have little effect on H ÷ efflux from ' o p e n ' particles. In their recent review dealing with the role of H + translocation in the m i t o c h o n d r i a l e n e r g y - c o u p l i n g process, W i k s t r 6 m and Krab x set the following questions:
References
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I 2 3 4 5 6 7 8 9 10
Williams, R. J. P. (1961)J. Theor. Biol. 1, 1 17 Mitchell. P. ( 1961 ) Nature (London), 191, 144-148 Thayer, W. S. and Hinkle, P. C. (1975) J. Biol. Chem. 25(), 5330 5335 Thayer, W. S. and Hinkle, P. C. (1975)J. Biol. (item, 250, 5336-5342 Sorgatu, M. C., Branca. D. and Fergusom S. J. (1980) Biochem. J. 188, 945-948 Pullman, M. E. and Monroy, G. C. (19631J. Biol. Chem. 238, 3762-3769 Ernster, L. ( 19771Annu. Rev. Biochem. 46. 981-995 Storey, B.T., Scott, D. M. and Lee, C.-P. (1980)J. Biol. ('hem. 255, 5224 5229 Stoeckenius, W. and Lozier, R. H. (1974)Supramolec. Struct. 2, 769-774 Baum, H., Hall, G. S., Raider, J. and Beechey, R. B. (1971) in Energy Transduction in Respiration and Photosynthesis (Quagliariello, E.. Papa, S. and Rossi, C. S., eds), pp, 747-755, Adriatica Editrice. Bari
14drten Wik~trdm is at the Department o f Medical Chemistry, Universi O' o f Hel~inki, Sihavuorenpenger I OA, SF-O0170 HeL~inki 17, ["Tnland. Bayard T. Storey and (_Tman-pu Lee
(a) H o w is A/2H÷ g e n e r a t e d by the redox reactions of the respiratory chain carriers? (b) H o w is A/2w utilized by the A T P synthetase? (c) Is the H ÷ ' c o n n e c t i o n ' b e t w e e n the redox reactions and e n e r g y coupling osmotic (i.e. t r a n s m e m b r a n e ) or localized (i.e. i n t r a m e m b r a n e ) ? They p o i n t e d out that these questions have not been a n s w e r e d at the m o l e c u l a r level, and we fully agree with this. H o w e v e r , we feel that the existence of ' o p e n ' f r a g m e n t s of the inner m i t o c h o n d r i a l m e m b r a n e which retain the capacity for e n e r g y coupling does supply an a n s w e r to the third question: the connection is definitely localized, not osmotic. This may not be an a n s w e r in terms of a m o l e c u l a r m e c h a n i s m . But it is an a n s w e r which e m p h a s i z e s the key role of H + transfer reactions b e t w e e n molecules within the m e m b r a n e r a t h e r than H + e x c h a n g e s between vesicular c o m p a r t m e n t s and the medium, with the m e m brane acting only as p e r m e a b i l i t y barrier. It is o u r hope that these and further studies of energy coupling in 'open' fragments of the m i t o c h o n d r i a l m e m b r a n e , in which the c o m p l i c a t i o n of t r a n s m e m b r a n e A/2w is lacking, will refocus a t t e n t i o n on the m e m b r a n e phase as the d y n a m i c locus of protein reactions which m e d i a t e e n e r g y conservation and utilization.
References 1 Wikstr6m, M. and Krab, K. (1980) in Current Topics in Bionergetm~ (Sanadi, D. R., ed.), Vol. 1(1,pp. 51-101~ Academic Press, New York 2 Mitchell, P. (1966) Biol. Rev. 41,455 502 3 Williams, R. J. P. (19791 Biochim. Biophys. Acta, 505, 1~:14 4 Ernster, L. (1977) Annu. Rev. Biochem. 46,981 995 5 Lee, C. P. and Ernster, L. (1966)BBA (Biochim. Biophys. Acta) Libr. 7, 218-236 6 Storey, B.T., Lee, C. P.. Papa, S.. Rosen, S. G. and Simon, G. (1976) Biochemisto,, 15,928-933 7 Lee, C. P., Ernstcr, L. and Chance. B. (1969) Eur. J. Biochem. 8, 153 163 8 Baum. H.. Hall. G. S., Raider, J. and Beechey, R. B. (19711 in: Energy Transduction in Respiration and Photosynthesis (Quagliariell, E., Papa. S. and Rossi, C. S, eds), pp. 747-755, Adriatica Editrice, Barri 9 Ernster, L, Juntti, K. and Asauri, K. (1973)J. Bioenerg. 4, 148-159 10 Ferguson. S. J., Lloyd. W. J. and Radda, G. K. (19761 Biochim. Biophys. Acta, 423, 174-188 11 Von Jagow, G. and Klingenberg, M, (197(I) Eur. J. Biochem. 12,247 252 12 Storey, B. F., Scott. D. M. and Lee, C. P. (198(I) J. Biol. Chem. 255, 5224 5229 Bayard 72 Storey ts at the Departments oJ Physiology and Ohstetric~ and Gynecology, Universi O' o]" Pennsylvania School o f Medicine. PhihMelphia, PA 19104, U.S.A. and Chuan-pu Lee i~ at the Department o f Biochemistry, Wayne State University School o f Medicine, Detroit, %4148201. U.S.A.
TIBS -Jtt[y 1981
Discussion Forum Is transmembrane A/Xn+essential to mitochondrial energy coupling? Bayard T. Storey and Chuan-pu Lee Transmembrane A/2w is not obligatory
Bayard 1". Storey. Ph. D., is m:~ociate research profes:sor o f obstetrics and gynecology, physiology, and physical biochemistry at the University o f Pennsylvania School o f Medicine. He has worked on the mechanisms o f electron transport in plant mitochondria and more recently has been studying the mechanism o f energy coupling in skeletal muscle mitochondria, using submitochondrial particles, and its relation to O~ and CO= handling in this tissue. His interests also include the energy metabolism o f mammalian spermatozoa and the reactions leading to fertilization in rnammaL~.
c. P. Lee, Ph.D., D.Phil., is professor o f biochemist O' at the Wayne State University School o f Medicine. She has long been interested in the process o f mitochondrial energy transduction: her work with Professor Lars Ernster in the 60~ establL*hed submitochondrial particles as a powerful tool for the study o f this process. Her present research deals with the chemical mechanism o f the mimchondrial energy coupling proce~w and its implications in neuromuscular d&eases.
The inner mitochondrial membrane acts as a fuel cell. The multi-enzyme assembly of the respiratory chain mediates the isothermal combustion of hydrogen from N A D H and succinate generated in the mitochondrial matrix and from L-3-glyeerol phosphate (L-3-GP) in the cytosol. This isothermal combustion is accomplished in a series of electron transport steps, in which electrons removed from the substrate are transported to oxygen by the various electron transport carriers of the respiratory chain. The actual reduction of oxygen to water is carried out by the enzyme complex, eytochrome oxidase. The combustion of substrate hydrogen occurs with a large positive free energy change which is conserved as A T P by coupling between electron transport and the A T P synthetase in the inner mitochondrial membrane. The question which has exercised mitochondrial biochemists for many years is: what is the mechanism by which this free energy change of electron transport is conserved in the inner mitochondrial membrane and then utilized to make ATP? For convenience, this process is referred to as 'energy coupling'. Two main hypotheses have
M~trten Wikstr6m - The question of localized or delocalized proton circuits ~ldrten Wikstrom. ~I.D., Dr.Med.Sci., born in Helsinki, Finland in 1945, is docent in medical biochemistry at the University o f Helsinki since 1973 and presently carries out his second five-year term as senior lecturer at the Department o f Medical Chemistry o f this University. He was a post-doctoral fellow at the University o f Amsterdam in 1971 72 and visiting aswociate professor a~ the University o f Pennsylvania in 1975-76. His particular research interests are in bioenergeties with emphasis on the mechanZsms o f electron transfer and energy conservation by cytochrome comph,xes. In 1977 he was awarded the ["EELS Anniversa O, Prize for achievements in the field o f cytochrome hioenergetics.
While it is widely agreed that an electrochemical gradient of protons(A/2w) serves as an obligatory intermediate thermodynamic force that links mitochondrial (as well as photosynthetic and bacterial) electron transfer to phosphorylation, the more precise nature of this force is still uncertain. A coupling mechanism of oxidative phosphorylation involving localized proton circuits was proposed by Williams ~ at about the same time as MitchelP proposed the chemiosmotic theory. In the latter, the coupling between respiration (or electron transfer, more generally) and phosphorylation is achieved through A/2w which is delocalized as a gradient between the bulk aqueous phases on each side of the inner mitochondrial (bacterial or photosynthetic) membrane. In contrast, 'localized proton' theories (several variations on this theme have been proposed since Williams' proposal) postulate that the coupling occurs through a non-osmotic A/2H- that is strictly localized to the membrane (to proteins within the membrane or to the membrane/water interphases). Thus, the proton circuitry that connects electron transfer complexes, which generate A~w, with (
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been proposed to account for both the energy conservation and the energy utilization in this process. The H + ion is central to these hypotheses. Considerable evidence has accumulated to indicate that some form of H + transfer is a key component of the primary reaction of energy couplingL This H + transfer can be formulated as a transmembrane reaction, generating an H + electrochemical potential, A/2w, across the inner mitochondrial membrane, as required by the chemiosmotic hypothesis 2. It can also be formulated as an intramembrane H + fluxa: more specifically, a direct H + transfer between membrane proteins occurring in a 'limited energy transfer domain' in the membrane 4. These two hypotheses are here designated the transmembrane and intramembrar~e hypotheses for convenience. A schematic view of them is shown in Fig. 1. It appears to be easy in principle to differentiate between these two hypotheses. In practice, it has proved very difficult experimentally. A suspension of intact mitochondria will release H + into the suspending medium on 'energization' of the inner mitochondrial membrane, leading to Ap.w as predicted by the chemiosmotic hypothesis. Energization may be accomplished by the addition of oxidizable substrate to mitochondria in the presence of 02 or by addition of 02 to mitochondria which have become anaerobic in the presence of substrate. The rate and extent of H + release, and the stoichiometry of H + released per 0 atom consumed, are all critically dependent on the exact reaction conditions employed for energizationL Submitochondrial particles, prepared by sonication of intact mitochondria, have also been extensively studied with regard to energy coupling, because these are a purified preparation of fragments of the inner mitochondrial membrane. These particles have the properties of sealed vesicles of 'inverted' configuration: the side of the membrane facing the matrix space in the intact mitochondrion becomes the outer surface of the vesicle, while the side originally facing the cytosol becomes the inner face 5. The chemiosmotic hypothesis predicts that H + transfer in submitochondrial particles leading to At2w should be opposite to that observed in intact mitochondria. This is what is observed: H + uptake occurs on energization of these submitochondrial particles. The rate, extent, and stoichiometry of this process are again critically dependent upon the experimental conditions. which has resulted in much controversyL In addition, these par-
the ATP synthase that consumes it, will be delocalized through the aqueous phases in the chemiosmotic theory but localized to the membrane in the 'localized proton' theory. The difference between the two ideas is not merely an academic one, but is reflected in rather different structural and catalytic requirements of the system, e.g. in terms of protein/ protein interactions in the membrane, possible requirement of H+-conducting catalysts, etc. It has also bearings on the other unsolved aspects of oxidative phosphorylation, viz. the mechanisms by which At~LH is generated by electron transfer complexes and is used by ATP synthase. The consensus on a kind of At2w as the coupling parameter is an important achievement, but clearly only the beginning to our understanding of the molecular mechanism. Against this background the question addressed by Storey and Lee is a fundamental one. But before discussing their thesis I should like to give a very brief experimental background to the problem. In my mind the qocalized proton' idea has a weaker position at present. As demonstrated quite frequently today, A/2H* is indeed generated between the bulk aqueous phases as a result of electron transfer. While proponents of the 'localized proton' idea usually regard this as a consequence of side reactions with little or no direct significance for the phosphorylation of ADP, the application of an artificial bulk phase A#H* has been shown to lead to ATP synthesisa. The problem therefore becomes a kinetic one, but as Thayer and Hinkle 4 have shown, the artificial bulk phase Ap,H*induces ATP production at least as fast, if not faster, than does respiration. On the other hand, Sorgato and her collaborators 5 (and see cited Refs for other related work) have shown that under steady state conditions the rate of ATP synthesis seems to be quite insensitive to the measured bulk phase At2w. This could mean that the main proton current is of a localized character in the steady state. But alternatively the rate of ATP synthesis may be a complicated function of (bulk phase) Ai2w. For instance, there may be a threshold A/2w (due to the opening of the H + gate in the ATP synthase or to the removal of its natural inhibito&?) above which the rate of ATP synthesis may be extremely sensitive to small variations in the absolute magnitude of A/2w that may be difficult to detect experimentally. The reported dependence of the rate of ATP synthesis on an artificial A0,w is not incompatible with this possibility4. There is also another group of experimental findings (see review by Ernster 7) that have been interpreted to indicate that the transfer of energy (protons) occurs in discrete domains of the membrane rather than in a delocalized osmotic fashion. 1 will briefly comment on these experiments later because they are closely related to the data 8 discussed by Storey and Lee. The interpretation by Storey and Lee is subject to general criticism that also applies to other related data. The chemiosmotic notion relates to coupling between respiration a n d p h o s p h o r y lation by means of a delocalized Ao,w. Therefore, this notion cannot be disproved by demonstrations of (i) respiratory control. (ii) the related uncoupler-induced oxidation of cytochrome b in the aerobic steady state, or (iii), 'energy-linked' changes in fluorescence of extrinsic probes, even though the submitocbondrial particles could be shown unequivocally not to sustain At2w between the aqueous phases due to excessive leakiness of the membrane. It is self-evident that energy conservation occurs primarily (in terms of separation of charge with associated protolytic effects) at the local level of the molecular A#w generators, i.e. electron transfer complexes. The question
168
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Bayard 72 Storey and Chuan-pu Lee
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ticles are permeable to sucrose, unlike the intact mitochondria6; this question of differing permeability is, unfortunately, us0ally ignored. While these results are all consistent with the transmembrane hypothesis, they do not differentiate between the two: the observed H + movements to and from the suspending medium could well be reactions secondary to the primary reaction of energy coupling. There are several experimental findings obtained with beef heart submitochondrial particles which favor the intramembrane hypothesis, however. These are: (a) oligomycin-induced respiratory control is unaffected by partially inhibitory concentrations of the membrane-bound inhibitors of electron transport, rotenone and antimycinT; (b) the titration profile of oligomycin inhibition of ATPsupported NAD + reduction by succinate is unaffected by partially inhibitory concentrations of either rotenone or malonateL (c) the ATP-driven NADH/NADP + transhydrogenase is inhibited by the mitochondrial ATPase inhibitor protein with titration in parallel with ATPase activity even when the mitochondrial ATPase activity is not rate-limiting to the transhydrogenase reaction g. A similar result is observed with the energy-linked enhancement of 8-anilino-napthalene sul-
addressed here is whether delocalization of A/Zw is necessary to achieve coupling to ATP synthesis (chemiosmotic), or whether the main contact to the ATP synthase is established more locally in the membrane. By analogy, bacteriorhodopsin undergoes primary light-induced chemical/structural changes that are much faster than the subsequent light-insensitive translocation of protonsL If, as claimed by Storey and Lee, the membranes in their preparation are indeed fully permeable to H + (but see below), the rate-limiting step of electron transfer may be delocalization of H + from the primary A/2H+generators under their experimental conditions. This would explain the findings i-iii without disproving the idea that ATP synthesis may still require the delocalization of A/2w. Also other findings (reviewed in Ref. 7) indicating 'energization' effects in localized domains near the ATP synthase or respiratory chain complexes are really not unexpected, and do not in my opinion provide any evidence for a localized mode of coupling between respiration and phosphorylation. Baum et al. lo studied the combined inhibitory effects of the ATPase inhibitor oligomycin and the respiratory chain inhibitor rotenone on the rate of the ATP-linked reduction of NAD + by succinate. The results were suggested to imply localized domains of energy transfer between ATPase and respirat-
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fonate (ANS) supported by A T P when 7-chloro-4nitrobenzofurazan (Nbf-Cl) is the ATPase inhibitoP °. These results are compatible with the intramembrane hypothesis but not with the transmembrane hypothesis. A n o t h e r approach to distinguishing between the hypotheses was sought using a different submitochondrial membrane preparation: 'open' fragments of inner membrane of skeletal muscle mitochondria which are incapable of sustaining a transmembrane A/2w. Skeletal muscle mitochondria have an active L-3-GP oxidase activity; the L-3-GP dehydrogenase is located on the outside of the inner mitochondrial membrane which is impermeable to the substrate L-3-GP n. Both the succinate and N A D H dehydrogenases are located on the inside of the inner mitochondrial membrane which is impermeable to the substrate N A D H . The particular set of permeability properties and dehydrogenase locations possessed by the inner membrane of skeletal muscle mitochondria allows one to differentiate between 'right-side-to', "inside-out', and 'open' submitochondrial particles (Fig. 2). The 'right-side-to' particles should oxidize L-3-GP but not N A D H , while the 'inside-out' particles should oxidize N A D H but not L-3-GP, if the permeability barriers native to the inner membrane of these mitochondria remain intact. If those barriers are broken down such that N A D H and L-3-GP are freely permeant, then we have the 'open' particle, able to oxidize both substrates. Note that, for the "open' particle, geometry is unimportant. Such a particle may have the shape of a vesicle but be infinitely 'leaky'; it may have the shape of a m e m b r a n e strip, as shown in Fig. 2; it may be a M6bius strip, or its three-dimensional equivalent, a Kline bottle. The important point is that there exists no functional 'inside' or 'outside', and so no transmembrane gradient can be generated or sustained. Preparations of submitochondrial particles prepared from rabbit skeletal muscle mitochondria by high pressure disruption (rather than by sonication) have the properties of "open' fragments of the inner mitochondrial membrane, yet retain the capacity for energy coupling TM. (a) They show respiratory control and uncoupler-induced cytochrome b oxidation with N A D H , L-3-GP, and succinate as oxidative substrates. (B) The uncoupled rate of N A D H oxidation by the particles is the same as that observed with mitochondria treated with II.4% cholate and is itself not affected by cholate. Intact mitochondria do not oxidize N A D H . The uncoupled rate of succinate and L-3-GP oxidation by the particles is the same as that by uncoupled intact mitochondria, and is not further increased by 0.4% cholate. (c) Cytochromes b and c in the particles are reduced to the same degree by each of the three oxidizable substrates in anaerobiosis. No further reduction of cytochrome is observed when one substrate is added to particles whose cytochrome is already reduced with another. (d) Energization of the particles by adding 02 pulses to a particle suspension, rendered anaerobic with either succinate of L-3-GP, results in H + efflux to the medium; this efflux is little affected by the presence of K + plus either the permeant anion, SCN-, or the ionophore, valinomycin. (e) An energy-linked decrease in fluorescence emission of the fluorescent probe quinacrine, enhanced by the anion SCN=, is observed. This reaction is essentially the same as that observed with beef heart sonic submitochondrial par-
ory chain complexes. However, there are alternative explanations consistent with delocalized coupling TM. Most of these could be tested if Ap,,+ could be measured simultaneously in this type of experiment. Ernster et al. (see Ref. 7) showed that while the ATP-linked nicotinamide nucleotide transhydrogenase reaction is much less sensitive to oligomycin and uncoupling agents than is the ATP-linked reduction of N A D + by succinate (the latter is indeed expected to require a much higher A/~w than the former), the two activities are inhibited in parallel by the natural ATPase inhibitor of Pullman and Monroy ~. While these results show no evidence of localized coupling between the respiratory chain and the ATPase, they certainly strongly suggest such an interaction between the latter and the transhydrogenase. Now moving back to the results of Storey and Lee, a verification of the proposed 'openness' of the m e m b r a n e s in their preparation seem necessary. If it consists of membrane sheets, this should be seen by electron microscopy. But i f E M shows closed vesicles the suggested infinite membrane 'leakiness' should be verified biochemically by probing for the presence of a waterpermeable but sucrose-impermeable space. Light scattering measurements (see Ref. 6 of Storey and Lee) are indirect and do not seem sufficient for this purpose due to the unspecificity of this technique in determining the size of submitochondrial particles or mitochondria. If a solute-impermeable space is found by direct biochemical measurements, the membranes" permeability to H + could be measured directly. These controls are essential in my opinion because the observed unusual substrate accessibility need not necessarily reflect a 'generalized' permeability of the membrane. Instead, it may represent a more specific effect on the organization of the enzymes in the membrane. Assuming now that these checks reveal open membrane sheets or the equivalent, the reported experiments would still not prove the proposed thesis, as 1 already concluded above. Only a demonstration of oxidative phosplaorylation in such a preparation might provide such proof. But even if A T P were produced the notion of localized coupling would not be proven unless the P/O ratio were comparable to that in intact mitochondria, or at least of a considerable magnitude. As indicated in the beginning, the 'localized H +' hypothesis must allow for the finding that some A T P may be produced by the delocalized AtiH'. By the same token the chemiosmotic theory would allow for the possibility of a small fraction of H + current being carried more locally, e.g. over short distances at the membrane/water interphases. From this it is clear that a compromise is possible between the two notions. 50% of the current may be localized and 50% carried through the aqueous phases. It is, of course, also possible that this proportion may vary depending on the conditions (transient and steady states, for instance; cf. above). However, at the present time, these speculations are ahead of experimental reality. While I think that the data of Storey and Lee (as well as the other data cited) provide interesting directions for future experimental work, they are not presently sufficient to motivate a departure from the chemiosmotic interpretation of A/2w. This interpretation is supported by the available data, and there are no absolute contradictions. In contrast, the indications of more localized proton circuits are, at least so far. more ambiguous. ~,,,+,,,~,,,~ot,,r,~l,,~,,,,,,
171)
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Bayard 7". Storey and Chuan-pu Lee
%4{trten WikYtrom
ticles, in which the reaction has b e e n d e m o n s t r a t e d to be a m o n i t o r of i n t r a m e m b r a n e H + transfer to the probe: an a p p a r e n t i n t r a m e m b r a n e 'acidification'. The inner m i t o c h o n d r i a l m e m b r a n e is i m p e r m e a b l e to the substrates N A D H and L - 3 - G P **. The ability of these subm i t o c h o n d r i a l particles to oxidize N A D H at the same rate as m i t o c h o n d r i a t r e a t e d with cholate (which destroys the m e m brane's p e r m e a b i l i t y barrier), shows that the N A D H dehydrogenase of the particles is fully accessible to this substrate, in contrast to intact mitochondria. Yet the L - 3 - G P d e h y d r o g e n ase is also equally accessible. The particles c a n n o t be a m i x e d p o p u l a t i o n of finside-out' and 'right-side-to' particles: the c y t o c h r o m e s are c o m p l e t e l y reduced by e i t h e r N A D H or L-3-GP; in a mixed population, each substrate could achieve only partial reduction (Fig. 2). The conclusion s e e m s clear: open f r a g m e n t s of the inner m i t o c h o n d r i a l m e m b r a n e oxidize substrates with e n e r g y coupling, yet totally lack the g e o m e t r y for sustaining a t r a n s m e m brane A~w. They d e m o n s t r a t e i n t r a m e m b r a n e H + transfer to the f u o r e s c e n t p r o b e quinacrine in an e n e r g y - l i n k e d reaction which is identical to that observed in sonic particles, which are 'inside-out' vesicles. The direction of e n e r g y - l i n k e d H + movement with respect to the s u s p e n d i n g m e d i u m is opposite in the two particles: e n e r g y q i n k e d H + efflux is o b s e r v e d with ' o p e n ' skeletal muscle particles while e n e r g y - l i n k e d H + u p t a k e is o b s e r v e d with 'inside-out' b e e f heart particles. This H + u p t a k e by the 'inside-out' particles is m a r k e d l y e n h a n c e d in the presence of K ÷ and v a l i n o m y c i n or S C N - - conditions which have little effect on H ÷ efflux from ' o p e n ' particles. In their recent review dealing with the role of H + translocation in the m i t o c h o n d r i a l e n e r g y - c o u p l i n g process, W i k s t r 6 m and Krab x set the following questions:
References
lnside- Out S M P
NADH
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Right-Side-To SMP ..----~ DHAP L-B-GP I~))4. 2. Geornen2v o f inside-out, right-side-to, and open fragment submitockondrial particles (SMP). The NA DH-dehydrogenase i~ symbolized by the shaded squares, L-3-GP dehydrogenase by the filled circles. The A r P synthetase is symbolized by stalk and ball (FO.
I 2 3 4 5 6 7 8 9 10
Williams, R. J. P. (1961)J. Theor. Biol. 1, 1 17 Mitchell. P. ( 1961 ) Nature (London), 191, 144-148 Thayer, W. S. and Hinkle, P. C. (1975) J. Biol. Chem. 25(), 5330 5335 Thayer, W. S. and Hinkle, P. C. (1975)J. Biol. (item, 250, 5336-5342 Sorgatu, M. C., Branca. D. and Fergusom S. J. (1980) Biochem. J. 188, 945-948 Pullman, M. E. and Monroy, G. C. (19631J. Biol. Chem. 238, 3762-3769 Ernster, L. ( 19771Annu. Rev. Biochem. 46. 981-995 Storey, B.T., Scott, D. M. and Lee, C.-P. (1980)J. Biol. ('hem. 255, 5224 5229 Stoeckenius, W. and Lozier, R. H. (1974)Supramolec. Struct. 2, 769-774 Baum, H., Hall, G. S., Raider, J. and Beechey, R. B. (1971) in Energy Transduction in Respiration and Photosynthesis (Quagliariello, E.. Papa, S. and Rossi, C. S., eds), pp, 747-755, Adriatica Editrice. Bari
14drten Wik~trdm is at the Department o f Medical Chemistry, Universi O' o f Hel~inki, Sihavuorenpenger I OA, SF-O0170 HeL~inki 17, ["Tnland. Bayard T. Storey and (_Tman-pu Lee
(a) H o w is A/2H÷ g e n e r a t e d by the redox reactions of the respiratory chain carriers? (b) H o w is A/2w utilized by the A T P synthetase? (c) Is the H ÷ ' c o n n e c t i o n ' b e t w e e n the redox reactions and e n e r g y coupling osmotic (i.e. t r a n s m e m b r a n e ) or localized (i.e. i n t r a m e m b r a n e ) ? They p o i n t e d out that these questions have not been a n s w e r e d at the m o l e c u l a r level, and we fully agree with this. H o w e v e r , we feel that the existence of ' o p e n ' f r a g m e n t s of the inner m i t o c h o n d r i a l m e m b r a n e which retain the capacity for e n e r g y coupling does supply an a n s w e r to the third question: the connection is definitely localized, not osmotic. This may not be an a n s w e r in terms of a m o l e c u l a r m e c h a n i s m . But it is an a n s w e r which e m p h a s i z e s the key role of H + transfer reactions b e t w e e n molecules within the m e m b r a n e r a t h e r than H + e x c h a n g e s between vesicular c o m p a r t m e n t s and the medium, with the m e m brane acting only as p e r m e a b i l i t y barrier. It is o u r hope that these and further studies of energy coupling in 'open' fragments of the m i t o c h o n d r i a l m e m b r a n e , in which the c o m p l i c a t i o n of t r a n s m e m b r a n e A/2w is lacking, will refocus a t t e n t i o n on the m e m b r a n e phase as the d y n a m i c locus of protein reactions which m e d i a t e e n e r g y conservation and utilization.
References 1 Wikstr6m, M. and Krab, K. (1980) in Current Topics in Bionergetm~ (Sanadi, D. R., ed.), Vol. 1(1,pp. 51-101~ Academic Press, New York 2 Mitchell, P. (1966) Biol. Rev. 41,455 502 3 Williams, R. J. P. (19791 Biochim. Biophys. Acta, 505, 1~:14 4 Ernster, L. (1977) Annu. Rev. Biochem. 46,981 995 5 Lee, C. P. and Ernster, L. (1966)BBA (Biochim. Biophys. Acta) Libr. 7, 218-236 6 Storey, B.T., Lee, C. P.. Papa, S.. Rosen, S. G. and Simon, G. (1976) Biochemisto,, 15,928-933 7 Lee, C. P., Ernstcr, L. and Chance. B. (1969) Eur. J. Biochem. 8, 153 163 8 Baum. H.. Hall. G. S., Raider, J. and Beechey, R. B. (19711 in: Energy Transduction in Respiration and Photosynthesis (Quagliariell, E., Papa. S. and Rossi, C. S, eds), pp. 747-755, Adriatica Editrice, Barri 9 Ernster, L, Juntti, K. and Asauri, K. (1973)J. Bioenerg. 4, 148-159 10 Ferguson. S. J., Lloyd. W. J. and Radda, G. K. (19761 Biochim. Biophys. Acta, 423, 174-188 11 Von Jagow, G. and Klingenberg, M, (197(I) Eur. J. Biochem. 12,247 252 12 Storey, B. F., Scott. D. M. and Lee, C. P. (198(I) J. Biol. Chem. 255, 5224 5229 Bayard 72 Storey ts at the Departments oJ Physiology and Ohstetric~ and Gynecology, Universi O' o]" Pennsylvania School o f Medicine. PhihMelphia, PA 19104, U.S.A. and Chuan-pu Lee i~ at the Department o f Biochemistry, Wayne State University School o f Medicine, Detroit, %4148201. U.S.A.