In Gram-positive bacteria and other prokaryotes containing
succinate:
menaquinone reductases, it has previously been shown that the
succinate oxidase and
succinate:
menaquinone reductase activities are lost when the transmembrane electrochemical
proton potential, Deltap, is abolished by the
rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of
menaquinone by
succinate is driven by the electrochemical
proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of
protons upon the reduction of
menaquinone and their release upon
succinate oxidation, with the two reactions linked by the transfer of two electrons through the
enzyme. However, it has previously been argued that the observed Deltap dependence is not associated specifically with the
succinate:
menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound
succinate:
menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into
proteoliposomes, and functional characterization of the diheme-containing
succinate:
menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of
quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Deltap-dependent catalysis of
succinate oxidation by
quinone as well as for Deltap generation upon catalysis of
fumarate reduction by
quinol.