A mutation in the Escherichia coli F0F1-ATP synthase rotor, γE208K, perturbs conformational coupling between transport and catalysis

Cross-Linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving gamma and epsilon subunits in F-1 and subunit c in F-0 (Watts, S. D., Tang, C., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 28341-28347), To assess the function of these interactions, we introduced random mutations in this region of the gamma subunit (gamma 194-213). One mutation, gamma Glu-208 to Lys (gamma E208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the gamma E208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 degrees C. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was Linear from 20 to 50 degrees C, Analysis of this plot revealed a significant increase in the activation energy of the catalytic transition state to a value very similar to soluble, epsilon subunit-inhibited F-1 and suggested that the mutation blocked normal release of epsilon inhibition of ATP hydrolytic activity upon binding of F-1 to F-0. The difference in temperature dependence suggested that the gamma E208K mutation perturbed release of inhibition via a different mechanism than it did energy coupling. Suppressor mutations in the polar loop of subunit c restored ATP-dependent H+ pumping and transition state thermodynamic parameters close to wild-type values indicating that interactions between gamma and c subunits mediate release of epsilon inhibition and communication of coupling information.