A simple strategy to differentiate between H⁺- and Na⁺-transporting NADH:quinone oxidoreductases

We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na⁺-translocating (NQR) and H⁺-translocating (NDH-1) enzymes of the soil bacterium Azotobactervinelandii as the models. Submillimolar concentrations of Na⁺ and Li⁺ increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na⁺. These findings testified a primary Na⁺-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H⁺ under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H⁺-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na⁺ or H⁺ transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na⁺ transport in nitrogen fixation by A. vinelandii.