Single Molecule Behavior of Inhibited and Active States of Escherichia coli ATP Synthase F1 Rotation

ATP hydrolysis-dependent rotation of the F-1 sector of the ATP synthase is a successive cycle of catalytic dwells (similar to 0.2 ms at 24 degrees C) and 120 degrees rotation steps (similar to 0.6 ms) when observed under V-max conditions using a low viscous drag 60-nm bead attached to the gamma subunit (Sekiya, M., Nakamoto, R. K., AlShawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401-22410). During the normal course of observation, the gamma subunit pauses in a stochastic manner to a catalytically inhibited state that averages similar to 1 s in duration. The rotation behavior with adenosine 5'-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 mu M) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F-1 requires similar to 2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric gamma subunit, the inhibitor of F-1-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the gamma subunit has little effect on the transition between active and inhibited states. Rather, the gamma subunit confers lower activation energy of steady-state rotation. These results suggest that the gamma subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.