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The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni2+ chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The Fo liposomes catalysed 22Na+ uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N′-dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the Fo complexes catalysed 22Na+out/Na+in-exchange. After F1 addition the F1Fo complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na+ pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional Fo hybrids were reconstituted with recombinant subunits a and b from P. modestum and c11 from Ilyobacter tartaricus. These Fo hybrids had Na+ translocation activities that were not distinguishable from that of P. modestum Fo.

The most prominent residue of subunit a of the F 1 F o ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum ), which was reported to permit no substitution with retention of ATP synthesis or H + -coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P. modestum a subunit catalyse ATP-driven Na + transport above or below pH 8.0, respectively. Reconstituted F o with either mutation catalysed 22 Na + out /Na + in exchange with similar pH profiles as found in ATP-driven Na + transport. ATP synthase with an aR227A substitution catalysed Na + -dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na + transport. This suggests that in the mutant the dissociation of Na + becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na + concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na + from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, F o with the aR227A mutation did not catalyse 22 Na + out /Na + in exchange at high internal sodium concentrations, and (ii) at a constant ΔpNa + , 22 Na + uptake was inhibited at elevated internal Na + concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na + even into compartments of high Na + concentration.

The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane. The site-specific introduction of the cQ32I and cS66A substitutions in the putative vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system. This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P. modestum c subunit. The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+. ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt. Both activities were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed. The ATPase of the E. coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl. The mutant ATPase exhibited a 5-fold higher Km for NaCl but no change in the Km for LiCl in comparison to that of the parent strain. These results demonstrate that the binding of Na+ to the c subunit of P. modestum requires liganding groups provided by Q32, E65, and S66. For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone.