The ATP-waiting conformation of rotating F₁-ATPase revealed by single-pair fluorescence resonance energy transfer

F₁-ATPase is an ATP-driven rotary motor in which a rod-shaped γ subunit rotates inside a cylinder made of α₃β ₃ subunits. To elucidate the conformations of rotating F ₁, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three βs and an acceptor on γ in single F ₁ molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of γ. In the ATP-waiting state, the FRET yields indicated a γ position ≈40° counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F₁, suggesting that the crystal structures mimic a metastable state before product release.