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Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

Nature volume 410, pages 898904 (19 April 2001) | Download Citation

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Abstract

The enzyme F1-ATPase has been shown to be a rotary motor in which the central γ-subunit rotates inside the cylinder made of α3β3 subunits. At low ATP concentrations, the motor rotates in discrete 120° steps, consistent with sequential ATP hydrolysis on the three β-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120° step consists of roughly 90° and 30° substeps, each taking only a fraction of a millisecond. ATP binding drives the 90° substep, and the 30° substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed (130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.

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Acknowledgements

We thank T. Ariga for sample preparation; A. Kusumi for colloidal gold; T. Hisabori, E. Muneyuki, T. Nishizaka, K. Adachi, C. Gosse, M. Y. Ali, S. Ishiwata and G. W. Feigenson for critical discussions; and H. Umezawa for laboratory management. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.

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Author notes

    • Ryohei Yasuda

    Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

Affiliations

  1. *CREST ‘Genetic Programming’ Team 13, Teikyo University Biotechnology Center 3F, Nogawa 907, Miyamae-Ku, Kawasaki 216-0001, Japan

    • Ryohei Yasuda
    • , Hiroyuki Noji
    • , Masasuke Yoshida
    • , Kazuhiko Kinosita Jr
    •  & Hiroyasu Itoh
  2. †Department of Physics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan

    • Ryohei Yasuda
    •  & Kazuhiko Kinosita Jr
  3. ‡Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan

    • Masasuke Yoshida
  4. §Tsukuba Research Laboratory, Hamamatsu Photonics KK, Tokodai, Tsukuba 300-2635, Japan

    • Hiroyasu Itoh

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Correspondence to Kazuhiko Kinosita Jr.

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https://doi.org/10.1038/35073513

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