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

• Published in: Archives of biochemistry and biophysics 2020-03, Vol.681, p.108266-108266, Article 108266
• Main Authors: Bertsova, Yulia V., Baykov, Alexander A., Bogachev, Alexander V.
• Format: Article
• Language: English
• Subjects: Azotobacter vinelandii - metabolism; Azotobactervinelandii; Bacterial Proteins - metabolism; H+ transport; Hydrogen - metabolism; Na+ transport; NAD(P)H Dehydrogenase (Quinone) - metabolism; NADH:quinone oxidoreductase; Nitrogen Fixation; Respiratory protection; Sodium - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2020.108266

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. •Azotobacter vinelandii has H+-transporting and Na+-transporting NADH:quinone oxidoreductases.•These enzymes were differentiated by using Δψ and ΔpH measurements in membrane vesicles.•Strains of A. vinelandii deficient in either of these enzymes were engineered.•The Na+-transporting NADH:quinone oxidoreductase has a role in nitrogen fixation by A. vinelandii.