Single-Molecule Observation of Rotation of F1-ATPase Through Microbeads

F₀ F₁ -ATP synthase catalyzes the synthesis of ATP using proton-motive force across a membrane. When isolated, the F₁ sector, composed of five polypeptide chains with a stoichiometry of α₃ β₃ γδε, solely hydrolyzes ATP into ADP and phosphate, and is thus called F₁ -ATPase. Rotation of a shaft domain in F₀ F₁ -ATP synthase has been hypothesized by Paul Boyer, and ultimately was confirmed by direct observation as rotation of the γ-subunit in an isolated α₃ β₃ γ subcomplex. Unitary turnover of ATP induces 120� steps, consistent with the configuration of three catalytic sites arranged 120� apart around γ. We have shown the relationships between chemical and mechanical events by imaging individual F₁ molecules under an optical microscope. A new scheme emerges: as soon as a catalytic site binds ATP, the γ-subunit always turns the same face (interaction surface) to the β hosting that site; ∼80� rotation is driven by ATP binding; ∼40� rotation is induced by completion of hydrolysis [and/or phosphate release] in the site that bound ATP one step earlier.