F1 is an ATP-driven rotary molecular motor in which three catalytic sites, primarily hosted by a β subunit, hydrolyze ATP sequentially to power the rotation of γ subunit. The rotation occurs in steps of 120° per ATP, and the 120° step is further resolved into 80-90° and 40-30° substeps. In the basic coupling scheme,1 ATP binding starts rotation from one of three ATP-waiting angles at 0°, and the ATP is cleaved into ADP and Pi at ~200°, and the ADP is released around 240° after a third ATP is bound. The timing of Pi release is yet unsettled: either at 200° or 320°. The timing of ATP cleavage, too, is not yet unequivocal.
To complete the scheme, we observed, with single fluorophore imaging, the rotation in the presence of fluorescently (Cy3) labeled ATPγS and unlabeled ATP. Only 200° dwell after the binding of Cy3-ATPγS was extended remarkably. ATPγS would retard not only the cleavage but also the thio-Pi release, because addition of thio-Pi increased dwell time at 80° and induced frequent backward substeps from ATP-waiting angles at much lower concentrations compared to Pi. We observed the high-speed rotation of 40-nm gold bead with ATP and a small amount of unlabeled ATPγS. Not two consecutive elongated dwells but an elongated dwell alone appeared. Furthermore, the binding of Cy3-AMPPNP halted rotation driven by ATP at 200° after binding. These results imply that both the cleavage and the Pi release occur at 200° after the ATP binds at 0°.