Monitoring γ-Subunit Movement in Reconstituted Single EF°F1 ATP Synthase by Fluorescence Resonance Energy Transfer

The membrane-bound enzymes H+ ATP synthases contain two coupled rotary motors that drive catalysis. We applied a single molecule spectroscopy approach to monitor the internal rotation of the γ-subunit of the F₁ part against its static counterpart, the b-subunits of the F₀ part. We specifically attached two fluorophores to H+ ATP synthase from E. coli, namely Cy5 at the γ-subunit and tetramethylrhodamine at one b-subunit. After reconstitution into liposomes, these enzymes regained their full catalytic activity as measured by ATP synthesis rates. Fluorescence resonance energy transfer (FRET) was monitored in photon bursts of freely diffusing proteoliposomes using a confocal setup for single molecule detection. Incubation with non-hydrolyzable AMPPNP resulted in stable intensity ratios within a photon burst. This corresponds to a fixed γ-subunit orientation. We detected three different FRET efficiencies, i.e., γ-subunit orientations. After addition of ATP a consecutive order of three distinguishable FRET efficiencies was observed within the bursts, indicating a stepwise unidirectional γ-subunit movement against the b-subunits.