Mechanism of Inhibition by C-terminal α-Helices of the ε Subunit of Escherichia coli FoF1-ATP Synthase

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1 epsilon Delta C), and ATP synthesis driven by acid-base transition (Delta pH) and the K+-valinomycin diffusion potential (Delta Psi) was compared in detail with that of the wild-type enzyme (FoF1 epsilon WT). The turnover numbers (k(cat)) of FoF1 epsilon WT were severalfold lower than those of FoF1 epsilon Delta C.FoF1 epsilon WT showed higher Michaelis constants (K-m). The dependence of the activities of FoF1 epsilon WT and FoF1 epsilon Delta C on various combinations of Delta pH and Delta Psi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1 epsilon WT also showed lower k(cat) and higher K-m values for ATP hydrolysis than the corresponding values of FoF1 epsilon Delta C. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.