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.     

Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch

F(1)-ATP synthase (F(1)-ATPase) is equipped with a special mechanism that prevents the wasteful reverse reaction, ATP hydrolysis, when there is insufficient proton motive force to drive ATP synthesis. Chloroplast F(1)-ATPase is subject to redox regulation, whereby ATP hydrolysis activity is regulated by formation and reduction of the disulfide bond located on the γ subunit. To understand the molecular mechanism of this redox regulation, we constructed a chimeric F(1) complex (α(3)β(3)γ(redox)) using cyanobacterial F(1), which mimics the regulatory properties of the chloroplast F(1)-ATPase, allowing the study of its regulation at the single molecule level. The redox state of the γ subunit did not affect the ATP binding rate to the catalytic site(s) and the torque for rotation. However, the long pauses caused by ADP inhibition were frequently observed in the oxidized state. In addition, the duration of continuous rotation was relatively shorter in the oxidized α(3)β(3)γ(redox) complex. These findings lead us to conclude that redox regulation of CF(1)-ATPase is achieved by controlling the probability of ADP inhibition via the γ subunit inserted region, a sequence feature observed in both cyanobacterial and chloroplast ATPase γ subunits, which is important for ADP inhibition (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865).     

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.     

ATPase activity of a highly stable alpha(3)beta(3)gamma subcomplex of thermophilic F(1) can be regulated by the introduced regulatory region of gamma subunit of chloroplast F(1)

A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.     

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.     

C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.     

Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit

The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the gamma regulatory segment and its function in ATPase regulation via its interaction with the inhibitory epsilon subunit. Mutations were designed using a homology model of the chloroplast gamma subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the epsilon subunit but did not abolish epsilon binding. The results suggest a mechanism by which the gamma and epsilon subunits interact with each other to induce the latent state of the enzyme.     

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.     

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.     

F1-ATPase, the C-terminal end of subunit gamma is not required for ATP hydrolysis-driven rotation

ATP hydrolysis by the isolated F(1)-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3). The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F(1)-ATPase). The ATP hydrolysis activity of a load-free ensemble of F(1) with 12 residues deleted decreased to 24% of the control. EF(1) with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF(1) when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF(1) (full-length gamma) to 34 kJ/mol for EF(1)(gamma-12). These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.     

Regulation of the F0F1-ATP synthase: the conformation of subunit epsilon might be determined by directionality of subunit gamma rotation

F(0)F(1)-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the gamma epsilon c(approximately 10)-subunits complex relative to the rest of the enzyme. In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit epsilon also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F(1). The latter form inhibits ATP hydrolysis (but not synthesis). We propose that the directionality of the coiled-coil subunit gamma rotation determines whether subunit epsilon is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit epsilon. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit epsilon into a consistent picture.     

Electrostatic interactions in catalytic centers of F1-ATPase

The first part of this paper is a brief review of works concerned with the mechanisms of functioning of F0F1-ATP synthases. F0F1-ATP syntheses operate as rotating molecular machines that provide the synthesis of ATP from ADP and inorganic phosphate (Pi) in mitochondria, chloroplasts, and bacteria at the expense of the energy of electrochemical gradient of hydrogen ions generated across energy-transducing mitochondrial, chloroplast or, bacterial membranes. A distinguishing feature of these enzymes is that they operate as rotary molecular motors. In the second part of the work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), reaction products (MgADP and Pi), and charged amino acid residues of the F1-ATPase molecule to energy changes associated with the binding of ATP and its chemical transformations in the catalytic centers located at the interface of the alpha- and beta-subunits of the enzyme (oligomer complex alpha 3 beta 3 gamma of bovine mitochondrial ATPase). The catalytic cycle of ATP hydrolysis considered in the work includes conformational changes of alpha- and beta-subunits caused by unidirectional rotations of the central gamma-subunit. The results of our calculations are consistent with the idea that the energetically favorable process of ATP binding to the "open" catalytic center of F1-ATPase initiates the rotation of the gamma-subunit followed by ATP hydrolysis in another ("closed") catalytic center of the enzyme.     

The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit

In F(1)-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260-14263), and the role of this motif remained unknown. Here, we found that the epsilon subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F(1)-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the epsilon subunit in the mutant F(1)-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the epsilon subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the beta subunit or of the basic residues in the C-terminal alpha-helix of the epsilon subunit induced a partial effect. We here conclude that the epsilon subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the beta subunit.     

A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis

The F(0)F(1)-ATP synthase couples the functions of H(+) transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located gamma, epsilon, and c subunits. To understand the gamma subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F(1)-ATPase enzyme that uniquely fits the pre-steady state and steady state data ( Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797 ). Here we directly test the model using two single cysteine mutants, betaD380C and betaE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gammaCys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg.ATP conditions retained the burst of hydrolysis but was not able to release P(i). These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P(i) release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the betaGlu-381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.     

Mutations within the C-terminus of the gamma subunit of the photosynthetic F1-ATPase activate MgATP hydrolysis and attenuate the stimulatory oxyanion effect

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to gamma subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit were changed to leucine and alanine, respectively. Each mutant gamma was assembled together with alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg2+ ions, but is not essential for cooperative nucleotide exchange.     

Replacement of arginine 246 by histidine in the beta subunit of Escherichia coli H+-ATPase resulted in loss of multi-site ATPase activity

A mutant strain KF43 of Escherichia coli defective in the beta subunit of H+-translocating ATPase (F0F1) was examined. In this mutant, replacement of Arg246 by His was identified by DNA sequencing of the mutant gene and confirmed by tryptic peptide mapping. The mutant F1-ATPase was defective in multi-site hydrolysis of ATP but was active in uni-site hydrolysis. Studies on the kinetics of uni-site hydrolysis indicated that the k1 (rate of ATP binding) was similar to that of the wild-type, but the k-1 (rate of release of ATP) could not be measured. The mutant enzyme had a k3 (rate of release of inorganic phosphate) about 15-fold higher than that of the wild-type and showed 3 orders of magnitude lower promotion from uni- to multi-site catalysis. These results suggest that Arg246 or the region in its vicinity is important in multi-site hydrolysis of ATP and is also related to the binding of inorganic phosphate. Reconstitution experiments using isolated subunits suggested that hybrid enzymes (alpha beta gamma complexes) carrying both the mutant and wild-type beta subunits were inactive in multi-site hydrolysis of ATP, supporting the notion that three intact beta subunits are required for activity of the F1 molecule.     

ATP-dependent rotation of mutant ATP synthases defective in proton transport

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.     

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).     

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H(+)-F(O)F(1)-ATP synthase couples proton flow through its membrane portion, F(O), to the synthesis of ATP in its headpiece, F(1). Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gammaLys23 with the DELSEED region of subunit beta stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation.     

Interactions among gamma R268, gamma Q269, and the beta subunit catch loop of Escherichia coli F1-ATPase are important for catalytic activity

Removal of the ability to form a salt bridge or hydrogen bonds between the beta subunit catch loop (beta Y297-D305) and the gamma subunit of Escherichia coli F1Fo-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues beta T304, beta D305, beta D302, gamma Q269, and gamma R268 were found to be very important for ATP hydrolysis catalyzed by soluble F1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue beta D305 would not tolerate substitution with Val or Ser and had extremely low activity as beta D305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the gamma subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which alpha beta gamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.