F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F₁ domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)₃δab₂. Subunits c10 or (αβ)₃ alone are rotationally symmetric. However, symmetry is broken by the b₂ homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F₁ 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 b₂δ in F₁ and with b₂a in FO. 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.
Keywords: Escherichia coli; FOF1 ATP synthase; cryo-EM structure; elasticity; single-molecule fluorescence; subunit rotation; symmetry.