Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer

The EF0F1-ATP synthase mutants bQ64C and gammaT106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b-subunit and with a cyanine (Cy5) at the gamma-subunit. After reconstitution into liposomes, these double-labelled enzymes catalyzed ATP synthesis at a rate of 33 s(-1). Fluorescence of TMR and Cy5 was measured with a confocal set-up for single-molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET-states were identified, indicating stepwise rotation of the gamma-subunit of EF0F1. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.