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

Abstract ATP hydrolysis-dependent rotation of the F1 sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under Vmax conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401–22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5′-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μm) 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 F1 requires ∼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 ϵ subunit, the inhibitor of F1-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ϵ subunit has little effect on the transition between active and inhibited states. Rather, the ϵ subunit confers lower activation energy of steady-state rotation. These results suggest that the ϵ 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.