Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride.
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.
Covès
J
J
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, CEA-Grenoble, DBMS/CB-CNRS-Université Joseph Fourier, 17 Rue des Martyrs, 38054 Grenoble Cedex 9, France. coves@cbcrb.ceng.cea.fr
Lebrun
C
C
Gervasi
G
G
Dalbon
P
P
Fontecave
M
M
D001483:Base Sequence; D001665:Binding Sites; D001713:Biphenyl Compounds / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D015870:Gene Expression; D013058:Mass Spectrometry; D009861:Onium Compounds / Q000494:pharmacology; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050864:Sulfite Reductase (NADPH)
D001713:Biphenyl Compounds; D017931:DNA Primers; D004791:Enzyme Inhibitors; D009861:Onium Compounds; D011994:Recombinant Proteins; C031291:diphenyliodonium; D005182:Flavin-Adenine Dinucleotide; D050862:Oxidoreductases Acting on Sulfur Group Donors; D050864:Sulfite Reductase (NADPH); C494314:sulfite reductase (NADPH), E coli