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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Movie 1 (mov 1.7 mb)
Stepping rotation of a 40-nm bead attached to the gamma subunit of F1-ATPase. ATP concentration, 20 micromolar. Close look at the movie will reveal 90- and 30-degree substeps. Images were recorded at 8,000 frames per second and are played at 11 frames per second. Diameter of the circular images, 320nm.
Movie 2 (mov 1 mb)
Stepping rotation of a 40-nm bead attached to the gamma subunit of F1-ATPase. ATP concentration, 2 mM. Images were recorded at 8,000 frames per second and are played at 11 frames per second. Diameter of the circular images, 320nm.
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Yasuda, R., Noji, H., Yoshida, M. et al. Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898–904 (2001). https://doi.org/10.1038/35073513
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DOI: https://doi.org/10.1038/35073513
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