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Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase*

  • Author Footnotes
    1 Both authors equally contributed to this work.
    Yoshihiro Minagawa
    Footnotes
    1 Both authors equally contributed to this work.
    Affiliations
    From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656,
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  • Author Footnotes
    1 Both authors equally contributed to this work.
    Hiroshi Ueno
    Footnotes
    1 Both authors equally contributed to this work.
    Affiliations
    the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551,
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  • Mayu Hara
    Affiliations
    From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656,
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  • Author Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Yoshiko Ishizuka-Katsura
    Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Affiliations
    the RIKEN Systems and Structural Biology Center, Yokohama 230-0045,
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  • Author Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Noboru Ohsawa
    Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Affiliations
    the RIKEN Systems and Structural Biology Center, Yokohama 230-0045,
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  • Author Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Takaho Terada
    Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
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    the RIKEN Systems and Structural Biology Center, Yokohama 230-0045,
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  • Author Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Mikako Shirouzu
    Footnotes
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    Affiliations
    the RIKEN Systems and Structural Biology Center, Yokohama 230-0045,
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  • Author Footnotes
    3 Present address: RIKEN Structural Biology Laboratory, Yokohama 230-0045, Japan.
    Shigeyuki Yokoyama
    Footnotes
    3 Present address: RIKEN Structural Biology Laboratory, Yokohama 230-0045, Japan.
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    the RIKEN Systems and Structural Biology Center, Yokohama 230-0045,
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  • Ichiro Yamato
    Affiliations
    the Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585,
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  • Eiro Muneyuki
    Affiliations
    the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551,
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  • Hiroyuki Noji
    Affiliations
    From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656,
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  • Takeshi Murata
    Correspondence
    To whom correspondence may be addressed: Dept. of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan. Tel.: 81-43-290-2794; Fax: 81-43-290-2794
    Affiliations
    the Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522

    Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Chiba 263-8522, Japan
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  • Ryota Iino
    Correspondence
    To whom correspondence may be addressed: Dept. of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: 81-3-5841-7241; Fax: 81-3-5841-1872
    Affiliations
    From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656,
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  • Author Footnotes
    * This work was supported in part by Grants-in-aid for Scientific Research 24651167 (to R. I.) and 23370047 (to T. M.) and the Target Proteins Research Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
    1 Both authors equally contributed to this work.
    2 Present address: Div. of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
    3 Present address: RIKEN Structural Biology Laboratory, Yokohama 230-0045, Japan.
Open AccessPublished:October 02, 2013DOI:https://doi.org/10.1074/jbc.M113.506329
      V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μm at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μm ATP (⪡Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 106 m−1 s−1. At 40 mm ATP (⪢Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.
      Background: The chemomechanical coupling scheme of the rotary motor V1-ATPase is incompletely understood.
      Results: Enterococcus hirae V1-ATPase (EhV1) showed 120° steps of rotation without substeps, as commonly seen with F1-ATPase.
      Conclusion: The basic properties of rotary dynamics of EhV1 are similar to those of Thermus thermophilus V1-ATPase.
      Significance: This study revealed the common properties of V1-ATPases as rotary molecular motors, distinct from those of F1-ATPases.

      Introduction

      V-ATPase is a rotary molecular motor that couples ion transport to ATP hydrolysis and synthesis. The main function of V-ATPase in eukaryotes is to transport protons across a membrane by using the energy derived from ATP hydrolysis (
      • Nishi T.
      • Forgac M.
      The vacuolar (H+)-ATPases–nature's most versatile proton pumps.
      ,
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ,
      • Marshansky V.
      • Futai M.
      The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function.
      ). V-ATPase also catalyzes ATP synthesis, harnessing the energy of proton flow in certain eubacteria such as Thermus thermophilus. V-ATPases are composed of V1-ATPase (V1),
      The abbreviations used are: V1, V1-ATPase; V0, V0-ATPase; TtV1, T. thermophilus V1-ATPase; F1, F1-ATPase; TF1, thermophilic Bacillus PS3 F1-ATPase; EF1, E. coli F1-ATPase; EhV1, E. hirae V1-ATPase; Ni-NTA, nickel-nitrilotriacetic acid; fps, frames/s; rps, revolutions/s.
      a water-soluble moiety that hydrolyzes and synthesizes ATP, and a membrane-embedded moiety (V0) that translocates ions. The V1 and V0 domains are connected by a rotary shaft and peripheral stalks (
      • Nishi T.
      • Forgac M.
      The vacuolar (H+)-ATPases–nature's most versatile proton pumps.
      ,
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ,
      • Marshansky V.
      • Futai M.
      The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function.
      ). The V1 complex is composed of A, B, D, and F subunits, in which the three A and three B subunits are alternately arranged, forming a hexameric stator A3B3 ring (
      • Numoto N.
      • Hasegawa Y.
      • Takeda K.
      • Miki K.
      Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase.
      ,
      • Maher M.J.
      • Akimoto S.
      • Iwata M.
      • Nagata K.
      • Hori Y.
      • Yoshida M.
      • Yokoyama S.
      • Iwata S.
      • Yokoyama K.
      Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus.
      ,
      • Arai S.
      • Saijo S.
      • Suzuki K.
      • Mizutani K.
      • Kakinuma Y.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Iwata S.
      • Yamato I.
      • Murata T.
      Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.
      ,
      • Nagamatsu Y.
      • Takeda K.
      • Kuranaga T.
      • Numoto N.
      • Miki K.
      Origin of asymmetry at the intersubunit interfaces of V-ATPase from Thermus thermophilus.
      ). ATP hydrolysis and synthesis occur on the catalytic sites that are located at the interfaces of the A and B subunits, with the majority of the catalytic residues residing in the A subunits. The rotary shaft is composed of D and F subunits penetrating into the central cavity of the A3B3 ring (
      • Arai S.
      • Saijo S.
      • Suzuki K.
      • Mizutani K.
      • Kakinuma Y.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Iwata S.
      • Yamato I.
      • Murata T.
      Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.
      ,
      • Nagamatsu Y.
      • Takeda K.
      • Kuranaga T.
      • Numoto N.
      • Miki K.
      Origin of asymmetry at the intersubunit interfaces of V-ATPase from Thermus thermophilus.
      ).
      The rotation of V1 has been visualized using optical microscopy by attachment of a probe to the rotary shaft (
      • Imamura H.
      • Nakano M.
      • Noji H.
      • Muneyuki E.
      • Ohkuma S.
      • Yoshida M.
      • Yokoyama K.
      Evidence for rotation of V1-ATPase.
      ,
      • Hirata T.
      • Iwamoto-Kihara A.
      • Sun-Wada G.H.
      • Okajima T.
      • Wada Y.
      • Futai M.
      Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
      ,
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ). V1 of T. thermophilus (TtV1), which functions as an ATP synthase, rotates stepwise in a counterclockwise direction (
      • Imamura H.
      • Nakano M.
      • Noji H.
      • Muneyuki E.
      • Ohkuma S.
      • Yoshida M.
      • Yokoyama K.
      Evidence for rotation of V1-ATPase.
      ). The basic step size is 120°, and similar to F1-ATPase (F1), the water-soluble moiety of F0F1-ATP synthase (
      • Iino R.
      • Noji H.
      Operation mechanism of FoF1-adenosine triphosphate synthase revealed by its structure and dynamics.
      ), each step is coupled to the consumption of a single ATP molecule (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ). Although no substeps have yet been resolved in the rotation of TtV1 (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ), the 120° steps of F1 from the thermophilic Bacillus PS3 (TF1) and Escherichia coli (EF1) have been shown to be further divided into 80° and 40° substeps and into 85° and 35° substeps, respectively (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      ,
      • Shimabukuro K.
      • Yasuda R.
      • Muneyuki E.
      • Hara K.Y.
      • Kinosita Jr., K.
      • Yoshida M.
      Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation.
      ,
      • Bilyard T.
      • Nakanishi-Matsui M.
      • Steel B.C.
      • Pilizota T.
      • Nord A.L.
      • Hosokawa H.
      • Futai M.
      • Berry R.M.
      High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
      ). The 80° and 85° substeps are triggered by ATP binding and ADP release, whereas the 40° and 35° substeps are known to occur after ATP cleavage and release of inorganic phosphate. Accordingly, the pauses before the 80° and 85° substeps are referred to as ATP-binding (ATP-waiting) pauses, and those prior to the 40° and 35° substeps are known as catalytic pauses. As described above, the chemomechanical coupling scheme of TtV1 appears to be distinct from that of F1. However, to date, the stepping rotations of V1 complexes other than TtV1 have not been described, and the chemomechanical coupling scheme of V1 remains unclear (
      • Hirata T.
      • Iwamoto-Kihara A.
      • Sun-Wada G.H.
      • Okajima T.
      • Wada Y.
      • Futai M.
      Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
      ).
      Enterococcus hirae V-ATPase functions as a primary ion pump, similar in nature to eukaryotic V-ATPases (
      • Kakinuma Y.
      • Igarashi K.
      Purification and characterization of the catalytic moiety of vacuolar-type Na+-ATPase from Enterococcus hirae.
      ,
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ). We recently solved the crystal structures of the V1 component of E. hirae V-ATPase (EhV1) and proposed a model of its rotation mechanism (
      • Arai S.
      • Saijo S.
      • Suzuki K.
      • Mizutani K.
      • Kakinuma Y.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Iwata S.
      • Yamato I.
      • Murata T.
      Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.
      ). In this study, to characterize the stepping rotation of EhV1, we analyzed and compared the basic properties of EhV1 rotary dynamics with those of TtV1, TF1, and EF1. As was the case with TtV1, no substeps were resolved in the rotation of EhV1, suggesting that 120° stepping rotation without substeps is a common property of V1 complexes.

      DISCUSSION

      In this study, using a single-molecule assay, we have shown that EhV1 is a rotary molecular motor. To our knowledge, this is the first report showing that a eubacterial V1 functions as an ATP-driven ion pump under physiological conditions. EhV1 exhibited two rotational states, namely clear and unclear (Fig. 4). Assuming that the clear rotational state represents the tight chemomechanical coupling of EhV1, we analyzed this state to elucidate the basic rotational properties of EhV1. Our hypothesis that the unclear state is caused by unstable interactions between the rotor and stator of EhV1 must be examined by rotation assay of the entire E. hirae V-ATPase complex, in which the interactions between the rotor and stator are stabilized by two peripheral stalks. To perform this study, we are currently designing an E. coli expression system in which an appropriately tagged recombinant V-ATPase complex can be produced for a rotation assay.
      In the clear rotational state, at all ATP concentrations ranging from below to above the Km, EhV1 rotated unidirectionally in a counterclockwise direction, exhibiting three pauses separated by 120° (Fig. 5). No substeps were resolved, as has been reported for TtV1 (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ). In contrast, in the region of their respective Km values, TF1 and EF1 have been reported to rotate with six pauses/turn (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      ,
      • Shimabukuro K.
      • Yasuda R.
      • Muneyuki E.
      • Hara K.Y.
      • Kinosita Jr., K.
      • Yoshida M.
      Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation.
      ,
      • Bilyard T.
      • Nakanishi-Matsui M.
      • Steel B.C.
      • Pilizota T.
      • Nord A.L.
      • Hosokawa H.
      • Futai M.
      • Berry R.M.
      High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
      ). Recently, the overall crystal structures of TtV1 and EhV1 were shown to be similar (
      • Numoto N.
      • Hasegawa Y.
      • Takeda K.
      • Miki K.
      Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase.
      ,
      • Maher M.J.
      • Akimoto S.
      • Iwata M.
      • Nagata K.
      • Hori Y.
      • Yoshida M.
      • Yokoyama S.
      • Iwata S.
      • Yokoyama K.
      Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus.
      ,
      • Arai S.
      • Saijo S.
      • Suzuki K.
      • Mizutani K.
      • Kakinuma Y.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Iwata S.
      • Yamato I.
      • Murata T.
      Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.
      ,
      • Nagamatsu Y.
      • Takeda K.
      • Kuranaga T.
      • Numoto N.
      • Miki K.
      Origin of asymmetry at the intersubunit interfaces of V-ATPase from Thermus thermophilus.
      ), especially with respect to the interaction sites between the rotor and stator. These structures are distinct from the structure of F1 (
      • Abrahams J.P.
      • Leslie A.G.
      • Lutter R.
      • Walker J.E.
      Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria.
      ), although many amino acid residues associated with catalysis in the binding pocket are conserved between V1 and F1. These results imply that the degree of similarity in the interactions between the rotor and stator determines the presence or absence of substeps in the rotation.
      Table 1 contains a comparison of the kinetic parameters determined by the single-molecule assay for EhV1, showing values for TtV1, TF1, and EF1. Despite the difference in physiological function between EhV1 and TtV1 and notwithstanding the large difference (>30 °C) in the optimal growth temperatures between E. hirae and T. thermophilus, the values for EhV1 are closer to those for TtV1 than for TF1 and EF1. This result implies that the basic properties of rotary dynamics are determined by their overall structures and that the difference in the physiological function derives from regulatory mechanisms such as MgADP inhibition.
      TABLE 1Kinetic parameters of V1-ATPases and F1-ATPases from different sources
      Protein/measurement temperaturekon(ATP)
      The second-order binding rate constant for ATP (kon(ATP)) was determined from the distribution of the duration of the ATP-waiting pause.
      kon(ATP) (3 × Vmax/Km)
      The second-order binding rate constant for ATP (kon(ATP)) was determined from the distribution of the duration of the ATP-waiting pause.
      τ12
      The second-order binding rate constant for ATP (kon(ATP)) was determined from the distribution of the duration of the ATP-waiting pause.
      Ref.
      m−1 s−1m−1 s−1ms
      EhV1
      The values are the mean ± S.E. of fitting.
      26 ± 1 °C(2.3 ± 0.03) × 106(2.2 ± 0.4) × 1062.6 ± 0.1/0.5 ± 0.02
      The values were obtained at 40 mm ATP.
      This work
      TtV1
      23 °C1.5 × 1060.84 × 1062.8/2.8
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      24 ± 1 °C1.39 × 106
      • Uner N.E.
      • Nishikawa Y.
      • Okuno D.
      • Nakano M.
      • Yokoyama K.
      • Noji H.
      Single-molecule analysis of inhibitory pausing states of V1-ATPase.
      TF1
      23 °C3.0 × 1072.6 × 1071.6/0.71
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      25 ± 1 °C1.34/0.29
      • Ueno H.
      • Nishikawa S.
      • Iino R.
      • Tabata K.V.
      • Sakakihara S.
      • Yanagida T.
      • Noji H.
      Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.
      25 ± 1 °C2.2 × 107
      • Tanigawara M.
      • Tabata K.V.
      • Ito Y.
      • Ito J.
      • Watanabe R.
      • Ueno H.
      • Ikeguchi M.
      • Noji H.
      Role of the DELSEED loop in torque transmission of F1-ATPase.
      EF1
      23 °C4.7 × 1076.4 × 1070.41/0.29
      • Bilyard T.
      • Nakanishi-Matsui M.
      • Steel B.C.
      • Pilizota T.
      • Nord A.L.
      • Hosokawa H.
      • Futai M.
      • Berry R.M.
      High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
      b The second-order binding rate constant for ATP (kon(ATP)) was determined from 3 × Vmax/Km.
      c Time constants were determined from the distribution of the duration of the catalytic pause, which corresponds to ATP cleavage and either ADP or phosphate release (or both).
      a The second-order binding rate constant for ATP (kon(ATP)) was determined from the distribution of the duration of the ATP-waiting pause.
      d The values are the mean ± S.E. of fitting.
      e The values were obtained at 40 mm ATP.
      During the unclear rotational state, the centroids of the gold colloid showed wide fluctuations toward the rotation center. It should be noted that EhV1 nevertheless rotated unidirectionally, implying that even if the interactions between the rotor and stator are not perfect, EhV1 maintains unidirectional and cooperative rotary catalysis. Recently, rotary catalysis of the rotor-less stator α3β3 ring of TF1 was demonstrated by high-speed atomic force microscopy (
      • Uchihashi T.
      • Iino R.
      • Ando T.
      • Noji H.
      High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase.
      ), and we speculate that the stator A3B3 ring also likely exhibits rotary catalysis in the absence of the rotor DF subunits.
      The chemomechanical coupling scheme of TF1 has been extensively studied by advanced single-molecule techniques such as a rotation assay of hybrid molecules and single-molecule manipulation with magnetic tweezers (
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits.
      ,
      • Adachi K.
      • Oiwa K.
      • Nishizaka T.
      • Furuike S.
      • Noji H.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation.
      ,
      • Watanabe R.
      • Iino R.
      • Noji H.
      Phosphate release in F1-ATPase catalytic cycle follows ADP release.
      ). For a single catalytic site of TF1, after ATP binding at 0°, ATP cleavage, ADP release, and phosphate release occur at 200°, 240°, and 320°, respectively (
      • Watanabe R.
      • Iino R.
      • Noji H.
      Phosphate release in F1-ATPase catalytic cycle follows ADP release.
      ). Further studies on EhV1 using advanced single-molecule techniques and high-resolution structural analysis will provide details on its chemomechanical coupling scheme. Moreover, comparison of the schemes of V1 and F1 from various species will shed light on the general mechanism of rotary molecular motors.

      Acknowledgments

      We thank Mio Inoue and Ken Ishii for preparation of the plasmids and Shoichi Toyabe for providing the data analysis software. We also thank all members of the laboratory for valuable discussions and comments.

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