Most FF type ATP synthases, including that in Escherichia coli, use H as the coupling ion for ATP synthesis. However, the structurally related FF ATP synthase in Propionigenium modestum uses Na instead. The binding site for Na resides in the F sector of the P. modestum enzyme. We postulated that Na might interact with subunit c of F. Subunit c of P. modestum and E. coli are reasonably homologous (19% identity) but show striking variations around the H-translocating, dicyclohexylcarbodiimide-reactive carboxyl (Asp in E. coli). Several hydrophobic residues around Asp were replaced with polar residues according to the P. modestum sequence in the hope that the polar replacements might provide liganding groups for Na. One mutant from 31 different mutation combinations did generate an active enzyme that binds Li, the combination being V60A, D61E, A62S, and I63T. Li binding was detected by Li inhibition of ATP-driven H transport, Li inhibition of FF-ATPase activity, and Li inhibition of F-mediated H transport. The Li effects were observed with membrane vesicles prepared from a nhaA, nhaB mutant background which lacks Na/H antiporters, and with purified, reconstituted preparations of F prepared from this background strain. Li inhibition was observed at pH 8.5 but not at pH 7.0. H thus appears to compete with Li for the binding site. Li binding was abolished by replacement of Glu by Asp or Ser by Ala. The side chains at Ala and Thr may act in a supporting structural role by providing a more flexible conformation for the Li binding cavity. Thr does not appear to provide a liganding group since H transport in two other mutants, with Gly or Ala in place of Thr, was also inhibited by Li. We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding, and that the flexibility provided by residues X and Y will prove crucial to this structure.

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