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

SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphit e reductase flavoprotein component (SiR-FP), has been overexpressed and cha racterized. It folds independently, retaining FAD as a cofactor and the cat alytic properties associated with the presence of this cofactor. Iodonium d iphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP 43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K-i = 18.5 +/- 5 mu cM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalen t binding of a phenyl group to FAD exclusively, in marked contrast with pre vious results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses ar e in agreement with the inhibition mechanism demonstrated with CPR [Tew (19 93) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts we re isolated and, for the first time, two FAD phenylated adducts were identi fied directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicato r for a flavin radical intermediate in catalysis.