Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase

V-1-ATPase (V-1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis?derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V-1 from the pathogen Enterococcus hirae (EhV(1)). We found that 120? steps in each ATP hydrolysis event are divided into 40 and 80? substeps. In the main pause before the 40? substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 ? 10(7) m(?1) s(?1). At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATP?S, indicating that ATP is cleaved during the main pause. In another subpause before the 80? substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, ?80 and ?40? backward steps were observed. The time constants of the pauses before ?80? backward and +40? recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80? substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV(1), including substeps largely different from those of F-1-ATPases.