PMID:15175326

Citation	Kao, MC, Di Bernardo, S, Perego, M, Nakamaru-Ogiso, E, Matsuno-Yagi, A and Yagi, T (2004) Functional roles of four conserved charged residues in the membrane domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli. J. Biol. Chem. 279:32360-6
Abstract	The H(+)(Na(+))-translocating NADH-quinone (Q) oxidoreductase (NDH-1) of Escherichia coli is composed of 13 different subunits (NuoA-N). Subunit NuoA (ND3, Nqo7) is one of the seven membrane domain subunits that are considered to be involved in H(+)(Na(+)) translocation. We demonstrated that in the Paracoccus denitrificans NDH-1 subunit, Nqo7 (ND3) directly interacts with peripheral subunits Nqo6 (PSST) and Nqo4 (49 kDa) by using cross-linkers (Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388 and Kao, M.-C., Matsuno-Yagi, A., and Yagi, T. (2004) Biochemistry 43, 3750-3755). To investigate the structural and functional roles of conserved charged amino acid residues, a nuoA knock-out mutant and site-specific mutants K46A, E51A, D79N, D79A, E81Q, E81A, and D79N/E81Q were constructed by utilizing chromosomal DNA manipulation. In terms of immunochemical and NADH dehydrogenase activity-staining analyses, all site-specific mutants are similar to the wild type, suggesting that those NuoA site-specific mutations do not significantly affect the assembly of peripheral subunits in situ. In addition, site-specific mutants showed similar deamino-NADH-K(3)Fe(CN)(6) reductase activity to the wild type. The K46A mutation scarcely inhibited deamino-NADH-Q reductase activity. In contrast, E51A, D79A, D79N, E81A, and E81Q mutation partially suppressed deamino-NADH-Q reductase activity to 30, 90, 40, 40, and 50%, respectively. The double mutant D79N/E81Q almost completely lost the energy-transducing NDH-1 activities but did not display any loss of deamino-NADH-K(3)Fe(CN)(6) reductase activity. The possible functional roles of residues Asp-79 and Glu-81 were discussed.
Links	PubMed Online version:10.1074/jbc.M403885200
Keywords	Amino Acid Sequence; Animals; Aspartic Acid/chemistry; Blotting, Western; Cell Division; Cell Membrane/metabolism; Cloning, Molecular; Cytoplasm/metabolism; DNA/chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Genetic Vectors; Glutamic Acid/chemistry; Immunoblotting; Immunohistochemistry; Mice; Mice, Knockout; Models, Genetic; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport; Protons; Quinone Reductases/chemistry; Sequence Homology, Amino Acid; Transgenes
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