FoF1-ATP synthase (FoF1) couples H+ flow in Fo domain and ATP synthesis/hydrolysis in F1 domain through rotation of the central rotor shaft, and the H+/ATP ratio is crucial to understand the coupling mechanism and energy yield in cells. Although H+/ATP ratio of the perfectly coupling enzyme can be predicted from the copy number of catalytic β subunits and that of H+ binding c subunits as c/β, the actual H+/ATP ratio can vary depending on coupling efficiency. Here, we report actual H+/ATP ratio of thermophilic Bacillus FoF1, whose c/β is 10/3. Proteoliposomes reconstituted with the FoF1 were energized with ΔpH and Δψ by the acid-base transition and by valinomycin-mediated diffusion potential of K+ under various [ATP]/([ADP]⋅[Pi]) conditions, and the initial rate of ATP synthesis/hydrolysis was measured. Analyses of thermodynamically equilibrated states, where net ATP synthesis/hydrolysis is zero, show linear correlation between the chemical potential of ATP synthesis/hydrolysis and the proton motive force, giving the slope of the linear function, that is, H+/ATP ratio, 3.3 ± 0.1. This value agrees well with the c/β ratio. Thus, chemomechanical coupling between Fo and F1 is perfect.
Keywords: ATPase; FoF1-ATP synthase; chemiosmotic coupling theory; electrochemical potential; proton motive force.