Purified F1F0 ATPase of Propionigenium modestum was rapidly inactivated by dicyclohexylcarbodiimide (DCCD) with k2 = 1.2 x 10(5) M-1 min-1 at pH 5.6 and 0-degrees-C. Na+ ions provided specific protection from the modification by DCCD while protons stimulated the reaction. Plots of pseudo-first-order rate constants of inactivation (k(obs)) against pH yielded titration curves with pK(H+) = 7.0 in the absence of Na+ and pK(H+) = 6.2 in the presence of 0.5 mM Na+. From the dependencies of k(obs) on Na+, pK(Na+) of about 2.5 and 3.3 were obtained at pH 6.5 and 8.0, respectively. These results indicate that DCCD reacts with a protonated group of the enzyme that dissociates with pK(H+) = 7.0 in the absence of Na+, and that Na+ ions promote the dissociation of this group. Additionally, higher Na+ concentrations, were required at more acidic pH values to yield half-maximal protection from inactivation. These effects fit a competitive binding model for Na+ or H+ at the DCCD-reactive conserved acidic amino acid of subunit c (Glu-65). The active-site carboxylate could either be protonated and modified by DCCD or bind Na+ which then provides protection. Complementary results were obtained from the effects of Na+ and H+ on ATPase activity. The pH-rate profile of v(max) (with saturating Na+) indicated an increase of activity with apparent pK = 6.8, an optimum around pH 7.5, and decreasing activity with apparent pK = 8.7. The remarkable activation of the enzyme by Na+ ions (20-fold at pH 7.5 and above) indicates that a step involving Na+ binding to F(o) is rate limiting and therefore that the pH effect on v(max) reflects an effect of pH on the Na+ binding site. Maximal ATPase activities were about the same at pH 6.5 and 9.0, but about 10 times higher Na+ concentrations were required at the acidic pH to saturate the enzyme. These results can be explained by an interference of protons (H3O+) with Na+ binding at the active site. The ATPase at pH 9.0 showed positive cooperativity (n(H) = 2.6) with respect to Na+ transport and Na+-activated ATPase activity, indicating an interaction of at least three Na+ binding sites. The multiple c subunits present in the enzyme could readily provide binding sites for three or more Na+ ions at a time. A new model for ion translocation involving formation and breakdown of salt bridges at the membrane-embedded Glu- residue is discussed that can account for Na+ and H3O+ transport.
