F1F0 H+-Atpase/ATP Synthases (Energy Transduction/Membrane Transport/Conformational Change) GENE A

Home , ATPase, ATP synthase

Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3688-3692, June 1986 Biochemistry A chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases (energy transduction/membrane transport/conformational change) GENE A. SCARBOROUGH Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514 Communicated by Mary Ellen Jones, January 27, 1986

ABSTRACT A general hypothesis for the molecular mech- changes in the ATPase molecule, and models employing this anism of membrane transport based on current knowledge of general coupling strategy are commonly termed conforma- protein structure and the nature of ligand-induced protein tional coupling models. Such models have also been termed conformational changes has recently been proposed indirect coupling models (10), because the effects ofA7ZH+ are [Scarborough, G. A. (1985) Microbiol. Rev. 49, 214-231]. visualized as being exerted "at a distance" from the site of According to this hypothesis, the essential reaction undergone the chemical bond rearrangements (11). The other school of by all proteinaceous transport catalysts is a ligand-induced thought on this matter, headed by Mitchell (10), visualizes the hinge-bending-type conformational change that results in the interconversion of A7ZH+ and the chemical bond energy of transposition of binding-site residues from access on one side of ATP largely, if not solely, via atomic interactions occurring the membrane to access on the other side. Subsequent release at the catalytic site of the ATPase and, accordingly, models and/or alteration of the ligand or ligands that induce the of this ilk have been termed direct coupling models (10). conformational change facilitates the converse conformational In a recent general treatment of the problem of membrane change, which returns the binding-site residues to their original transport (12), which of course includes the problem of the position. With this simple cyclic ligand-dependent gating F1Fo ATPase mechanism, I endeavored to show how trans- process as a central feature, biochemically orthodox mecha- porters might operate at the molecular level of dimensions, nisms for virtually all known transporters are-readily con- for the most part employing only already known modes of ceived. In this article, a chemically explicit model for the protein behavior and established principles of enzymatic molecular mechanism of the F1Fo H+-ATPase/ATP synthases catalysis. An essential feature of every model that resulted ofmitochondria, bacteria, and chloroplasts, formulated within from this treatment is that all of the key reactions of the the guidelines ofthis general transport paradigm, is presented. membrane transport process occur at the transporter active At least three points of potential interest arise from this site, or more generally at the transporter ligand binding site, exercise. First, with the aid of the model, it is possible to which clearly categorizes these models as direct ones. Nev- visualize how energy transduction catalyzed by these enzymes ertheless, a sine qua non of every model presented is also a might proceed, with no major events left unspecified. Second, conformational change in the transporter protein, through explicit possibilities as to the molecular nature of electric field which the necessary alternating access change is accom- effects on the transport process are raised. And finally, it is plished. This is not a paradox of any sort, but simply serves shown that enzyme conformational changes, energy-dependent to demonstrate that direct coupling mechanisms and trans- binding-affinity changes, and several other related phenomena porter conformational changes are not mutually exclusive, a as well, need not be taken as evidence of "action at a distance" point that Mitchell has also made (27, 28). But importantly, or indirect energy coupling mechanisms, as is sometimes from this it follows that transporter conformational changes assumed, because such events are also integral features of the are not in any way diagnostic of an indirect coupling mech- mechanism presented, even though all of the key reactions anism. By using an F1Fo H+-ATPase/ATP synthase model as proposed for both ATP-driven proton translocation and proton a specific example, one purpose ofthis paper is to make again translocation-driven ATP synthesis occur at the enzyme active the point that transporter conformational changes, and most site. of the other phenomena commonly put forth in favor of indirect coupling as well, do not support indirect coupling any The F1F0 H'-ATPase/ATP synthases of mitochondria, bac- more or less than they support direct coupling. A second teria, and chloroplasts serve a vital role in cellular energetics more important purpose of this paper is to elaborate, in acting both as ATP hydrolysis-driven generators of a greater detail than before (12), a model for the molecular transmembrane electrochemical difference mechanism of the F1Fo ATPases that involves no major protonic potential undescribed molecular activities. With attention to the chem- (ASH+), and as A7ZH+-driven generators ofATP from ADP and istry of the catalyzed reaction and known characteristics of Pi, depending on the needs of the cell. Accordingly, a great enzyme-ligand interactions, and recognition of the probable deal of experimental effort has been expended in efforts to importance of electric field effects on several steps of the elucidate the molecular mechanism of these enzymes (1-7). process, it is possible to describe the entire energy transduc- At least in part from the accretion ofbiochemical information tion sequence in relatively simple terms, without invoking about these enzymes that has resulted from this intense ''action at a distance" energy coupling phenomena. experimental attention, two fundamentally different schools of thought as to how the F1F0 H+-ATPase/ATP synthases The General Transport Paradigm might function have evolved. One school, championed by Boyer (8, 9), holds that the energies of ASH+ and ATP are Understanding the essential features of the F1Fo ATPase transduced through the intermediacy of conformational model to be described requires an understanding of the fundamental transport postulates that have been proposed The publication costs of this article were defrayed in part by page charge elsewhere (12). These will now be briefly reiterated, but the payment. This article must therefore be hereby marked "advertisement" serious reader is urged to refer to the original treatment. in accordance with 18 U.S.C. §1734 solely to indicate this fact. According to the general transport paradigm, transporter 3688 Downloaded by guest on October 1, 2021 Biochemistry: Scarborough Proc. Natl. Acad. Sci. USA 83 (1986) 3689 proteins comprise at least two relatively rigid domains or proteinases, where a proton is bound with an abnormally high lobes connected by relatively flexible portions of the poly- pKa as a result of electrostatic repulsion between two peptide chain (or possibly by covalent or noncovalent inter- juxtaposed carboxylate anions in the active site (21). A actions between monomers of a dimer or higher multimer) similar effect would operate if the R residue of the ATP with ligand binding-site residues present in each lobe in a cleft binding site is a divalent cation that must give up a bound region that separates the lobes. In the absence of the hydroxide ion (22) before it effectively binds to its part of transporter's specific ligand or ligands, the lobes are gener- ATP (not shown). In this case, dehydroxylation of R rather ally (with possible exceptions) poised relatively distant from than protonation of R would promote ATP binding and vice one another rendering the binding site incomplete. Upon the versa. In stages 2-3, the cleft is driven closed as the ATP binding of a suitable ligand, there ensues a rapid ligand- binding reaction is completed, and the hydrolytic water induced and -stabilized interdomain movement or cleft clo- molecule moves into its binding site represented by the two sure leading to sequestration of the bound ligand or ligands proton binding residues, designated B to recognize their that induce the conformational change away from the aque- general base character (14). This site could comprise residues ous medium on the side of the membrane from which the from the y subunit, which would explain the requirement for ligand approached, and the concomitant opening of a new this subunit for hydrolytic activity. However, it is also pathway for diffusion in the transporter molecule from the possible that the active site is made up entirely of /3-subunit binding site to the aqueous medium on the side of the residues. In this case, the figure would be drawn slightly membrane opposite that from which the ligand approached. differently with the Bs in the cleft, but the essence of the The bound ligand facing the opposite side is then displaced in model would not change. Such a model would, however, a variety of different processes specific for the transporter in necessitate a somewhat less direct explanation for why question, and it is the precise nature of this displacement isolated A3 subunits can bind substrates and products (13) but reaction that determines the species that are actually trans- cannot hydrolyze ATP. As the reaction proceeds from stage ported and what the direction of that transport is. Finally, 3 to stage 4, the transition state configuration with ADP as the following the displacement reaction, in most cases, the leaving group and H20 as the incoming group is attained. This transporter conformation changes back to the conformation is assisted by electron withdrawal by the various positive or favored in the absence of ligands, whereupon another round hydrogen-bonding centers (14, 23-25) in the active site, of the transport cycle can ensue. In general terms, that is which makes the y phosphorus atom more electrophilic, and essentially all that need be involved in transport, and as was by proton withdrawal by the Bs, which makes the water proposed (12), virtually all transporters could operate ac- oxygen atom more nucleophilic. In stages 4-5, the transition cording to this fundamental sequence. The model for the state configuration breaks down at a rate exceeding 1012 per molecular mechanism of the F1F0 H+-ATPase/ATP sec (26). As a result, the hydrolytic water protons become synthases that follows is simply an elaboration ofthis general associated with the bases that withdrew them, the water transport scheme. oxide ion is transferred to the y phosphorus atom, and ADP is displaced from the y phosphorus atom. Because the Molecular Mechanism of the F1F0 H+-ATPase/ATP presence of ATP facilitates protonation of the R, the break- Synthases down of ATP decreases the affinity of the R for its bound proton, and the proton is released from the R (or the R is Fig. 1 depicts the proposed reaction cycle of the F1F0 rehydroxylated, leaving behind a proton) into the F0 sector as ATPases. Only the active-site region, most or all of which indicated in stages 5-6. As a result of the covalent bond may be in an interdomain cleft in each P subunit (13), is exchange that has occurred, the forces tending to hold the shown. It is assumed that the experimentally observed cleft closed are diminished, and the cleft tends to reopen apparent nonequivalence of the three catalytic sites in the F1 (stages 6-7), leaving the three protons behind. Mutual elec- sector is a regulatory feature and/or is related to the inherent trostatic repulsion between ADP and Pi assists the process. asymmetry of the complex arising as a result of only single After the cleft reopens, ADP and Pi are released (stages 7-8), copies of the y, 8, and e subunits (using the Escherichia coli and during release, the Pi becomes protonated. Concomi- terminology), but is not, as Boyer apparently agrees (8), tantly, as long as the protonic potential on the trans-ATP side central to the question of the molecular mechanism ofenergy remains reasonably low, the protons diffuse via F0 to the transduction. It may simply be that the only catalytically trans-ATP side of the membrane (not shown). The overall active P3 subunit in the complex at any given time is the one result is the generation ofthree protons on the trans-ATP side that is in the proper position to interact productively with one at the expense of the hydrolysis of one ATP molecule. It is or more ofthe single copy subunits. Rotation of certain ofthe important to mention that the chemistry of this proposed subunits relative to the others (15, 16) may also be a mechanism is similar to that suggested by Mitchell for these necessary consequence of such asymmetry, but this is enzymes for many years (27-30). likewise not viewed as central to the catalytic mechanism. The process of A/uH+-driven ATP synthesis catalyzed by Whether or not such rotation occurs, a requirement for the F1F0 ATPases can also be described with the aid of Fig. specific interactions between the F0 sector and the subunits 1, by following the diagrams in the counterclockwise direc- bearing the individual active sites, presumably via the single tion. The imposed protonic potential difference is high on the copy subunits of F1, probably explains why certain pertur- trans-ATP (right hand) side of each diagram and low on the bations ofF0 can influence the catalytic activity ofF1 (17-20). ATP side. To bring to light several possible trans-active-site The major events occurring during the process of ATP electric field effects, as well as protonic activity effects on hydrolysis-driven proton translocation are depicted in stages proton binding reactions, it is assumed that regardless of 1-8 in a clockwise direction. In stage 1, the cleft is predom- whether the transmembrane A7H+ is applied in the form of an inantly open awaiting the approach of ATP [or the divalent electrical potential difference (Aqi) or a ApH, the F0 sector cation complex of ATP (not shown)]. If the divalent cation and the F1-F0 interface region act to convert it approximately complex of ATP had been used in this specific formulation, isopotentially (27) to a trans-active-site /.H+ with significant the active site would contain two less positive charges. In contributions ofboth A& and ApH via a proton relay and trap stages 1-2, ATP binds to a part of its binding site. ATP mechanism such as that visualized by Mitchell (27-30). binding is facilitated by protonation ofthe residue designated Several possibilities have been suggested for the molecular R and, conversely, protonation of the R is facilitated by ATP nature of the proton relay system (29, 31-33). In this binding. This may be likened to the situation with the aspartic formulation, the Bs comprise at least a part ofthe proton trap. Downloaded by guest on October 1, 2021 3690 Biochemistry: Scarborough Proc. Natl. Acad. Sci. USA 83 (1986)

0 0 H B-&,

0D A-- By- 0+ +

H P+ ,H0 _1 0 ~ ~ 0 7 2H 0 0 A+ \ If ON p ++ -o o- + +H

FIG. 1. Reaction cycle of the F1Fo H+-ATPase/ATP synthases. The larger structure in each of the numbered diagrams represents the nucleotide and Pi binding part of the active site. The smaller structure at the right of each diagram represents the interface region between the active sites and the F1 end of the FO sector. Although not shown, the F0 sector extends further to the right of each diagram across the coupling membrane. The pH on the ATP side of the membrane (left hand side of each diagram) is -8. The probable participation of a divalent cation in the phosphoryl transfer chemistry is acknowledged but has been omitted for the purpose of clarity. Counterions not participating directly in the mechanism have also been omitted for the same reason. Although not drawn in the conventional way (14), the transition state of the phosphoryl transfer rbaction (stage 4) is that of an associative in-line nucleophilic displacement reaction with trigonal bipyramidal geometry. See text for additional details. A slightly different version of this model was first presented at the International Symposium on Achievements and Perspectives in Mitochondrial Research in Ostuni, Italy, September, 1985.

In stage 8, the cleft is predominantly open, awaiting collisions and/or stabilized by this increment of the total electrical with ADP and P1. In stages 8-7, ADP and Pi enter their potential drop. The voltage effects on stages 8-7 and 7-6 binding sites. Pi binding causes liberation of its proton. The together amount to an energy-driven increase in the affinity binding sites are phases distinct from the bulk aqueous of the enzyme for ADP and Pi. The possibilities of such medium; if this were not true, ADP and Pi could not electric field effects on binding reactions and protein accumulate in their sites as they do. Therefore, it is possible conformational changes were mentioned before (12) and have that some of the total electrical potential drop between the occurred to many others (34-39). Continuing, after the cleft proton trap at the F1 end of the FO sector and the aqueous is closed (stage 6), the ADP and Pi binding part of the active medium on the ATP side occurs between the binding sites and site is in communication with the bottom ofthe Fo sector, and the aqueous medium on the ATP side. If so, because both the high protonic activity in this region favors protonation (or binding processes are electrogenic as formulated, this frac- dehydroxylation) of the R (stages 6-5). This, as well as tion of the potential drop will promote ADP and Pi binding. /LH+-driven protonation of the Bs, promotes the formation of Moreover, a substantial increment of the total electrical the transition state configuration, as signified by stages 5-4. potential drop between the proton trap and the aqueous In stages 4-3, the transition state breaks down at a rate of medium on the ATP side occurs between the proton trap and approximately the same magnitude as its rate of breakdown the ADP and Pi binding sites when the enzyme is in its open in the other direction, and enzyme-bound ATP and water are configuration. Therefore, because the ADP-Pi binding-site formed. Although not shown, at this point the water molecule complex shown in stage 7 bears a net charge of -2 (by leaves (possibly via FO) and the Bs become reprotonated. The design), cleft closure (i.e., stage 7 to stage 6) will be driven ATP-binding site complex now bears a net positive charge, Downloaded by guest on October 1, 2021 Biochemistry: Scarborough Proc. Natl. Acad. Sci. USA 83 (1986) 3691 and the electrical potential drop that drove the cleft closed steps shown in Fig. 1 may be such that the ATPase molecules with ADP and Pi bound now drives cleft reopening (stages tend to accumulate around stages 3-5. Under such condi- 3-2). The overall process ofATP release is electroneutral and tions, this state of affairs might well allow the experimental is therefore unaffected by any electrical potential drop demonstration of energy-independent synthesis of bound between the binding site and the aqueous medium on the ATP ATP from ADP and Pi, high-affinity ATP binding, approxi- side. However, such a field will favor the deprotonation (or mately equal amounts ofbound ATP and bound ADP plus Pi, hydroxylation) of the R, which will facilitate ATP release and uncoupler-insensitive exchange reactions, but such re- relative to the unenergized situation (stages 2-1). The effects sults can by no means, as proved by the fact that the direct of the voltage drop on stages 3-2 and 2-1 amount to an coupling model shown in Fig. 1 can explain such phenomena energy-driven decrease in the affinity of the enzyme for ATP. equally well, be taken as evidence in support of the notion The result of all of the energy effects, exerted solely on the that the energy input in F1F0 H+-ATPase/ATP synthases is participants of the chemical reaction and enzyme active-site indirectly coupled to ATP synthesis "at a distance" via residues, is a facile turning ofthe cycle in the direction of ATP enzyme conformational changes. And finally, as pointed out synthesis at the expense of the transmembrane A H+. Al- above in the detailed description of the model of Fig. 1 though not emphasized here, it should be pointed out that operating in the ATP synthesis mode, the voltage effects on electric field effects on the covalent bond rearrangement per stages 8-7 and 7-6 amount to an energy-dependent increase se are also entirely possible, as explained by Mitchell & in the affinity of the ATPase for ADP and Pi, and the voltage Koppenol (30). effects on stages 3-2 and 2-1 amount to an energy-driven Several points regarding the specific formulation of Fig. 1 decrease in the affinity of the enzyme for ATP. Therefore, merit brief additional discussion. The stoichiometry of the again because all of the key reactions of the model described reaction as arbitrarily drawn is 3 protons generated, con- in Fig. 1 occur in or near the enzyme active site, the dem- sumed, or translocated per ATP hydrolyzed or synthesized. onstration of energy-dependent binding affinity changes also The actual stoichiometry may be more or less depending on cannot be taken as evidence for the existence of an indirect the ATPase in question and, importantly, upon the experi- coupling mechanism. From these considerations, it can be mental conditions. Thus, certain F1F0 ATPases may not have concluded that most, if not all, of the evidence normally an R group capable of protonation or hydroxylation, in which offered in support of an indirect coupling mechanism for the case the proton stoichiometry would be 2. Others may have F1F0 H+-ATPase/ATP synthases (e.g., see refs. 8, 9, 40, and two such R groups with a stoichiometry of 4, and more of 41) essentially fails to discriminate between the direct and such groups could raise the stoichiometry even somewhat indirect coupling possibilities. Indeed, as argued before (12), higher. Moreover, for the ATPases that have such R groups, it is unlikely that any solid evidence in favor of indirect under certain experimental conditions it is entirely possible coupling exists for any transporter. that the protonation-deprotonation or dehydroxylation-hy- In conclusion, what is offered in this paper is a hypothetical droxylation reactions of the R groups may not occur at a reaction sequence for the operation of the F1F0 H+- significant rate, even though the other reactions of the cycle ATPase/ATP synthases that takes into account the major do. In this case, the condition-dependent stoichiometry known properties of these enzymes, and enzymes in general, would approach 2. And intermediate conditions would pro- and that in addition, puts forth specific proposals for the duce intermediate stoichiometries. It is thus not very sur- pathway of proton flow and the mechanisms by which A/.LH+ prising that there is so little agreement among experimenters and the trans-enzyme proton flow could bring about the facile as to the "true" reaction stoichiometry for the F1F0 ATPases synthesis of ATP. It is understood that the provision of such (and many other transporters as well) because there may be detail virtually ensures some inaccuracy in the overall no such thing. scheme, but the purpose of theories in general is to provide A second point to be made about the model of Fig. 1 a temporary framework for the design and interpretation of regards the arbitrary charge assignments that are shown. The experiments, and in accord with this notion, it is hoped that net charge of the ADP-Pi binding-site complex of -2 was the model presented in this article might promote progress in chosen to make the point that with a proton stoichiometry of the F1F0 ATPase field by providing a viable alternative to 3 per ATP, both cleft closure with ADP and Pi bound and cleft currently fashionable conformational coupling models. opening with ATP bound would be driven by the electrical potential drop between the proton trap at the F1 end of the F0 This work was supported by U.S. Public Health Service National sector and the nucleotide and Pi binding sites. Other charge Institutes of Health Grants GM 24784 and GM 32166. assignments and/or stoichiometries would tend to increase or decrease the voltage dependence of each of these two steps to the point where one or the other, but not both, could 1. Kozlov, I. A. & Skulachev, V. P. (1977) Biochim. Biophys. Acta 463, 29-89. become voltage independent. The same holds for the other 2. Kagawa, Y., Sone, N., Hirata, H. & Yoshida, M. (1979) J. voltage-dependent steps in the model. It should also be Bioenerg. Biomembr. 11, 39-78. emphasized that in general, the contribution of electric field 3. Fillingame, R. H. (1980) Annu. Rev. Biochem. 49, 1079-1113. effects to the energy transduction mechanism depends en- 4. Amzel, L. M. & Pedersen, P. L. (1983) Annu. Rev. Biochem. tirely on the molecular anatomy of the F1F0 ATPase and the 52, 801-824. shape of the field in which the enzyme resides. Parts of the 5. Futai, M. & Kanazawa, H. (1983) Microbiol. Rev. 47, 285-312. enzyme in a field can be influenced by it, and any parts 6. Senior, A. E. & Wise, J. G. (1983) J. Membr. Biol. 73, 105-124. outside the field are less likely to be. 7. Walker, J. E., Saraste, M. & Gay, N. J. (1984) Biochim. A final important topic for discussion regards the experi- Biophys. Acta 768, 164-200. 8. Boyer, P. D. (1979) in Membrane Bioenergetics, eds. Lee, mental distinction between direct and indirect coupling C. P., Schatz, G. & Ernster, L. (Addison-Wesley, Reading, mechanisms. Clearly, the transport mechanics shown in Fig. MA), pp. 461-479. 1 include important ATPase conformational changes, but all 9. Boyer, P. D., Kohlbrenner, W. E., McIntosh, D. B., Smith, of the key reactions occur at the enzyme active site. This L. T. & O'Neal, C. C. (1982) Ann. N. Y. Acad. Sci. 402, shows that ATPase conformational changes per se do not 65-83. indicate an indirect coupling mechanism any more than they 10. Mitchell, P. (1979) Eur. J. Biochem. 95, 1-20. indicate a direct coupling mechanism. Furthermore, it is 11. Tanford, C. (1982) Proc. Natl. Acad. Sci. USA 79, 2882-2884. entirely possible that under certain experimental and/or 12. Scarborough, G. A. (1985) Microbiol. Rev. 49, 214-231. physiological conditions, the rate constants of the various 13. Khananshvili, D. & Gromet-Elhanan, Z. (1985) Proc. Natl. 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