Characterization Of Clinically Identified Mutations In Peripheral Arm Subunits NDUFV1, And NDUFS1 Of Respiratory Complex I, Using an E. coli Model System

Complex I (NADH: ubiquinone oxidoreductase) is the largest enzyme of the mammalian mitochondrial and bacteria respiratory chain. During the oxidation of NADH, hydrogen ions are translocated across the mitochondrial membrane, leading to the synthesis of ATP, the chemical energy currency of cells. Complex I in E. coli contains 13 different subunits, encoded by the nuo operon, which form an L‐shaped structure: a peripheral arm, containing 6 subunits involved in electron transport, and a membrane arm containing 7 subunits involved in pumping hydrogen ions. Peripheral arm subunits E, F, and G, form an NADH binding domain, and B, CD, and I interact with membrane subunits. Mutations in Complex I genes in humans lead to loss of vision, brain or muscle dysfunction, or other disorders. Here, we have modeled clinical mutations that have been reported to cause Complex I dysfunction in humans, using our bacterial system. Mutations have been constructed in subunit F (NDUFV1), the Flavin‐binding subunit: R358A, R358H, T173A, T173P, R102A, R102L, E349A, E349K, and in subunit G (NDUFV1), an Fe‐S protein: I154A, I154T, R219A, R219W. Analysis of additional mutations is underway. Expression levels of the mutant proteins have been determined by western blotting, and the NADH oxidase activities of the mutants have been measured. In conclusion, we have identified several mutations that are highly deleterious to the function or assembly of the enzyme. These results can be rationalized by consideration of the 3D structure of the Complex I.