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

F-1-ATPase is an ATIP-driven rotary motor in which a rod-shaped gamma subunit rotates inside a cylinder made Of alpha(3)beta(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 betas and an acceptor on gamma in single F-1 molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of gamma. In the ATP-waiting state, the FRET yields indicated a gamma position approximate to40degrees counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F-1, suggesting that the crystal structures mimic a metastable state before product release.