Large conformational changes of the ε subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F1F0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F-1 part and c subunit ring in the F-0 part, rotates relative to a stator composed of alpha (3)beta (3)delta ab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the E 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-alpha -helix hairpin structure that extends away from the alpha (3)beta (3) region, and toward the position of the c subunit ring in the intact F1F0. The second arrangement was observed in a structure determination of a complex of the gamma and E subunits of the Escherichia coli F-1-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the a and P 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 F1F0. confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon 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 epsilon subunit in the F1F0 complex and argue for a ratchet function of this subunit.