Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I).
Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.
Euro
Liliya
L
Helsinki Bioenergetics Group, Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Finland.
Belevich
Galina
G
Verkhovsky
Michael I
MI
Wikström
Mårten
M
Verkhovskaya
Marina
M
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D010641:Phenotype; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; D009245:NADH Dehydrogenase; C476247:NuoM protein, E coli; D042967:Electron Transport Complex I; D008239:Lysine