The Lower γ Region Ensures Unidirectional Rotation and Torque Generation in the Latter Half of the 80° Substep of F1-ATPase

F1-ATPase achieves unidirectional rotation of its {gamma} shaft through coordinated conformational cycling of the 3{beta}3 ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF1 lacking the lower half of the rotor shaft by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six pauses per turn, indicating the presence of an additional intermediate during rotation. This previously unreported intermediate dwell was observed at 40{degrees} between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps confined to the 40{degrees} transition between the intermediate and catalytic dwells. Analysis of the dwell-time distributions indicated an approximately zero free energy bias between these two states, consistent with thermally driven interconversion. Cryo-EM resolved three corresponding intermediates--binding, intermediate, and catalytic dwells--showing a major {beta} conformational change from 0{degrees} to 40{degrees}, but minimal {beta} rearrangement between 40{degrees} and 80{degrees}, suggesting that the {beta} power stroke is not operative in this interval. Analysis of the {gamma} rotational scheme indicates that the driving force changes across the 0-80{degrees} step: the 0-40{degrees} advance proceeds via a power stroke to an intermediate dwell, whereas the subsequent 40{degrees} transition to the catalytic dwell lacks a power stroke and is dominated by thermal fluctuations. Taken together, these findings suggest that the lower {gamma} region contributes to unidirectional rotation and torque generation specifically in the latter half of the 0-80{degrees} step, while the initial half can proceed even without contributions from the lower {gamma} region.