Stimulation of Menaquinone-Dependent Electron Transfer in the Respiratory Chain ofBacillus subtilisby Membrane Energization

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Abstract
At a pH of ≤7, respiration ofBacillus subtiliscells on endogenous substrates shut down almost completely upon addition of an uncoupler (carbonyl cyanidem-chlorophenylhydrazone [CCCP]) and a K+-ionophore (valinomycin). The same effect was observed with cell spheroplasts lacking the cell wall. The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K+-valinomycin was below 100 nM. Either CCCP or valinomycin alone was much less efficient than the combination of the two. The inhibitory effect was easily reversible and depended specifically on the H+and K+concentrations in the medium. Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) andN,N,N′,N′-tetramethyl-p-phenylenediamine cation (TMPD+), which intercept reducing equivalents at the level of menaquinol. Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling. The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores. We conclude that the menaquinone-dependent electron transfer in theB. subtilisrespiratory chain is facilitated, thermodynamically or kinetically, by membrane energization. A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant ofB. subtilislacking succinate:quinone reductase as well as for substrates other than succinate. Possible mechanisms of the energy-dependent regulation of menaquinone-dependent respiration inB. subtilisare discussed.