F1-ATPase (F1), a soluble portion of F oF1-ATP synthase (FoF1), is an ATP-driven motor in which γε subunits rotate in the α3β3 cylinder. Activity of F1 and FoF1 from Bacillus PS3 is attenuated by the ε subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of ε in single F1 molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F1 with hairpin ε directly determines the fraction of active F1 at any ATP concentration. Next, mechanical activation and stiffness of ε-inhibited F1 were examined by the forced rotation of magnetic beads attached to γ. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from ε inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if FoF1 is resting in the ε-inhibited state, Fo motor must transmit to γ a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.
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