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Abstract

F₁-ATPase, the catalytic domain of ATP synthase, synthesizes most of the ATP in living organisms. Running in reverse powered by ATP hydrolysis, this hexameric ring-shaped molecular motor formed by three αβ-dimers creates torque on its central γ-subunit. This reverse operation enables detailed explorations of the mechanochemical coupling mechanisms in experiment and simulation. Here, we use molecular dynamics simulations to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (P_i) release coupled to the rotation. In response to torque-driven rotation of the γ-subunit in the hydrolysis direction, the nucleotide-free αβ_E interface forming the “empty” E site loosens and singly charged P_i readily escapes to the P loop. By contrast, the interface stays closed with doubly charged P_i. The γ-rotation tightens the ATP-bound αβ_TP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged P_i from the αβ_E interface 120° after ATP hydrolysis closely matches the ∼1-ms functional timescale. Conversely, P_i release from the ADP-bound αβ_DP interface postulated in earlier models would occur through a kinetically infeasible inward-directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of P_i exit, its pathway and kinetics, associated changes in P_i protonation, and changes of the F₁-ATPase structure in the 40° substep. Important elements of the molecular mechanism of P_i release emerging from our simulations appear to be conserved in myosin despite the different functional motions.