Kinetics and Activation Parameters for Oxidations of Styrene by Compounds I from the Cytochrome P450BM-3 (CYP102A1) Heme Domain and from CYP119

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• 作者：Xinting Yuan ; Qin Wang ; John H. Horner ; Xin Sheng ; Martin Newcomb
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：September 29, 2009
• 年：2009
• 卷：48
• 期：38
• 页码：9140-9146
• 全文大小：770K
• 年卷期：v.48,no.38(September 29, 2009)
• ISSN：1520-4995

文摘

Cytochrome P450 (CYP or P450) enzymes are ubiquitous in nature where they catalyze a vast array of oxidation reactions. The active oxidants in P450s have long been assumed to be iron(IV)−oxo porphyrin radical cations termed Compounds I, but P450 Compounds I have proven to be difficult to prepare. The recent development of an entry to these transients by photo-oxidation of the corresponding iron(IV)−oxo neutral porphyrin species (Compounds II) permits spectroscopic and kinetic studies. We report here application of the photo-oxidation method for production of Compound I from the heme domain of CYP102A1 (cytochrome P450_BM-3), and product and kinetic studies of reactions of styrene with this Compound I transient and also Compound I from CYP119. The studies were performed at low temperatures in 1:1 (v:v) mixtures of glycerol and phosphate buffer. Single-turnover reactions at 0 °C gave styrene oxide in good yields. In kinetic studies conducted between −10 and −50 °C, both Compounds I displayed saturation kinetics permitting determinations of binding constants and first-order oxidation rate constants. Temperature-dependent functions for the binding constants and rate constants were determined for both Compounds I. In the temperature range studied, the Compound I transient from the CYP102A1 heme domain bound styrene more strongly than Compound I from CYP119, but the rate constants for oxidations of styrene by the latter were somewhat larger than those for the former. The temperature-dependent functions for the first-order oxidation reactions are as follows: log k = 13.2 − 15.2/2.303RT and log k = 13.3 − 14.6/2.303RT (kilocalories per mole) for Compounds I from the CYP102A1 heme domain and CYP119, respectively.