Cross-linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving γ and ε subunits in F1 and subunit c in F0 (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 γ subunit (γ194-213). One mutation, γGlu-208 to Lys (γE208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the γE208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 °C. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was linear from 20 to 50 °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, ε subunit-inhibited F1 and suggested that the mutation blocked normal release of E inhibition of ATP hydrolytic activity upon binding of F1 to F0. The difference in temperature dependence suggested that the γE208K 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 γ and γ subunits mediate release of ε inhibition and communication of coupling information.
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