F-1-ATPase is a highly efficient molecular motor that can synthesize ATP driven by a mechanical torque. Its ability to function reversibly in either direction requires tight mechanochemical coupling between the catalytic domain and the rotating central shaft, as well as temporal control of substrate binding and product release. Despite great efforts and significant progress, the molecular details of this synchronized and fine-tuned energy conversion mechanism are not fully understood. Here, we use extensive molecular dynamics simulations to reconcile recent single-molecule experiments with structural data and provide a consistent thermodynamic, kinetic and mechanistic description of the main rotary substep in the synthetic cycle of mammalian ATP synthase. The calculated free energy profiles capture a discrete pattern in the rotation of the central gamma-shaft, with a metastable intermediate located consistently with recent experimental findings-at 70 degrees relative to the X-ray position. We identify this rotary step as the ATP-dependent substep, and find that the associated free energy input supports the mechanism involving concurrent nucleotide binding and release. During the main substep, our simulations show no significant opening of the ATP-bound beta subunit; instead, we observe that, mechanical energy is transmitted to its nucleotide binding site, thus lowering the affinity for ATP. Simultaneously, the empty subunit assumes a conformation that enables the enzyme to harness the free energy of ADP binding to drive ATP release. Finally, we show that ligand exchange is regulated by a checkpoint mechanism, an apparent prerequisite for high efficiency in protein nanomotors.