PMID:10455035
| Citation |
Covès, J, Lebrun, C, Gervasi, G, Dalbon, P and Fontecave, M (1999) Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride. Biochem. J. 342 ( Pt 2):465-72 |
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| 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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| Keywords |
Base Sequence; Binding Sites; Biphenyl Compounds/pharmacology; Chromatography, High Pressure Liquid; DNA Primers/genetics; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Flavin-Adenine Dinucleotide/metabolism; Gene Expression; Mass Spectrometry; Onium Compounds/pharmacology; Oxidoreductases Acting on Sulfur Group Donors/antagonists & inhibitors; Oxidoreductases Acting on Sulfur Group Donors/genetics; Oxidoreductases Acting on Sulfur Group Donors/metabolism; Recombinant Proteins/antagonists & inhibitors; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sulfite Reductase (NADPH) |
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