MOLECULAR-BASIS FOR THE COUPLING ION SELECTIVITY OF F1F0 ATP SYNTHASES - PROBING THE LIGANDING GROUPS FOR NA-SUBUNIT OF THE ATP-SYNTHASE FROM PROPIONIGENIUM-MODESTUM( AND LI+ IN THE C)

The conserved glutamate residue at position 65 of the Propionigenium m odestum 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 gro wth of the single-site mutants on succinate minimal agar, indicating t hat 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, re vealed a change in the coupling ion specificity from Na+ to H+. ATP hy drolysis by this enzyme was therefore not activated by NaCl, and ATP-d riven H+ transport was not affected by this alkali salt. Both activiti es were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites with in this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/c L87V), the specificity of the ATPase was further restricted to H+ as t he exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated t he 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 K-m for NaCl but no change in the K-m 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.