Structural basis of unisite catalysis of bacterial F0F1-ATPase

Abstract

Abstract 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 α3β3 in three different conformations (referred to as βE, βTP, and βDP). 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 βTP to adopt a closed form independent of the nucleotide binding to βTP. The structure of F0F1 under conditions that permit only a single catalytic β subunit per enzyme to bind ATP is referred to as unisite catalysis and reveals that ATP hydrolysis unexpectedly occurs on βTP instead of βDP, 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.