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 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.