Abstract
Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as − 50 °C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77 K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step gave a 6:1 mixture of Compound II and ferric CPO, the photolysis step gave an approximate 1:1 mixture of active oxidant and ferric CPO, and the final mixture after reaction with excess styrene contained ferric CPO in 80% yield. In single turnover reactions at − 50 °C, styrene was oxidized to styrene oxide in high yield. Kinetic studies of styrene oxidation at − 50 °C displayed saturation kinetics with an equilibrium constant for formation of the complex of K bind = 3.8 × 10 4 M − 1 and an oxidation rate constant of k ox = 0.30 s − 1 . UV–Visible spectra of mixtures formed in the photo-oxidation sequence at ca. − 50 °C did not contain the signature Q-band absorbance at 690 nm ascribed to CPO Compound I prepared by chemical oxidation of the enzyme, indicating that different species were formed in the chemical oxidation and the photo-oxidation sequence.
Highlights
The cytochrome P450 (CYP or P450) enzymes are heme-containing enzymes with thiolate from protein cysteine as the fifth ligand to iron [1,2]
Heme enzyme Compound II intermediates are iron(IV)-oxo species, but we show an iron(IV) hydroxide structure in Scheme I because this type of structure was indicated by long Fe-O bonds in the extended X-ray absorption fine structure (EXAFS) spectra of both CPO Compound II [26] and Compound II from the P450 enzyme CYP119 [27]
horseradish peroxidase (HRP) Compound II photolysis gave the known Compound I species [14], and the expectation was that the same reaction sequence would be found for CPO
Summary
The cytochrome P450 (CYP or P450) enzymes are heme-containing enzymes with thiolate from protein cysteine as the fifth ligand to iron [1,2]. The predominant role of P450s in nature is to serve as oxidation catalysts, and these enzymes have been modeled by simple porphyrin-. The high-valent iron-oxo complexes formed in peroxidase and catalase enzymes are iron(IV)oxo porphyrin radical cations [7] termed Compounds I, and for many years similar intermediates have been assumed to be the active oxidants in P450s. An especially important peroxidase enzyme for modeling P450s has been chloroperoxidase (CPO) from Caldariomyces fumago [8], which, similar to P450s, contains a cysteine thiolate ligand to iron instead of the histidine nitrogen ligand in typical peroxidases. Upon reaction with hydrogen peroxide or peroxy acids, CPO forms a Compound I transient [9] that is relatively stable (t1/2 ≈ 1 s at 22 ° C [10]), and CPO Compound I has served as a surrogate for P450 Compounds I in various spectroscopic and kinetic studies. The UV-visible spectrum of CPO Compound I observed in acidic solutions at room temperature [9,11] resembles that of Compound I from catalase [7, 12] with a Soret band absorbance at 367 nm and a Q-band absorbance at 690 nm, and it is commonly assumed that the UV-visible spectra of P450 Compounds I will resemble that of CPO Compound I [13]
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