Sequencing and preliminary characterization of the Na+-translocating NADH : ubiquinone oxidoreductase from Vibrio harveyi

The Na+-translocating NADH: ubiquinone oxidoreductase (Na+-NQR) generates a n electrochemical Na+ potential driven by aerobic respiration. Previous stu dies on the enzyme from Vibrio alginolyticus have shown that the Na+-NQR ha s six subunits, and it is known to contain FAD and an FeS center as redox c ofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second f lavin, tentatively identified as FMN, was discovered to be covalently attac hed to the NqrC subunit. The purified V, harveyi Na+-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na+, resistant to the protonophore CCCP, and sensitive to the sodium ionophore E TH-157, showing that the enzyme operates as a primary electrogenic sodium p ump. Interior alkalinization of the inside-out proteoliposomes due to the o peration of the Na+-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquin ol formation must be taken up from the outside of the liposomes, which corr esponds to the bacterial cytoplasm. The Na+-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously rep orted Na+-NQR operons from V. alginolyticus and Haemophilus influenzae. Hom ology studies show that a number of other bacteria, including a number of p athogenic species, also have an Na+-NQR operon.