The 24-kDa iron-sulphur subunit of complex I is required for enzyme activity

We have cloned the nuclear gene encoding the 24-kDa iron-sulphur subunit of complex I from Neurospora crassa. The gene was inactivated in vivo by repe at-induced point-mutations, and mutant strains lacking the 24-kDa protein w ere isolated. Mutant nuo24 appears to assemble an almost intact complex I o nly lacking the 24-kDa subunit. However, we also found reduced levels of th e NADH-binding, 51-kDa subunit of the enzyme. Surprisingly, the complex I f rom the nuo24 strain lacks NADH:ferricyanide reductase activity. In agreeme nt with this, the respiration of intact mitochondria or mitochondrial membr anes from the mutant strain is insensitive to rotenone inhibition. These re sults suggest that the nuo24 complex is not functioning in electron transfe r and the 24-kDa protein is absolutely required for complex I activity. Thi s phenotype may explain the findings that the 24-kDa iron-sulphur protein i s reduced or absent in human mitochondrial diseases. In addition, selected substitutions of cysteine to alanine residues in the 24-kDa protein suggest that binding of the iron-sulphur centre is a requisite for protein assembl y.