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 a-type cytochromes and partial reduction of c/h-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 6. 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.In 1961 Mitchell [l] suggested that membrane-linked energy conservation is due to the splitting of hydrogen atoms into protons and electrons separated by a membrane. As a result of charge separation, the electrochemical H ' potential difference (proton potential, A&+) is formed. Recently this suggestion was directly proved for the bacterial photosynthetic-reaction-center complex (for review, see [2]). The very fact that it is the proton potential which is generated by such mechanisms is an inevitable consequence of the chemistry of the process (H + H + + e-). However, it is also quite clear that some membrane-linked energy transductions are organized

Submicromolar Ag+ increases passive Na+ permeability and inhibits the respiration‐supported formation of Na+ gradient in Bacillus FTU vesicles

“…As shown by Asano et al [4], a very low [Ag+] specifically inhibits the Na'-motive NADHquinone reductase of Vibrio alginolyticus. As it was shown in our laboratory in the experiments on the Bacillus FTU vesicles, Ag + strongly suppresses the respiration-supported uphill transport of Na+ at a concentration as low as 1 x 10e8 M, while that of H+ is not affected by Ag+ taken even at a lOO-fold higher concentration [3].…”

“…(ii) HQNO (which inhibits the Na+-motive NADHquinone reductase but has no effect on the terminal oxidase [3]) was found to prevent Ag+ inhibition of the Na+ uptake coupled to ascorbate oxidation (Fig. IC).…”

Kinetics of CO binding to putative Na+‐motive oxidases of the o‐type from Bacillus FTU and of the d‐type from Escherichia coli

“…FTU grown under low 4iir.i+ conditions [6] were competent in Na' transport when TMPD and ascorbate were used as electron donors. The process was resistant to low concentrations of KCN [5,7,8]. The present data reveal a high degree of similarity between these oxidases.…”

Appearance of the Na+‐motive terminal oxidase in Bacillus FTU grown under three different conditions lowering the Δ\̄gmH+ level

“…The cluale wasdiluted IO-fold with bidistilled water and ma*] was measured with a PFM flnme photometer. The above procedure was found to decrease extravesicular ma.1 by a factor of 5x10' [3]. Oxygen consumption was monitored with a standard Clark-typ oxygen electrode.…”