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

• Published in: Mitochondrion Vol. 9; no. 6; pp. 394 - 401
• Main Authors: Maliniemi, Pilvi, Kervinen, Marko, Hassinen, Ilmo E
• Format: Journal Article
• Language: English
• Published: Netherlands 01.11.2009
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Escherichia coli; 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
• ISSN: 1567-7249; 1872-8278
• DOI: 10.1016/j.mito.2009.07.001

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.