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

F-1, a water-soluble portion of FoF1-ATP synthase, is an ATIP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 801 substep rotation, interim dwell, and rapid 40degrees 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 ATIP binding, correspond to these events. To discover which steps, we analyzed rotations of F-1 subcomplex (alpha(3)beta(3)gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATIP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F, and hydrolysis of a slowly hydrolyzable substrate ATPgammaS (adenosine 5'-[gamma-thio]triphosphate) by wild-type F-1. 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 ATPgammaS hydrolysis by the mutant F-1 ensure that cleavage of ATIP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F-1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.