Obligatory Intermolecular Electron-Transfer from FAD to FMN in Dimeric P450BM-3

详细信息    查看全文

• 作者：Tatsuya Kitazume ; Donovan C. Haines ; Ronald W. Estabrook ; Baozhi Chen ; Julian A. Peterson
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 23, 2007
• 年：2007
• 卷：46
• 期：42
• 页码：11892 - 11901
• 全文大小：506K
• 年卷期：v.46,no.42(October 23, 2007)
• ISSN：1520-4995

文摘

Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resultingin the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygenatom to water. The two electrons required for the reaction are normally provided by another redox activeprotein, 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 fusednaturally to a diflavin reductase homologous to CPR. From the original characterization of the enzymeby Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzymeconcentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence ofsubstrate 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 formwith significant activity. Further experiments selectively knocking out the activity of individual domainswith site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that theelectron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during asingle turnover, traversing from the FAD domain of one molecule into the FMN domain of the othermolecule before passing to the heme domain. Analysis of our results combined with other analyses in theliterature suggests that the heme domain of either monomer may accept electrons from the reduced FMNdomain.