Disruption of iron-sulphur cluster N2 from NADH: ubiquinone oxidoreductase by site-directed mutagenesis.
We have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3 kDa subunit of complex I (EC 1.6.5.3) from Neurospora crassa, the homologue of the bovine PSST polypeptide. Mitochondria from mutant nuo19.3 lack the peripheral arm of complex I while its membrane arm accumulates. Transformation with wild-type cDNA rescues this phenotype and assembly of complex I is restored. To interfere with assembly of a proposed bound iron-sulphur cluster, site-directed mutants were constructed by introducing cDNA with altered codons for two adjacent cysteines, Cys-101 and Cys-102. The mutant complexes were purified and their enzymic activities and EPR and UV/visible spectra were analysed. Either of the mutations abolishes assembly of iron-sulphur cluster N2, showing that this redox group is bound to the 19.3 kDa protein. We also observed an interference with the reduction of redox group X, suggesting that cluster N2 is the electron donor to this high-potential redox group.
Duarte
Margarida
M
Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Pópulo
Helena
H
Videira
Arnaldo
A
Friedrich
Thorsten
T
Schulte
Ulrich
U
D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D010084:Oxidation-Reduction; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013053:Spectrophotometry
D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I