Chapter 23 The Role of Electrogenic Proton Translocation in Mitochondrial Oxidative Phosphorylation

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 16

Chapter 23 The Role of Electrogenic Proton Translocation in Mitochondrial Oxidative Phosphorylation JANNA P . WEHRLEI Department of Physiological Chemistry The Johns Hopkins University School of Medicine Baltimore, Maryland

Introduction ....................................................................................... Respiration-Dependent Proton Pumping ........... ....................................... A. Structure of the Mitochondrial Respiratory Chain ................................. B. Models for Respiration-Dependent Proton Pumping .............................. C. Data on Respiratory Chain Proton Pumping ........................................ 111. Reversible Electrogenic Proton Translocation by the Fl-Fo ATPase ................. A. Structure of the Mitochondrial F,-F, ATPase Complex ......................... B. Models for Synthesis of ATP ............................................................ C. Electrogenic Proton Pumping by the Mitochondrial F,-Fo ATPase Complex .......................................................................... IV. The Role of Proton Translocation in Mitochondrial Oxidative Phosphorylation .................................................................................. A. Models for Energy Coupling ............................................................ B. Correlation between Phosphorylation Potential and AKH+ .................................................................................... C. Synthesis of ATP by Artificial Proton Gradients ................................... V. Electrophoretic Metabolite Transport ....................................................... VI. Summary ........................................................................................... References ............................................. ....................................... I. 11.

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I Present address: Department of Chemistry, University of Maryland-Baltimore County, Catonsville, Maryland 21228

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Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153316-6

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I.

INTRODUCTION

Oxidative phosphorylation in eukaryotes is accomplished through the simultaneous operation of no less than four electrogenic proton “pumps” embedded within the inner membrane of the mitochondrion. Three of these ordinarily operate in the forward direction, producing net translocation of protons from the mitochondrion during substrate oxidation. In contrast, the F,-F, ATPase functions in reverse, as a proton-driven ATP synthetase during oxidative phosphorylation. Like its counterpart in bacteria, the ATPase complex of mitochondria can also function as a proton-pumping ATPase, resulting in the accumulation of cations or reversal of electron flow through the respiratory chain. In addition to the proton pumps, mitochondria also contain a number of other transport sytems that catalyze net transfer of charge. These are electrophoretic systems, driven by the membrane potential created by the proton pumps, but do not function electrogenically under physiological conditions. The two major electrophoretic transport systems are the ATP4-ADP3- exchange translocator and the Ca2+ uniporter. Mitochondria also contain proton-driven translocators which are electrically neutral and therefore are driven solely by the proton concentration gradient. In the early 1960s Mitchell (1961) and Williams (1961) independently introduced the concept that proton movement stabilized by the special structure of the biological membrane might be responsible for storing energy released during substrate oxidation and transferring this energy to the synthesis of ATP. Although hypotheses about the precise nature of the proton movement vary and are currently the subject of much research and debate, it now seems likely that this general mechanism is involved not only in oxidative phosphorylation in mitochondria but in oxidative phosphorylation in bacteria and in photophosphorylation in chloroplasts as well. In comparing models for oxidative phosphorylation it is important to realize that there are at least three separate major problems involved: (1) the mechanism of respiratory chain proton pumping, (2) the mechanism of ATPase proton pumping and ATP synthesis, and (3) the mechanism of conversion of redox to phosphate bond energy. Not all models address all three problems, and more importantly most experimental data address only one of these problems. In this article I have attempted to isolate the three problems of oxidative phosphorylation insofar as possible, to present in each case several models currently under consideration, and to present the most pertinent data which may lead to an understanding of the molecular mechanism and the true role of electrogenic proton pumping in mitochondria.

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11.

RESPIRATION-DEPENDENT PROTON PUMPING

A. Structure of the Mitochondria1 Respiratory Chain An abbreviated view of the respiratory chain is shown in Fig. 1. A more detailed picture may be found in Hatefi and Galante (1978). The points marked “ATP” are the so-called sites of oxidative phosphorylation. This term refers to electron transfers which release sufficient energy for synthesis of one molecule of ATP, and not to the locution of ATP synthesis, which is the F,-F, ATPase. Thus substrates whose oxidations produce NADH cause electron transfer through all three sites and have P/O ratios of 3, because nearly three ADPs are phosphorylated per oxygen consumed, that is, per two electrons transferred. Electrons from succinate (and other FAD-linked substrates) cross sites 2 and 3 only, and so have P/O ratios near 2. Certain artificial substrates which reduce cytochrome c have P / O ratios of 1 or less. Experimental values are typically extrapolated to the next highest integer, but the existence of a precise stoichiometry relation between phosphorylation and electron transport has not been unequivocally established. The sequence of electron transfer components shown in Fig. 1 was developed on the basis of studies with site-specific inhibitors and a variety of electron donors and acceptors of known redox potential. Its physical reality can be demonstrated when the membrane is disrupted with detergent. Certain sections of the chain persist as well-defined, functional multipolypeptide complexes, indicating relative internal cohesiveness. These complexes, designated I-IV, correspond rather strikingly to the sites of oxidative phosphorylation demonstrated classically. (For a review, see Hatefi and Galante, 1978.) Complex V, isolated similarly, is the F,-F, ATPase. Complex I contains the proteins for the reduction of quinones by NADH (site I). Complex I1 contains succinic dehydrogenase and other proteins necessary for the reduction of quinone by succinate. This section is not a site of phosphorylation. Complex I11 (cytochromes b and c1and other

NADH+~Qp,&cyti DEHYDRO

.&02

succinato

FIG. 1. Organization of the mitochondria1 respiratory chain.

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proteins) transfers electrons from quinones to cytochrome c and so includes site 2. Complex IV (cytochrome oxidase) catalyzes the oxidation of cytochrome c by oxygen (site 3). This complex includes cytochromes a and a, and copper.

B. Models for Respiration-DependentProton Pumping Figure 2 illustrates the orientation of proton pumping systems of the inner membrane. In intact mitochondria (Fig. 2A) the F,-F, ATPase faces the internal compartment, the matrix. The side of the membrane bearing the ATPase is referred to as the M (matrix)-side. The opposite side is the C (cytoplasmic)-side. These names are retained even in submitochondrial vesicles, which are “inside-out” (Fig. 2B). These vesicles, prepared by sonicating the inner membrane, played an important role in examinations of the spatial arrangement of the inner membrane. Models for proton pumping fall into two categories, the “direct chemiosmosis” model of Mitchell (1979) and the molecular proton pump model, which includes many variations. In Mitchell’s hypothesis the protons that are translocated are formed from redox hydrogens. Redox carriers of hydrogen and carriers of electrons alternate throughout the chain folded back and forth across the membrane so that only hydrogens move from the M-side to the C-side, and only electrons from the C-side to the M-side (Fig. 3A). Hydrogens donated at the M-face discard their protons at the C-face before the electron travels back across the membrane during its next transfer, and then an M-side proton must be absorbed before hydrogen can recross the membrane in the subsequent step. This model has several testable predictions. First, the number of protons translocated per electron must equal the number of complete “loops” traversed. Second, the structure of the respiratory chain must consist of alternating hydrogen

B

A

FIG.2. Orientation of proton translocation during oxidative phosphorylation in (A) mitochondria and (B) submitochondrial vesicles.

U

INTACT MITOCHONDRION

SUBMITOCHONDRIAL VESICLE

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41 1

FIG.3. Models for respiratory chain proton translocation. (A) Mitchell’s (1974) direct chemiosmosis. (B) Proton pumping due to protein conformational changes accompanying the redox transition. See text for details.

and electron carriers appropriately disposed at the M- and C-faces and suitable for transmembrane electron transfer. Specifically, regions of pure electron transfer are not candidates for proton pumping according to this scheme. In contrast to the direct formulation of Mitchell are the mechanisms described as molecular proton pumps (Fig. 3B). According to any of these relatively undefined models, redox reactions cause conformational changes in protein components of the chain, resulting in net proton translocation. Often invoked is the Bohr effect in hemoglobin, where binding of oxygen causes a change in the protonation state of the protein. In the case of mitochondria, a “vectorial Bohr effect’’ has been suggested, in which a proton is taken up at the M-face on reduction but released at the C-face upon oxidation. These models in general lack enough detail to permit complete experimental evaluation. However, one may expect a pH-dependence of certain redox transitions, and probably a stoichiometric relationship between protons pumped and electrons translocated, although the ratio could be any number. Structural features might resemble known molecular ion pumps or be similar at all three sites of proton pumping in the respiratory chain. However, the possibility of three unique molecular pumps is a real one. It should be noted that a chemiosmotic scheme for energy conservation does not depend upon the mechanism of proton pumping, but upon equilibration of the translocated protons with the aqueous phase before their use for ATP synthesis.

C. Data on Respiratory Chain Proton Pumping Electrogenic proton translocation during coupled respiration has been observed under a wide variety of conditions almost since the idea was first suggested (Mitchell, 1961). Although it has not yet been possible to observe a substantial respiration-dependent, interior negative transmembrane potential in mitochondria using microelectrodes (Bowman et al., 1978), a wide variety of other techniques have produced results consistent with the existence of such a potential. During respiration cations are accumulated

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by intact mitochondria when provided with either endogenous or exogenous pathways for electrophoretic transport. These include Ca*+, K+ in the presence of the ionophore valinomycin, and lipid-soluble cations such as tetraphenylphosphonium (Brand et al., 1976; Kamo et al., 1979). Respiration-dependent changes in certain potential-sensitive indicator dyes are identical to those observed in the presence of artificially imposed diffusion potentials which are inside negative (Jasaitis et al., 1971). The results obtained with these and other techniques, although not quantitatively identical (Azzone et al., 1978a,b), support the conclusion that respiration induces formation of a proton electrochemical gradient of between 180 and 220 mV, alkaline and negative inside (Nicholls, 1974; Kamo et al., 1979). Sorgato et al. (1978) measured a gradient of 185 mV positive and acid inside in submitochondrial vesicles. For the present it does not seem useful to disregard the existence of a respiration-dependent potential on the basis of the microelectrode data alone. For a discussion, see Tedeschi (1979) and Rottenberg (1979). The observation of respiration-dependent proton extrusion by mitochondria is dependent upon the presence of a permeant ion to provide charge compensation, indicating that development of a transmembrane electrical potential opposes further H+ translocation. (But for an alternative explanation see Kell, 1979.) Uncoupling, the state in which respiration is maximized and its ability to do work is abolished, occurs only when a path for proton recycling is created by protonophores or by a combination of ionophores which provide a complete electrophoretic proton return pathway (e.g., valinomycin plus nigericin) or by disruption of the membrane. Taken together, these results indicate that electrogenic proton translocation is a necessary part of energy-conserving respiration. Two approaches have been taken in the search for the location and molecular mechanisms of proton pumping by the respiratory chain. In the first approach inhibitors and artificial redox donors and acceptors are used to bracket isolated spans of the chain in mitochondria or submitochondrial vesicles. In the second approach the components of the chain are physically isolated, purified, and then reconstituted into phospholipid vesicles and assayed for respiration-dependent charge and proton transfer. The combination of these two approaches is yielding a more unified picture of the proton-translocating systems of the respiratory chain. 1. TRANSLOCATION OF PROTONS BY THE INTACT RESPIRATORY CHAIN Knowledge of the location and stoichiometry of H+ and e- movements during respiration is a necessary prerequisite for an adequate model of electrogenic proton translocation. Unfortunately, knowledge of the specific

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redox transfers associated with H + translocation is only beginning to emerge, and there is no general agreement on proton stoichiometry at any of the energy-conserving sites. Recently determined stoichiometries from several laboratories are summarized in Table I. Early values for H+ transfer were very close to 2 H + / 2 e- per site for a variety of substrates in mitochondria (Mitchell and Moyle, 1967) and in submitochondrial particles (Hinkle and Horstman, 1971), in keeping with the hypothesis of Mitchell. The same ratio has continued to receive supporting data from Mitchell's laboratory (Moyle and Mitchell, 1978). Other laboratories have shown, however, that respiration-dependent uptake of even small amounts of phosphate (which is coupled with H+ uptake) can cause a substantial underestimation of the number of protons ejected by mitochondria during respiration in the presence of permeant cations. Measurements of H translocation where phosphate movement has been inhibited or where endogenous phosphate has been removed (Brand et al., 1976) give values of at least 3 H+/2 e- per site for all substrates tested. Indeed, steady state measurements, as contrasted with the usual oxygen pulse measurements, yield H+/2 e- per site ratios approaching 4 in experiments with a variety of techniques and substrates. Net charge transfer ratios of 4 K+ /2 e- per site were measured in the presence of valinomycin whether phosphate moved or not (Reynafarge and Lehninger, 1978). In many cases the observation of different values by different laboratories can be rationalized in terms of experimental conditions, but whether the highest values are the physiologically significant ones is not easy to say. Although observations of Hf per site ratios greater than 2 are now widely reported, it is not clear whether all three coupling sites actually function with the same ratio. Brand et al. (1978) have titrated mitochondria with low levels of substrates and determined the dependence of the resul+

TABLE I PROTONS TRANSLOCATED PER TWO ELECTRONS DURING RESPIRATION Site

Mitchell and Moyle"

Lehninger et at.6

Brand et al.=

Wikstrom et

Azzone et aLe

1 2 3

Two Two Two

Four Four Four

Two Two Four

Two or three Two Four

Four Four Four

' Mitchell (1979).

Lehninger et al. (1979). Brand et at. (1978). Wikstrom and Krab (1979). Azzone et at. (1979a); Pozzan et al. (1979).

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tant membrane potential upon the rate of oxidation, using safranine dye to track the potential. Different substrates were used to bracket either all three sites (3-hydroxybutyrate), or site 2 plus site 3 (succinate), or only site 3 (isoascorbate). In all cases plots of potential versus the rate of oxygen consumption were linear, with slopes in the ratio 2.0: 1.5: 1.O, respectively. The result is consistent with the translocation of different numbers of protons at the three sites: e.g., 2, 2, and 4 at sites 1, 2, and 3, respectively. One broadly accepted result is the stoichiometry of Ca2+uptake to oxygen consumption. Both Reynafarge and Lehninger (1977) and Moyle and Mitchell (1977a) agree that two Ca2+are taken up per electron pair per site. However, disagreement over the net charge transfer associated with Caz+ uptake (Section V) has allowed this to be used as evidence for the ejection of either two or four H + per site (Moyle and Mitchell, 1977b; Fiskum et al., 1979). Perhaps the greatest controversy at present involves the possibility of proton translocation below cytochrome c. Because the redox reactions through this region are exclusively electron transfers they cannot form a Mitchell loop. Mitchell has positioned the third proton translocation as part of a stepwise reduction of ubiquinone (Q cycle, Mitchell, 1979). This would leave the last transmembrane electron transfer alone at the third site, but a number of laboratories (Wikstrom, 1975; Azzone et al., 1979; Alexandre et al., 1979) have found that electron transfer from ferrocyanide or reduced cytochrome c is in fact accompanied by translocation of protons. Although the stoichiometry varies from 2 to 4, depending on the experimental conditions, the results of Wikstrom and others appear clearly incompatible with a direct Mitchell loop at site 3. Some kind of protonpumping conformation change almost certainly is involved. More evidence on this subject is found in studies on purified cytochrome oxidase (see below). 2. PROTON PUMPING BY PURIFIED RECONSTITUTED RESPIRATORY CHAINCOMPLEXES Each respiratory chain complex associated with a site of oxidative phosphorylation has been shown to function as an oxidation-driven, electrogenic proton pump when purified and reconstituted into phospholipid vesicles. In contrast, the succinic dehydrogenase complex does not appear to pump protons during oxidation. The NADH dehydrogenase-containing complexes from rat liver and from beef heart mitochondria have been shown by Lawford and Garland (1972) to couple reduction of the ubiquinone analog Q, by NADH to an uncoupler-sensitive translocation of protons into the interior of phospho-

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lipid vesicles. H +/2 e- ratios of 0.75 and 1.4 were obtained for the liver and heart enzyme, respectively. Ragan and Hinkle (1975) obtained similar results with beef heart complex I. Oxidation was stimulated threefold by uncoupling with a protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a combination of K+ plus valinomycin plus nigericin, suggesting that the development of A&, + inhibited oxidation. Proton uptake was dependent on charge compensation, in this case K+ plus valinomycin. Oxidation also drove the accumulation of a lipophilic anion, tetraphenylboron (TPB-), indicating a positive potential in the interior of the vesicles. (Operational asymmetry in this system is obtained because the substrate NADH cannot itself enter the vesicles.) The components responsible for proton translocation have not been identified. Complex I has both hydrogen and electron carriers, as required for a Mitchell loop. The pH dependence of component midpoint potentials in this region has not been thoroughly examined; it is known, however, that the electron acceptor ubiquinone has a pH-dependent midpoint potential (Dutton and Wilson, 1974), as would be necessary for a redox change in equilibrium with proton binding or release, and that two of the iron-sulfur centers appear to have midpoint potentials sensitive to the presence of ATP (Ohnishi and Pring, 1974). For a review on complex I, see Ragan (1976). Purified complex I11 has been shown by Leung and Hinkle (1975) to translocate protons when reduced Q2 is oxidized by exogenous cytochrome c. These authors observed outward translocation of approximately 2 H+/2 e-. In the same experiments oxidation was stimulated 10-fold by uncoupling with either protonophores or K+ plus valinomycin and nigericin. While the ubiquinone-to-cytochrome c step provides one site of Mitchelltype proton release in this region, the absence of any hydrogen carriers after ubiquinone has lead Mitchell to postulate the Q cycle (Mitchell, 1975), by which a two-stage oxidation of ubiquinone would produce net translocation of two protons per election, leaving only the final transmembrane electron transport to complex IV (cytochrome oxidase). Potential sites for proton pumping by other mechanisms are also limited. Cytochrome c, has a pH-insensitive midpoint potential, but one form of cytochrome b (b566) appears to have a midpoint potential which is changed by deprotonation (von Jagow, 1979) and is sensitive to the presence of ATP (Dutton and Wilson, 1974). In addition, evidence that the reduction induces a conformational change in complex 111 is substantial (e.g., Rieske et al., 1967). For a review on complex 111, see Rieske (1976). Extensive study of purified complex IV (cytochrome oxidase) confirms the results of studies with the intact respiratory chain, suggesting that protons are translocated during the reduction of cytochrome c by oxygen (Krab and Wikstrom, 1978; Sigel and Carafoli, 1979; Wrigglesworth and

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Nicholls, 1979; Casey et al., 1979). Cytochrome oxidase catalyzes an inhibitor and uncoupler-sensitive H+ ejection equivalent to 2 H+/2 e-, but nearly 4 K+/2 e- are taken up, compensating both for protons consumed to form water (2 H +12 e-) and for protons translocated (apparently 2 H+12 e-). A wide variety of substrates have been used. Translocation is strictly proportional to the number of turnovers of the enzyme and is in excess of the amount of any prereduced components of complex I11 which might be present. (See the arguments of Moyle and Mitchell, 1978; and Lorusso et a[., 1979.) In the purified system stoichiometries higher than 2 H + / 2 ehave not been observed. Whether the higher stoichiometries observed in the laboratories of Lehninger and of Azzone in intact mitochondria represent artifacts or whether the coupling in intact mitochondria is much better than in the purified complexes is not known at the present time. Candidates for proton-releasing redox proteins in complex IV include cytochromes a and a,, both of which have pH-sensitive midpoint potentials. In contrast, the midpoint potential of the copper appears not to be influenced by pH (Dutton and Wilson, 1974). (For a review on cytochrome oxidase, see Wikstrom and Krab, 1979.) 111. REVERSIBLE ELECTROGENIC PROTON TRANSLOCATION BY THE F,-F, ATPase A. Structure of the Mitochondria1 F,-F, ATPase Complex The F,-F, ATPase of mitochondria, like its counterpart in bacteria and chloroplasts, has a structure much more complex than that of many other electrogenic ion pumps (Soper et al., 1979; Kagawa, 1978; Nelson, 1976). F,-F, ATPase complexes of mitochondria from a variety of sources appear to consist of at least 9 types of polypeptide chains and as many as 20 peptides per functional unit (Serrano et al., 1976; Stiggal et al., 1978; Soper et af., 1979). Located in the inner mitochondria1 membrane, the ATPase complex has traditionally been described as a “ball-and-stalk” arrangement (Fig. 2) where the “ball” represents the chemical catalytic portion of the complex, designated F, F, has been suggested to be attached to the membrane sector (designated F,) by an ill-defined protein “stalk.” The appropriateness of this well-used model has recently been confirmed experimentally. Soper et al. (1979) have shown electron micrographs of a dispersed preparation of ATPase complex (“oligomycin-sensitive ATPase” in that reference) from rat liver mitochondria in which the individual enzyme complexes bear an unmistakable resemblance to the artists’ versions published earlier. The F, portion of the ATPase complex can be dissociated from the membrane-bound peptides by a variety of techniques. The F, ATPase alone

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contains five types of polypeptide chains and has a molecular weight in the range 350,000-380,000 (Catterall and Pedersen, 1972). Isolated F, contains all the catalytic sites involved in the non-energy-coupled hydrolysis of ATP, but its activity is no longer inhibited by oligomycin or dicyclohexylcarbodiimide (DCCD), which inhibit both ATP synthesis and ATP hydrolysis when the enzyme is bound to its proper membrane sector. Less well characterized is the membrane or F, sector of the ATP synthetase complex, consisting of highly hydrophobic membrane proteins and coupling proteins which bind F, to the membrane. The major component of F, is the DCCD-binding protein. This small, CHC1,-MeOH-soluble protein is the binding site for both DCCD and oligomycin. F, appears to form a proton-conducting channel through the mitochondrial membrane. When reconstituted into phospholipid vesicles, it induces a proton-specific conductance which can be inhibited by DCCD or oligomycin or by rebinding F, (Shchipakin et al., 1976). In addition to the peptides of F, and F, the ATP synthetase complex appears to contain a small inhibitor or regulator peptide. First reported by Pullman and Monroy (1963), the peptide inhibits ATP hydrolysis under certain conditions and appears not to inhibit ATP synthesis under conditions which favor linear rates of synthesis (Cintron and Pedersen, 1979), but its mode of action is far from clear. For a review on the mitochondrial ATPase complex, see Pedersen et al., 1978. B. Models for Synthesis of ATP Models for ATP synthesis may discuss only catalysis or only coupling or may include both. Most models which consider only catalysis only invoke an energy-dependent conformational change of unspecified origin to drive phosphate bond formation or release of preformed ATP. Two such models are illustrated in Fig. 4. The work of Tiefert and Moudrianakis (1979) on the analogous chloroplast ATPase system has lead these authors to suggest that enzyme-bound AMP, rather than ADP, may be the acceptor of the new phosphodiester bond, this first step being followed by an adenylate kinase-type transfer of P to an acceptor ADP. The quite different model of Boyer et af. (1973) shares the idea that addition of Pi to ADP may not be the energy-requiring step in phosphorylation. According to the “alternating catalytic site” mechanism, tightly bound ADP is the primary acceptor but reacts nearly at equilibrium to form ATP. ATP is then expelled from its binding site in an energy-requiring reaction, promoting recycling of the enzyme. In fact Rosing et al. (1977) have reported isotope exchange data consistent with the notion that formation of the P,y-phosphodiester bond is relatively insensitive to protonophore uncouplers.

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FIG.4. Conformational models for catalysis of ATP hydrolysis and synthesis. (A) Boyer et al. (1974) have suggested an alternating catalytic site mechanism. (B) Tiefert and Moudrianakis (1979) have suggested that AMP rather than ADP may be the primary acceptor in phosphorylation by chloroplast F,-F, ATPase. See text for details.

Other models for synthesis describe explicitly a role for protons in ATP synthesis. These are therefore also models for ATP-dependent H+ pumping and ultimately must take some position on the nature of energy coupling. These models may be divided into two categories, described below. 1. CHEMIOSMOTIC MECHANISMS OF SYNTHESIS

Mitchell has suggested direct participation in the chemical reactions of ATP synthesis as the simplest mechanism by which translocation of protons can aid in the dehydration of ADP and Pi (Mitchell, 1973). As illustrated in Fig. 5A, his model designates the proton-conducting channel of the F, as a “proton well” in which the proton concentration increases as the thickness of the membrane is traversed, parallel to the reduction in A$. At the M-surface, where the concentration of protons corresponds to the total A,!iH+, the active site of F, is organized in space so that protonation of the oxygen departing from phosphate is favored, without permitting protonation of ADPO-. This is followed by an attack of ADP on the phosphorus, with phosphodiester bond formation and the release of H,O.

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41 9

B

2H*

FIG. 5 . Models for energy coupling to ATP synthesis. (A) Mitchell’s (1974) direct chemical involvement mechanism for proton-driven ATP synthesis. (B) Protonation of the protein may induce a conformational change resulting in ATP synthesis by mechanisms such as those shown in Fig. 4.See text for details.

ATP is thought to leave the catalytic site in a protonation state, which differs from the protonation of ADP + Pi by exactly the number of protons used in the protonation steps, so that release of ATP into solution on the M-side completes translocation of the protons. Mitchell has frequently suggested that two protons are involved (Moyle and Mitchell, 1973) but, as discussed below and as mentioned by Mitchell (1977), the number may be three, which does not eliminate the possibility of direct chemical involvement. Still consistent with a role for A,iiH+ as the driving force for ATP synthesis are the conformational model of Boyer (1975) and the magnesium complex model of Racker (1977). These are indirect, by comparison with Mitchell’s model, since the electrochemical gradient for protons would only serve to release energy (in the form of ATP) from storage. To distinguish further between direct and indirect chemiosmotic models would require detailed molecular information not currently available. Now that F,-F, ATPase complexes have been purified from several mitochondria1 sources (Serrano et al., 1976; Stigall et al., 1978; Soper et al., 1979), it is to be hoped that such necessary information will soon be forthcoming.

2. NONCHEMIOSMOTIC MECHANISMS OF SYNTHESIS Nonchemiosmotic models for energy coupling differ substantially in the role assigned to proton translocation. Few contain any specific predictions about the mechanism of ATP synthesis. R. J. P. Williams (1975) has suggested that a high local concentration of protons in some nonaqueous phase of the membrane assists in the dehydration by immediate protonation of the newly formed water, removing it from the active site. In this

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model protons in the bulk aqueous phase-and therefore proton pumping as it is commonly conceived-play no role. Direct protein-protein conformational interactions are still considered necessary to explain all the available data, according to some authors (Slater, 1977).

C. Electrogenic Proton Pumping by the Mitochondria1 F,-F, ATPase Complex As discussed earlier in connection with respiratory chain-linked activity (Section II,A), the generation of a substantial membrane potential due to ATP hydrolysis by the F,-F, ATPase complex has not been observed directly using microelectrodes. Nonetheless it is an absolute prediction of the chemiosmotic hypothesis that the ATP complex is an electrogenic H + pumping device. Therefore this proposal has been examined by other techniques. The evidence that the F,-F, ATPase translocates protons during ATP hydrolysis and that such translocation is electrogenic falls into two categories. Kinetic studies have measured the rate of appearance of protons during ATP hydrolysis. Other studies measure the establishment of a steady state transmembrane electrochemical potential difference in protons generated during ATP hydrolysis, using a wide variety of mobile ions or pH- or potential-sensitive dyes. Although the quantitative results are not identical, every technique used so far has produced data consistent with net electrogenic translocation of protons from the M-face aqueous phase to the C-face aqueous phase during coupled ATP hydrolysis in intact mitochondria, in submitochondrial particles, and in liposomes containing the purified F,-F, ATPase complex. 1. PROTONPUMPING BY F,-F, IN INTACT MITOCHONDRIA AND SUBMITOCHONDRIAL VESICLES

Hydrolysis of added ATP, by respiration-inhibited mitochondria in the presence of K+ plus valinomycin, is accompanied by the appearance of protons in the external medium (Mitchell and Moyle, 1968; Brand and Lehninger, 1977). The ejection is blocked by oligomycin (which also inhibits ATP hydrolysis) and also by protonophores, which actually stimulate ATP hydrolysis. This suggests that translocation rather than net production of protons is occurring. In the absence of a mobile cation neither proton ejection nor substantial ATP hydrolysis occurs, which suggests the need for charge compensation in order to obtain repeated cycles of ATP hydrolysis. Reynafarge and Lehninger (1978) report the inward movement of K+ equivalent to the outward movement of protons. The quantitative aspect of ATP-driven proton pumping is still the sub-

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ject of debate. Mitchell and Moyle (1968) and Brand and Lehninger (1977), using small pulses of ATP, observed a proton/ATP ratio of about 2. More recently Alexandre et al. (1978) have reported steady state rate ratios of three protons ejected per ATP hydrolyzed, as well as three K + taken up per ATP hydrolyzed in the presence of valinomycin (Reynafarge and Lehninger, 1978). This suggests fully electrogenic translocation of three H+ per ATP. Translocation ratios were equivalent when Ca2+was used for charge compensation (Alexandre et al., 1979). Ca2+has an endogenous system for rapid electrophoretic uptake, avoiding the sometimes criticized practice of using ionophores. Measurements of the steady state A,%”+generated by intact mitochondria hydrolyzing ATP are not as numerous as those measuring the respirationdependent state, although qualitative indications of the development of an internally negative transmembrane potential have been observed using a variety of methods. Nicholls (1974), using the distribution of Rb+ in the presence of valinomycin and HOAc, reported a A$ of 125 m V and a ApH corresponding to 85 mV in rat liver mitochondria hydrolyzing ATP. More recently, using trace amounts of the permanent cation tetraphenylphosphonium, Kamo et al. (1979) measured values of A$= 150 mV and ApH = 30 mV. This method, which does not employ valinomycin, demonstrates that a potential is generated by the low “state 4-like” ATP hydrolysis which occurs under tightly coupled conditions, as well as during hydrolysis facilitated by rapid charge compensation as with K+ plus valinomycin. ATP hydrolysis by the F,-F, ATPase in submitochondrial vesicles is accompanied by protonophore-sensitive proton uptake (Moyle and Mitchell, 1973; Thayer and Hinkle, 1973). Because submitochondrial vesicles are inverted relative to intact mitochondria, the ATPase has direct access to ATP without intervention of the electrogenic ATP-ADP exchange translocator. Measurements were carried out at pH 6.2-6.3 in both studies to avoid scalar proton release due to inorganic phosphate, which in these vesicles is released into the external medium. Under these conditions ratios of H+ uptake to ATP hydrolysis of 1.7 or slightly less were obtained in both laboratories. In submitochondrial vesicles protons leak through the membrane via F, sectors from which F, has been removed. Respiration rates can be depressed by blocking F,, with oligomycin (Thayer and Hinkle, 1973). For this reason the authors concluded that H+/ATP ratios were somewhat underestimated, and they suggested that a ratio of 2 H+/ATP might be the correct value. Because of the poor coupling in submitochondrial vesicles due to uncovered F,, values for A&+ developed by ATP hydrolysis alone are rarely reported. Qualitative indications for the development of a membrane

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potential during ATP hydrolysis (uptake of the lipid-soluble anion tetraphenylboron) have been reported by Grinius et al. (1970). As observed during the measurement of respiration-dependent H+ translocation, the movement of ions or metabolites may complicate interpretations of ATP-dependent proton transport data in intact mitochondria or submitochondrial vesicles. Study of the F,-F, ATPase in better resolved systems has therefore assisted substantially in clarifying the behavior of the complex. 2. PROTON PUMPING BY PURIFIED F,-F,

The most straightforward evidence that the F,-F, ATPase complex can function as an electrogenic proton pump has come from the study of the purified enzyme complex. The F,-F, ATPase purified from bovine heart mitochondria has been reconstituted into artificial phospholipid vesicles (Serrano et al., 1976). In the presence of valinomycin, to allow rapid charge compensation by K+ movement, uptake of protons during ATP hydrolysis has been demonstrated. Proton uptake is sensitive to rutamycin, a form of oligomycin, but also to the protonophore uncoupler FCCP, indicating that translocation of protons rather than scalar production of OH- is occurring. Enhancement of the fluorescence of 1-anilino-&naphthalene sulfonate can also be observed, indicating the development of a membrane potential. Now that several other preparations of ATPase complexes are available, confirmation of the results of Serrano et al. (1976) may be forthcoming. In the case of the purified, reconstituted thermophilic bacterium enzyme complex TF,-F,, Sone et al. (1976) have demonstrated that the hydrolysis of ATP leads to creation of a AilH+ of at least 280 mV, with substantial contributions from both A$ and ApH.

IV. THE ROLE OF PROTON TRANSLOCATION IN MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION The last of the three problems of oxidative phosphorylation is the mechanism of energy coupling between respiration and ATP synthesis. It has been established that both the respiratory chain and the F,-F, ATPase can function as electrogenic proton pumps under certain conditions. What is the role of this translocation in energy transfer? This question can be addressed in two ways. One is to reexamine the relationship between AilH+ generated by respiration and ATP synthesis. The other is to assay directly for synthesis of ATP by an artificial proton gradient in intact mitochondria and in better resolved systems.

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A. Models for Energy Coupling 1. CHEMIOSMOTIC MECHANISMS The term “chemiosmosis” refers specifically to Mitchell’s hypothesis about the mechanism of energy coupling and not to his proposals for the mechanisms of proton pumping discussed above. Chemiosmosis proposes that energy is stored and used in the form of a true transmembrane gradient in the electrochemical potential of osmotically active protons. It is only protons in equilibrium with the bulk aqueous phase which can be described by the now familiar expression for the total electrochemical potential difference in protons (AFH+)across a membrane, or the proton motive force @P):

AFH+ = FAp

AP = A$ - ZApH

(1)

where A$ is the electrical potential difference, ApH is the pH difference across the membrane, and Z is 2.303RT/F, equal t o about 59 mV at 25°C (Mitchell, 1961). According to this formulation, the only effect of the membrane potential is to increase the potential energy difference between protons on the M-side and protons on the C-side of the membrane. All models in which ApH+ is considered the “high-energy intermediate” are chemiosmotic mechanisms, regardless of the mechanism proposed for generation of the proton gradient. The energy of formation of ATP under a given set of circumstances is given by the so-called phosphorylation potential AG,: AG,=AG,O +2.303RTlog ([ATP]/[ADPl [Pi])

(2)

In a chemiosmotic system the energy available to synthesize ATP is given by the number of protons moved multiplied by the potential energy difference through which they are moved; thus AG,=nAji,+

(3)

Both the prediction of linearity and the stoichiometry can be tested experimentally, as can the prediction that only total A,iiH+ is directly related to phosphorylation and not its separate components. 2. NONCHEMIOSMOTIC MECHANISMS In contrast to the hypothesis of Mitchell are a wide variety of other models, some of which involve protons and some of which do not. In the first category are the models in which protons play a role but are never in full equilibrium with either aqueous phase between relocation by respira-

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tion and utilization for ATP synthesis. The model of R. J. P. Williams (1975) conceives of protons as separated from their charge-compensating electrons but remaining within the membrane, later to be used to dehydrate ADP + Pi, still within the membrane, rather than equilibrating with either aqueous phase. Kell (1979) has suggested that protons released by respiration may be restricted to the membrane by the energy barrier associated with penetration of the first layer of organized water at the membrane surface. Other proton-driven but nonchemiosmotic models have been proposed by Azzone et al. (1977), Robertson and Boardman (1975), and Rottenberg (1978). Models for energy coupling in which high-energy protons are not considered the sole intermediate are still under consideration. Direct protein-protein interactions between respiration and phosphorylation are considered necessary by some authors in order to explain certain inhibitor data which suggests that the ATP synthetase and the respiratory chain are not completely independent (Slater, 1977).

B. Correlation between Phosphorylation Potential and ApH+ The AGp/AiH+ ratio has now been measured under a wide variety of conditions. Values of n, the number of protons translocated per ATP synthesized, may be compared to the direct proton flux measurements during ATP hydrolysis. Evaluations of ApH and A$ have proved difficult and strongly dependent upon the indicators used (Azzone et al., 1978a,b). Nonetheless many types of studies have yielded similar results. Nicholls (1974), using SCN- and OAc-, determined that respiring rat liver mitochondria maintained a AFH+ ranging from - 180 to - 230 mV, depending upon the medium, but that in all cases AGp was to approximately -7.9 kcal/mole, which would correspond to 270-290 mV if two protons were translocated per ATP synthesized. Kamo et al. (1979), who measured a ApH+ of 200 mV during respiration or ATP hydrolysis, calculated AGP/ApH+= 2 . 7 and suggested that the true value might be n = 3. In submitochondria1 vesicles Rottenberg measured a AG,/AFH+ of 2.9. Sorgato et al. (1978), who measured a AFH+ in submitochondrial vesicles of 185 mV, found AGp/AFH+ ratios of 3.1, 3.0, and 3.3 in various media. Where it has been measured, AFH+ has been found to decline during ATP synthesis, consistent with its proposed role as the energy source (Nicholls, 1974; Sorgato et al., 1978). Although the possibility remains that ApH+ is being seriously underestimated in all cases, it appears that a value of n = 3 for ATP synthesis is widely supported both by flux measurements during ATP hydrolysis (Sec-

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tion 111, C ) and by measurements of the ratio of A $ H + generated to the AG, maintained. This is incompatible with a pumping of 2 H + / 2 e- per site by the respiratory chain and so with Mitchell’s direct chemiosmosis hypothesis of how the proton gradient is generated; it is certainly not incompatible with a chemical role for protons in ATP synthesis, nor generally with the chemiosmotic hypothesis for energy coupling. A more serious problem for the chemiosmotic hypothesis is presented by the work of Azzone and co-workers (e.g., Azzone el al., 1978a,b), who have made an extensive study of the relationship between A P H + and AG, as A$H + is varied by different reagents (salts, respiratory inhibitors, protonophores, ionophores, and combinations of ionophores). According to their results, not all methods of changing A,iiH+ have the same effect on AGp (Azzone et al., 1977). Thus, using nigericin plus valinomycin-so that A$ and ApH are lowered independently-produces a simultaneous fall in AhH+ and AG,, with a constant ratio of n = 3 . In contrast, protonophore uncouplers such as FCCP, which lower A,iiH+ directly, give a value of n approaching infinity at a low A/ZH+, which suggests that AG, can be maintained by a very large number of low-energy protons. It is unfortunate that AG, and AFH+in these studies were not measured under identical conditions for each addition of special reagent. However, the results nevertheless require explanation if the chemiosmotic hypothesis is to be accepted.

C. Synthesis of ATP by Artificial Proton Gradients In order t o answer the question of whether proton pumping or some other function of respiration is responsible for ATP synthesis, a more direct physical approach has also been taken. This involves the use of artificial gradients of proton concentration and charge to synthesize ATP in the complete absence of respiration and ultimately in the absence of all respiratory chain components. As early as 1967 Cockrell et al. (1967) had obtained evidence that a small amount of ATP could be synthesized by mitochondria during K + efflux facilitated by valinomycin and driven by a K + concentration gradient. Glynn (1967) suggested that this might be due to the development of a membrane potential which could drive ATP synthesis. Rossi and Azzone (1970) demonstrated that, by increasing the internal K + , substantial amounts of ATP could be synthesized in respiration-inhibited mitochondria using valinomycin-facilitated K + efflux. They noted that the addition of acid t o the external medium increased the amount of ATP formed, and they attributed the ATP synthesis t o reversal of a proton-cation exchange pump. Azzone and Massari (1971) showed that the effluent ion could be Rb+ as well as K + . In their study several ratios of ApH to A$ (described as

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ApK+) could synthesize ATP, provided that the total gradient exceeded 2.5 units (150 mV). The K+,,u,/ATP,, stoichiometry was found to vary from 2 to 4 as the pH gradient decreased at a constant K + gradient. In 1975 Thayer and Hinkle (1975a) reported the synthesis of ATP in response to additive pH and pK+ gradients in respiration-inhibited submitochondrial vesicles treated with valinomycin. They also demonstrated (Thayer and Hinkle, 1975b) that ATP synthesis using artificial gradients could proceed at a rate faster than ATP synthesis energized by respiration, indicating that proton gradient-driven ATP synthesis was kinetically competent t o transfer energy from respiration t o synthesis. Varying the ratio of A$ to ApH at a constant Ah,+, they found similar rates of ATP synthesis, supporting the proposal of Mitchell that the effect of A$ is to increase Ah,+ rather than to produce some separate effect. Net synthesis of ATP by purified, reconstituted mitochondrial F1-Fo ATPase using only chemical gradients and ionophores has not yet been reported, although this has been accomplished for the more stable complex from thermophilic bacteria (Sone el al., 1977; Kagawa, this volume). The bovine heart mitochondrial F,-F, ATPase enzyme has been reconstituted with other purified respiratory chain complexes to give phosphorylating proteoliposomes showing P/O ratios as high as 0.5. A simpler protein, bacterial rhodopsin reconstituted into liposomes, can be shown to pump protons inward in response to light. Simultaneous incorporation of mitochondrial F,-F, ATPase plus bacteriorhodopsin into liposomes produces a light-dependent, uncoupler- and rutamycin-sensitive ATP synthesis, as summarized by Racker el al. (1975). Although the results are consistent with synthesis being dependent upon Afi, + generated by respiratory or photosynthetic pumps, the presence of other proteins in the liposomes leaves open the possibility of direct protein-mediated energy transfer. Thus, the situation with mitochondrial ATPase complexes cannot be considered as well defined as for the analogous system in thermophilic bacteria. Nonetheless, the evidence is quite convincing that ATP can be synthesized by an imposed AhH+ via the F,-Fo ATPase complex, independent of the redox activity or even of the presence of respiratory chain components. Although it is not impossible that the in vivo pathway may be different, certainly the burden of proof rests with those who suggest pathways which do not involve Ah, + .

V.

ELECTRO PH0 R ETlC METABO LlTE TRANSPORT

A large variety of metabolites and inorganic ions are transported both in and out of mitochondria. For a review, see LaNoue and Schoolwerth

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(1979). The transport of most of these substances has been shown to be dependent upon the mitochondrial energy state. Electrophoretic transport of cations or anions involves net transfer of charge and can be driven by the electrical potential gradient. Electroneutral transport by proton symport or antiport can be driven by ApH. Electroneutral exchange diffusion of substrates also occurs. Electrogenic transport of ions other than protons has not been demonstrated in coupled, respiring mitochondria. However, given the necessary concentration gradients, systems which function electrophoretically under energized conditions can be identified, because they will function electrogenically under deenergized conditions. The natural permeability of mitochondria to cations is quite limited, with the exception of Ca 2+,for which an extremely rapid endogenous electrophoretic carrier system exists (Bygrave, 1978). Ca2+ uptake appears to be fully electrogenic (Fiskum eta/., 1979; Akerman, 1978). It stimulates increased respiration and causes H ejection necessary to reestablish the membrane potential decreased by cation uptake. This permits Ca2+ to be used experimentally as a replacement for K + plus valinomycin, and for observation of the maximal events of H + translocation. Ca2+uptake in the absence of added permeant weak acid anion is clearly limited (40 ng. atoms/mg of protein), and Moyle and Mitchell (1977a,b) claim that all Ca2+is taken up by obligatory symport with Pi, thus reducing the effective charge transfer to C a + . However, direct measurements during Ca2+ uptake (Fiskum el a/., 1979) have not shown translocation of phosphate. Resolution of this conflict is critical to an understanding of charge translocation in mitochondria. Another very active endogenous transport system that results in net charge transfer is the ATP4--ADP3- exchange system (Klingenberg et a/., 1977). This process, which promotes an antiport of ATP4- for ADP3-, is specific for free nucleotides (rather than Mg2+-boundnucleotides), in contrast t o virtually every other cell reaction. The energy cost of translocating one negative charge for ATP out of the matrix against the membrane potential is a substantial fraction of the cost of oxidative phosphorylation (Lehninger et al., 1979). There are a variety of mitochondrial transport systems which catalyze electroneutral ion flux. One of the most active is H,POa.H+ symport (Coty and Pedersen, 1975). Lipophilic weak acids, e.g., acetate and 6-hydroxybutyrate are accumulated in a similar manner, except that their transport occurs by unmediated diffusion of the neutral free acid (Chappell and Haarhoff, 1967). The Na +-H+ system and the less active K+- H+ exchange system may function in cation extrusion (Brierley, 1976). These systems are all driven under appropriate conditions by the proton concentration gradient alone. Another type of neutral exchange transporter responsive to +

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neither All/ nor ApH is the anion-exchange mediator. These systems catalyze exchange of anionic metabolites for other metabolites of the same charge. (See LaNoue and Schoolwerth, 1979.) VI.

SUMMARY

The substantial evidence that electrogenic proton translocation is responsible for oxidative phosphorylation in mitochondria makes more important than ever elucidation of the molecular mechanisms involved. If direct chemiosmosis must be abandoned because of well-substantiated measurements of proton movements in excess of the number of electrons transferred, then the mechanism of proton pumping by the respiratory chain is completely unknown. The proton-pumping complexes bear no striking resemblance to simple ion-pumping ATPases, to the mitochondria1 F,-F, ATPase complex, or to each other. Knowledge of the precise pathway of electron transfer within each complex is essential. Experimental verification must be sought for each of the conformational changes so casually invoked. Indeed study of the proton pumps of oxidative phosphorylation has just begun. ACKNOWLEDGMENTS The author would like to thank Dr. Gary Fiskum, for helpful discussions regarding ion translocations, and Dr. Peter L. Pedersen for critical reading of the manuscript. This article was written while the author was supported by National Science Foundation grant PCM 7813249 awarded to Dr. Peter L. Pedersen.

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