Mechanism of ATP hydrolysis dependent rotation of bacterial ATP synthase.

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α3ß3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αß dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain in FoF1 synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic αß dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at αDßD, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

Structural basis of unisite catalysis of bacterial F0F1-ATPase.

Adenosine triphosphate (ATP) synthases (F0F1-ATPases) are crucial for all aerobic organisms. F1, a water-soluble domain, can catalyze both the synthesis and hydrolysis of ATP with the rotation of the central γε rotor inside a cylinder made of α 3 ß 3 in three different conformations (referred to as ß E, ß TP, and ß DP). In this study, we determined multiple cryo-electron microscopy structures of bacterial F0F1 exposed to different reaction conditions. The structures of nucleotide-depleted F0F1 indicate that the ε subunit directly forces ß TP to adopt a closed form independent of the nucleotide binding to ß TP. The structure of F0F1 under conditions that permit only a single catalytic ß subunit per enzyme to bind ATP is referred to as unisite catalysis and reveals that ATP hydrolysis unexpectedly occurs on ß TP instead of ß DP, where ATP hydrolysis proceeds in the steady-state catalysis of F0F1. This indicates that the unisite catalysis of bacterial F0F1 significantly differs from the kinetics of steady-state turnover with continuous rotation of the shaft.