The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.

Amphotericin B is an antibiotic that forms ion channels in the membrane of a host cell. The change in permeability produced by these channels is greatly improved by sterols; nevertheless, the single channel conductivity remains invariant. Hence, it is proposed that sterols do not act directly, but rather through the modulation of the membrane phase. We look at the formation of these channels in the bacterial membrane to determine the mechanism of its known antibiotic resistance. We found that channels can indeed be formed in this membrane, but a substantial amount of amphotericin B is required. We also study the effects of the antibiotic concentration needed for channel expression as well as the dynamics of channels affected by both sterol and temperature in phosphatidylcholine membranes. The results support the idea that membrane structure is a determining factor in the action of the antibiotic.

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The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores of Rhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 Mg2+ or Pi for the enzyme; Mn2+ and Co2+ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg2+; other divalent cations like Zn2+ or Ca2+ do not support the exchange reaction. In the hydrolytic reaction, Zn2+, at low concentration, substitutes for Mg2+ as substrate, and Co2+ also substitutes in limited amount (50%). Other cations (Ca2+, Cu2+, Fe2+, etc.) do not act as substrates in complex with PPi. The Zn2+ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn2+. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn2+ greater than Mn2+ greater than Ca2+ greater than or equal to Co2+ greater than Fe2+ greater than Cu2+ greater than Mg2+. The data in this work suggest that H+ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.