Detection and Kinetic Characterization of a Highly Reactive Heme–Thiolate Peroxygenase Compound I

Abstract

The extracellular heme-thiolate peroxygenase from Agrocybe aegerita (AaeAPO) has been shown to hydroxylate alkanes and numerous other substrates using hydrogen peroxide as the terminal oxidant. We describe the kinetics of formation and decomposition of AaeAPO compound I upon its reaction with mCPBA. The UV–vis spectral features of AaeAPO–I (361, 694 nm) are similar to those of chloroperoxidase–I and the recently–described cytochrome P450–I. The second–order rate constant for AaeAPO–I formation was 1.0 (±0.4) ×107 M−1s−1 at pH 5.0, 4 °C. The relatively slow decomposition rate, 1.4 (±0.03) s−1, allowed the measurement of its reactivity toward a panel of substrates. The observed rate constants, k2’, spanned five orders of magnitude and correlated linearly with bond dissociation enthalpies of strong C–H bond substrates with a log k2’ vs. BDE slope of ~ 0.4. However, the hydroxylation rate was insensitive to C–H BDE below 90 kcal/mol, similar to the behavior of the t-butoxy radical. The shape and slope of the Brønsted-Evans-Polanyi plot indicate a symmetrical transition state for the stronger C–H bonds and suggest entropy control of the rate in an early transition state for weaker C–H bonds. The AaeAPO–II FeIVO–H BDE was estimated to be ~ 103 kcal/mol. All results support the formation of a highly reactive AaeAPO oxoiron(IV) porphyrin radical cation intermediate that is the active oxygen species in these hydroxylation reactions.

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

“…An observed 2 H KIE for Ole-I decay at this H 2 O 2 concentration, which represents a lower limit for the unmasked value, is provided by a ratio of the fast RRTs, [ H k app / D k app = RRT 1 (H 39 -EA)/RRT 1 (D 39 -EA)], giving a KIE ≥ 8.1 ± 1.1. The large KIE, which has not been previously measured for a P450-I generated with a bound substrate, is in accord with unmasked KIEs for steady-state P450 hydroxylations (12), rapid mixing studies of P450-I (10), and similar thiolateligated heme iron−oxo species (41), and theoretical considerations (43). This confirms that the distinctive C−Cα cleavage catalyzed by OleT most likely commences in a very similar way to P450 monooxygenations.…”

“…The observation of a compound I species in OleT, optically indistinguishable from those that promote hydroxylations (10,41) (Table S1 and Fig. S1A).…”

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

“…Accordingly, we anticipated that these water-soluble nitroxyl radicals might be able to reduce APO-I readily but would not reduce APO-II efficiently. Furthermore, since cyclohexane carboxylic acid was found to be an excellent substrate for APO (29), the similar molecular topographies of 4-carboxy-TEMPO and 3-carboxy-PROXYL suggested that they would fit into the relatively small, conical active site of APO (25). We tested the reaction of 3-carboxy-PROXYL with the well-studied enzyme CPO in a single-turnover experiment.…”

“…We have recently shown that the APO from Agrocybe aegerita (AaeAPO) forms an oxoiron(IV) porphyrin radical cation (APO-I) that can be detected by UV-visible (UV-vis) spectroscopy (29). This intermediate was shown to be highly competent for the hydroxylation of even strong C−H bonds.…”

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

“…They constitute a distinct (super)family of fungal heme‐thiolate proteins that catalyze efficient and selective oxygen‐transfer from peroxides to diverse organic substrates including non‐activated compounds 11. UPOs are relatively stable and versatile biocatalysts, and combine the catalytic cycle of heme peroxidases with the “peroxide shunt” of P450s 12, 13. The first UPO was discovered in the wood‐dwelling agaric fungus Agrocybe aegerita ( Aae UPO); two similar enzymes were described in the mushrooms Coprinellus radians ( Cra UPO) and Marasmius rotula ( Mro UPO) 14, 15, 16.…”