Cytochrome P450 Compound I

Newcomb, Martin; Zhang, Rui; Chandrasena, R. Esala P.; Halgrimson, James A.; Horner, John H.; Makris, Thomas M.; Sligar, Stephen G.

doi:10.1021/ja060048y

Cited by 139 publications

(183 citation statements)

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“…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).…”

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confidence: 99%

“…For a model compound and horseradish peroxidase enzyme, photoejection of an electron from compound II derivatives, iron(IV)-oxo neutral porphyrin species, gave the known compound I species (12), and this method was extended to studies of a cytochrome P450 enzyme (9). In the P450 study, the putative compound II species from cytochrome P450 119 (CYP119) was formed by reaction of the resting enzyme with peroxynitrite (PN), a reaction analogous to those previously reported for other heme-containing enzymes (13) including another P450 enzyme, P450 BM3 (or CYP102) (14), and the heme-thiolate protein chloroperoxidase (CPO) (9,14,15). Recently, however, Green and coworkers (16) reported that reaction of CYP102 with PN gave a nitrosyl species instead of a compound II derivative.…”

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confidence: 99%

“…The interest arises mainly because P450-II is related to the transients or transition structures that are formed in the course of a carbon-hydrogen bond hydroxylation reaction catalyzed by a P450 enzyme. Unfortunately, the only evidence for and characterization of P450-II species are the UV-visible spectra of the products from reactions of CYP102 and CYP119 with PN (9,14) and the photochemistry of the thus produced CYP119 derivative (9). For most heme enzymes, the compound II species are thought to be conventional ferryl-oxo species containing iron-oxygen double bonds, but Green and coworkers (17,18) reported that the compound II derivative of CPO has a long iron-oxygen bond reflecting an Fe-OH moiety; i.e., a basic ferryl group that is protonated at physiological pH.…”

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confidence: 99%

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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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confidence: 99%

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confidence: 99%

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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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“…More recently, Newcomb and coworkers used x-ray absorption spectroscopy to identify the reaction intermediate observed upon interaction of PN with cytochrome P450 as a Cpd-II species (13). The photo-oxidation of this intermediate apparently gave rise to the buildup of a Cpd-I species (14,17,50,51), similar to what was reported for chloroperoxidase (16). Such species showed some competency for substrates oxidation reinforcing the first assignment of I435 as an oxoferryl intermediate.…”

Section: Nature Of the Nos Intermediate I435 Observed During The Actimentioning

confidence: 62%

“…1, red arrows) with the concomitant buildup of a reaction intermediate identified as an oxoferryl species. This intermediate was later assigned to a compound II species (Cpd-II) by Newcomb and colleagues when analyzing the interaction of PN with various cytochromes P450s (13)(14)(15)(16). This putative Cpd-II generated by the reaction of various cytochromes P450 with PN was photoreduced to an intermediate that was somewhat competent for catalysis (15,17).…”

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Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases

Lang

Maréchal

Couture

et al. 2016

Biophysical Journal

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The activation of the peroxynitrite anion (PN) by hemoproteins, which leads to its detoxification or, on the contrary to the enhancement of its cytotoxic activity, is a reaction of physiological importance that is still poorly understood. It has been known for some years that the reaction of hemoproteins, notably cytochrome P450, with PN leads to the buildup of an intermediate species with a Soret band at~435 nm (I435). The nature of this intermediate is, however, debated. On the one hand, I435 has been presented as a compound II species that can be photoactivated to compound I. A competing alternative involves the assignment of I435 to a ferric-nitrosyl species. Similar to cytochromes P450, the buildup of I435 occurs in nitric oxide synthases (NOSs) upon their reaction with excess PN. Interestingly, the NOS isoforms vary in their capacity to detoxify/activate PN, although they all show the buildup of I435. To better understand PN activation/detoxification by heme proteins, a definitive assignment of I435 is needed. Here we used a combination of fine kinetic analysis under specific conditions (pH, PN concentrations, and PN/NOSs ratios) to probe the formation of I435. These studies revealed that I435 is not formed upon homolytic cleavage of the O-O bond of PN, but instead arises from side reactions associated with excess PN. Characterization of I435 by resonance Raman spectroscopy allowed its identification as a ferric iron-nitrosyl complex. Our study indicates that the model used so far to depict PN interactions with hemo-thiolate proteins, i.e., leading to the formation and accumulation of compound II, needs to be reconsidered.

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Freeze‐Quench Kinetics

Vries

2005

Encyclopedia of Inorganic Chemistry

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Rapid‐mixing, rapid‐freezing techniques are employed for the study of enzyme or chemical catalytic mechanisms. The techniques aim to provide electronic and molecular structures of transient intermediates formed during the reaction by means of spectroscopic analyses of the rapidly frozen samples. Briefly, the mixing‐sampling procedure consists of the rapid mixing of two reactants, for example, enzyme and substrate, followed by rapid freezing of the mixture in a liquid cryo‐medium or on a cold plate to quench the reaction progress. The resulting frozen powder is subsequently sampled and made available for further low‐temperature spectroscopic analyses such as UV‐Vis spectroscopy, multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy, Electron Nuclear Double Resonance (ENDOR) spectroscopy, Electron Spin Echo Envelope Modulation (ESEEM), Magnetic Circular Dichroïsm (MCD) spectroscopy, Mössbauer spectroscopy, resonance Raman spectroscopy, or X‐ray Absorption Spectroscopy. By preparing a series of samples freeze‐quenched after various times, a full kinetic profile of the catalytic cycle of an enzyme or any other chemical reaction is obtained. The structures of intermediates are assigned in conjunction with (a selection of) the spectroscopic techniques listed above. This article details various aspects of continuous‐flow and stopped‐flow rapid‐mixing techniques, considering mixer design, freeze‐quenching methodology, and sampling. The scope and limitations to (bio)chemical research of the well‐established rapid freeze‐quench (RFQ) and the recently developed microsecond freeze‐hyperquenching (MHQ) technologies are specifically highlighted.

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Cytochrome P450 Compound I

Cited by 139 publications

References 33 publications

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

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

Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases

Freeze‐Quench Kinetics

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