Requirement of Bacillus subtilis succinate:menaquinone oxidoreductase activity for membrane energization depends on the direction of catalysis.

Succinate:quinone oxidoreductases (SQR) from Bacilli catalyze reduction of menaquinone by succinate, as well as the reverse reaction. The direct activity is energetically unfavorable and lost upon ΔμН dissipation, thus suggesting ΔμН to be consumed during catalysis. Paradoxically, the generation of ΔμН upon fumarate reduction was never confirmed. Thus, the exact role of ΔμН in the operation of bacillary-type SQRs remained questionable. The purpose of this work was to clarify this issue. We have described the different operating modes of the membrane-bound SQR from Bacillus subtilis. Tightly coupled membrane vesicles from both wild-type cells and the mutant containing cytochrome bd as the only terminal oxidase were studied. This made it possible to compare the respiratory chains with 2 versus 1H/e stoichiometry of ΔμН generation. Direct and reverse activities of SQR were determined under either energized or deenergized conditions. The wild-type membranes demonstrated high succinate oxidase activity very sensitive to uncoupling. On the contrary, the mutant showed extremely low succinate oxidase activity resistant to uncoupling. ΔμН generation at the cost of ATP hydrolysis restored the uncoupling sensitive succinate respiration in the mutant. Membranes of the both types effectively reduced fumarate by menaquinol. This activity was not affected by energization or uncoupling, neither it was followed by ΔμН generation. Thus, B. subtilis SQR demonstrates two regimes: ΔμН-coupled and not coupled. This behavior can be explained by assuming the presence of two menaquinone binding sites which drastically differ in affinity for the oxidized and reduced substrate.