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. 1999 Sep 1;342(Pt 2):465–472.

Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride.

J Covès 1, C Lebrun 1, G Gervasi 1, P Dalbon 1, M Fontecave 1
PMCID: PMC1220485  PMID: 10455035

Abstract

SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.

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Selected References

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