Uncovering the Role of Hydrophobic Residues in Cytochrome P450鈭扖ytochrome P450 Reductase Interactions

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• 作者：Cesar Kenaan ; Haoming Zhang ; Erin V. Shea ; Paul F. Hollenberg
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：May 17, 2011
• 年：2011
• 卷：50
• 期：19
• 页码：3957-3967
• 全文大小：951K
• 年卷期：v.50,no.19(May 17, 2011)
• ISSN：1520-4995

文摘

Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4鈭扖PR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450鈭扖PR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K_d of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K_m values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450鈭扖O adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K_m values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450鈭扖O formation.