Theoretical Investigation of the Proton Assisted Pathway to Formation of Cytochrome P450 Compound I

Harris, Danni L.; Loew, Gilda H.

doi:10.1021/ja981059x

Cited by 201 publications

(317 citation statements)

References 38 publications

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“…In fact, as we made clear in our earlier rR study of the dioxygen adducts of CYP17A1 (27), the lowered ν(Fe-16 O) frequency of the peroxo-form of the 17-OH PREG sample (546 cm −1 ), relative to the peroxo-form of the PREGbound sample (554 cm −1 ), suggests that an H-bonding interaction occurs between the hydroxyl group of the substrate and the proximal oxygen atom of the Fe-O p -O t peroxo-fragment [i.e., the H-bonding seen in the dioxygen adduct persists in the peroxo-species, as we had suggested in our earlier work (27)]. This finding is important, because such a specifically directed H-bonding interaction to the O p of the peroxo-fragment is expected to facilitate its involvement in the lyase phase of catalysis (30,31). This result suggests the 17-OH PREG-bound peroxo-intermediate is poised for attack upon the susceptible electrophilic C 20 carbon of the bound substrate.…”

Section: Resultsmentioning

confidence: 62%

“…1), only 17-OH PREG was properly positioned to donate an H-bond to the proximal oxygen of the Fe-O-O fragment, an interaction that was suggested likely to persist in the subsequent peroxo-intermediate, a suggestion that is now shown to be valid by the data presented herein (vide infra). This finding was important, showing that the particular substrate efficiently processed in the lyase step of metabolism is also the only one that adopts an orientation that effectively intercepts the fleeting peroxo-intermediate and facilitates its attack on the juxtaposed electrophilic C 20 atom (30,31). Although the rR data obtained for the dioxygen intermediate of 17-OH PREG-bound CYP17A1 provided evidence suggesting the existence of a "poised" Fe-O p -O t peroxo-fragment, confirmation of the proposed lyase pathway demands the trapping and structural characterization of this complex and the following crucial intermediate shown in the center of Fig.…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Unveiling the crucial intermediates in androgen production

Mak

Gregory

Denisov

et al. 2015

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

189

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Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C 17 -C 20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C 20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.T he excessive production of androgen, which effectively fuels the progression of cancer, especially prostate cancer, was first treated by surgical methods (1), whereas more modern approaches are focused on the discovery and development of pharmaceuticals that can selectively inhibit androgen synthesis (2, 3). A member of the cytochrome P450 superfamily (4, 5), cytochrome P450 17A1 (CYP17A1), occupies a central role in the biosynthesis of steroid hormones in humans. As was first reported by Nakajin and Hall (6) and Nakajin et al. (7) for CYP17 from pig testis, this enzyme catalyzes two fundamentally different types of chemical transformations (5-11), with the first being the efficient hydroxylation of both of its primary substrates, pregnenolone (PREG) and progesterone (PROG), to 17-hydroxypregnenolone (17-OH) PREG and 17-hydroxypregnenolone, respectively (Fig. 1). Importantly, 17-OH PREG is further processed in a second step in which CYP17A1 now catalyzes, not another hydroxylation reaction, but a complex 17,20 carbon-carbon (C-C) bond cleavage (lyase) reaction. This step converts 17-OH PREG to dehydroepiandrosterone (DHEA), a process that represents a critical branch point in human steroidogenesis by providing the essential precursor to androgens and various corticosteroids (5-9). Although a similar C-C bond cleavage of 17-OH PROG is also mediated by CYP17A1, it is of less importance in humans because its efficiency is only about 2% of the efficiency of the physiologically important lyase reaction involving 17-OH PREG (10). Recognizing the intensive efforts currently underway to design and test substances that selectively inhibit CYP17A1 (2, 3), there is a pressing need to enhance our understanding of the relevant reaction mechanisms, a task that typically entails identification of key reaction intermediates. Dir...

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Section: Resultsmentioning

confidence: 62%

Section: Significancementioning

confidence: 99%

Unveiling the crucial intermediates in androgen production

Mak

Gregory

Denisov

et al. 2015

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

189

View full text Add to dashboard Cite

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“…Experimental (15) and theoretical (8) studies have proved the need for reduction of 3 by a second electron before double protonation of the distal oxygen atom, in which crystallography revealed waters play a major role. After distal oxygen protonation, OOO bond cleavage and water formation, as observed in small theoretical models (9), results in a highly exothermic (by Ϸ80 kcal͞mol) process leading to the oxyferryl species. This proposed oxidant is well known as compound I (4 in Fig.…”

mentioning

confidence: 83%

Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450

Guallar

Baik

Lippard

et al. 2003

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

144

185

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We elucidate the hydroxylation of camphor by cytochrome P450 with the use of density functional and mixed quantum mechanics͞ molecular mechanics methods. Our results reveal that the enzyme catalyzes the hydrogen-atom abstraction step with a remarkably low free-energy barrier. This result provides a satisfactory explanation for the experimental failure to trap the proposed catalytically competent high-valent heme Fe(IV) oxo (oxyferryl) species responsible for this hydroxylation chemistry. The primary and previously unappreciated contribution to stabilization of the transition state is the interaction of positively charged residues in the active-site cavity with carboxylate groups on the heme periphery. A similar stabilization found in dioxygen binding to hemerythrin, albeit with reversed polarity, suggests that this mechanism for controlling the relative energetics of redox-active intermediates and transition states in metalloproteins may be widespread in nature.T he family of cytochrome P450 monooxygenases is ubiquitous in human biology, playing a key role in the metabolism of pharmaceutical agents and other ingested exogenous compounds (1). These enzymes insert an oxygen atom from O 2 into a wide variety of substrates, with substrate specificity determined by the nature of the protein active-site cavity. All members of the family are believed to share a common mechanism for such hydroxylation (2). In the catalytic cycle, dioxygen bound to an iron porphyrin undergoes a series of transformations to produce an intermediate capable of hydroxylating a COH bond of the substrate. The currently accepted steps in this cycle are depicted in Fig. 1. We focus here on the bacterial isozyme cytochrome P450cam, for which camphor is the substrate, to exploit the wealth of experimental data measured for this system.Because of its prominent role in biology and medicine, an exceptionally large amount of experimental and theoretical work has been invested in understanding the various steps of the cytochrome P450 reaction cycle (1-16). A complete atomic-level picture of the key steps, particularly the hydroxylation reaction, has yet to be produced, however. Such knowledge is essential in building quantitative models of P450-based contributions to drug metabolism and toxicity. This deficiency is due in part to the difficulties in carrying out incisive experimental and theoretical studies. Experimental investigation of the catalytically competent species in the hydroxylation reaction is hindered by the fact that the presence of substrate is necessary to initiate the reaction cycle. This situation can be contrasted with that in soluble methane monooxygenase (MMO). Formation of the key catalytic intermediate in the reaction cycle of the MMO hydroxylase can be initiated by dioxygen and then trapped in the absence of substrate. Such a result has yet to be duplicated in P450, as is discussed in more detail below. From a theoretical point of view, the P450 core chemistry is larger and more dispersed in the active site than that of MMO, ren...

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“…Py5 ) 2,6-bis(methoxy(di(2-pyridyl))methyl)pyridine; pz ) pyrazole; SCE ) saturated calomel electrode; SOR ) superoxide reductase; TACN ) 1,4,7-triazacyclononane; TACNPy2 ) 1-di(2-pyridyl)methyl-4,7-dimethyl-1,4,7-triazacyclononane;tpen )N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,2-diaminoethane; TPP ) meso-tetraphenylporphyrin dianion; Tp 3,5-iPr2 ) hydrotris(3,5-diisopropylpyrazolyl)borate; trispicMeen ) [33][34][35][36][37] Treatment of these low-spin Fe-OOH complexes with base converts them into new high-spin iron(III) species, 20,29-32, 38 and mixed isotope resonance Raman experiments strongly implicate a side-on bound peroxo ligand. 38 The only other established (η 2 -peroxo)iron complexes prior to these studies are those of Fe III (EDTA) 39 and Fe III (porphyrin).…”

Section: -Carboxylic Acid N-[2-(4′-imidazolyl)ethyl]amide;mentioning

confidence: 99%

“…5b If such a species were to be involved in the enzyme mechanism, the lack of oxidative reactivity of corresponding biomimetic complexes suggests that such a species may require subsequent activation, perhaps by protonation, to effect substrate oxidation, as also proposed for heme enzymes. 33,34 Indeed Chen et al have found the first iron catalysts for the cis-dihydroxylation of olefins and proposed the participation of Fe III -η 2 -OOH species in these reactions. 75,76 Another example of an iron-peroxo intermediate observed in a non-heme enzyme is that associated with superoxide reductase (SOR), the enzyme responsible for converting superoxide to H 2 O 2 in anaerobes.…”

Section: Summary and Perspectivementioning

confidence: 99%

End-On and Side-On Peroxo Derivatives of Non-Heme Iron Complexes with Pentadentate Ligands: Models for Putative Intermediates in Biological Iron/Dioxygen Chemistry

et al. 2003

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Mononuclear iron(III) species with end-on and side-on peroxide have been proposed or identified in the catalytic cycles of the antitumor drug bleomycin and a variety of enzymes, such as cytochrome P450 and Rieske dioxygenases. Only recently have biomimetic analogues of such reactive species been generated and characterized at low temperatures. We report the synthesis and characterization of a series of iron(II) complexes with pentadentate N5 ligands that react with H 2 O 2 to generate transient low-spin Fe III −OOH intermediates. These intermediates have low-spin iron(III) centers exhibiting hydroperoxo-to-iron(III) charge-transfer bands in the 500−600-nm region. Their resonance Raman frequencies, ν O-O , near 800 cm -1 are significantly lower than those observed for high-spin counterparts. The hydroperoxo-to-iron(III) charge-transfer transition blue-shifts and the ν O-O of the Fe−OOH unit decreases as the N5 ligand becomes more electron donating. Thus, increasing electron density at the low-spin Mononuclear iron(III) peroxide species are implicated as intermediates in the mechanisms of oxygen activating biomolecules such as cytochrome P450, 1 heme oxygenase, 2 the antitumor drug bleomycin, 3 and Rieske dioxygenases, 4,5 as well as superoxide reductases from anaerobic bacteria. [6][7][8][9] Experimental evidence for some of these intermediates has

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Theoretical Investigation of the Proton Assisted Pathway to Formation of Cytochrome P450 Compound I

Cited by 201 publications

References 38 publications

Unveiling the crucial intermediates in androgen production

Unveiling the crucial intermediates in androgen production

Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450

End-On and Side-On Peroxo Derivatives of Non-Heme Iron Complexes with Pentadentate Ligands: Models for Putative Intermediates in Biological Iron/Dioxygen Chemistry

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