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 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 F₀F₁ exposed to different reaction conditions. The structures of nucleotide-depleted F₀F₁ 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 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 β _TP instead of β _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.

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