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A highly reactive p450 model compound I.

ABSTRACT: The detection and kinetic characterization of a cytochrome P450 model compound I, [OFe(IV)-4-TMPyP](+) (1), in aqueous solution shows extraordinary reaction rates for C-H hydroxylations. Stopped-flow spectrophotometric monitoring of the oxidation of Fe(III)-4-TMPyP with mCPBA revealed the intermediate 1, which displays a weak, blue-shifted Soret band at 402 nm and an absorbance at 673 nm, typical of a porphyrin pi-radical cation. This intermediate was subsequently transformed into the well-characterized OFe(IV)-4-TMPyP. Global analysis afforded a second-order rate constant k(1) = (1.59 +/- 0.06) x 10(7) M(-1) s(-1) for the formation of 1 followed by a first-order decay with k(2) = 8.8 +/- 0.1 s(-1). (1)H and (13)C NMR determined 9-xanthydrol to be the major product (approximately 90% yield) of xanthene oxidation by 1. Electrospray ionization mass spectrometry carried out in 47.5% (18)OH(2) indicated 21% (18)O incorporation, consistent with an oxygen-rebound reaction scenario. Xanthene/xanthene-d(2) revealed a modest kinetic isotope effect, k(H)/k(D) = 2.1. Xanthene hydroxylation by 1 occurred with a very large second-order rate constant k(3) = (3.6 +/- 0.3) x 10(6) M(-1) s(-1). Similar reactions of fluorene-4-carboxylic acid and 4-isopropyl- and 4-ethylbenzoic acid also gave high rates for C-H hydroxylation that correlated well with the scissile C-H bond energy, indicating a homolytic hydrogen abstraction transition state. Mapping the observed rate constants for C-H bond cleavage onto the Brønsted-Evans-Polanyi relationship for similar substrates determined the H-OFe(IV)-4-TMPyP bond dissociation energy to be approximately 100 kcal/mol. The high kinetic reactivity observed for 1 is suggested to result from a high porphyrin redox potential and spin-state-crossing phenomena. More generally, subtle charge modulation at the active site may result in high reactivity of a cytochrome P450 compound I.

SUBMITTER: Bell SR

PROVIDER: S-EPMC2760262 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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A highly reactive p450 model compound I.

Bell Seth R SR Groves John T JT

Journal of the American Chemical Society 20090701 28

The detection and kinetic characterization of a cytochrome P450 model compound I, [OFe(IV)-4-TMPyP](+) (1), in aqueous solution shows extraordinary reaction rates for C-H hydroxylations. Stopped-flow spectrophotometric monitoring of the oxidation of Fe(III)-4-TMPyP with mCPBA revealed the intermediate 1, which displays a weak, blue-shifted Soret band at 402 nm and an absorbance at 673 nm, typical of a porphyrin pi-radical cation. This intermediate was subsequently transformed into the well-chara ...[more]

PMID: 19552441

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Project description: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.

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A highly reactive p450 model compound I.

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A highly reactive p450 model compound I.

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