Role of Subunit D in Ubiquinone-Binding Site of Vibrio cholerae NQR: Pocket Flexibility and Inhibitor Resistance.

Abstract

The ion-pumping NADH: ubiquinone dehydrogenase (NQR) is a vital component of the respiratory chain of numerous species of marine and pathogenic bacteria, including Vibrio cholerae. This respiratory enzyme couples the transfer of electrons from NADH to ubiquinone (UQ) to the pumping of ions across the plasma membrane, producing a gradient that sustains multiple homeostatic processes. The binding site of UQ within the enzyme is an important functional and structural motif that could be used to design drugs against pathogenic bacteria. Our group recently located the UQ site in the interface between subunits B and D and identified the residues within subunit B that are important for UQ binding. In this study, we carried out alanine scanning mutagenesis of amino acid residues located in subunit D of V. cholerae NQR to understand their role in UQ binding and enzymatic catalysis. Moreover, molecular docking calculations were performed to characterize the structure of the site at the atomic level. The results show that mutations in these positions, in particular, in residues P185, L190, and F193, decrease the turnover rate and increase the Km for UQ. These mutants also showed an increase in the resistance against the inhibitor HQNO. The data indicate that residues in subunit D fulfill important structural roles, restricting and orienting UQ in a catalytically favorable position. In addition, mutations of these residues open the site and allow the simultaneous binding of substrate and inhibitors, producing partial inhibition, which appears to be a strategy used by Pseudomonas aeruginosa to avoid autopoisoning.