Disruption of iron-sulphur cluster N2 from NADH : ubiquinone oxidoreductase by site-directed mutagenesis

• Type: Article in International Scientific Journal
• Date: 2002

Authors

duarte, m (Author) ICBAS; populo, h (Author) Other; videira, a (Author) ICBAS; friedrich, t (Author) Other; schulte, u (Author) Other

Journal

Title: Biochemical Journal

Vol. 364

Pages: 833-839

ISSN: 0264-6021

Publisher: PORTLAND PRESS LTD

Indexing

ISI Web of Knowledge - 31 Citations

Scopus - 31 Citations

Other information

Authenticus ID: P-000-P0Z

DOI: 10.1042/BJ20011750

Abstract (EN): 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 7