Quinone Reduction by the Na + -Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production in Vibrio cholerae

Abstract

ABSTRACT 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. cholerae 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 H 2 O 2 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.