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FoF1-ATP synthase (FoF1) produces most of the ATP in cells, uniquely, by converting the proton motive force (pmf) into ATP production via mechanical rotation of the inner rotor complex. Technical difficulties have hampered direct investigation of pmf-driven rotation, which are crucial to elucidating the chemomechanical coupling mechanism of FoF1. Here we develop a novel supported membrane system for direct observation of the rotation of FoF1 driven by pmf that was formed by photolysis of caged protons. Upon photolysis, FoF1 initiated rotation in the opposite direction to that of the ATP-driven rotation. The step size of pmf-driven rotation was 120°, suggesting that the kinetic bottleneck is a catalytic event on F1 with threefold symmetry. The reaction equilibrium was slightly biased to ATP synthesis like under physiological conditions, and FoF1 showed highly stochastic behaviour, frequently making a 120° backward step. This new experimental system would be applicable to single-molecule study of other membrane proteins. [ABSTRACT FROM AUTHOR]