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

Wang, Xiaoshi; Peter, Sebastian C.; Kinne, Matthias; Hofrichter, Martin; Groves, John T.

doi:10.1021/ja3049223

Cited by 120 publications

(135 citation statements)

References 37 publications

Supporting

Mentioning

122

Contrasting

Order By: Relevance

“…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.…”

Section: Significancesupporting

confidence: 77%

See 1 more Smart Citation

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

Grant

Mitchell

Makris

2016

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

OleT is a cytochrome P450 that catalyzes the hydrogen peroxidedependent metabolism of C n chain-length fatty acids to synthesize C n-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe 4+ −oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe 4+ −OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT.C ytochrome P450 (CYP) enzymes catalyze an extraordinary breadth of physiologically important oxidations for xenobiotic detoxification and specialized biosynthetic pathways (1, 2). The metabolic diversity of CYP enzymes originates from a sophisticated interplay of substrate molecular recognition with precise tuning of metal oxygen species formed at the enzyme active site. CYPs use a thiolate-ligated heme iron cofactor to activate molecular oxygen and produce short-lived ferric superoxo (3-5), ferric (hydro)peroxo (6-8), and ferryl (9, 10) intermediates. Coordinated efforts over several decades have resulted in isolation of each intermediate, including recent characterization of the principal oxidant thought to be responsible for the vast majority of P450 oxidations, the Fe 4+ −oxo pi−cation radical species commonly referred to as compound I (or P450-I) (10).The archetypal P450 hydroxylation involves C−H bond abstraction by P450-I and ensuing rapid oxygen insertion through a recombination process termed "oxygen rebound" (11, 12). The hydrogen abstraction/rebound mechanism describes the strategy used for most P450 transformations, and is thought to describe catalysis by numerous metal-dependent oxygenases and synthetic bio-inspired metal−oxo complexes (e.g., refs. 13-17). Despite the ubiquity of this mechanism, in some metalloenzymes, a metal−oxo species is formed that is not ultimately destined for incorporation into a substrate. Some characterized examples include the nonheme dinuclear iron ribonucleotide reductase (RNR R2) (18,19) and mononuclear iron halogenase SyrB2 (20-22). The mechanisms for RNR and SyrB2 highlight the importance of quaternary structural elements, an extensive 35-Å proton-coupled electron transfer pathway in RNR (23), and extremely subtle (subangstrom) substrate positional tuning (SyrB2) (21,22), to enable efficient circumvention from the monooxygenation reaction coordinate.P450-I has also been link...

show abstract

Section: Significancesupporting

confidence: 77%

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

Section: Significancementioning

confidence: 91%

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

Grant

Mitchell

Makris

2016

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Significancementioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Wang

Ullrich

Hofrichter

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapidmixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pHjump spectrophotometry revealed a basic pK a of 10.0 for the Fe IV −O−H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C−H and phenolic substrates, with second-order rate constants for C−H and O−H bond scission in the range of 10-10 7 M −1 ·s −1 . Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C−H and O−H bond scission.

show abstract

“…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.…”

Section: Introductionmentioning

confidence: 99%

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

et al. 2017

Self Cite

View full text Add to dashboard Cite

Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

show abstract

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

Cited by 120 publications

References 37 publications

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

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

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

Contact Info

Product

Resources

About