Modeling of human pathogenic mutations in Escherichia coli complex I reveals a sensitive region in the fourth inside loop of NuoH

Mitochondrion. 2009 Nov;9(6):394-401. doi: 10.1016/j.mito.2009.07.001. Epub 2009 Jul 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Humans
  • Kinetics
  • Malates / metabolism
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation, Missense*
  • NADH Dehydrogenase / metabolism
  • Protein Structure, Quaternary
  • Proton Pumps / metabolism
  • Ruthenium Compounds / metabolism
  • Ubiquinone / analogs & derivatives
  • Ubiquinone / metabolism


  • Escherichia coli Proteins
  • Malates
  • Membrane Proteins
  • NuoH protein, E coli
  • Proton Pumps
  • Ruthenium Compounds
  • Ubiquinone
  • hexammineruthenium
  • 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone
  • malic acid
  • NADH Dehydrogenase