Functional Roles of Four Conserved Charged Residues in the Membrane Domain Subunit NuoA of the Proton-translocating NADH-Quinone Oxidoreductase from Escherichia coli

• Published in: The Journal of biological chemistry 2004-07, Vol.279 (31), p.32360-32366
• Main Authors: Kao, Mou-Chieh, Di Bernardo, Salvatore, Perego, Marta, Nakamaru-Ogiso, Eiko, Matsuno-Yagi, Akemi, Yagi, Takao
• Format: Article
• Language: English
• Subjects: 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; 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; Paracoccus denitrificans; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport; Protons; Quinone Reductases - chemistry; Sequence Homology, Amino Acid; Transgenes
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M403885200

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.