Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Schünemann, Volker; Lendzian, Friedhelm; Jung, Christiane; Contzen, Jörg; Barra, Anne Laure; Sligar, Stephen G.; Trautwein, Alfred X.

doi:10.1074/jbc.m307884200

Cited by 96 publications

(126 citation statements)

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“…The curve shows a strong temperature dependence especially at low temperature, as reported for cytochrome c (50). reported for similar organic radicals that coupled to the ferryl heme iron in other peroxidase systems (51,52). …”

Section: Ghz Epr Spectroscopy-insupporting

confidence: 50%

“…Although the hyperfine structure observed in conventional X-band EPR can be rather similar for different types of organic radicals, it has been demonstrated that the principal values (g xx,yy,zz ) of the g-tensor, which are resolved for such radicals only in high-field EPR, can serve as a molecular fingerprint for identification of the type of radical (48,49,(51)(52)(53). In particular, tyrosine and tryptophan radicals are difficult to distinguish in X-band (9.4 GHz) EPR, whereas at 94 GHz EPR, they are clearly distinguished based on the much larger g xx and g yy values observed for tyrosyl radicals exhibiting large spin density on the oxygen, which has a large spin orbit coupling constant, compared with tryptophan radicals with spin density only on carbons and nitrogen, both being nuclei with only small spin orbit coupling constants (51)(52)(53). For a tryptophan radical, the field corresponding to 94 GHz EPR (3.3 tesla) does still not represent the so-called high-field limit, where the g-tensor shifts exceed the hyperfine splitting and all three g-tensor components are separated in the spectrum.…”

Section: Ghz Epr Spectroscopy-inmentioning

confidence: 99%

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A Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from Pleurotus eryngii

Pogni

Baratto

Teutloff³

et al. 2006

Journal of Biological Chemistry

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118

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Versatile peroxidases are heme enzymes that combine catalytic properties of lignin peroxidases and manganese peroxidases, being able to oxidize Mn 2؉ as well as phenolic and non-phenolic aromatic compounds in the absence of mediators. The catalytic process (initiated by hydrogen peroxide) is the same as in classical peroxidases, with the involvement of 2 oxidizing equivalents and the formation of the so-called Compound I. This latter state contains an oxoferryl center and an organic cation radical that can be located on either the porphyrin ring or a protein residue. In this study, a radical intermediate in the reaction of versatile peroxidase from the ligninolytic fungus Pleurotus eryngii with H 2 O 2 has been characterized by multifrequency (9.4 and 94 GHz) EPR and assigned to a tryptophan residue. Comparison of experimental data and density functional theory theoretical results strongly suggests the assignment to a tryptophan neutral radical, excluding the assignment to a tryptophan cation radical or a histidine radical. Based on the experimentally determined side chain orientation and comparison with a high resolution crystal structure, the tryptophan neutral radical can be assigned to Trp 164 as the site involved in long-range electron transfer for aromatic substrate oxidation.Different heme peroxidases are considered to be involved in the lignin biodegradation process, a key step for carbon recycling in terrestrial ecosystems. These are lignin peroxidase (LiP) 4 and manganese peroxidase (MnP), first described in Phanerochaete chrysosporium (1-3), and the versatile peroxidase (VP), more recently described in fungi from the genera Pleurotus (4 -6) and Bjerkandera (7,8). VP is characterized by combining catalytic properties of the other two ligninolytic peroxidases, MnP and LiP. This enzyme is able to oxidize Mn 2ϩ to Mn 3ϩ and also exhibits manganese-independent activity toward veratryl alcohol and p-dimethoxybenzene. Furthermore, it oxidizes hydroquinones and substituted phenols that are not efficiently oxidized by LiP or MnP in the absence of veratryl alcohol and Mn 2ϩ , respectively. VP is even able to degrade directly high redox potential dyes, which can be eventually oxidized by LiP only in the presence of veratryl alcohol (9, 10).Two genes encoding VP isoenzymes VPL and VPS1, expressed in liquid-and solid-state fermentation cultures, respectively, have been cloned from Pleurotus eryngii (11,12). The deduced amino acid sequences for both isoenzymes were used to build molecular models by homology modeling, taking advantage of sequence identity to P. chrysosporium LiP and MnP and Coprinopsis cinerea (synonym Coprinus cinereus) peroxidase (13). Very recently, the crystal structure of recombinant P. eryngii VP expressed in Escherichia coli and activated in vitro (14) has been determined at 1.33-Å resolution (Protein Data Bank code 2BOQ).Catalytically, VP would follow the classical heme peroxidase cycle, in which hydrogen peroxide is the final electron acceptor, acting as a 2-electron oxidizing substrate f...

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Section: Ghz Epr Spectroscopy-insupporting

confidence: 50%

Section: Ghz Epr Spectroscopy-inmentioning

confidence: 99%

A Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from Pleurotus eryngii

Pogni

Baratto

Teutloff³

et al. 2006

Journal of Biological Chemistry

Self Cite

118

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“…Spolitak et al recently showed similar behavior with substratefree ferric cytochrome P450CAM using mCPBA as an oxidant, and also demonstrated that peracetic acid could generate this intermediate that absorbed at ~406 nm [8]. They have tentatively assigned this species as a CcP Cpd ES-like intermediate in which a protein radical is formed by electron transfer to Cpd I. Freeze-Quench EPR and ENDOR experiments using wild-type and mutant forms of P450 CAM suggested that the position of the protein radical was either Tyr96 or Tyr75, because mutation of both residues completely eliminated the Tyr EPR radical signal, whereas the single mutation at Tyr96 did not [11]. In contrast to P450 CAM , P450 BM3 has a Trp residue directly adjacent to the heme in the active site, as shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Schünemann et al, using EPR and Mössbauer techniques and peracetic acid as an oxygen donor, could not detect any Cpd I with P450 CAM , but they did identify a species containing a tyrosine radical and a Fe IV =O [11,12]. This type of species, often called Compound ES (Cpd ES) [8], is well known in cytochrome c peroxidase (CcP).…”

Section: Introductionmentioning

confidence: 99%

“…In CcP, Cpd ES has been shown by a variety of techniques to contain an Fe IV =O prosthetic heme coupled to an active site Trp radical cation [13]. Recent reports on P450 [7,8] seemed to indicate that Cpd I was only observable when mCPBA was the oxidant, and that only Cpd ES was observable when peracetic acid was used [11,12,14]. Spolitak et al recently addressed this discrepancy using detailed stopped-flow spectroscopic analysis of the reaction between substrate-free ferric P450 CAM and either peracetic acid or mCPBA at several different pH values [8].…”

Section: Introductionmentioning

confidence: 99%

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Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors☆

Raner

Thompson

Haddy

et al. 2006

Journal of Inorganic Biochemistry

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Iron: Heme Proteins, Mono‐ & DioxygenasesBased in part on the article Iron: Heme Proteins, Mono‐ & Dioxygenases by Masanori Sono & John H. Dawson which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

Wong

Bell

2005

Encyclopedia of Inorganic Chemistry

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Heme (iron protoporphyrin IX) proteins and enzymes play crucial roles in all living organisms. Iron is very tightly bound to the porphyrin and does not dissociate under physiological conditions. Indeed the heme group is almost like a separate element altogether. It is redox‐active, and heme proteins such as cytochrome c have important electron‐transfer functions. The strong π donor nature of heme iron(II) also means that heme proteins are involved in the binding and activation of small molecules such as oxygen. The P450 heme monooxygenases use two electrons and two protons to activate O 2 , giving one molecule of water and a high‐valent iron‐oxo intermediate that is sufficiently reactive to attack aliphatic CH bonds in a diverse range of organic molecules. The heme dioxygenases, as the name implies, insert both oxygen atoms of the O 2 molecule into the substrate. The known heme dioxygenases are indoleamine ring cleavage enzymes but have not been studied in detail, and no crystal structure of the enzymes is available. It is known that the active form of the enzyme has Fe(II), and that both oxygen and superoxide could be the source of the two oxygen atoms inserted into substrates. The main focus of this article is on the cytochrome P450 monooxygenases. The P450 (CYP) superfamily of enzymes is found in virtually all organisms where they catalyze the oxidation of endogenous and exogenous organic compounds. These reactions are vital steps in the biosynthesis of steroids and other hormones, and the detoxification and oxidative removal of xenobiotics. The primary activity of P450 enzymes is CH bond oxidation, but other activities such as alkene epoxidation, CC bond cleavage, heteroatom oxidation, and even reductive reactions are known. These reactions and their mechanisms are surveyed. The major sections review and summarize the steps in the P450 catalytic cycle, the nature of the rate‐limiting step, the chemical properties and electronic structure of the heme in the intermediates, and the mechanism of CH bond oxidation. The unique activity of P450 enzymes does not arise solely from the heme group; the pivotal roles of the protein environment and dynamics are discussed. The mechanism of aliphatic CH bond oxidation is one of the great challenges in chemistry. The nature of the active intermediate in P450 catalysis and the mechanism of action remain controversial. The results of mechanistic and theoretical approaches are brought together and reviewed.

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Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Cited by 96 publications

References 75 publications

A Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from Pleurotus eryngii

A Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from Pleurotus eryngii

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors☆

Iron: Heme Proteins, Mono‐ & DioxygenasesBased in part on the article Iron: Heme Proteins, Mono‐ & Dioxygenases by Masanori Sono & John H. Dawson which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

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