The H(+)-motive and Na(+)-motive respiratory chains in Bacillus FTU subcellular vesicles.

Abstract

Respiration-dependent pumping of Na+ and H+ into the inside-out subcellular vesicles of alkalotolerant and halotolerant Bacillus FTU grown at alkaline pH was studied. The vesicles were shown to be competent in Na+ and H+ transport coupled to ascorbate oxidation via N,N,N',N'-tetramethyl-p-phenylenediamine or diaminodurene. The uphill Na+ uptake is strongly stimulated by either protonophores or valinomycin, whereas H+ uptake is stimulated by valinomycin and completely inhibited by protonophores. The salt of a penetrating weak base and of the penetrating weak acid, diethylammonium acetate, potentiates the stimulating effect of protonophores on Na+ uptake and abolishes H+ uptake. Na+ transport, supported by ascorbate oxidation, is resistant to 2-heptyl-4-hydroxyquinoline N-oxide, but sensitive to Ag+ and Na+ ionophore, N,N'-dibenzyl-N,N'-diphenyl-1,2-phenylenediacetamide. Micromolar concentrations of cyanide specifically inhibit the H+ uptake but does not affect Na+ uptake. These cyanide concentrations are shown to cause 70% inhibition of respiration, complete reduction of alpha-type cytochromes and partial reduction of c/b-type cytochromes. To inhibit the remaining respiratory activity and Na/ uptake, approximately 100-fold higher cyanide concentrations are necessary. High cyanide concentrations cause some additional increase in absorbance in the region of cytochromes c and/or b. In the presence of a high cyanide concentration, Na+ uptake can be supported by NADH oxidation by fumarate. This Na+ transport is stimulated by protonophores and diethylammonium acetate, being sensitive to very low concentrations of 2-heptyl-4-hydroxyquinoline N-oxide and Ag+. The NADH-fumarate reductase reaction is also found to be competent in H+ uptake, which is inhibited by protonophores and by much higher 2-heptyl-4-hydroxyquinoline N-oxide concentrations, and is resistant to Ag+. It is inferred that Bacillus FTU possesses two respiratory chains: the H(+)-motive and the Na(+)-motive, which strongly differ in their inhibitor sensitivities. Each chain comprises at least two energy-coupling sites which are localized in their initial and terminal segments. It has been indicated that common redox carrier(s) are present in the two chains.