Hypothesis Paper. Rotary Movements within the ATP Synthase do not Constitute an Obligatory Element of the Catalytic Mechanism

IUBMB Life, 55(8): 473 481, August 2003 Hypothesis Paper Rotary Movements within the ATP Synthase do not Constitute an Obligatory Element of the Catalytic Mechanism Jan A. Berden Swammerdam InstituteforLifeSciences, BioCentrum, UniversityofAmsterdam, PlantageMuidergracht12, 1018TV, Amsterdam, The Netherlands Summary After a brief history of the proposals for the mechanism of the ATP synthase, the main experimental arguments for a rotational mechanism of catalysis are analyzed and on the basis of this analysis it is concluded that no evidence has been provided for rotation as an obligatory element of the catalytic mechanism. On the other hand, the experimental evidence in favor of a two-sites catalytic mechanism, derived from various approaches and not compatible with a three-sites rotary mechanism, appear to be very solid. Finally a brief characterization of the various nucleotide binding sites is provided and a suggestion is made how the enzyme has evolutionarily developed from a rotating machine into an asymmetrical device for energy conservation. IUBMB Life, 55: 473 481, 2003 Keywords ATP synthase; binding change mechanism; rotary mechanism; dual-site mechanism; nucleotide binding sites; kinetic analysis; crosslinking. A BRIEF HISTORY OF THE PROPOSED MECHANISM OF CATALYSIS OF THE ATP SYNTHASE After the development of a reliable isolation procedure for mitochondrial F 1 in the early seventies (1) the groups of Slater and Penefsky discovered the presence of tightly bound nucleotides in isolated MF 1 (2 4). At the same time Boyer and colleagues developed, on the basis of 18 O exchange experiments with coupled SMP, the concept of a near equilibrium between ATP + water and ADP + Pi at a tight Received 16 June 2003; accepted 12 March 2003 Address correspondence to Jan A. Berden, Swammerdam Institute for Life Sciences, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018, TV Amsterdam, The Netherlands. E-mail: jan.berden@science.uva.nl Abbreviations: F 1, hydrophilic part of the ATP synthase; F 0, hydrophobic membrane part of the ATP synthase; MF 1, mitochondrial F 1 ; CF 1, chloroplast F 1 ; TF 1, F 1 from the thermophylic bacterium PS3; NbfCl, 7-chloro-4-nitro-2-oxa-1,3-diazole. catalytic site, the required energy for ATP synthesis not being needed for the synthesis reaction itself, but for the dissociation of bound ATP (5). On this basis the so called binding change mechanism was proposed (6). This mechanism requires the contribution of two interacting sites to catalysis, each of them alternating between a conformation with a tightly bound nucleotide, responsible for the catalytic reaction, and a conformation with a low binding affinity. The affinity of the bound product at the tight site is decreased when a nucleotide is bound at the low-affinity site and this latter site then becomes the new tight site. In the process of ATP synthesis the energy for the conformational change of the binding sites is delivered by the proton gradient, in the process of ATP hydrolysis by the binding energy of ATP (cf. (7)). After the subunit stoichiometry of MF 1 had been established as 3 : 3 : 1 : 1 : 1 (8, 9) and the group of Penefsky in the early eighties had discovered the phenomenon of single-site catalysis and enhancement of the catalysis at one site by binding of substrate to more sites (10 12), Boyer and Cross and others combined the binding change mechanism with the proposal that not two sites, but all three b sites are involved in (sequential) catalysis, concomitant with rotation of part of the enzyme (13). In spite of our challenges of the rotary three-sites mechanism (14, 15) it attracted substantial support and this support strongly increased when Abrahams et al. in 1994 reported the crystal structure of the mitochondrial F 1 ATPase (16) and chose to interpret their data on the basis of this model, proposing rotation of the g subunit within the ring of a/b subunits. Acceptance of rotation as an obligatory element of the catalytic mechanism was then further boosted by the publication in 1997 of the by now famous experiment of Noji et al. (17), in which it was shown that in a population of molecules, consisting of the a 3 b 3 g moiety of TF 1, the g subunit of some of them was actually seen to rotate on addition of ATP and when in ISSN 1521-6543 print/issn 1521-6551 online # 2003 IUBMB DOI: 10.1080/15216540310001612318

474 BERDEN the same year the Nobel prize for Chemistry was awarded to Drs Boyer and Walker for their work on the ATP synthase, the rotary model was considered to be proven and was ever since as such presented in nearly all biochemistry textbooks. The new evidence for the presence of a separate stator, in addition to the stalk (18), as a connecting element between the F 1 moiety of the enzyme and the membranous F 0 part (see Figure 1), was easily incorporated into the model and even considered as further evidence. But what, then, is the interpretation of the data, old and recent, that seem to be in conflict with the threesites rotary model as envisaged by Boyer and colleagues? Are they wrong? Or can they be incorporated into an adapted rotary model? Or is the rotary model itself a misinterpretation? In the present paper I will first discuss the main arguments in favor of rotation and provide an alternative interpretation of the data. After that I will provide a solution for the apparent contradictions between some data on the rotary mechanism and finally I will summarize the data in favor of a two-sites mechanism of catalysis and describe some characteristics of such a mechanism. Figure 1. Subunit model of the E. coli ATP synthase. The model is taken from Dunn et al. (58) with permission. For the comparison with the mitochondrial enzyme it should be kept in mind that the mitochondrial equivalent of the bacterial d subunit is the OSCP, while the mitochondrial d subunit is the equivalent of the bacterial e subunit. The number of c subunits in the E. coli enzyme may be 10 instead of 12 (59) and this number equals 10 in the enzyme from yeast mitochondria (60) and 14 in the chloroplast enzyme (52). ANALYSIS OF THE MAIN EXPERIMENTAL ARGUMENTS IN FAVOR OF ROTATION The Rotational Model before 1994 The Main Argument for Equivalency of the Three b Sites. In his review in 1993 (19) Boyer lists all arguments in favor of a form of the binding change mechanism that includes three equivalent catalytic sites. The arguments for a binding change mechanism as such do not have to be discussed here, since on this point no real controversy exists: the question to be discussed here is whether two or three catalytic sites are directly involved in the catalytic mechanism and whether rotation is feasible. For the equivalency of the three potentially catalytic b sites Boyer presents in fact only one argument and on the basis of this assumed equivalency other data are interpreted. This one argument for equivalency of all three b sites is the conclusion from 18 O experiments that one single reaction pathway exists for all product Pi (hydrolysis reaction) or ATP (synthesis reaction). This conclusion itself seems justified, but does it mean that all three b sites are equivalent? When only two subunits of a trimer are catalytically active, Boyer argues, they cannot have identical interactions with the third subunit and therefore not follow the same reaction pathway (19). At present it is known, however, that the main interactions do not occur between the b subunits as was envisaged by Boyer at the time, but between the single g subunit and the catalytic b subunits, and between the a and b subunits, the b-b interactions being largely indirect. Equivalency of the three b subunits, therefore, is not required for the presence of a single catalytic pathway. Also the three a subunits are not equivalent. Interpretation of other Mechanistically Relevant Data. Because of the experimental finding that only one tightly bound nucleotide exchanges during catalysis (14, 20) Boyer and colleagues had to assume that at any moment only one b site contains a tightly bound nucleotide, the site where ADP is converted into ATP or vice versa. The two other presumed catalytic b sites should either be empty or contain a loosely bound nucleotide and so the model with a T(ight), L(oose) and O(pen) conformation of the three catalytic sites was born. The concomitant presence of a T, L and O conformation, however, has actually been disproved by the recent data presented by Weber and Senior (21) and Menz et al. (22). As will be explained later, these data imply that O and L are two conformations of one site, while the two other b sites contain a tightly bound nucleotide. One of them cannot be catalytic, therefore. The exchange data of Cross and Nalin (23), showing three rapidly exchangeable and three not- or slowly-exchangeable nucleotides were interpreted as indicating that the nucleotides at any of the three non-catalytic a sites of MF 1 are not exchangeable or only very slowly exchangeable, while on the three b sites all nucleotides are rapidly exchangeable. The

ROTARY MOVEMENTS WITHIN THE ATP SYNTHASE 475 finding by Kironde and Cross (20, 24) that one non-catalytic site contains a slowly exchangeable, quite tightly bound nucleotide, in addition to the presence on the enzyme of two non-exchangeable nucleotides, was then taken to imply that the three a subunits contain the two non-exchangeable nucleotides and this slowly exchangeable nucleotide. But Edel et al. (25) demonstrated the presence of a low-affinity noncatalytic regulatory site, whose occupation with a ligand (e.g. 8-nitreno-ATP) influences the Km for substrate ATP. This latter result implied that one of the two non-exchangeable tightly bound nucleotides could not be located on an a subunit. Similarly, the finding that of the four nucleotide binding sites available for modification with 8-nitreno-ATP and/or 8- nitreno-adp two were located on a subunits and two on b subunits, while the two non-exchangeable nucleotides remained bound during the whole procedure (26), also implied that only one of the latter was bound at an a site and the other at a b site. More recently similar data were obtained with the 2-azido-analogues [see below]. The enhancement of catalysis at one site, induced by binding of substrate to other catalytic sites (10 12) showed cooperation of at least two sites in rapid catalysis, but it was assumed that the measured single-site catalysis by MF 1 was occurring at the first b site (the site with the highest affinity), the other two b sites supposedly being empty in the absence of added nucleotides. However, if in MF 1 one b site is occupied with a non-exchangeable nucleotide (see above), the single-site catalysis is performed by the second b site and the catalysis at this site was then enhanced by providing substrate to the third b site, in agreement with the basic binding change mechanism. With CF 1 Fromme and Gra ber had shown (27) that the slow hydrolysis of ATP bound at a single site, only one b site being occupied in this case, remains slow after addition of more ATP that induces rapid hydrolysis. This finding was dismissed by Boyer (19), but is recently confirmed by the very accurate kinetic analysis of ATP hydrolysis by CF 1 reported by Berger et al. (28): at all concentrations of ATP two independent hydrolytic reactions occur with the kinetic characteristics of the slow single site catalysis and the rapid multi-site (two-site) catalysis, respectively. The Lineweaver-Burk plot for ATP hydrolysis by MF 1 shows negative cooperativity and two Km values (12, 25, 29). The curvature of the plot was interpreted by e.g. Gresser et al. (29) as due to binding of ATP to a third catalytic site, causing a higher Km and a higher rate of catalysis. The K D should be around 55 mm. This interpretation can be excluded by the data of Edel et al. (25) who showed that the change in Km can be induced by covalent binding of 8-nitreno-ATP or 8-nitreno- ADP to a low-affinity non-catalytic site. In later years Boyer assumed that the K D of the third site is very much higher (19, 30), thereby implicitly rejecting his former interpretation of the Lineweaver-Burk plot. The Crystal Structure of Bovine Heart F 1 The MF 1 structure presented by Abrahams et al. (16) was interpreted by the authors as indicative for a rotational movement during catalysis. But the structural possibility of rotation, as evident from the structure, does not imply that rotation is an obligatory element of catalysis. The presence of one empty b site and three occupied a sites suggested, indeed, that Boyer s model with one empty b site and three high-affinity a sites was correct. Without discussing in detail the observed occupation of the nucleotide binding sites, it is evident, however, that the authors overlooked one essential point: both the ADP-containing and the AMPPNP-containing b sites are described as high-affinity sites, but as mentioned above, only one catalytic site with high affinity should be present on the basis of exchange experiments. Since the bound AMPPNP at one b site was not exchanged with the added ADP, I suppose that the site with AMPPNP is not catalytic, in agreement with the later finding by our group of a b site containing a tightly bound, nonexchangeable adenosine triphosphate (31). The proposal that the AMPPNP-containing site in the structure of Abrahams et al. is not the loose site of the Boyer model (the ADP-containing site is clearly the high-affinity catalytic site) is confirmed by the structure more recently reported by the group of Walker (22). This structure shows again two tightly bound nucleotides at b sites, but now the third b site contains a loosely bound nucleotide. These data show that the empty and loose site of the Boyer model are just one site and that only one of the other two sites (both with a tightly bound nucleotide) can additionally participate in catalysis. Support for Rotation Presented after 1994 In the recent literature three papers are mainly cited as providing evidence for rotation: Duncan et al. (32), Sabbert et al. (33) and Noji et al. (17). Do the data in these papers really provide evidence for rotation or at least participation of three equivalent catalytic sites in catalysis? The set up of the experiments by Duncan et al. (32) is well designed. Using a b-d380c mutant the authors introduced a disulfide linkage between the g subunit and one of the b subunits. By subsequent dissociation and reconstitution with radioactively labeled enzyme a preparation was obtained that contained one non-radioactive b subunit, cross-linked to the g subunit, and two radioactive b subunits. With this preparation it was possible to investigate a change in the orientation of the g subunit relative to the b subunits during catalysis by determining the radioactivity of the cross-linked band formed upon reoxidation after a period of catalysis that was started by reduction of the preformed crosslink. The authors state that upon reoxidation similar reactivities of unlabeled and radio labeled b subunits are found and that therefore the g subunit rotates relative to the b subunits. Is this statement correct?

476 BERDEN Upon reoxidation after catalysis the found radioactivity of the crosslinked band at 86 kda was 88% of the value expected in case of equivalency of all three b sites. When only two sites are involved in catalysis, the level of radioactivity should have been 75%. The result, therefore, is exactly in between the two predicted possible values. Although the measurement of label in a band on a gel is not very accurate, an error of 12% is quite high and not very likely and such an error should make the experiment non-discriminatory. If we, on the other hand, take the measured value as an accurate one, the rotational model can in fact be dismissed, since there should have been full randomization. But does the result fit with a two-sites model? During the period of catalysis (at least several tens of seconds) the supposedly non-participating first b site performs slow (single-site) catalysis, and this may result in some randomization of the g subunit relative to the b subunits, although it has been reported that single-site catalysis does not require mobility of the g subunit relative to the b subunits (34). More important, in the absence of catalysis (by the absence of either Mg 2+ or ATP or the presence of azide) 25% of the expected value of radioactivity in case of full randomization was already found, much higher than the value found in the presence of only buffer (3%). This finding indicates partial randomization in the presence of ATP and/or Mg 2+, independent of turnover, significantly increasing the expected value for the radioactivity in the b/g crosslinked band in case of participation of only two sites in rapid catalysis. In addition, Xiao and Penefsky have reported (35) that preparations of E. coli F 1 contain a mixture of d-deficient and d- containing enzyme and while in enzyme without d subunit the catalysis at the first b site appeared to be strongly enhanced upon addition of more substrate -indicating that this first site participates in multi-site catalysis-, in d-containing enzyme no such enhancement was observed, indicating that the first site in this form of the enzyme does not take part in multi-site catalysis. The higher than expected randomization (on the basis of a two-sites mechanism) in the experiment of Duncan et al. is, therefore, well explained if in some enzyme molecules the d subunit was lacking. Aggeler et al. (36) performed a similar experiment, based on the identification of crosslinks between a, d, g and e in a mutant with two engineered cysteines in the a (one enabling a quantitative crosslink with d and one with g or e) and one in the e, enabling a crosslink with the a. The authors conclude that g/e is essentially randomly distributed relative to the a subunits after a period of ATP hydrolysis, but this conclusion is very questionable. The expected result, assuming rotation, is that one third of the g a crosslinks also contains d and the same holds for the e a links. And one third of the a-crosslinks with d will contain g. In the presence of azide 40% of the a d crosslinks contained g and after hydrolysis this value was 30%. This indeed indicates some randomization, but certainly not quantitative. And the percentage of a d crosslinks that also contains e changed only from 22 to 20, not very convincing for a random distribution of the g/e subunits relative to the a 3 b 3 d domain! The data of the experiment by Sabbert et al. (33) show depolarization of the fluorescence of g-bound dye at the timescale of turnover, while the enzyme is immobilized. The depolarization is partial (the anisotropy decreases from 0.10 to 0.018) and the authors conclude that a rotation over more than 200 degrees or a motion in three steps are required to explain the depolarization effect and that therefore all three sites take part in catalysis. The calculated rotational motion is in between the expected value for participation of three sites in catalysis (rotation over 360 degrees) and the value expected for participation of only two sites (rotation over 120 degrees). The fact that the calculated value is lower than the expected 360 degrees indicates that no real rotation occurs, but that some phenomenon increases the apparent rotational movement to more than 120 degrees. Because of the low turnover of the enzyme (the t 1/2 is 100 ms) the turnover at the first site (0.5 s 71 (27)) is relatively fast and this may result in a mean rotational mobility in between the 120 and 360 degrees. Also some randomization independent of catalysis may occur (see above). With highly active enzyme, the calculated rotational movement may be closer to 120 degrees. A similar explanation can be given for the data presented later by Ha sler et al. (37). The experiment by Noji et al. (17) has already been mentioned above. To see a rotation is impressive and the authors earn much praise for this experiment, but scientifically it does not provide any evidence for an obligatory link between catalysis and rotation. The finding that in a preparation devoid of subunits d and e a few percent of the molecules rotate, does not show that in intact enzyme rotation and activity are coupled. On the contrary: since it is likely that also the wobbling molecules hydrolyse ATP, the data indicate that in the preparation studied no obligatory connection between activity and rotation is present. Since we know that in the E. coli enzyme the first site participates in multi-site catalysis when the d subunit is absent (35), we may assume that also in this incomplete TF 1 the first site can take part in multi-site catalysis. The structure of the enzyme then allows rotation to occur incidentally. One year later Kato-Yamada et al. performed a similar experiment, also the e-subunit (but not the d subunit) being incorporated in the enzyme and this subunit contained the flag (38). Again rotation was observed, but now less than 1% of the molecules showed rotation. Again, such an experiment shows that rotation is possible when the d subunit is absent, but at the same time it indicates that hydrolysis is not obligatory linked with rotation. Many experiments were subsequently carried out in a similar way with several systems, and occasional rotation was indeed observed, but in no case an obligatory link between ATP hydrolysis and rotation was demonstrated. Probably all

ROTARY MOVEMENTS WITHIN THE ATP SYNTHASE 477 molecules in these experiments catalyzed ATP hydrolysis (but this was never determined) and when only a few molecules show rotation it is evident that catalysis and rotation are not obligatorily linked. The observation of occasional rotation in preparations of complete enzyme is probably due to the presence of some d-deficient molecules. CONTROVERSIES AROUND THE ROTATING MODEL Weber et al. (21) have shown, using quenching of tryptophan fluorescence, that the ATP-hydrolyzing activity of E. coli F 1 fully matches the occupation of the third site with substrate. When site 1 or sites 1 and 2 are occupied, the activity is extremely slow (single site catalysis). The model of Boyer (39) is in clear conflict with these data, but is another interpretation of Boyer s own data possible? Milgrom et al. (40) have analyzed the kinetic features of the ATP hydrolysis reaction and they reach the conclusion that catalysis under most conditions is performed by two sites. As a consequence Boyer (39) suggests a reaction pattern in which near maximal reaction velocity of ATP synthesis and hydrolysis is attained when only two catalytic sites are filled. He assumes that the two participating sites are the b sites with the highest affinity and that the third b site is usually empty. Its K D is assumed to be in the millimolar range (30, 41) and it should participate in catalysis only at very high ATP concentrations (at deviation from the earlier conclusions of Gresser et al. (29)). Boyer proposes a site with a very high K D because Murataliev and Boyer (30) did not find a competition between TNP-ATP and ATP at a concentration of ATP of around 100 mm. I like to suggest that their problem is due to the fact that TNP-ATP binds to the enzyme also when the catalytic ATP sites on the b subunits are occupied, since the TNP moiety has itself a high affinity for a region of the protein that is near the ATP site (causing the dissociation constant of TNP ATP to be very low). When bound ATP dissociates while ATP TNP is present, the ATP moiety of the bound TNP ATP then binds in its place and therefore is competition of added ATP with TNP ATP only observed when the ATP concentration is very high. Accepting the thorough kinetic analysis of Milgrom et al. (40) we have to conclude that rapid catalysis is indeed performed by two sites, but these are not the sites 1 and 2, as is assumed by Boyer, but sites 2 and 3. Site 1, then, is not involved in the rapid catalysis and Weber et al. (21) indeed only show that all three sites have to be occupied with ligand, not that all three sites participate in catalysis. catalysis implies that all three b sites show a rapid turnover in the presence of a high concentration of ATP. For the chloroplast enzyme, whether isolated CF 1 or CF 0 F 1 or the enzyme in intact chloroplasts, Gräber and Fromme have shown already long ago (27) that the release of ADP from the first site is only 0.4 0.5 s 71, also at high ATP concentrations. Under these latter conditions the ATPase activity of the enzyme is 80 100 s 71, much faster than the turnover at the first catalytic site. The recent data reported by Berger et al. (28) confirm that at all ATP concentrations the system with a low Km and low Vm (similar to the properties of single-site catalysis) remains active. For the E. coli enzyme Xiao and Penefsky (35) have shown that an increased turnover at the first site upon addition of a high ATP concentration is only observed when the enzyme is deficient in the d subunit. In the full enzyme the turnover of the first site remains slow when the enzyme as a whole displays rapid catalysis at high ATP concentrations. In the mitochondrial F 1 with its very tightly bound nonexchangeable nucleotides, the first b site does not turn over at all (31). In the famous experiments by the Penefsky group on single-site catalysis (10 12) the tightly bound nucleotide at the first b site was never removed and the measured singlesite catalysis, enhanced upon addition of more ATP, occurred at the second b site. Also this (second) site is in the isolated enzyme occupied with a tightly bound (but exchangeable) nucleotide (31) and this site was partly emptied by treatment with phosphate and then available for single-site catalysis. In this respect is TF 1 quite different, since it does not contain tightly bound nucleotides and the dissociation of product from the first site may be fast enough for participation of this site in multi-site catalysis under certain conditions. Demonstration of the Presence of a Low-affinity Noncatalytic Site in MF 1. Edel et al. (19) have shown that modification of a low-affinity non-catalytic site with 8- nitreno-atp or 8-nitreno-ADP linearizes the Lineweaver- Burk plot for ATP hydrolysis by MF 1 showing a single high Km value. Binding of ATP to this site appeared to be responsible for the apparent negative cooperativity of catalysis displayed by the Lineweaver-Burk plot. Since MF 1 also contains a medium-affinity non-catalytic site (20, 24, 42) and two sites with non-exchangeable nucleotides, one of the latter nucleotides can not be bound at an a site (there are only 3 a sites) and is apparently bound at a b site, not participating in catalysis. SUMMARY OF DATA IN SUPPORT OF A DUAL-SITE MECHANISM OF CATALYSIS Kinetic Arguments No Enhancement of Catalysis at the First b Site Upon Addition of ATP. Involvement of three sites in multi-site Biochemical Evidence 2 a Sites and 2 b Sites are Available for Binding of Azidoadenine Nucleotides. We have shown in the eighties, using the 8-azido-analogues of the adenine nucleotides, that in mitochondrial F 1 4 nucleotide binding sites, 2 a sites and 2 b sites, are available for binding of these analogues, while two

478 BERDEN non-exchangeable adenine nucleotides remain bound to the enzyme (26). Since the enzyme contains 3 a and 3 b sites, one of them is bound at an a site and one at a b site. Similar experiments were more recently performed with 2-azido-ADP (43), an analogue that has the same anti-configuration as ADP and of which the site of binding can be unequivocally determined by analysis of the site of modification after illumination (44, 45). Also in this case 2 a sites and 2 b sites could be occupied with the analogue, while the two nonexchangeable nucleotides were still bound at their original sites (43), apparently one a site and one b site. Activity Parallels Occupation of the Third b-site. The ATPase activity of the E. coli enzyme parallels the occupation of the third b site (21). Two sites, then, contain a tightly bound nucleotide, and since experiments with the mitochondrial enzyme show that only one tightly bound nucleotide exchanges during catalysis, one tightly bound nucleotide does not exchange. In the E. coli and chloroplast enzyme the slow catalysis by the first site (see above) is rapid enough to be responsible for some exchange with medium nucleotides on the scale of a few seconds, but this site does not participate in rapid catalysis. The Tightly Bound Nucleotide at One b Site of MF 1 Does Not Exchange During Catalysis. In MF 1 Hartog and Berden (31) have replaced all the bound adenine nucleotides, apart from one non-exchangeable ADP at an a site, with 2-azidoadenine nucleotides. Their final enzyme contained, after precipitation and column centrifugation, one bound ADP and three bound 2-azido-adenine nucleotides. Of the latter, one was bound at an a site and two (one ATP and one ADP) at b sites. The enzyme was fully active and after 2 min of ATP hydrolysis the 2-azido-adenine nucleotide at one b site was exchanged, the others were not. This implies that one b site (with a non-exchanging nucleotide) does not take part in catalysis. Functional Evidence In 1987 Nieboer et al. (46) showed that the level of reactivation of Nbf-inhibited enzyme upon dissociation with LiCl, followed by reconstitution, could only be fitted with the assumption that for an active enzyme the b subunit in one of the three possible positions was allowed to contain Nbf, this subunit not being directly involved in catalysis. A very similar result was obtained by Miwa et al. (47) using TF 1 that contained two native b subunits and one mutated b subunit (E210Q) that could not bind any adenine nucleotide. The activity was one third of the activity of native enzyme, when corrected for the scrambling that was later shown to occur under these conditions (48). This result implies that in one of the three possible positions of the b subunits the mutation does not affect activity. Using an enzyme that contained one differently mutated b subunit, in which the mutation resulted in the very tight binding of a nucleotide without catalytic activity (E190Q), no activity was observed (47, 48). Apparently some modifications of one b subunit are allowed (binding of NbfCl, the E210Q mutation), others are not (E190Q). Structural Evidence Birkenha ger et al. (49) have shown that at the interface between F 1 and F 0 some members of the ring of c subunits are in contact with F 1 while others are available for antibodies. The binding of antibodies did not interfere with the binding of F 1 to F 0, nor with the activity of the enzyme. Since rotation of the ring of c subunits is expected to be fully inhibited by the binding of antibodies because of the presence of the stator, these data indicate that rotation is not required for catalysis. The possibility that the antibody continuously dissociates and associates, thereby not inhibiting rotation (50), seems very implausible. Another structural argument against rotation comes from crosslinking experiments. In 1992 Zanotti et al. (51) established that diamide induces a crosslinking in the ATP synthase of SMP between the g subunit of F 1 and the b subunit of F 0. The ATP hydrolysis and the concomitant proton pumping were not affected by this cross-link. Since the g subunit belongs to the stalk and the b subunit to the stator, the crosslinking data imply that catalysis can go on without rotation of the stalk relative to the stator. A final argument against rotation might be the number of c subunits in relation with the proton/atp stoicheiometry. If the reported number of c subunits in the chloroplast enzyme (52) is correct, the proton/atp stoicheiometry should be 4.67 (14 divided by 3) according to the current rotational model (rotation of the ring of c subunits is coupled with the passage of one proton through each of the c subunits and with rotation of the g subunit without slip), while this number has been determined as being 4.0 (53, 54). A movement over 120 degrees, however, can accommodate this number quite well. THE ALTERNATING DUAL-SITE MECHANISM The data described in section IV are not compatible with rotation as an essential element of the catalytic mechanism of the ATP synthases, and they provide compelling evidence in favor of a dual-site mechanism of catalysis. In addition, all data presented as arguments for rotation, appear to fit very well with a two-sites mechanism. The evidence is most compelling for MF 1, containing very tightly bound nucleotides. But also for the E. coli and the chloroplast enzyme the kinetic data prove that the b site with the highest affinity does not participate in multi-site catalysis. TF 1 does not contain any tightly bound nucleotide, and for this enzyme the evidence for a dual-site mechanism is only provided by the experiment of Miwa et al. (50) with the enzyme containing the E210Q mutation in one b subunit. Actual rotation may occur in some molecules when the turnover at the first b site is in the same order as the turnover at the other sites. In the E. coli and chloroplast enzyme this

ROTARY MOVEMENTS WITHIN THE ATP SYNTHASE 479 may be the case when the d subunit is missing or the turnover is very slow, respectively. In native MF 1 this is never the case since turnover at the first b site is absent. The binding change mechanism, therefore, operates with two sites and not with three. Of the two catalytic sites one is in the closed conformation in which bound ATP + water are in near equilibrium with ADP + phosphate, the other in an open or semi-open conformation in which the product dissociates and new substrate binds. The interaction between the two sites is mediated mainly by the g subunit, but also by the a subunits. The ligand at the first (not catalytically involved) b site is in active MF 1 an ATP, ADP being inhibitory and this finding explains the phenomenon of the so called hysteretic inhibition by ADP (31). Also the chloroplast enzyme is inactive when the first b site contains ADP and activation includes dissociation of this ADP (55). In both the E. coli enzyme and the chloroplast enzyme the first b site catalyses single-site catalysis, but in the mitochondrial enzyme the bound ATP remains tightly bound as ATP. In MF 1 the ligand at the low-affinity a site determines the affinity of the open/loose catalytic site for its substrate. The medium-affinity a site is probably important for the direction of the catalysis: with ADP bound at this site the activity of ATP hydrolysis is about 40% lower than when ATP is bound (56) and this form of the enzyme may be more suited for ATP synthesis than for ATP hydrolysis. For the non-exchangeable ADP at the high-affinity a site so far only a structural role (stability of the enzyme) has been demonstrated. In the chloroplast enzyme the a sites bind only ATP and no ADP (55). A schematic representation of the six nucleotide binding sites of MF 1 is presented in Figure 2. WHY A DUAL-SITE MECHANISM IN COMBINATION WITH THE POSSIBILITY OF ROTATION? Many experiments have shown that in various F 1 preparations molecules have the possibility of rotation, in agreement with the structural data of Abrahams et al. (16). But since rotation is not an element of the catalytic mechanism, as has been concluded in the present analysis, the question arises why the capability of rotation has been developed? The answer might be that rotation was part of the original catalytic mechanism of hydrolysis, but that for (adequate regulation of) ATP synthesis a non-rotating asymmetrical device proved more satisfactory. If we consider the various available preparations of ATP synthase, it is evident that the TF 1 represents the most symmetrical form and the MF 1 the least symmetrical form of the catalytic part of the enzyme. TF 1 does not contain any tightly bound nucleotide, E. coli F 1 contains tightly bound, but well removable, bound nucleotides, CF 1 contains one very tightly bound nucleotide and MF 1 contains 2 non-exchangeable nucleotides. The final evolutionary stage, then, is that one of the three ab couples contains two very tightly bound, nonexchangeable nucleotides, stabilizing the enzyme, at the same time providing an asymmetrical structure. It seems plausible that at this ab couple OSCP is bound. The reason for the development towards an asymmetrical structure might be the fact that rotation is principally dissipative and for the development of an efficient system, working in two directions, that of synthesis and that of hydrolysis, such dissipation had to be avoided. If this argumentation is correct, it might be possible that the V-type ATPases, working in only one direction in higher organisms (that of ATP hydrolysis), have maintained to a larger extent the original symmetrical structure and possibly still can rotate (57). Figure 2. Schematic characterization of the 6 nucleotide binding sites of MF 1. Sites 1 and 4 contain a tightly bound non-exchangeable nucleotide. 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