F1-ATPase of Escherichia coli THE ε-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS, IS DISTINCT FROM THE ADP-INHIBITED STATE, AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS

F-1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit epsilon, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F-1.epsilon binding and dissociation, we show that formation of the extended, inhibitory conformation of epsilon (epsilon(X)) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the epsilon(X) state, and post-hydrolysis conditions stabilize it. We also show that epsilon inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of epsilon is responsible for initial binding to F-1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the epsilon N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F-1-bound epsilon suggest dynamic conformational and rotational mobility in F-1 that is paused near the catalytic dwell position.