title: Modeling of human pathogenic mutations in Escherichia coli complex I reveals a sensitive region in the fourth inside loop of NuoH.
abstract: Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations. Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K(m) value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.
authors.affiliation: Department of Medical Biochemistry and Molecular Biology, Institute of Biomedicine, University of Oulu, Finland.
authors.forename: Pilvi
authors.initials: P
authors.lastname: Maliniemi
authors.forename: Marko
authors.initials: M
authors.lastname: Kervinen
authors.forename: Ilmo E
authors.initials: IE
authors.lastname: Hassinen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009245:NADH Dehydrogenase / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017494:Proton Pumps / Q000378:metabolism; D017975:Ruthenium Compounds / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008293:Malates; D008565:Membrane Proteins; C540656:NuoH protein, E coli; D017494:Proton Pumps; D017975:Ruthenium Compounds; D014451:Ubiquinone; C031584:hexammineruthenium; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; C030298:malic acid; D009245:NADH Dehydrogenase