In Escherichia coli F1Fo ATP synthase, γT273 mutants that eliminate the ability to form a hydrogen bond to βV265 were incapable of ATP synthase-dependent growth and ATPase-dependent proton pumping, had very low rates of ATPase activity catalyzed by purified F1, and had significantly decreased sensitivity to inhibition by Mg2+-ADP- AlFn species, while γT273D and γT273D mutants which maintained or increased the hydrogen bond strength maintained or increased catalytic activity. The βP262G mutation that increases the potential flexibility of the rigid sleeve that surrounds the γ subunit C-terminus also virtually eliminated ATPase activity and susceptibility to Mg 2+-ADP-AlFn inhibition. The γE275 mutants that retained the ability to form the βV265 hydrogen bond had higher ATPase activity than those that eliminated the hydrogen bond. These results provide evidence that the ability to form hydrogen bonds between βV265 and the γ subunit C-terminus contributes significantly to the rate-limiting step of catalysis and to the ability of the F1Fo ATP synthase to use a proton gradient to drive ATP synthesis. The loss of activity observed with βP262G may result from increased flexibility conferred by glycine that decreases the efficiency of communication between the γ subunit-βV265 hydrogen bonds and the Walker B aspartate at the catalytic site. The partial loss of coupling observed with γT273 mutants that eliminate the βV265 hydrogen bond is consistent with participation of this hydrogen bond in the escapement mechanism for ATP synthesis in which interactions between the γ subunit and (αβ)3 ring prevent rotation until the empty catalytic site binds substrate.
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