LHON (
Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human
mtDNA (
mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (
NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from
deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine
ruthenium), whereas
ubiquinone reductase activity was determined using DB (
decylubiquinone) as a substrate. Mutant growth in minimal medium with
malate as the main
carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on
malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of
ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the
enzyme's catalytic properties contribute to LHON pathogenesis is discussed.