Quinone reduction by the Na+-translocating NADH dehydrogenase promotes extracellular superoxide production in Vibrio cholerae

The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). To study the function of the Na+-NQR in the respiratory chain of V cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na+-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na+-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na+ to 0.4 mM at 14.7 mM Na+, indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. choterae membranes. During respiration, V cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min(-1) mg(-1) in the wild type compared to 3.1 nmol min(-1) mg(-1) in the NQR deletion strain. Raising the Na+ concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V cholerae strain by at least 70%. Rates of respiratory H2O2 formation by wild-type V. cholerae cells (30.9 nmol min(-1) mg(-1)) were threefold higher than rates observed with the mutant strain lacking the Na+-NQR (9.7 nmol min(-1) mg-(1)). Our study shows that environmental Na+ could stimulate ubisemiquinone formation by the Na+-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones.