Reactive Intermediates in Cytochrome P450 Catalysis

Abstract

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...

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

“…5), the signal-to-noise ratio of 18 O-incorporated acetate was three times greater than when (8,9). For clarity throughout the text, compound I is referred to interchangeably with FeO 3ϩ , and ferric peroxide is referred to interchangeably with FeO 2 Ϫ .…”

“…8 and 9). Ideally, the compound I form of P450 17A1 could be prepared using the approaches that Green and co-workers (8,55,64,77) have used with two bacterial P450s (64), and the reaction could be investigated directly. Nevertheless, in considering all of the literature in this field and that presented here in this article, the iodosylbenzene and H 2 O 2 results (Fig.…”

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

“…The widely distributed heme-thiolate monooxygenases, such as cytochrome P450 (CYP), serve similar roles in catalyzing C−H hydroxylation reaction. CYP enzymes also participate in the primary pathways for oxidative steroid and prostaglandin biosynthesis as well as phase I drug metabolism (8)(9)(10)(11)(12). For example, 20 isoforms of CYP with significant physiological functions exist in Mycobacterium tuberculosis, making them potential drug targets (13).…”

Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases

“…In this environment, the Fe III NO complex is not stable and decays back to the ferric state within seconds, suggesting that I435 disappearance may be linked to other secondary reactions as well. How photoreduction of such a complex mixture may trap a Cpd-I state at relatively high yield is not clear (52,55). Beyond the dispute between the Newcomb's and Green's groups concerning the spectroscopic fingerprints of Cpd-I and Cpd-II complexes, the major outcome of our work concerns the reaction of PN with hemoproteins and the link between PN activation and I435 intermediate formation.…”

“…In addition, using Mossbauer spectroscopy, they suggested that the initial I435 intermediate would not be a Cpd-II but rather an iron-nitrosyl species. However, the differences in the Mossbauer parameters for all these compounds remain minor and in the absence of additional spectroscopic characterization of the I435 intermediate, the dispute might go on (52,55).…”