A double mutation in subunit c of the Na(+)-specific F1F0-ATPase of Propionigenium modestum results in a switch from Na+ to H(+)-coupled ATP synthesis in the Escherichia coli host cells.

The in vivo synthesis of an F1F0-ATPase hybrid in Escherichia coli strain PEF42 which harbours the genes for the Propionigenium modestum subunits a, b, c, and delta, a gene for hybrid alpha subunit with the N-terminal portion (amino acids 1 to 173) of P. modestum and the C-terminal region (amino acids 176 to 513) from E. coli, and the genes for the E. coli subunits beta, gamma and epsilon, yielded a functional enzyme complex. This hybrid ATPase coupled ATP synthesis to Na+ transport and required Na+ for growth on succinate. After random mutagenesis of the P. modestum genes of strain PEF42, clones were selected that grew on succinate in the absence of Na+. A double-mutation cPhe84Leu, cLeu87Val that was found in several of these clones, was introduced by site specific mutagenesis into the parent strain PEF42. The resulting strain E. coli MPC8487 also exhibited Na(+)-independent growth on succinate, showing that the double mutation is the only reason for the new phenotype. The mutation causes a change of the coupling ions of the hybrid ATPase from Na+ in strain PEF42 to H+ in strain MPC8487. This conclusion was supported by the biochemical properties of the ATPase from strain MPC8487. Unlike the parent enzyme, the mutated ATPase was not activated by Na+, but retained activation by Li+. The pH optimum of the mutated ATPase (in the absence of Na+ or Li+) was shifted from pH 6.5 to pH 7.5, and the specific ATPase activity of the cell membranes increased about fourfold over that found in membranes of the parent cells. The mutated ATPase pumped protons or Li+ after reconstitution into proteoliposomes, and the transport of both cations was not affected by Na+. The double mutation in the c subunit thus results in the loss of Na+ binding, retention of Li+ binding and an improvement of H+ binding.