Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3
Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3
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DOI：
10.1021/bi701031r
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发表时间：
2007-10-23
期刊：
影响因子：
2.9
通讯作者：
Peterson, Julian A.
中科院分区：
文献类型：
--
作者：
Kitazume, Tatsuya;Haines, Donovan C.;Peterson, Julian A.
细胞色素P450通常催化疏水性化合物的单加氧作用，导致一个双氧原子插入有机底物中，另一个氧原子还原为水。反应所需的两个电子通常由另一种氧化还原活性蛋白质提供，例如哺乳动物内质网膜中的细胞色素P450还原酶（CPR）。来自巨大芽孢杆菌的P450 BM-3是广泛研究的P450细胞色素，其中P450天然融合到与CPR同源的二黄素还原酶。根据Fulco实验室对酶的原始表征，该酶显示出反应速率对酶浓度的非线性依赖性。在最近的实验中，我们观察到酶在稀释时失活，底物的存在可以减少这种失活。因此，我们进行了酶动力学，交联实验，和分子量测定，建立酶是能够在溶液中二聚化。在大多数条件下，二聚体在较高浓度下是主要形式，并且是具有显著活性的唯一形式。进一步的实验用定点诱变选择性地敲除单个结构域的活性并测量异源二聚体中的酶活性，确定P450 BM-3中的电子转移途径在单个周转期间穿过二聚体中的两个蛋白质分子，在传递到血红素结构域之前从一个分子的FAD结构域穿过另一个分子的FMN结构域。结合文献中的其他分析，我们的结果分析表明，血红素结构域的单体可以接受电子从减少FMN域。
Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.