Catalysis and rotation of F₁ motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation

F₁, a water-soluble portion of F₀F₁-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central γ-subunit rotates in the α₃β₃ cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80° substep rotation, interim dwell, and rapid 40° substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F₁ subcomplex (α₃β₃γ) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F₁ and hydrolysis of a slowly hydrolyzable substrate ATPγS (adenosine 5′-[γ -thio]triphosphate) by wild-type F₁. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATPγS hydrolysis by the mutant F₁ ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F₁ occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.