Spectra and kinetic studies of the compound I derivative of cytochrome P450 119.
The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which was first prepared by reaction of the resting enzyme with peroxynitrite. The UV-vis spectrum of the Compound I species contained an asymmetric Soret band that could be resolved into overlapping transitions centered at approximately 367 and approximately 416 nm and a Q band with lambda(max) approximately 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxidized (alkene oxidation) and hydroxylated (C-H oxidation) products, as demonstrated by product studies and oxygen-18 labeling studies. The kinetics of oxidations by CYP119 Compound I were measured directly; the reactions included hydroxylations of benzyl alcohol, ethylbenzene, Tris buffer, lauric acid, and methyl laurate and epoxidations of styrene and 10-undecenoic acid. Apparent second-order rate constants, equal to the product of the equilibrium binding constant (K(bind)) and the first-order oxidation rate constant (k(ox)), were obtained for all of the substrates. The oxidations of lauric acid and methyl laurate displayed saturation kinetic behavior, which permitted the determination of both K(bind) and k(ox) for these substrates. The unactivated C-H positions of lauric acid reacted with a rate constant of k(ox) = 0.8 s(-1) at room temperature. The CYP119 Compound I derivative is more reactive than model Compound I species [iron(IV)-oxo porphyrin radical cations] and similar in reactivity to the Compound I derivative of the heme-thiolate enzyme chloroperoxidase. Kinetic isotope effects (kH/kD) for oxidations of benzyl alcohol and ethylbenzene were small, reflecting the increased reactivity of the Compound I derivative in comparison to models. Nonetheless, CYP119 Compound I apparently is much less reactive than the oxidizing species formed in the P450 cam reaction cycle. Studies of competition kinetics employing CYP119 activated by hydrogen peroxide indicated that the same oxidizing transient is formed in the photochemical reaction and in the hydrogen peroxide shunt reaction.
Sheng
Xin
X
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607, USA.
Horner
John H
JH
Newcomb
Martin
M
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D013053:Spectrophotometry; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology
D019843:Archaeal Proteins; D030421:Peroxynitrous Acid; D003577:Cytochrome P-450 Enzyme System; C408874:CYP119 protein, Sulfolobus solfataricus
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