Cooperative Three-step Motions In Catalytic Subunits of F1-ATPase Drive 80° And 40° Substep Rotations

Abstract

Detecting conformational changes in enzymes is crucial for understanding their functions. Concerning F1-ATPase, the smallest rotary molecular motor ever known, domain motions in three catalytic β-subunits coupled with transitions between chemical states during ATP hydrolysis are assumed to be responsible for rotation of the central shaft γ-subunit. Nevertheless, the extent and timing of motions in β in rotating F1-ATPase still remain largely unknown. Here we directly observe motions in β and rotation of γ simultaneously in the same single molecules; changes in lateral orientation of a single fluorophore fixed to the C-terminal helix of β and substep rotations of beads attached to γ were detected. Furthermore, asymmetric stepping patterns of γ in hybrid F1-ATPase containing one or two mutant β-subunits enabled identification of chemical steps coupled to these motions. The 80° substep of γ is caused by cooperative bending motions of two β-subunits; a ∼40° counterclockwise motion of the first β upon ATP-binding and a clockwise ∼20° motion of the second β upon ADP-release (viewed from Fo side). Meanwhile, the 40°substep of γ is driven by another ∼20° clockwise motion of the third β after ATP-cleavage. Thus three-step cooperative bending of β-subunits causes two substep rotations of γ. Moreover, these results indicate that the initial crystal structure mimics the conformation in the catalytic dwells, and that the conformation in the ATP-waiting dwells contains a novel set of β-subunits; Open, Closed, and partially closed β-subunits. Thus the present study bridges the gap between chemical steps and mechanical work in motor proteins. The present approach to illustrate the enzymatic functions through detection of motions will give insights into mechanisms of diverse enzymes.