Covès J, Lebrun C, Gervasi G, Dalbon P, Fontecave M
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.
Biochem J. 1999 Sep 1;342 ( Pt 2)(Pt 2):465-72.
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.
大肠杆菌亚硫酸盐还原酶黄素蛋白组分（SiR-FP）的NADPH和FAD结合结构域SiR-FP43已被过量表达并进行了表征。它能独立折叠，保留FAD作为辅因子以及与该辅因子存在相关的催化特性。二苯基氯化碘鎓（IDP）被证明是SiR-FP43和SiR-FP60（SiR-FP的单体形式，含有FMN和FAD作为辅因子）的一种非常有效的抑制剂（K(i)=18.5±5微摩尔，最大失活速率=0.053±0.005秒(-1)）。在这两种情况下，失活均显示是由于苯基仅与FAD共价结合所致，这与之前用细胞色素P450还原酶（CPR）获得的结果形成显著对比，在CPR中FMN和一个色氨酸被苯基化，但FAD未被苯基化。然而，我们的动力学分析与CPR所证明的抑制机制一致[Tew(1993)生物化学32,10209 - 10215]。分离出了九种不同的FAD苯基化加合物，并且首次在从蛋白质中提取后直接鉴定出了两种FAD苯基化加合物。综上所述，我们的结果表明，IDP导致黄素蛋白失活并非催化过程中黄素自由基中间体的可靠指标。
