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

F ₁ -ATPase achieves unidirectional rotation of its γ shaft through coordinated conformational cycling of the α ₃ β ₃ ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF ₁ 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 TF ₁ exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF ₁ exhibited six pauses per turn, indicating the presence of an additional intermediate during rotation. This previously unreported intermediate dwell was observed at 40° between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps confined to the 40° 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 β conformational change from 0° to 40°, but minimal β rearrangement between 40° and 80°, suggesting that the β power stroke is not operative in this interval. Analysis of the γ rotational scheme indicates that the driving force changes across the 0–80° step: the 0–40° advance proceeds via a power stroke to an intermediate dwell, whereas the subsequent 40° transition to the catalytic dwell lacks a power stroke and is dominated by thermal fluctuations. Taken together, these findings suggest that the lower γ region contributes to unidirectional rotation and torque generation specifically in the latter half of the 0–80° step, while the initial half can proceed even without contributions from the lower γ region.