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Principal Role of the Arginine Finger in Rotary Catalysis of F1-ATPase*

Open AccessPublished:March 08, 2012DOI:https://doi.org/10.1074/jbc.M111.328153
      F1-ATPase (F1) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α3β3 stator ring. The 3 catalytic sites of F1 reside on the interface of the α and β subunits of the α3β3 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 F1 remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F1 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 F1 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 F1 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.

      Introduction

      F1-ATPase (F1)
      The abbreviations used are: F1
      F1-ATPase
      GAP
      G-protein-activating protein
      ATPγS
      adenosine 5′-(γ-thio)triphosphate
      AMP-PNP
      adenosine-5′-(β,γ-imino)-triphosphate
      EF1
      F1-ATPase from Escherichia coli
      MF1
      F1-ATPase from bovine or yeast mitochondria
      TF1
      F1-ATPase from thermophilic Bacillus PS3
      F1(αR364K)
      F1 carrying three copies of the αR364K subunit
      F1(1×αR364K)
      hybrid F1 carrying single copy of the αR364K subunit.
      is a water-soluble portion of the FoF1-ATP synthase, and a rotary motor protein driven by ATP hydrolysis (
      • Kinosita Jr., K.
      • Yasuda R.
      • Noji H.
      F1-ATPase: a highly efficient rotary ATP machine.
      ,
      • Nakamoto R.K.
      • Baylis Scanlon J.A.
      • Al-Shawi M.K.
      The rotary mechanism of the ATP synthase.
      ,
      • Senior A.E.
      • Nadanaciva S.
      • Weber J.
      The molecular mechanism of ATP synthesis by F1F0-ATP synthase.
      ,
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ATP synthase-a marvelous rotary engine of the cell.
      ,
      • Düser M.G.
      • Zarrabi N.
      • Cipriano D.J.
      • Ernst S.
      • Glick G.D.
      • Dunn S.D.
      • Börsch M.
      36 degrees step size of proton-driven c-ring rotation in FoF1-ATP synthase.
      ). Bacterial F1 is composed of α, β, γ, δ, and ϵ subunits with a stoichiometry of 3:3:1:1:1. The minimum complex that functions as a rotary motor is the α3β3γ subcomplex. Three β subunits and 3 α subunits form the stator ring, in which these subunits are alternately arranged. The rotary shaft, namely, the γ subunit, is accommodated in the central cavity of the α3β3 stator ring. The catalytic reaction centers are located at the α/β interfaces. Although most of the residues forming the catalytic site are on the β subunits, the α subunits contain 1 catalytically critical and well-conserved arginine residue, termed the arginine finger, which is the focus of the present study.
      The rotation of F1 can be visualized by optical microscopy (
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      Direct observation of the rotation of F1-ATPase.
      ,
      • Spetzler D.
      • York J.
      • Daniel D.
      • Fromme R.
      • Lowry D.
      • Frasch W.
      Microsecond time scale rotation measurements of single F1-ATPase molecules.
      ,
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      ,
      • Omote H.
      • Sambonmatsu N.
      • Saito K.
      • Sambongi Y.
      • Iwamoto-Kihara A.
      • Yanagida T.
      • Wada Y.
      • Futai M.
      The γ-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli.
      ). The γ subunit of F1, from various species studied to date, rotates in a counterclockwise direction. The unidirectionality of the rotation is supported by the intrinsic cooperative torque generation of the 3 β subunits (
      • Uchihashi T.
      • Iino R.
      • Ando T.
      • Noji H.
      High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase.
      ). The step size of the rotation is 120°, each coupled with single turnover of ATP hydrolysis (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      ). The 120° step is further divided into 80° and 40° substeps. The 80° substep is triggered by ATP binding and ADP release, each of which occurs on different β subunits. The 40° substep is initiated by ATP hydrolysis and release of inorganic phosphate (Pi), which also occurs on different β subunits. The angular positions of the dwell before the 80° and 40° substeps are referred to as the ATP-binding (ATP-waiting) angle and the catalytic angle, respectively. The basic reaction scheme for the rotation and catalysis of F1 has been recently established (
      • Watanabe R.
      • Iino R.
      • Noji H.
      Phosphate release in F1-ATPase catalytic cycle follows ADP release.
      ), although several uncertainties do remain (
      • Weber J.
      Structural biology: Toward the ATP synthase mechanism.
      ). Each β subunit binds to ATP when the γ subunit is oriented at a specific angle, and the binding angles for the individual β subunits differ by 120°. Each β subunit catalyzes ATP hydrolysis at 200° from its ATP-binding angle (
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits.
      ). Subsequently, ADP and Pi are released at 240° (
      • Adachi K.
      • Oiwa K.
      • Nishizaka T.
      • Furuike S.
      • Noji H.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      Coupling of rotation and catalysis in F(1)-ATPase revealed by single-molecule imaging and manipulation.
      ) and 320° (
      • Watanabe R.
      • Iino R.
      • Noji H.
      Phosphate release in F1-ATPase catalytic cycle follows ADP release.
      ), respectively. When the γ subunit returns to the original position, the β subunits initiate the next round of catalysis by binding to a new ATP.
      The crystal structure of F1 reveals the structural basis of the chemomechanical coupling of F1 (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.
      ). The 3 β subunits have distinct ligand-bound states and conformations: one β is bound to an ATP analog, AMP-PNP; a second is bound to ADP and azide (
      • Bowler M.W.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      How azide inhibits ATP hydrolysis by the F-ATPases.
      ); and the third binds to none. These 3 states of β are referred to as βTP, βDP, and βempty, respectively. A cross-linking experiment in a single-molecule rotation assay established that the crystal structure represents the catalytic dwell state (
      • Sielaff H.
      • Rennekamp H.
      • Engelbrecht S.
      • Junge W.
      Functional halt positions of rotary FOF1-ATPase correlated with crystal structures.
      ), and that βTP, βDP, and βempty represent the 80°, 200°, and 320° states of the above mentioned reaction scheme, respectively (
      • Okuno D.
      • Fujisawa R.
      • Iino R.
      • Hirono-Hara Y.
      • Imamura H.
      • Noji H.
      Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation.
      ). Both the βTP and βDP states assume a closed conformation in which the C-terminal domain rotates inward toward the γ subunit and enclose the bound nucleotide. On the other hand, βempty assumes an open conformation by the outward rotation of the C-terminal domain. Therefore, the widely accepted view is that the β subunit undergoes a conformational transition between the open and closed states during nucleotide binding/dissociation, inducing unidirectional rotation of the central γ subunit (
      • Masaike T.
      • Koyama-Horibe F.
      • Oiwa K.
      • Yoshida M.
      • Nishizaka T.
      Cooperative three-step motions in catalytic subunits of F(1)-ATPase correlate with 80 degrees and 40 degrees substep rotations.
      ,
      • Oster G.
      • Wang H.
      Reverse engineering a protein: the mechanochemistry of ATP synthase.
      ).
      The βTP and βDP conformations closely resemble each other. Both β subunits bind to the ATP analog MgADP-BeF3 (
      • Kagawa R.
      • Montgomery M.G.
      • Braig K.
      • Leslie A.G.
      • Walker J.E.
      The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride.
      ) or the transition state analog MgADP-AlF4 (
      • Menz R.I.
      • Walker J.E.
      • Leslie A.G.
      Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis.
      ), simultaneously. A recent single-molecule experiment has indicated that βDP is the catalytically active state for ATP cleavage (
      • Okuno D.
      • Fujisawa R.
      • Iino R.
      • Hirono-Hara Y.
      • Imamura H.
      • Noji H.
      Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation.
      ). The conformations of the catalytic sites on βTP and βDP are different at the α/β interfaces. βTP forms a relatively open interface with the neighboring α subunit (αTP), whereas βDP forms a closed interface with αDP, suggesting this is the highest affinity site. However, based on nucleotide titration experiments, it was shown that βTP has the highest affinity for nucleotide (
      • Mao H.Z.
      • Weber J.
      Identification of the βTP site in the x-ray structure of F1-ATPase as the high-affinity catalytic site.
      ). This positional rearrangement of the α subunit induces proximity of the well-conserved arginine residue to the β- and γ-phosphates of the bound ATP (Fig. 1). The stereogeometric position of this arginine residue is similar to that of the catalytically crucial arginine residue found in the α subunit of trimeric G-proteins and in GTPase-activating proteins (GAPs) for small G-proteins (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.
      ,
      • Noel J.P.
      • Hamm H.E.
      • Sigler P.B.
      The 2.2 A crystal structure of transducin-α complexed with GTPγS.
      ,
      • Scheffzek K.
      • Ahmadian M.R.
      • Kabsch W.
      • Wiesmüller L.
      • Lautwein A.
      • Schmitz F.
      • Wittinghofer A.
      The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants.
      ). The latter is termed the “arginine finger,” and upon binding to small G-proteins, GAPs present this residue to the β- and γ-phosphate of bound GTP, and remarkably accelerate their hydrolysis by stabilizing the transition state.
      Figure thumbnail gr1
      FIGURE 1Crystal structure of MF1 viewed from the side, αDPDP catalytic interface (PDB code: 1W0J). The α, β, and γ subunits are shown in yellow, green, and gray, respectively. The “arginine finger” in the α subunit (αR373 in MF1) is shown by red space-filling model. ADP, BeF3, and magnesium ion bound to the catalytic site are shown by blue, cyan, and purple space-filling models, respectively. The arginine finger of TF1, αR364, was mutated into lysine (αR364K) in this study.
      In a genetic screen, the conserved arginine of Escherichia coli F1 (EF1) was originally found to be one of the catalytically important residues on the α subunit (
      • Soga S.
      • Noumi T.
      • Takeyama M.
      • Maeda M.
      • Futai M.
      Mutational replacements of conserved amino acid residues in the α subunit change the catalytic properties of Escherichia coli F1-ATPase.
      ). Biochemical studies then confirmed a critical role for this arginine (
      • Soga S.
      • Noumi T.
      • Takeyama M.
      • Maeda M.
      • Futai M.
      Mutational replacements of conserved amino acid residues in the α subunit change the catalytic properties of Escherichia coli F1-ATPase.
      ,
      • Le N.P.
      • Omote H.
      • Wada Y.
      • Al-Shawi M.K.
      • Nakamoto R.K.
      • Futai M.
      Escherichia coli ATP synthase α subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state.
      ,
      • Nadanaciva S.
      • Weber J.
      • Wilke-Mounts S.
      • Senior A.E.
      Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization.
      ,
      • Turina P.
      • Aggeler R.
      • Lee R.S.
      • Senior A.E.
      • Capaldi R.A.
      The cysteine introduced into the α subunit of the Escherichia coli F1-ATPase by the mutation α R376C is near the αβ subunit interface and close to a noncatalytic nucleotide binding site.
      ). In particular, a nucleotide titration study with a transition state analog, MgADP-fluoroaluminate, showed that this arginine stabilizes the transition state of hydrolysis and accelerates the rate of hydrolysis (
      • Nadanaciva S.
      • Weber J.
      • Wilke-Mounts S.
      • Senior A.E.
      Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization.
      ). Several theoretical studies also support the view that this arginine contributes to lowering the energy level of the post-hydrolysis state compared with the pre-hydrolysis state (
      • Dittrich M.
      • Hayashi S.
      • Schulten K.
      On the mechanism of ATP hydrolysis in F1-ATPase.
      ,
      • Yang W.
      • Gao Y.Q.
      • Cui Q.
      • Ma J.
      • Karplus M.
      The missing link between thermodynamics and structure in F1-ATPase.
      ). A crystal structure of mitochondrial F1 (MF1) revealed that the guanidinium group of the arginine finger is positioned at 1 Å closer to the β-phosphate in the βDP than that in the βTP (Fig. 1) (
      • Kagawa R.
      • Montgomery M.G.
      • Braig K.
      • Leslie A.G.
      • Walker J.E.
      The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride.
      ). This positional rearrangement is proposed to initiate ATP hydrolysis. The arginine finger of EF1 is αR376, and corresponds to αR373 in MF1 and αR364 in the thermophilic Bacillus PS3 F1 (TF1).
      The cysteine mutant of the arginine finger of F1 was severely impaired in multisite catalysis (
      • Soga S.
      • Noumi T.
      • Takeyama M.
      • Maeda M.
      • Futai M.
      Mutational replacements of conserved amino acid residues in the α subunit change the catalytic properties of Escherichia coli F1-ATPase.
      ), which is the highly active mode where 3 catalytic sites sequentially hydrolyze ATP to reach the maximum hydrolysis rate. However, this mutation only slightly impaired unisite catalysis, a slow catalysis mode of F1 that proceeds only when one of the catalytic sites is occupied by a nucleotide and the others are unoccupied. Unisite catalysis is considered indicative of the intrinsic catalytic ability of F1 that is independent of the allosteric interactions with other catalytic sites. Subsequent biochemical research showed that alanine or lysine mutations of the arginine finger also caused almost complete loss of multisite catalysis (by a factor of 103), although unisite catalysis was not affected (
      • Le N.P.
      • Omote H.
      • Wada Y.
      • Al-Shawi M.K.
      • Nakamoto R.K.
      • Futai M.
      Escherichia coli ATP synthase α subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state.
      ). From these observations, it was concluded that the arginine finger is not directly involved in the enhancement of ATP hydrolysis rate of F1, but it has a critical role in mediating cooperativity among the 3 catalytic sites to facilitate multisite catalysis.
      Thus, although the importance of the arginine finger in catalysis is well recognized, its principal role is still unclear. In this study, we investigated the role of the arginine finger of TF1 by analyzing the rotational behavior of the lysine mutant. This allowed us to elucidate the impact of this mutation on the intrinsic catalytic power and the efficiency of allosteric cooperative communication among the catalytic sites of F1.

      DISCUSSION

      Our results verify the crucial role of the arginine finger of F1 (Fig. 1) in the ATP cleavage step. The lysine mutation caused drastic suppression of the rate constant of the ATP cleavage step in the rotary catalysis of F1. This is shown by the following results: F1(αR364K) showed a 350-times longer pause than wild-type F1 at the catalytic angle where ATP cleavage and Pi release occur (FIGURE 3, FIGURE 4). Moreover, this catalytic pause was lengthened by a slowly hydrolyzed ATP analog, ATPγS (Fig. 5), as expected from the impact of ATPγS on the catalysis of the wild-type. Hybrid F1 carrying a single copy of the αR364K subunit, F1(1×αR364K), was found to specifically induce a long catalytic pause only at +200° from the binding angle of the introduced αR364K, where the ATP cleavage step occurs (Fig. 6). On the other hand, the lysine mutation did not affect the unidirectionality of the rotation that is sustained by the sequential torque generated by the 3 β subunits. In addition, the mutation did not affect the coupling ratio of 3 ATPs per turn that is found in the wild-type. Thus, the present study establishes that the principal role of the arginine finger of F1 is not cooperative signal transmission among catalytic sites, but acceleration of the ATP cleavage rate.
      The αR364K mutation also affected other aspects of F1. The other remarkable effect was severe ADP inhibition (Fig. 2); the mutant sustained successive rotation for only 13 s and for up to 80 s observed. The reason for this severe inhibition is unknown. One possibility, considering that the arginine finger is also involved in Pi release, is that irregular Pi release increases the extent of ADP inhibition in the mutant over that in the wild-type, (
      • Ahmad Z.
      • Senior A.E.
      Identification of phosphate binding residues of Escherichia coli ATP synthase.
      ,
      • Kabaleeswaran V.
      • Puri N.
      • Walker J.E.
      • Leslie A.G.
      • Mueller D.M.
      Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase.
      ).
      The binding rate constant for ATP is also affected by the αR364K mutation (Fig. 3). This implies that the arginine finger is also involved in the ATP binding process to some extent. However, the magnitude of this suppression is 15-fold, and the impact is lower than that on the ATP cleavage step. Although many qualitative attributes of αR364K mutation described in this study are consistent with those in previous biochemical studies, decrease in the binding rate constant is not apparently consistent with that in a biochemical study that showed KD is not affected by the mutation (
      • Nadanaciva S.
      • Weber J.
      • Wilke-Mounts S.
      • Senior A.E.
      Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization.
      ). This could be because the rotation assay evaluates the ATP-binding rate at the binding angle (0°), whereas the titration experiment in bulk likely assesses the binding at the catalytic angle of 320° under saturation conditions for measuring the third affinity site, KD3 (
      • Shimo-Kon R.
      • Muneyuki E.
      • Sakai H.
      • Adachi K.
      • Yoshida M.
      • Kinosita Jr., K.
      Chemo-mechanical coupling in F(1)-ATPase revealed by catalytic site occupancy during catalysis.
      ). Considering the angle-dependent modulation of the ATP-binding kinetics, the angular difference likely causes the quantitative difference of the αR364K mutation on the ATP binding process. A stall experiment (
      • Watanabe R.
      • Okuno D.
      • Sakakihara S.
      • Shimabukuro K.
      • Iino R.
      • Yoshida M.
      • Noji H.
      Mechanical modulation of catalytic power on F(1)-ATPase.
      ) on the ATP binding of F1(αR364K) would provide evidence for the role of the arginine finger in this process.
      The arginine finger principally contributes to hydrolysis by accelerating the ATP cleavage step, corroborating the recent findings of structural, biochemical, and theoretical studies (
      • Kagawa R.
      • Montgomery M.G.
      • Braig K.
      • Leslie A.G.
      • Walker J.E.
      The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride.
      ,
      • Nadanaciva S.
      • Weber J.
      • Wilke-Mounts S.
      • Senior A.E.
      Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization.
      ,
      • Dittrich M.
      • Hayashi S.
      • Schulten K.
      On the mechanism of ATP hydrolysis in F1-ATPase.
      ,
      • Yang W.
      • Gao Y.Q.
      • Cui Q.
      • Ma J.
      • Karplus M.
      The missing link between thermodynamics and structure in F1-ATPase.
      ). As suggested by these studies, the arginine finger likely stabilizes the transiently formed penta-coordinated state of γ-phosphate. Recently, the impact of the lysine mutation on ATP cleavage was studied using the quantum mechanics/molecular mechanics method.
      S. Hayashi, H. Ueno, A. R. Shaikh, M. Umemura, M. Kamiya, Y. Ito, M. Ikeguchi, Y. Komoriya, R. Iino, and H. Noji, unpublished data.
      This theoretical data reproduced the present experimental data on the suppression of the ATP cleavage rate well, revealing that the shorter side chain of the lysine residue remarkably reduces F1's catalytic power for ATP cleavage, in addition to differences in chemistry between the primary amino group and the guanidium group. Nadanaciva et al. (
      • Nadanaciva S.
      • Weber J.
      • Wilke-Mounts S.
      • Senior A.E.
      Importance of F1-ATPase residue α-Arg-376 for catalytic transition state stabilization.
      ) previously investigated a glutamine substitution mutant of the arginine finger. They showed that the glutamine substitution completely impaired the ability to attain transition state stabilization, whereas the lysine substitution slightly retained this ability. This suggests that the positive charge at the arginine finger position is required for catalysis. We have also tried an alanine substitution of the arginine finger. However, the ATP hydrolytic activity was not detected in the bulk ATPase assay, and the rotary motion of F1 was not found under the present conditions.
      There is apparent discrepancy between the present work and the previous biochemical study by Le et al., who concluded that the principal role of the arginine finger is not rate enhancement of ATP cleavage, but sustenance of cooperative signal transmission among the 3 catalytic sites (
      • Le N.P.
      • Omote H.
      • Wada Y.
      • Al-Shawi M.K.
      • Nakamoto R.K.
      • Futai M.
      Escherichia coli ATP synthase α subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state.
      ). Their conclusion was drawn on the basis of the observation that the arginine mutants diminished chase promotion, which assesses whether unisite catalysis is enhanced by the binding of ATP to other catalytic sites; thus, chase promotion is used as a barometer of the cooperative interactions among the catalytic sites. The apparent discrepancy between the present and previous conclusions is attributable to not only the largely slowed turnover rate of multisite catalysis, but also the severe level of ADP inhibition. The strong tendency of the mutant to be inhibited by bound ADP would mask the apparent chase promotion.
      Unisite catalysis was reported not to be impaired by the lysine mutation (
      • Soga S.
      • Noumi T.
      • Takeyama M.
      • Maeda M.
      • Futai M.
      Mutational replacements of conserved amino acid residues in the α subunit change the catalytic properties of Escherichia coli F1-ATPase.
      ,
      • Le N.P.
      • Omote H.
      • Wada Y.
      • Al-Shawi M.K.
      • Nakamoto R.K.
      • Futai M.
      Escherichia coli ATP synthase α subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state.
      ). On the other hand, the present study establishes that the same mutation remarkably lowers the ATP cleavage rate in multisite catalysis. Therefore, in conclusion, it is reasonable to assume that the arginine finger is not directly involved in unisite catalysis; the chemistry of unisite catalysis differs from that of the catalytically active site (200° state) in multisite catalysis, as thoroughly discussed previously (
      • Senior A.E.
      • Nadanaciva S.
      • Weber J.
      The molecular mechanism of ATP synthesis by F1F0-ATP synthase.
      ).

      Acknowledgments

      We thank all the members of Noji laboratory for valuable discussions and advice. We also thank Drs. K. Adachi (Gakushuin University), S. Toyabe (Munich University), E. Muneyuki (Chuo University), and M. Yoshida (Kyoto Sangyo University) for technical help in the preparation of hybrid F1 and data analysis.

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