Substrate Modulation of the Properties and Reactivity of the Oxy-Ferrous and Hydroperoxo-Ferric Intermediates of Cytochrome P450cam As Shown by Cryoreduction-EPR/ENDOR Spectroscopy

Davydov, Roman; Perera, Roshan; Jin, Shengxi; Yang, Tran Chin; Bryson, Thomas A.; Sono, Masanori; Dawson, John H.; Hoffman, Brian M.

doi:10.1021/ja045351i

Cited by 92 publications

(150 citation statements)

References 42 publications

(76 reference statements)

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“…For example, it has been shown that in cytochrome P450cam, camphor as substrate is hydroxylated by compound I, while methylenyl camphor can be hydroxylated both by P450 compound I and by hydroperoxo-ferri-P450. 6,7 In the case of HO we found that the hydroperoxo ferric-HO is the reactive state for heme hydroxylation, not compound I, 3,5 and this is confirmed by the recent demonstration that HO compound I is not reactive. 8…”

Section: Introductionsupporting

confidence: 77%

Distinct Reaction Pathways Followed upon Reduction of Oxy-Heme Oxygenase and Oxy-Myoglobin as Characterized by Mössbauer Spectroscopy

et al. 2007

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Activation of O 2 by heme-containing monooxygenases generally commences with the common initial steps of reduction to the ferrous heme and binding of O 2 followed by a one-electron reduction of the O 2 -bound heme. Subsequent steps that generate reactive oxygen intermediates diverge and reflect the effects of protein control on the reaction pathway. In this study, Mössbauer and EPR spectroscopies were used to characterize the electronic states and reaction pathways of reactive oxygen intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of oxyheme oxygenase (HO) and oxy-myoglobin (Mb). The results confirm that one-electron reduction of (Fe II -O 2 )HO is accompanied by protonation of the bound O 2 to generate a low-spin (Fe III -O 2 H − )HO that undergoes self hydroxylation to form the α-meso-hydroxyhemin-HO product. In contrast, oneelectron reduction of (Fe II -O 2 )Mb yields a low-spin (Fe III -O 2 2− )Mb. Protonation of this intermediate generates (Fe III -O 2 H − )Mb, which then decays to a ferryl complex, (Fe IV =O 2− )Mb, that exhibits magnetic properties characteristic of the compound II species generated in the reactions of peroxide with heme peroxidases and with Mb. Generation of reactive high-valent states with ferryl species via hydroperoxo intermediates is believed to be the key oxygen-activation steps involved in the catalytic cycles of P450-type monooxygenases. The Mössbauer data presented here provide direct spectroscopic evidence supporting the idea that ferric-hydroperoxo hemes are indeed the precursors of the reactive ferryl intermediates. The fact that a ferryl intermediate does not accumulate in HO underscores the determining role played by protein structure in controlling the reactivity of reaction intermediates.

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Section: Introductionsupporting

confidence: 77%

Distinct Reaction Pathways Followed upon Reduction of Oxy-Heme Oxygenase and Oxy-Myoglobin as Characterized by Mössbauer Spectroscopy

et al. 2007

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“…Reduction of the oxy complex yields a ferric peroxo species, which can be annealed at higher temperatures to allow for proton delivery, cleavage of the O-O bond, and P450-I formation. This technique has been coupled with electron nuclear double resonance spectroscopy and x-ray crystallography in hopes of obtaining electronic and structural characterizations of the intermediate (22)(23)(24)(25)(26)(27). These experiments provided clear evidence for cleavage of the dioxygen bond (i.e.…”

Section: Closing the Cycle: The Quest For Compound Imentioning

confidence: 99%

Reactive Intermediates in Cytochrome P450 Catalysis

Krest

Onderko

Yosca

et al. 2013

Journal of Biological Chemistry

173

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Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450 ST ). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis.A significant amount of research in the field of bioinorganic chemistry is focused on discerning the intimate details of enzyme catalysis. Critical to these efforts is the preparation of reactive intermediates that form the stations of the catalytic cycle of an enzyme. The study of these transient species is driven by the hope that insights gleaned from their electronic, structural, and kinetic characterizations will guide the design of next-generation catalysts or point the way to inhibitors that could serve as drugs for a variety of maladies. Although a thorough characterization of all the intermediates in a catalytic cycle is required for a detailed dissection of the catalytic mechanism, there are certain species that have special significance and, as such, are considered high-value targets for characterization. These species are generally the highly reactive intermediates that are ultimately responsible for the most important, difficult, or chemically interesting transformation in the catalytic mechanism.Recently, we reported the capture and characterization of one of the most highly sought intermediates in biological chemistry, P450 compound I (P450-I) 4 (1). This iron(IV)-oxo (or ferryl) radical species (7 in Fig. 1) had long been thought to be the principal intermediate in cytochrome P450 catalysis, but due to its highly reactive nature, it had eluded definitive characterization for over 40 years. The existence of P450-I was postulated based on the observation of a "shunt pathway" (Fig. 1) allowing the oxidation of substrates through the use of oxygen donors such as hydrogen peroxide and meta-chloroperbenzoic acid. These oxidants were known to generate high-valent iron-oxo species in heme peroxidases (2, 3), suggesting that a similar intermediate might be involved in P450 catalysis. However, 4 decades worth of searching for the elusive P450-I had led to questions about not only its competence as a hydroxylating agent but also its role in P450 catalysis (4 -6).Our investigations confirmed the existence and the reactive nature of the intermediate. P450-I is capable of hydroxylating unactivated C-H bonds with the remarkable rate constant of 1 ϫ 10 7 M Ϫ1 s Ϫ1 (1). Kinetic isotope effects support a mechanism in which P450-I abstracts hydrogen from substrate, forming an iron(IV)-hydroxide complex that rapidly recombines with substrate to yield hydroxylated product (7-9 in Fig. 1...

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“…Unlike other hemecontaining enzymes, P450s in nature are activated by a series of rapid processes that occur after substrate binding, reduction of iron from ϩ3 to ϩ2 oxidation state, reversible binding of oxygen, a second reduction, and two protonation reactions (1,3). High valent iron-oxo species in P450 enzymes are extremely shortlived and could not be observed under cryogenic reduction conditions that permitted detection of the immediate precursor to the oxidant(s), a hydroperoxy-iron(III) species (4,5). In fact, no P450 oxidant has been detected under natural conditions, although spectroscopic evidence exists for production of ironoxo species in low conversion under some unnatural conditions (6)(7)(8)(9), and the identities of the P450 oxidants remain in doubt (10,11).…”

mentioning

confidence: 99%

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative

Newcomb

Halgrimson

Horner

et al. 2008

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

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The cytochrome P450 enzyme CYP119, its compound II derivative, and its nitrosyl complex were studied by iron K-edge x-ray absorption spectroscopy. The compound II derivative was prepared by reaction of the resting enzyme with peroxynitrite and had a lifetime of Ϸ10 s at 23°C. The CYP119 nitrosyl complex was prepared by reaction of the enzyme with nitrogen monoxide gas or with a nitrosyl donor and was stable at 23°C for hours. Samples of CYP119 and its derivatives were studied by x-ray absorption spectroscopy at temperatures below 140 (K) at the Advanced Photon Source of Argonne National Laboratory. The x-ray absorption near-edge structure spectra displayed shifts in edge and pre-edge energies consistent with increasing effective positive charge on iron in the series native CYP119 < CYP119 nitrosyl complex < CYP119 compound II derivative. Extended x-ray absorption fine structure spectra were simulated with good fits for k ‫؍‬ 12 Å ؊1 for native CYP119 and k ‫؍‬ 13 Å ؊1 for both the nitrosyl complex and the compound II derivative. The important structural features for the compound II derivative were an iron-oxygen bond length of 1.82 Å and an iron-sulfur bond length of 2.24 Å, both of which indicate an iron-oxygen single bond in a ferryl-hydroxide, Fe IV OH, moiety.EXAFS ͉ ferryl-oxo ͉ XANES

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Substrate Modulation of the Properties and Reactivity of the Oxy-Ferrous and Hydroperoxo-Ferric Intermediates of Cytochrome P450cam As Shown by Cryoreduction-EPR/ENDOR Spectroscopy

Cited by 92 publications

References 42 publications

Distinct Reaction Pathways Followed upon Reduction of Oxy-Heme Oxygenase and Oxy-Myoglobin as Characterized by Mössbauer Spectroscopy

Distinct Reaction Pathways Followed upon Reduction of Oxy-Heme Oxygenase and Oxy-Myoglobin as Characterized by Mössbauer Spectroscopy

Reactive Intermediates in Cytochrome P450 Catalysis

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative

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