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


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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Figure 1: Observation of F1 rotation.
Figure 2: Relationship between rate of bead rotation and viscous friction on the bead.
Figure 3: Comparison of rotation and hydrolysis rates.
Figure 4: Unfiltered time courses of stepping rotation of 40-nm beads at varying [ATP].
Figure 5: Histograms of angular positions over 0.5 s runs.
Figure 6: Kinetics of substeps.
Figure 7: Proposed mechanism for F1 rotation.
Figure 8: Dwells between main steps.

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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).

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