title: Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol.
abstract: Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially nonexistent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono- and dideuterio isotopomers. Pseudo-first-order rate constants obtained at -50 degrees C with concentrations of substrates between 1.0 and 6.0 mM displayed saturation kinetics that gave binding constants for the substrate in the Compound I species (K(bind)) and first-order rate constants for the oxidation reactions (k(ox)). Representative results are K(bind) = 214 M(-1) and k(ox) = 0.48 s(-1) for oxidation of benzyl alcohol. For the dideuterated substrate C(6)H(5)CD(2)OH, kinetics were studied between -50 and -25 degrees C, and a van't Hoff plot for complexation and an Arrhenius plot for the oxidation reaction were constructed. The H/D kinetic isotope effects (KIEs) at -50 degrees C were resolved into a large primary KIE (P = 11.9) and a small, inverse secondary KIE (S = 0.96). Comparison of values extrapolated to 22 degrees C of both the rate constant for oxidation of C(6)H(5)CD(2)OH and the KIE for the nondeuterated and dideuterated substrates to values obtained previously in laser flash photolysis experiments suggested that tunneling could be a significant component of the total rate constant at -50 degrees C.
authors: Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607, USA.
authors: Qin
authors: Q
authors: Wang
authors: Xin
authors: X
authors: Sheng
authors: John H
authors: JH
authors: Horner
authors: Martin
authors: M
authors: Newcomb
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D019905:Benzyl Alcohol / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry; D004789:Enzyme Activation; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D008665:Metalloporphyrins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010782:Photolysis; D013057:Spectrum Analysis; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D007554:Isotopes; D008665:Metalloporphyrins; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; C408874:CYP119 protein, Sulfolobus solfataricus; D019905:Benzyl Alcohol