A pair of membrane-embedded acidic residues in the NuoK subunit of Escherichia coli NDH-1, a counterpart of the ND4L subunit of the mitochondrial complex I, are required for high ubiquinone reductase activity.
The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices. We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis. The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined. Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed. The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E. coli cells on malate as the main carbon source. The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes. Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant.
Kervinen
Marko
M
Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland.
Pätsi
Jukka
J
Finel
Moshe
M
Hassinen
Ilmo E
IE
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000737:chemistry*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D018698:Glutamic Acid; D009245:NADH Dehydrogenase; C476247:NuoM protein, E coli; D042967:Electron Transport Complex I