Three‐stepped rotation of subunits γ and ϵ in single molecules of F‐ATPase as revealed by polarized, confocal fluorometry

The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit gamma relative to the hexagonally arranged large subunits, (alphabeta)3. We investigated transition states of the rotatory drive by polarized confocal fluorometry (POCOF) as applied to single molecules of engineered, immobilized and load-free spinach-CF1. We found that the hydrolysis of ATP caused the stepped and sequential progression of subunit gamma through three discrete angular positions, with the transition states of gamma being too shortlived for detection. We also observed the stepped motion of epsilon, whereas delta was immobile as (alphabeta)3.