“Resolution of Distinct Rotational Substeps by Sub-Millisecond Kinetic Analysis of F1-ATPase”, Ryohei Yasuda, Hiroyuki Noji, Masasuke Yoshida, Kazuhiko Kinosita Junior, Hiroyasu Itoh2001-04-19 (, )⁠:

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 3 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 1ms, 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.

Figure 1: Observation of F1 rotation. (a) Atomic structure7 of F1-ATPase viewed from the Fo side (top in b). (b), Side view of the observation system. The 40-nm bead gave a large enough optical signal that warranted a sub-millisecond resolution; but the bead was small enough not to impede the rotation. (c) Laser dark-field microscopy for observation of gold beads. Only light scattered by the beads exited the objective and was detected. DFC, dark-field condenser. (d) Sequential images of a rotating bead at 2 mM ATP. Images are trimmed in circles (diameter 370 nm) to aid identification of the bead position; centroid positions are shown above the images at 3× magnification. The interval between images is 0.5 ms.
Figure 1: Observation of F1 rotation. (a) Atomic structure7 of F1-ATPase viewed from the Fo side (top in b). (b) Side view of the observation system. The 40-nm bead gave a large enough optical signal that warranted a sub-millisecond resolution; but the bead was small enough not to impede the rotation. (c) Laser dark-field microscopy for observation of gold beads. Only light scattered by the beads exited the objective and was detected. DFC, dark-field condenser. (d) Sequential images of a rotating bead at 2 mM ATP. Images are trimmed in circles (diameter 370 nm) to aid identification of the bead position; centroid positions are shown above the images at 3× magnification. The interval between images is 0.5 ms.

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Resolution of Distinct Rotational Substeps by Sub-Millisecond Kinetic Analysis of F1-ATPase