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 repeat-induced point-mutations, and mutant strains lacking the 24-kDa protein were isolated. Mutant nuo24 appears to assemble an almost intact complex I only lacking the 24-kDa subunit. However, we also found reduced levels of the NADH-binding, 51-kDa subunit of the enzyme. Surprisingly, the complex I from the nuo24 strain lacks NADH:ferricyanide reductase activity. In agreement with this, the respiration of intact mitochondria or mitochondrial membranes from the mutant strain is insensitive to rotenone inhibition. These results suggest that the nuo24 complex is not functioning in electron transfer and the 24-kDa protein is absolutely required for complex I activity. This phenotype may explain the findings that the 24-kDa iron-sulphur protein is 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 assembly.
Almeida
T
T
Instituto de Biologia Molecular e Celular, Porto, Portugal.
Duarte
M
M
Melo
A M
AM
Videira
A
A
D002467:Cell Nucleus / Q000235:genetics; D003001:Cloning, Molecular; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010101:Oxygen Consumption; D017354:Point Mutation; D012402:Rotenone / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D007506:Iron-Sulfur Proteins; D014475:Uncoupling Agents; D012402:Rotenone; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D009245:NADH Dehydrogenase