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

Abstract
The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit γ relative to the hexagonally arranged large subunits, (αβ)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 γ through three discrete angular positions, with the transition states of γ being too shortlived for detection. We also observed the stepped motion of ϵ, whereas δ was immobile as (αβ)3.