Subunit movements in membrane-integrated EF0F1 during ATP synthesis detected by single-molecule spectroscopy.

The H+ -ATPsynthase from E. coli was isolated and labelled at the gamma- or epsilon-subunit with tetramethylrhodamine, and at the b-subunits with bisCy5. The double labelled enzymes were incorporated into liposomes. They showed ATP hydrolysis activity, and, after energization of the membrane by DeltapH and Deltavarphi, also ATP synthesis activity was observed. Fluorescence resonance energy transfer (FRET) was used to investigate the movements of either the gamma-subunit or the epsilon-subunit relative to the b-subunits in single membrane-integrated enzymes. The results show that during catalysis, the gamma-epsilon complex rotates stepwise relative to the b-subunit. The direction of rotation during ATP synthesis is opposite to that during ATP hydrolysis. The stepwise motion is characterized by dwell times (docking time of the gamma-epsilon complex to one alphabeta pair) up to several hundred ms, followed by a rapid movement of the gamma- and epsilon-subunit to the next alphabeta pair within 0.2 ms. The same FRET levels (i.e., the same gamma-b and epsilon-b distances) are observed during proton transport-coupled ATP hydrolysis and ATP synthesis, indicating that the reaction proceeds via the same intermediates in both directions. Under non-catalytic conditions, i.e., in the absence of ATP or without energization also, three FRET levels are found, however, the distances differ from those under catalytic conditions. We conclude that this reflects a movement of the epsilon-subunit during active/inactive transition.