Highly purified microsomal P-450: The oxyferro intermediate stabilized at low temperature

Bonfils, Claude; Debey, Pascale; Maurel, Patrick

doi:10.1016/0006-291x(79)91122-7

Cited by 55 publications

(31 citation statements)

References 9 publications

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“…The spectrum of the oxy complex shows a Soret band at 418 nm and a single broad maximum at 552 nm in the visible region. This spectrum looks very similar to the spectra of bacterial oxygenated cytochromes P450 (15,35,36). The rate of autoxidation of substratefree CYP3A4 in Nanodiscs is very high, k ϭ 20 s Ϫ1 at 5°C and ϳ140 s Ϫ1 at 29°C.…”

Section: Resultssupporting

confidence: 55%

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

Denisov

Grinkova

Baas

et al. 2006

Journal of Biological Chemistry

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The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of ϳ30 ms at 5°C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.Cytochrome P450 CYP3A4 is the most prevalent P450 monooxygenase in human liver and is responsible for the metabolism of almost half of xenobiotics encountered by man (1). This isozyme has a large and flexible active site and is able to bind and catalytically convert multiple substrates, often displaying homotropic and heterotropic cooperativity in substrate binding and product formation. As a central player in human drug metabolism, CYP3A4 is one of the most intensely studied P450s, either in isolated human liver microsomes (2-5), detergent-solubilized form (6), or purified soluble aggregates (7,8). Recently, two groups have reported the crystal structure of a truncated CYP3A4 (9, 10). Despite this structural information, precise chemical and biophysical characterization of the human P450s has lagged that of the monomeric, soluble P450 isozymes isolated from bacteria. In particular, the critical intermediates in the reaction cycle of human P450s have not been precisely documented, due in part to the inability to form a robust, monomeric, and soluble entity that is amenable to the rapid reaction and spectroscopic methodologies successfully applied to the bacterial P450s. This lacuna is significant, as CYP3A4 and other human P450s display complex aspects of substrate recognition and catalytic mechanism that are not present in the simpler P450s such as CYP101 from Pseudomonas.The current version of the catalytic reaction cycle of cytochrome P450 monooxygenases involves intermediate reduction states of heme iron and atmospheric dioxygen and represents the result of over 30 years of intensive research (11,12). Traditionally, the cyclic process begins with the ferric low spin species and a water molecule occupying the sixth coordination site of the heme. In many cases the complementary fit of the native substrate into the pocket displaces this water ligand allowing the system to assume a predominantly hig...

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Section: Resultssupporting

confidence: 55%

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

Denisov

Grinkova

Baas

et al. 2006

Journal of Biological Chemistry

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“…Further evidence for this conclusion comes from the DynaFit simulations (Table III). The accumulated intermediate has a difference spectrum that resembles that attributed to the FeO 2 2ϩ -substrate species in previous work (55,56,67). The appearance of the complex was dependent upon the presence of a substrate or the inhibitor quinidine (Figs.…”

Section: Discussionmentioning

confidence: 67%

Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Guengerich

Miller²,

Hanna³

et al. 2002

Journal of Biological Chemistry

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Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction (ϳ30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3-and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3-or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular noncompetitive deuterium isotope effects of 3.1-3.8 were measured for k cat and k cat /K m for both O-demethylation reactions, implicating at least partially rate-limiting C-H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe 2؉ O 2 ؊ protonation (plus O-O bond scission) and (ii) C-H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron-oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than k cat , and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.

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“…Our attempts over the past few years to circumvent this high intrinsic reactivity have involved a collaborative effort with Davydov, Hoffman, and colleagues [9][10][11] to utilize cryoradiolytic methods to allow the isolation of active P450 heme-oxygen intermediates at low-temperature. As the isolation and proton mediated transformation of P450 peroxo states has been the subject of recent reviews [44,45], we will only cover some of the salient points of this line of study with respect to a highly suggestive result in trying to capture the putative Compound I species. Oxyferrous P450 represents the last easily isolatable step in P450 catalysis [44][45][46], and although the stability changes markedly as a function of isozyme [44], substrate, and distal pocket mutation [45] it can easily be generated and subsequently stabilized in CYP101 via chemical reduction and subsequent oxygenation.…”

Section: Oxoferryl Intermediates In the P450 Dioxygen Driven Reactionmentioning

confidence: 99%

“…As the isolation and proton mediated transformation of P450 peroxo states has been the subject of recent reviews [44,45], we will only cover some of the salient points of this line of study with respect to a highly suggestive result in trying to capture the putative Compound I species. Oxyferrous P450 represents the last easily isolatable step in P450 catalysis [44][45][46], and although the stability changes markedly as a function of isozyme [44], substrate, and distal pocket mutation [45] it can easily be generated and subsequently stabilized in CYP101 via chemical reduction and subsequent oxygenation. As the system is now usually poised to accept a second electron from its redox partner (the 2Fe-2S containing putidaredoxin in the case of CYP101 [46]) in the normal reaction scheme, one can utilize reducing equivalents as generated by the radiolysis of glycerol water mixtures at low temperatures at 77 K to initiate the reaction cycle.…”

Section: Oxoferryl Intermediates In the P450 Dioxygen Driven Reactionmentioning

confidence: 99%