The Proton-translocating a Subunit of F0F1-ATP Synthase Is Allocated Asymmetrically to the Peripheral Stalk

The position of the a subunit of the membrane-integral F0 sector of Escherichia coli ATP synthase was investigated by single molecule fluorescence resonance energy transfer studies utilizing a fusion of enhanced green fluorescent protein to the C terminus of the a subunit and fluorescent labels attached to specific positions of the ϵ or γ subunits. Three fluorescence resonance energy transfer levels were observed during rotation driven by ATP hydrolysis corresponding to the three resting positions of the rotor subunits, γ or ϵ, relative to the a subunit of the stator. Comparison of these positions of the rotor sites with those previously determined relative to the b subunit dimer indicates the position of a as adjacent to the b dimer on its counterclockwise side when the enzyme is viewed from the cytoplasm. This relationship provides stability to the membrane interface between a and b2, allowing it to withstand the torque imparted by the rotor during ATP synthesis as well as ATP hydrolysis.