Structural basis of unisite catalysis of bacterial F(0)F(1)-ATPase

Adenosine triphosphate (ATP) synthases (F(0)F(1)-ATPases) are crucial for all aerobic organisms. F(1), 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)β(3) in three different conformations (referred to as β(E), β(TP), and β(DP)). In this study, we determined multiple cryo-electron microscopy structures of bacterial F(0)F(1) exposed to different reaction conditions. The structures of nucleotide-depleted F(0)F(1) indicate that the ε subunit directly forces β(TP) to adopt a closed form independent of the nucleotide binding to β(TP). The structure of F(0)F(1) 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 β(TP) instead of β(DP), where ATP hydrolysis proceeds in the steady-state catalysis of F(0)F(1). This indicates that the unisite catalysis of bacterial F(0)F(1) significantly differs from the kinetics of steady-state turnover with continuous rotation of the shaft.