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Purified F1Fo 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 degree 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 (kobs) 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 kobs 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 numax (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.(ABSTRACT TRUNCATED AT 250 WORDS)

The purified ATPase of Propionigenium modestum (F1Fo) was incorporated into liposomes, and the F1 part was dissociated. The Fo-liposomes catalyzed proton uptake in response to a potassium diffusion potential (inside negative). Proton translocation was abolished by rebinding F1 to the Fo-liposomes or after incubation with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD). Proton uptake was also sensitive to the presence of external Na+ or Li+ ions and was completely abolished at 2 mM NaCl or 150 mM LiCl, respectively. However, the same concentrations of these salts in the internal volume of the Fo-liposomes were without effect, suggesting that the cation binding site is not accessible from both sides of the membrane simultaneously. An open channel-type of transport through Fo from P. modestum is therefore excluded. The Fo-liposomes also catalyzed Na+ influx or efflux in response to a K+ diffusion potential that was negative on the inside or outside, respectively. These Na+ fluxes could not be created, however, by delta pNa+ of about 60-180 mV. The initial rate of Na+ uptake depended strongly on the size of the membrane potential with no significant conductivity below -40 mV, followed by a proportional increase up to about -115 mV. In the absence of a membrane potential, the Fo-liposomes catalyzed 22Na+ counterflow against a 28-fold concentration gradient. Uptake of 22Na+ into Fo-liposomes against delta pNa+ (counterflow) was completely prevented by imposing an inside-positive potassium diffusion potential of 90 mV. The catalysis of 22Na+ counterflow by Fo from P. modestum is a clear indication of a carrier (transporter)-type mechanism and excludes a channel mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

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