The F1F0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the ␥ and subunits in the F1 part and c subunit ring in the F0 part, rotates relative to a stator composed of ␣3␤3␦ab2 during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the subunit plays a key role by acting as a switch of this motor. Two different arrangements of the subunit have been visualized recently. The first has been observed in beef heart mitochondrial F1-ATPase where the C-terminal portion is arranged as a two-␣-helix hairpin structure that extends away from the ␣3␤3 region, and toward the position of the c subunit ring in the intact F 1F0. The second arrangement was observed in a structure determination of a complex of the ␥ and subunits of the Escherichia coli F1-ATPase. In this, the two Cterminal helices are apart and extend along the ␥ to interact with the ␣ and ␤ subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F 1F0, confirming that both conformations of the subunit exist in the enzyme complex. With the C-terminal domain of toward the F0, ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F 1 part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the subunit in the F1F0 complex and argue for a ratchet function of this subunit.

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