Identification of the catalytic ubiquinone-binding site of Vibrio cholerae sodium-dependent NADH dehydrogenase: a novel ubiquinone-binding motif [Enzymology]

The sodium-dependent NADH dehydrogenase (Na+-NQR) is a key component of the respiratory chain of diverse prokaryotic species, including pathogenic bacteria. Na+-NQR uses the energy released by electron transfer between NADH and ubiquinone (UQ) to pump sodium, producing a gradient that sustains many essential homeostatic processes, as well as virulence factor secretion, and the elimination of drugs. The location of the UQ binding site has been controversial, with two main hypotheses that suggest that this site could be located in the cytosolic subunit A or in the membrane-bound subunit B. In this work, we performed Alanine scanning mutagenesis of aromatic residues located in transmembrane helices II, IV and V of subunit B, near glycine residues 140 and 141. These two critical glycine residues form part of the structures that regulate the site accessibility. Our results indicate that the elimination of phenylalanine residues 211 or 213 abolishes the UQ-dependent activity, produces a leak of electrons to oxygen, and completely blocks the binding of UQ and the inhibitor HQNO. Molecular docking calculations predict that UQ interacts with phenylalanine 211 and pinpoints the location of the binding site in the interface of subunits B and D. The mutagenesis and structural analysis allow us to propose a novel UQ-binding motif, which is completely different compared to the sites of other respiratory photosynthetic complexes. These results are essential to understanding the electron transfer pathways and mechanism of Na+-NQR catalysis

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