Analysis of compound heterozygous and homozygous mutations found in peripheral subunits of human respiratory Complex I, NDUFS1, NDUFS2, NDUFS8 and NDUFV1, by modeling in the E. coli enzyme.

Respiratory Complex I (NADH:ubiquinone oxidoreductase) is composed of 45 subunits, seven mitochondrially-encoded and 38 imported. Mutations in the nuclearly-encoded subunits have been regularly discovered in humans in recent years, and many lead to cardiomyopathy, Leigh Syndrome, and early death. From the literature, we have identified mutations at 17 different sites and constructed 31 mutants in a bacterial model system. Many of these mutations, found in NDUFS1, NDUFS2, NDUFS8, and NDUFV1, map to subunit interfaces, and we hypothesized that they would disrupt assembly of Complex I. The mutations were constructed in the homologous E. coli genes, nuoG, nuoCD, nuoI and nuoF, respectively, and expressed from a plasmid containing all Complex I genes. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity measured, which indicated a range of reduced activity. Some mutants were also analyzed using recently developed assays of assembly, time-delayed expression, and co-immunoprecipitation, which showed that assembly was disrupted. With compound heterozygotes, we determined which mutation was more deleterious. Construction of alanine mutations allowed us to distinguish between phenotypes that were caused by loss of the original amino acid or introduction of the mutant residue.