Biochemica I Reviews The Stoicheiometric Relationships between Electron Transport, Proton Translocation and Adenosine Triphosphate Synthesis and Hydrolysis in Mitochondria MARTIN D. BRAND Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1Q W, U.K.

The validity of the conclusion that the mitochondrial redox- and ATP-driven proton pumps translocate 2 H+ ions per ‘high-energy phosphate bond’ has been challenged on theoretical and experimental grounds. This review attempts to highlight the difficulties of the present position and to introduce an alternative model within the framework of the chemiosmotic hypothesis based on a stoicheiometry of 3 H+ (or possibly 4 H+) per ‘high-energy phosphate bond’.

H + translocatiorr It is now well-established that both mitochondria and bacteria are able to eject H+ions into the suspending medium during respiration, by a process intimately linked to transport of electrons from substrate down the respiratory chain to Ot. However, controversy still rages over the mechanism by which this H+ ejection occurs, and whether or not it is an obligatory step on the main pathway of oxidative phosphorylation of ADP to ATP. The chemiosmotic hypothesis (Mitchell, 1966) proposes a role for the electrochemical gradient of protons as the intermediate in oxidative phosphorylation, and support for this position is growing (see Mitchell, 1976). Several mechanisms for the redox proton pump have been put forward in the past 15 years. For example it has been proposed that H+ ejection is due to a vectorial arrangement in the inner mitochondrial membrane of alternating hydrogen and electron carriers of the respiratory chain [the ‘loops’ of Mitchell (1966)] and/or to more complex arrangements of these carriers [e.g. the protonmotive Q-cycle of Mitchell (1975)]. A number of authors reject the loop hypothesis and prefer to consider the redox proton pump in more general terms, with H+ translocation driven by electron-transport-dependent pK changes or other effects (see Papa, 1976; Ernster, 1975). Indirect models of the pump postulate an additional step between electron transport and H+ ejection, such as a ‘high-energy’ chemical intermediate (e.g. Chappell & Crofts, 1965) or an energy-linked conformational change driving an electroneutral cation-H+ exchange (Azzone & Massari, 1973). A basic requirement of any model of the redox proton pump is that it should be compatible with the observed stoicheiometry of H+ ejection. In a similar way, models which attempt to explain the molecular mechanism of the ATP-driven proton pump (Mitchell, 1974; Boyer, 1975; Racker, 1976) must accommodate the appropriate number of protons. A second test of the various models is the constancy of the stoicheiometry under different conditions of pH etc. Thus the loop hypothesis at present demands a constant H+/site ratio of 2.0 (see below for definitions); generalized proton-pump models allow the possibility of variable stoicheiometry and may have values other than 2.0; and the indirect models make varying predictions in this respect. From these considerations it is apparentthat a degree of confidence in the stoicheiometric relationships involved is a prerequisite for the understanding of both the mitochondrial proton pumps and oxidative phosphorylation. Vol. 5

1616

BIOCHEMICAL SOCIETY TRANSACTIONS

Definitions To avoid confusion, the various ratios I shall consider are defined as follows. H+/site ratio: the number of H+ ions ejected (by mitochondria) during passage of a pair of electrons through one of the three energy-conserving regions (sites) of the respiratory chain between NADH and 02.The H+/2e- and H+/O ratios are to be avoided in this context as they do not specify the number of sites involved. H+/ATP.,,. ratio : the number of H+ ions translocated by the (mitochondrial) ATPase* per ATP hydrolysed or synthesized. H+/ATP,,,,a,I ratio: the number of H+ ions translocated by the ATPase and the phosphate and adenine nucleotide porters acting together during oxidative phosphorylation when the rate of ATP synthesis equals the extramitochondrial rate of ATP hydrolysis (i.e. in the steady state). H+/- ratio : The number of H+ ions translocated per 'high-energy phosphate bond' equivalent; this is equal to the H+/site and H+/ATPOveraI, ratios, and denotes either or both. Measurement of H + / - : older values A large number of observations have led to the conclusion that the H+/site and H+/ATP.,,, ratios are 2.0 in both mitochondria and bacteria (see Papa, 1976). The basis of these experiments has been the oxygen-pulse technique introduced by Mitchell & Moyle (1965, 1967). In this simple and elegant method mitochondria are incubated anaerobically in the presence of substrate until pre-existing ion gradients are dissipated. They are then pulsed with a small, known, amount of 02,which is rapidly consumed, allowing electron transport to occur with concomitant H+ ejection. To prevent build-up of a potential gradient which would oppose further lH+ ejection, the process is electrically compensated by uptake of added Ca2+,or K+ in the presence of the K+ ionophore valinomycin. Measurement of the change in concentration of H+ in the extramitochondrial medium with a pH electrode then allows calculation of the H+ ejected per 0 atom consumed, and hence the H+/site ratio. The values of 2.0 for the H+/site ratio determined in this way have also been found for various parts of the electrontransport chain by the following methods: by using different substrates which feed in electrons at different points; by using electron acceptors other than 02,such as ferricyanide or quinone, thus taking electrons from the chain at different levels; by pulsing with reductant (quinol) rather than with oxidant (e.g. Lawford & Garland, 1973). Submitochondrial particles also give H+/site ratios approaching 2.0 (e.g. Hinkle & Horstman, 1971), as do bacteria (e.g. Lawford & Haddock, 1973) and systems in which isolated respiratory-chain complexes are reconstituted into phospholipid vesicles [e.g. Ragan & Hinkle (1975), who obtained an H+/site ratio of 1.4 for NADH-coenzyme Q reductase]. Analogous experiments have been carried out on the ATPase system to determine H+/ATP,,,.. In this case H+ ejection is caused by hydrolysis of a pulse of ATP. Although the calculation is more complex and the asscmptions more open to error, particularly in intact mitochondria, values of 2.0 have been reported for the H+/ATP,.,, ratio in both intact mitochondria and submitochondrial particles (Mitchell & Moyle, 1965, 1968; Moyle & Mitchell, 1973; Thayer & Hinkle, 1973). One tacit assumption made in this work is that H+/ATP.,,, during hydrolysis of ATP has the same value as H+/ATP..,. during ATP synthesis; the latter has yet to be measured. A second important assumption is that H+/ATP,,,, = H+/ATP,,,,,I,, i.e. H+ movements associated with phosphate and adenine nucleotide translocation contribute nothing to H+/ATP,,e,,II (but see Mitchell, 1966; Greville, 1969; Papa, 1976). This assumption is probably invalid (see below). Problems associated with an H + / - ratio of 2.0 Several lines of evidence have come to the fore in the past few years which might indicate that the value of 2.0 for the H+/- ratio is not correct. Taken individually these * Abbreviation: ATPase, adenosine triphosphatase. 1977

BIOCHEMICAL REVIEWS

1617

pieces of evidence are not insurmountable, provided that a certain amount of contortion is allowed, but taken together they make a more compelling case for an H+/- ratio of at least 3.0. (1) Perhaps the most pressing argument against the present version of the chemiosmotic hypothesis is the observation that the measured electrochemical gradient of H+ (protonmotive force, A&+) is substantially less under the same conditions than the measured phosphorylation potential (AGp = AGO’+ 1.361og [ATPI/[ADPl[P,I), assuming H+/- = 2.0 (Slater et al., 1973; Nicholls, 1974; Rottenberg, 1975; Wiechmann et al., 1975),whereas the chemiosmotic hypothesis proposes that Aj&+ and AGp are at or near equilibrium in State 4 (resting). This serious objection to the hypothesis is abolished at a stroke if it is assumed that H+/- = 3.0; the measured values then give good agreement with the chemiosmotic model. (2) Since ATP/site = 1.O (by definition), the chemiosmotic hypothesis demands that H+/ATPo,,,aII= H+/site. This condition is apparently fulfilled by H+/ATPenr.= 2.0; H+/site = 2.0. However, this assumes that H+/ATP,,,,. = H+/ATP,,,,alr, which is not true. During oxidative phosphorylation ADP and P, must enter the mitochondria and ATP must leave; this is achieved by the combined action of the H,P04--OH- antiporter and the ADP3--ATP4- antiporter in the mitochondria1 inner membrane. A single turnover of these two carriers in the physiological direction results in net inward transport of 1 H+ ion. To accommodate transport of PI and adenine nucleotides during oxidative phosphorylation in vivo the chemiosmotic hypothesis is forced to deny the demonstrated reactions of the metabolite carriers, or to postulate that the ATP/site ratio may be less than 1.0. Neither explanation is satisfactory. The problem is neatly resolved, however, if H+/site is set at 3.0 and H+/ATPOveraII is divided into 2 H+/ ATP,,,. for the synthesis of ATP and an additional 1 H+ per ATP for the transport reactions mentioned above, summing to 3 H+/ATPovera1, (see below). (3) Ca2+uptake by mitochondria takes precedence over oxidative phosphorylation and uses the same energy store. It has been known for many years that 1.7-2.0CaZ+ ions are accumulated per site (Lehninger et al., 1967); this corresponds to between 3.4 and 4.0 charges/site if CaZ+is accumulated by electrophoretic uniport, i.e. by inward transport of Ca2+in the absence of other ion movements, in response to the electric potential set up by H+ ejection by the respiratory chain. This suggests that more than 2 charges are ejected per site during electron transport, and therefore that the H+/site ratio may be greater than 2.0. (4) Stoicheiometry in photosynthetic systems has been studied by a number of groups. Although there is still a great deal of discussion, a consensus seems to be emerging in favour of H+/- ratios of more than 2.0, with most values around 3.0 (see, e.g., Witt, 1975). Measurement of H + / - : more recent findings In view of the problems discussed above Lehninger and I decided to reinvestigate the stoicheiometry of the redox and ATP-driven proton-pumping systems in mitochondria. The results obtained argue strongly for H+/siteand H+/ATP,,.,,lI ratios of 3.0 or even as high as 4.0. (1) Uptake of a weak acid during Ca2+ uptake gives a measure of preceding H+ ejection. By using this technique we have demonstrated ejection of 3.54.OH+/siteduring Ca2+accumulation (Brand et al., 19766,~).This method suffers from the drawback that any H+ movements due to a second mode of Ca2+transport (e.g. Caz+-H+ antiport) in addition to the known electrophoretic uniport may contribute to the observed values; however, any such pathway has yet to be identified. It has the advantage that relatively huge amounts of H+ translocation occur, thus greatly decreasing errors due to transport of endogenous metabolites or other unsuspected reactions that might affect the value of the measured H+/site ratio. (2) I have introduced a kinetic method for evaluation of the H+/siteratio. H+ ejection is initiated by addition of substrate to substrate-limited aerobic mitochondria, and the initial steady rates of H+ ejection and 0, consumption are then compared, giving the H+/

Vol. 5

1618

BIOCHEMICAL SOCIETY TRANSACTIONS

0 and hence H+/site ratios (Brand et al., 19766; Reynafarje et al., 1976). When precautions are taken to prevent false readings due to dissipation (by uptake on the H2P04--OH- antiporter of endogenous phosphate which had previously leaked from the mitochondria) of the H+ gradient being set up, H+/siteratios of 4.0 are obtained with either CaZ+or K+ plus valinomycin present to prevent build-up of potential gradients. Phosphate transport may be decreased either by inhibition of the carrier with N-ethylmaleimide, or by washing the mitochondria free of their endogenous phosphate pool. If these precautions are not taken an H+/site ratio of close to 2.0 is obtained. (3) We have repeated the oxygen-pulse experiments of Mitchell & Moyle (1967) under conditions in which re-uptake of endogenous phosphate, previously leaked into the medium, is prevented by addition of N-ethylmaleimide; by depleting the endogenous pool of phosphate by washing under appropriate conditions, or by lowering the temperature to 5°C so that the rate of phosphate transport is greatly decreased. Under these conditions the observed H+/site ratio is 3.0 (Brand et al., 1976a,b). (4) The H+/ATP,,,. ratio has also been re-evaluated to determine whether phosphate movements affect the value obtained. By using an improved method H+/ATP,,,. values of 2.0 are obtained when phosphate transport is prevented, although as expected higher values approaching 3.0 and approximating to H+/ATP,,c,all are observed at low pH when phosphate movements are permitted (Brand & Lehninger, 1977). Problems associated with an H + / - ratio of 3.0 or 4.0 Although these more recent experiments remove the problems associated with an H+/- ratio of 2.0 mentioned above, they bring in their wake a new set, which, however, may be less severe. (1) Why do the oxygen-pulse experiments give an H+/site ratio of 3.0, although the kinetic method gives a value of 4.01This question remains to be resolved. (2) Why do systems such as submitochondrial particles and respiratory complexes reconstituted in vesicles, which presumably contain little endogenous phosphate or weak acids, give H+/site ratios of G 2 . 0 rather than 3.01 This is possibly due to technical problems, for example those concerned with leakiness or the presence of populations with mixed polarity. Do the values of about 2 H+/site observed with bacteria also represent an underestimate for reasons similar to those operating in mitochondria ? (3) Why do the oxygen-pulse and kinetic methods give values of 2.0 in mitochondria so consistently when phosphate movements are not prevented; is this coincidence, due to the size of the endogenous phosphate pool, or does it reflect some more complex effect such as a functional interaction of the phosphate carrier with the electrontransport chain ? Conclusions The most reasonable interpretation of the results obtained on the value of the H+/site ratio is that the previous measurements were underestimates due to the uptake of phosphate during the oxygen pulse, with concomitant net H+ influx and decrease of the observed H+ ejection. This phosphate was present in the extramitochondrial medium having leaked during the anaerobic preincubation from the endogenous pool within the mitochondria. These earlier results must therefore be regarded as unsatisfactory. The true H+/site ratio using the oxygen-pulse technique appears to be 3.0, which should be compared with an H+/ATP,,,. ratio of 2.0 obtained from the very similar ATP pulse experiments. Using the kinetic method the H+/site ratio appears to be 4.0. On the basis of these results Lehninger and I presented a revised chemiosmotic scheme of oxidative phosphorylation based on an H+/site ratio of 3.0, an H+/ATP,.,. ratio of 2.0, and an H+/ATP,,,,,II ratio of 3.0 (Brand & Lehninger, 1977). It is important to point out that, should the kinetic method prove to be a more valid measure of these ratios, the values would then be 4.0,3.0 and 4.0 respectively. The scheme based on H+/- = 3 is shown in Fig. 1 . It contains elements previously proposed by Mitchell (1966), Greville (1969), Klingenberg et al. (1969) and Papa (1976), but differs in that H+/site is greater by 1 unit than H+/ATP,,,., thus allowing phosphate and adenine nucleotide translocation to occur during oxidative phosphorylation without decreasing the ATP/site 1977

1619

BIOCHEMICAL REVIEWS

I

2 H’

Fig. 1. Vectorial scheme for steudv-state oxidative phosphorylation in mitochondria

M, mitochondria1 inner membrane; I, redox proton pump, at one site; 11, HzP04--OHantiporter, shown for convenience as a H2P04--H+ symporter; 111, proton-translocating ATPase (as proton-driven ATP synthase); IVYadenine nucleotide translocase.

(or P/O) ratio. It should be emphasized that this scheme applies to the steady state, in which the rate of ADP and PIentry is equal to the rate of oxidative phosphorylation and ATP exit, and thus the concentrations of adenine nucleotides and phosphate on each side of the membrane are constant. This situation is most readily achieved by setting the rate of ATP synthesis within the mitochondria equal to the rate of ATP hydrolysis in the extramitochondrial medium, which presumably approximates the situation in vii’o. If these constraints are not imposed there may be net H+ translocation associated with net phosphate movements, with resulting variation in the apparent efficiency of ATP synthesis. Fig. 1 shows ejection of 3 H+ at one site in the respiratory chain by an unspecified mechanism. This could be a proton pump of the type discussed by Papa (1976), or one of the other models mentioned above, but it could not be one of Mitchell’s loops in their present form, which requires an H+/siteratio of 2.0. If the results we have obtained are valid, then the loop hypothesis must be severely modified in order to allow ejection of more than 2 H+ per 2 e- at each site. Mitchell’s (1975) protonmotive Q-cycle does in fact eject 4 H+ per 2 e- at the level of ubiquinone; to this would have to be added a hydrogen carrier at the level of cytochrome c, which could eject a further 2 H+ for a total of 3 H+/site from succinate (or a further 4 H+ for a total 4 H+/site from succinate). In addition, some means of ejecting 3 H+ (or 4 H+) at coupling-site 1 would have to be devised. These problems appear formidable. Leaving aside the mechanism of the redox proton pump, Fig. 1 shows how the electrochemical proton gradient it sets up may be used to drive ATP synthesis by two separate reactions occurring simultaneously. In the first, 1 H+ is translocated inwards together with HzP04- on the H2P04--OH- antiporter, which is shown as a HZPO4--Hf symporter in Fig. 1 for convenience (the two mechanisms of uptake are at present indistinguishable). This reaction is electrically neutral; no charge is carried across the membrane. Charge is carried by the adenine nucleotide antiporter, which exchanges incoming ADP3- for ATP4-, thus causing net influx of one positive charge per turnover. Vol. 5

1620

BIOCHEMICAL SOCIETY TRANSACTIONS

The result of these translocation steps is t o cause net influx of 1 H+ and to lower the ATP concentration and raise the A D P and Pi concentrations in the mitochondria1 matrix relative t o the extramitochondrial medium. This of course tends t o drive the reaction A D P + P i + ATP+H,Oto theright in thematrix(Klingenbergetaf., 1969). In thesecond reaction the remaining 2 H+ return to the matrix through the proton-translocating ATPase, causing it t o act as an ATP synthase, as postulated in thechemiosmotic hypothesis (Mitchell, 1966), and thus forming 1 ATP molecule by some uncharacterized mechanism (e.g. Mitchell, 1974; Boyer, 1975; Racker, 1976). I n this way, in contrast with the original form of the chemiosmotic hypothesis, about one-third of the energy for ATP synthesis by mitochondria is invested at the level of the phosphate and qdenine nucleotide porters, and only two-thirds is invested in the actual enzymic production and release of ATP from A D P and Pi. Awone, G. F. & Massari, S. (1973) Biochim. Biophys. Acta 301, 195-226 Boyer, P. D. (1975) FEBS Lett. 58, 1-6 Brand, M. D. & Lehninger, A. L. (1977) Proc. Natl. Acad Sci. U.S.A. 74, 1955-1959 Brand, M. D., Reynafarje, B. & Lehninger, A. L. (1976a)J. Biol. Chem. 251,5670-5679 Brand, M. D., Reynafarje, B. & Lehninger, A. L. (19766) Proc. Natl. Acad. Sci. U.S.A. 73, 437-441 Brand, M. D., Chen, C.-H. & Lehninger, A. L. (1976c)J. Biol. Chem. 251,968-974 Chappell, J. B. & Crofts, A. R. (1965) Biochem. J. 95, 393402 Erster, L. (1975) Proc. FEBS Meet. 40,253-276 Greville, G . D. (1969) Curr. Top. Bioenerg. 3, 1-78 Hinkle, P. C. & Horstman, L. L. (1971) J . Biol. Chem. 246, 6024-6028 Klingenberg, M., Heldt, H. W. & Pfaff, E. (1969) in The Energy Leveland Metabolic Control in Mitochondria (Papa, S . , Tager, J. M., Quagliariello, E. & Slater, E. C., eds.), pp. 237-253, Adriatica Editrice, Bari Lawford, H. G. & Garland, P. B. (1973) Biochem. J. 136, 711-720 Lawford, H. G. & Haddock, B. A. (1973) Biochem. J. 136,217-220 Lehninger, A. L,., Carafoli, E. & Rossi, C. S. (1967) Ado. Enzymol. 29,259-320 Mitchell, P. (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin Mitchell, P. (1974) FEBS Lett. 43, 189-194 Mitchell, P. (1975) FEBSLett. 59, 137-139 Mitchell, P. (1976) Biochem. SOC.Trans. 4, 399-430 Mitchell, P. & Moyle, J. (1!)65) Nature (London) 208, 147-151 Mitchell, P. & Moyle, J. (1967) Biochem. J. 105, 1147-1162 Mitchell, P. & Moyle, J. (1968) Eur. J. Biochem. 4, 530-539 Moyle, J. & Mitchell, P. (1973) FEBS Lett. 30, 317-320 Nicholls, D. G. (1974) Eur. J. Biochem. 50, 305-315 Papa, S. (1976) Biochim. Biophys. Acta 456,39-84 Racker, E. (1976) Trends Biochem. Sci. 1, 244-247 Ragan, C. I. & Hinkle, P. C. (1975) J. Biol. Chem. 250, 8472-8476 Reynafarje, B., Brand, M. D. & Lehninger, A. L. (1976) J. Biol. Chem. 251, 7442-7451 Rottenberg, H. (1975) J. Bioenerg. 7 , 61-74 Slater, E. C., Rosing, J. & Mol, A. (1973) Biochim. Biophys. Acta 292, 534-553 Thayer, W. S. & Hinkle, P. C. (1973) J. Biol. Chem. 248, 5395-5402 Wiechmann, A. H. C. A., Beem, E. P. & van Dam, K. (1975) in Electron Transport Chains and Oxidative Phosphorylution (Quagliariello, E., Papa, S., Palmieri, F., Slater, E. C. & Siliprandi, N., eds.), pp. 335-342, North-Holland, Amsterdam Witt, H. T. (1975) in Bioenergetics of Photosynthesis (Govindgee, ed.), pp. 493-554, Academic Press, New York

1977