期刊
BIOCHEMISTRY
卷 48, 期 38, 页码 9140-9146出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi901258m
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资金
- National Institutes of Health [GM48722]
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 degrees C gave styrene oxide in good yields. In kinetic studies conducted between -10 and -50 degrees 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 hem domain bound styrene in 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.
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