Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

F-ATP synthases use proton flow through the F(O) domain to synthesize ATP in the F(1) domain. In Escherichia coli, the enzyme consists of rotor subunits γεc(10) and stator subunits (αβ)(3)δab(2). Subunits c(10) or (αβ)(3) alone are rotationally symmetric. However, symmetry is broken by the b(2) homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded F(O) domain with the soluble F(1) domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)(3) catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b(2)δ in F(1) and with b(2)a in F(O). We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.