Large conformational changes of the ɛ subunit in the bacterial F(1)F(0) ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the γ and ɛ subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of α(3)β(3)δab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the ɛ subunit plays a key role by acting as a switch of this motor. Two different arrangements of the ɛ subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-α-helix hairpin structure that extends away from the α(3)β(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the γ and ɛ subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the γ to interact with the α and β 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(1)F(0), confirming that both conformations of the ɛ subunit exist in the enzyme complex. With the C-terminal domain of ɛ toward the F(0), 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 ɛ subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.