Kinetics and activation parameters for oxidations of styrene by Compounds I from the cytochrome P450(BM-3) (CYP102A1) heme domain and from CYP119.
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 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.
Yuan
Xinting
X
Department of Chemistry, University of Illinois, 845 West Taylor Street, Chicago, Illinois 60607, USA.
Wang
Qin
Q
Horner
John H
JH
Sheng
Xin
X
Newcomb
Martin
M
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020058:Styrene / Q000378:metabolism; D016945:Sulfolobus acidocaldarius / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006418:Heme; D020058:Styrene; D003577:Cytochrome P-450 Enzyme System; C408874:CYP119 protein, Sulfolobus solfataricus; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases