Characterization of the relationship between ADP- and epsilon-induced inhibition in cyanobacterial F1-ATPase

The ATPase activity of chloroplast and bacterial F(1)-ATPase is strongly inhibited by both the endogenous inhibitor ε and tightly bound ADP. Although the physiological significance of these inhibitory mechanisms is not very well known for the membrane-bound F(0)F(1), these are very likely to be important in avoiding the futile ATP hydrolysis reaction and ensuring efficient ATP synthesis in vivo. In a previous study using the α(3)β(3)γ complex of F(1) obtained from the thermophilic cyanobacteria, Thermosynechococcus elongatus BP-1, we succeeded in determining the discrete stop position, ～80° forward from the pause position for ATP binding, caused by ε-induced inhibition (ε-inhibition) during γ rotation (Konno, H., Murakami-Fuse, T., Fujii, F., Koyama, F., Ueoka-Nakanishi, H., Pack, C. G., Kinjo, M., and Hisabori, T. (2006) EMBO J. 25, 4596-4604). Because γ in ADP-inhibited F(1) also pauses at the same position, ADP-induced inhibition (ADP-inhibition) was assumed to be linked to ε-inhibition. However, ADP-inhibition and ε-inhibition should be independent phenomena from each other because the ATPase core complex, α(3)β(3)γ, also lapses into the ADP-inhibition state. By way of thorough biophysical and biochemical analyses, we determined that the ε subunit inhibition mechanism does not directly correlate with ADP-inhibition. We suggest here that the cyanobacterial ATP synthase ε subunit carries out an important regulatory role in acting as an independent "braking system" for the physiologically unfavorable ATP hydrolysis reaction.     

Redox regulation of the rotation of F(1)-ATP synthase

In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation.     

Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.     

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the epsilon subunit

The chloroplast-type F(1) ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor epsilon, tightly bound ADP, the membrane potential and the redox state of the gamma subunit. In order to understand the molecular mechanism of epsilon inhibition, we constructed an expression system for the alpha(3)beta(3)gamma subcomplex in thermophilic cyanobacteria allowing thorough investigation of epsilon inhibition. epsilon Inhibition was found to be ATP-independent, and different to that observed for bacterial F(1)-ATPase. The role of the additional region on the gamma subunit of chloroplast-type F(1)-ATPase in epsilon inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of gamma in epsilon inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the epsilon subunit of chloroplast-type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.     

Principal role of the arginine finger in rotary catalysis of F1-ATPase

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F(1) remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F(1) with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F(1) showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F(1) carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.     

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.     

Regulation of F0F1-ATPase from Synechocystis sp. PCC 6803 by gamma and epsilon subunits is significant for light/dark adaptation

The γ and ε subunits of F(0)F(1)-ATP synthase from photosynthetic organisms display unique properties not found in other organisms. Although the γ subunit of both chloroplast and cyanobacterial F(0)F(1) contains an extra amino acid segment whose deletion results in a high ATP hydrolysis activity (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865), its ε subunit strongly inhibits ATP hydrolysis activity. To understand the physiological significance of these phenomena, we studied mutant strains with (i) a C-terminally truncated ε (ε(ΔC)), (ii) γ lacking the inserted sequence (γ(Δ198-222)), and (iii) a double mutation of (i) and (ii) in Synechocystis sp. PCC 6803. Although thylakoid membranes from the ε(ΔC) strain showed higher ATP hydrolysis and lower ATP synthesis activities than those of the wild type, no significant difference was observed in growth rate and in intracellular ATP level both under light conditions and during light-dark cycles. However, both the ε(ΔC) and γ(Δ198-222) and the double mutant strains showed a lower intracellular ATP level and lower cell viability under prolonged dark incubation compared with the wild type. These data suggest that internal inhibition of ATP hydrolysis activity is very important for cyanobacteria that are exposed to prolonged dark adaptation and, in general, for the survival of photosynthetic organisms in an ever-changing environment.     

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.     

Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation

ATP hydrolysis-dependent rotation of the F(1) sector of the ATP synthase is a successive cycle of catalytic dwells (～0.2 ms at 24 °C) and 120° rotation steps (～0.6 ms) when observed under V(max) conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401-22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ～1 s in duration. The rotation behavior with adenosine 5'-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μM) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F(1) requires ～2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ε subunit, the inhibitor of F(1)-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ε subunit has little effect on the transition between active and inhibited states. Rather, the ε subunit confers lower activation energy of steady-state rotation. These results suggest that the ε subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.     

Determination of the partial reactions of rotational catalysis in F1-ATPase

Steady-state ATP hydrolysis in the F1-ATPase of the F(O)F1 ATP synthase complex involves rotation of the central gamma subunit relative to the catalytic sites in the alpha3beta3 pseudo-hexamer. To understand the relationship between the catalytic mechanism and gamma subunit rotation, the pre-steady-state kinetics of Mg x ATP hydrolysis in the soluble F1-ATPase upon rapid filling of all three catalytic sites was determined. The experimentally accessible partial reactions leading up to the rate-limiting step and continuing through to the steady-state mode were obtained for the first time. The burst kinetics and steady-state hydrolysis for a range of Mg x ATP concentrations provide adequate constraints for a unique minimal kinetic model that can fit all the data and satisfy extensive sensitivity tests. Significantly, the fits show that the ratio of the rates of ATP hydrolysis and synthesis is close to unity even in the steady-state mode of hydrolysis. Furthermore, the rate of Pi binding in the absence of the membranous F(O) sector is insignificant; thus, productive Pi binding does not occur without the influence of a proton motive force. In addition to the minimal steps of ATP binding, reversible ATP hydrolysis/synthesis, and the release of product Pi and ADP, one additional rate-limiting step is required to fit the burst kinetics. On the basis of the testing of all possible minimal kinetic models, this step must follow hydrolysis and precede Pi release in order to explain burst kinetics. Consistent with the single molecule analysis of Yasuda et al. (Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., and Itoh, H. (2001) Nature 410, 898-904), we propose that the rate-limiting step involves a partial rotation of the gamma subunit; hence, we name this step k(gamma). Moreover, the only model that is consistent with our data and many other observations in the literature suggests that reversible hydrolysis/synthesis can only occur in the active site of the beta(TP) conformer (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

Chemo-Mechanical Coupling in F1-ATPase Revealed by Catalytic Site Occupancy during Catalysis

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.     

Inhibition of mitochondrial F1-ATPase activity by an anti-alpha subunit monoclonal antibody which modifies interactions between catalytic and regulatory sites

A monoclonal antibody, 7B3, specific to the alpha subunit of the mitochondrial ATPase-ATP synthase inhibited the rate of ATP hydrolysis by either soluble F1 or electron transport particles up to a maximum of 75%. However, 7B3 did not modify the rate of ITP hydrolysis. In addition, the apparent Km for MgATP extrapolated at high ATP concentrations had the same value in the absence as in the presence of 7B3. The antibody did not change the inactivation rate of F1-ATPase induced by dicyclohexylcarbodiimide or 4-chloro-7-nitro-2,1,3-benzoxadiazole. These observations indicate that 7B3 did not directly interfere with the catalytic sites of ATP or ITP hydrolysis. On the contrary, 7B3 modified the interaction between nucleotide sites and therefore the regulation of the rate of ATP hydrolysis. Indeed, 7B3 changed into a positive cooperativity the negative cooperativity observed when measuring the rate of ATP hydrolysis as a function of ATP concentration. 7B3 also increased the binding of ADP to F1. 7B3 prevented the rapid phase of inactivation of F1 by 5'-p-fluorosulfonylbenzoyladenosine. This phase has been correlated to the binding of 5'-p-fluorosulfonylbenzoyladenosine to regulatory sites (Di Pietro, A., Godinot, C., Martin, J. C., and Gautheron, D. C. (1979) Biochemistry 18, 1738-1745). The inhibition of ATP hydrolysis is concomitant with the binding of 1 mol of IgG or of 2 mol of Fab fragments per mol of F1. However, by further increasing the ratio Fab/F1, only 1 mol of Fab remained bound to F1 without change in inhibition of ATPase activity. All these experiments strongly support the suggestion that F1 conformational changes occurring upon binding of 7B3 to alpha subunit induce a modification of interactions between nucleotide sites. This modification would be consecutive to a change in the normal interaction between the alpha and beta subunits which is required to observe an active rate of ATP hydrolysis or synthesis. In conclusion, the use of this monoclonal antibody demonstrates for the first time in mammalian F1 the role of the conformation of the alpha subunit in the regulation of the ATPase activity.     

Single-molecule Analysis of Inhibitory Pausing States of V1-ATPase

V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V(1)-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V(1) in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F(1), suggesting that LP represents the ADP-inhibited state of V(1)-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V(1)-ATPase rotation modulates the off rate of ADP.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

Modulation of the GTPase activity of the chloroplast F1-ATPase by ATP binding at noncatalytic sites

Although the binding of nucleotides at the noncatalytic sites of F1-ATPase has been regarded as probably having some type of regulatory function, only limited observations have been reported that support such a role. We present here results showing that the presence of ATP at noncatalytic sites can give a fivefold enhancement of the rate of GTP hydrolysis by the chloroplast F1-ATPase. Heat-activation of the chloroplast F1-ATPase in the presence of ATP, followed by column separation from the medium nucleotides gives an enzyme with two of the three noncatalytic sites filled with ATP. In contrast, heat-activation in the presence of ADP gives an enzyme with only one noncatalytic site filled with ADP. Such an enzyme with two noncatalytic sites empty catalyzes MgGTP hydrolysis only very slowly. The filling of a second noncatalytic site with ATP by exposure of the enzyme to ATP without Mg2+ present, followed by column separation, markedly increases the rate of GTP hydrolysis. A further increase occurs when a third noncatalytic site is filled by exposure to Mg2+ and ATP. The rate of MgATP hydrolysis is the same for the enzyme heat-activated in the presence of ATP or ADP, probably because MgATP, unlike MgGTP, rapidly binds to both catalytic and noncatalytic sites.     

Does the γ subunit move to an abortive position for ATP hydrolysis when the F1·ADP·Mg complex isomerizes to the inactive F1*·ADP·Mg complex?

F₁-ATPases transiently entrap inhibitory MgADP in a catalytic site during turnover when noncatalytic sites are not saturated with ATP. An initial burst of ATP hydrolysis rapidly decelerates to a slow intermediate rate that gradually accelerates to a final steady-state rate. Transition from the intermediate to the final rate is caused by slow binding of ATP to noncatalytic sites which promotes dissociation of inhibitory MgADP from the affected catalytic site. Evidence from several laboratories suggests that the γ subunit rotates with respect to α/β subunit pairs of F₁-ATPases during ATP hydrolysis. The α₃β₃ and α₃β₃δ subcomplexes of the TF₁-ATPase do not entrap inhibitory MgADP in a catalytic site during turnover, suggesting involvement of the γ subunit in the entrapment process. From these observations, it is proposed that the γ subunit moves into an abortive position for ATP hydrolysis when inhibitory MgADP is entrapped in a catalytic site during ATP hydrolysis.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.