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Haem-oxygen reactive intermediates: catalysis by the two-step T.M. Makris*, I.G. Denisov† and S.G. Sligar*†‡1 *The Center for Biophysics and Computational Biology, University of Illinois Urbana-Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, U.S.A., †Departments of Biochemistry and Chemistry, University of Illinois Urbana-Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, U.S.A., and ‡College of Medicine, University of Illinois Urbana-Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, U.S.A.
Abstract The catalytic schemes of a variety of haem enzymes, including the P450 mono-oxygenases, consist of a number of common reactive haem-oxygen adducts. The characterization of these intermediates by optical and EPR spectroscopies has reinforced the similarity of these intermediate states in a number of haem enzyme systems. Furthermore, the reactivity of these states in P450 and horseradish peroxidase, in which multiple potent oxidants are formed, provides a paradigm for many other haem enzymes.
Introduction The cytochrome P450 mono-oxygenases, thiolate-ligated haem enzymes which are ubiquitous in Nature, serve as a critical component in the oxidative transformation of a wide range of chemical substrates. These enzymes act both in physiological degradative pathways and in the metabolism of xenobiotics [1]. The oxygenation of a broad range of substrates, particularly by the mammalian P450s, is a feature indicative of both an active site that can accommodate a wide variety of chemical moieties, and the production of potent oxidants, electrophilic and nucleophilic in nature, which serve as effective and often indiscriminant catalysts. In the P450 reaction scheme, at least three potential oxidants are formed. These are designated the unprotonated ferric-peroxoanion, the ferric-hydroperoxo species and the high-valency ferryl-oxo π -cation radical or Compound I (Scheme 1). As these intermediates are short-lived in nature, their isolation and characterization has, until recently, proven to be an elusive task. These dioxygen intermediates are critical components of not only the P450 reaction scheme, but also of those of a wide variety of haem and non-haem metalloproteins, including oxidases [2], peroxidases [3,4], haem-oxygenases [5,6] and non-haem di-irons [7]. Likewise, the oxo-ferryl intermediate, or Compound I, serves as an intermediate common to P450 [8], catalase [9] and peroxidase [10] chemistries. Understanding active P450 haem-oxygen adducts in structural, spectroscopic and chemical detail is paramount not only in regard to the P450 mono-oxygenation reaction. Thus the P450 system can serve as a hub of bioinorganic catalysis.
Spectroscopic characterization of active haem-oxygen species Recently, the application of cryoradiolytic techniques has served as an essential tool in the isolation of the ferric-peroxo Key words: dioxygen activation, P450 mechanism. Abbreviation used: HRP, horseradish peroxidase. 1 To whom correspondence should be addressed at the Department of Biochemistry, University of Illinois Urbana-Champaign (e-mail
[email protected]).
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and ferric-hydroperoxo haem complexes of P450 [11–15]. The low-temperature exposure of the stable oxy-ferrous state to ionizing radiation, in the form of either β-radiation by 32 P or γ -radiation by 60 Co, effectively reduces the complex, delivering the second electron necessary for P450 catalysis. This two-electron-mediated reduced dioxygen species has been characterized by EPR as a low-spin (S = 12 ) ferricperoxo complex [11–13], with the ligand bound in an end-on (η1) fashion. This is consistent with comparable spectra from numerous systems, including radiolytic studies on other haem enzymes [6,16–18], and synthetically prepared alkyl- [19] and hydroperoxo model complexes [20]. Application of 1 H electron nuclear double resonance (ENDOR) spectroscopy, in conjunction with active-site mutants with altered proton delivery to the peroxo complex, has allowed a clear spectroscopic delineation between the unprotonated hydrogen-bonded complex and the protonated ferric-hydroperoxo complex, with a diagnostic broadening of the g-spread upon protonation of the distal oxygen [12]. The side-on (η2) bridged peroxo as seen in some model ferrihaem systems, differentiable by a high-spin (g ≈ 4) axial signal [21], is not observed in any of these cases, and may reflect active-site stabilization of the end-on conformation due to hydrogen-bonding interactions. The radiolytic methodologies have also enabled optical characterization of the peroxy complex, defining a 23 nm red shift of the Soret peak (to 440 nm) relative to the ferric protein [14,15]. These results are in remarkable agreement with spectra seen in pulse radiolytic studies on deuterohaem-substituted P450 [22] and with theoretical calculations [23], regarding both the diagnostic shape and position of the Soret band. On warming the cryoradiolytically prepared ferric-peroxo species to temperatures enabling molecular motion, and thus the enzymic reaction to proceed, one is able to spectroscopically ‘step through’ the P450 reaction scheme. At temperatures approaching the glass-transition temperature (around 190 K), one observes decay of the peroxo complex and formation of the hydroxylated product bound to the ferrihaem enzyme [13]. The functional cycling of the
Enzyme Mechanism – A Structural Perspective
Scheme 1 Reactive haem-oxygen intermediates in the P450 mono-oxygenation reaction
P450 mono-oxygenase is confirmed by gas chromatographic detection of the regio- and stereospecific alkane oxidation product. While neither the optical nor EPR studies have enabled direct detection of the Compound I state, detailed ENDOR analysis of exchangeable protons throughout the annealing profile strongly indicates the involvement of a highvalency intermediate [13]. The experiments outlined above, however, also suggest another role of the protonated peroxo complex in mediating between effective hydroxylation activity and unproductive hydrogen peroxide formation, serving as the effective branchpoint between these two pathways. The hydroperoxo ligand is thus subject to at least three fates: effective protonation and subsequent heterolytic cleavage to form Compound I, uncoupling to H2 O2 through ligand dissociation or an analogous mechanism, or direct reaction with ‘activated’ substrates, to be discussed below. The use of radiolytic reduction, in the form of longwavelength X-rays in crystallographic studies, has similarly elucidated the structural details of haem-enzyme reaction intermediates [24]. Irradiation and subsequent annealing of the P450 oxy-ferrous complex has revealed detailed facets regarding the participation of the enzyme active site in the evolution of active haem-oxygen complexes. For example, the radiolytic studies of Schlichting and colleagues [24] provided mechanistic details regarding the conserved acid– alcohol amino acid pair in the P450 distal pocket and subsequent phenotypes upon mutation, resulting in either complete uncoupling of the enzyme from H2 O2 [25,26], or a drastic decrease in the rate of electron consumption, respectively [27,28]. The position of both residues, whose localization is controlled by a conformational isomerization of the peptide backbone upon dioxygen binding, apparently stabilizes water molecules responsible for proton donation to the distal oxygen atom of the peroxo complex. The involvement of solvent in protonation reactions, such as the
comparable similarity in the intermediates themselves, serves as a common paradigm in haem-enzyme catalysis. Further annealing of the X-ray-reduced P450 oxy-ferrous complex reveals difference density maps in which a negative density is localized at the distal oxygen of the non-irradiated precursor and an expected shortening of the iron–oxygen distance from ˚ is seen, clearly indicating that dioxygen cleavage 1.80 to 1.65 A has occurred [24]. As with the optical and EPR annealing series, it is this intermediate that is responsible for substrate hydroxylation, demonstrated in the crystallographic studies as the formation of positive electron density on substrate with the correct enzymic stereospecificity [24]. Recent spectroscopic data have allowed definitive assignment of the optical signature of the P450 Compound I intermediate [29]. As with other haem enzyme and model systems, mixing of the protein in the ferric restingstate with peroxyacid results in heterolytic cleavage of the peroxide adduct to yield the high-valent iron-oxo species. Unfortunately, the instability of Compound I in most P450 systems has often precluded clear kinetic and spectral resolution, in comparison with analogous studies on other haem enzymes such as chloroperoxidase [30] and horseradish peroxidase (HRP) [31]. The use of a thermostable P450 enzyme from the archaeon Sulpholobus solfataricus, which stabilizes the intermediate at lower temperatures compared with its mesophilic counterpart, has allowed the direct observation and characterization of this intermediate [29]. The intermediate displays characteristics, with absorption maxima at 370, 610 and 690 nm, almost identical to those of the well-characterized Compound I in the thiolateligated chloroperoxidase [30] and transient P450 spectra [32]. The optical spectrum also shows remarkable similarity to spectra calculated by density functional theory (DFT) methods for a methylmercaptan-ligated model complex for the intermediate, demonstrating similar shifts from the C 2003
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spectrum of the ferric enzyme, particularly in the near-UV region [33]. Furthermore, titration of the P450 ferric resting state with the peroxyacid clearly demonstrates that only at stoichiometric ratios and below can the issue of protein degradation be avoided, which is necessary to allow clear assignment of both the enzymic intermediate, and its kinetics.
Involvement of the peroxo-ferric intermediate in P450-catalysed reactions The role of the peroxo-ferric intermediates in the direct mono-oxygenation of substrates, thus bypassing the Compound I intermediate, has been implicated in a number of P450 reactions. For example, P450 reactions which invoke the utilization of a nucleophilic reaction intermediate, such as nucleophilic attack of a carbonyl group in both the deformylation [34] and aromatization [35] reactions, implicate the peroxo-ferric intermediates as effective catalysts. The idea is not unique to P450, as both the reactions of NO synthases [36] and haem oxygenases [37,38] have relied on similar models. In the case of the P450 deformylation reaction, Coon and colleagues [34] have demonstrated the enhancement of this activity in cytochrome P450 2B4 upon mutation of the conserved threonine, known in P450s to impede the formation of the Compound I oxidant. Furthermore, this activity is often accompanied by the formation of an alkyl haem adduct, resulting from haemolytic cleavage of a peroxyhemiacetal formed during the reaction pathway [39]. Controversially, the peroxo-ferric intermediates have also been implicated in electrophilic reactions catalysed by P450. A mutation in which the conserved threonine is changed to an alanine still supports the epoxidation of alkenes, despite large impairments of this mutant in the hydroxylation reaction [40]. In particular, the T252A mutant of P450 CYP101, for which the hydroxylation activity with the native substrate camphor is abolished, still epoxidizes a number of olefinic substrates, albeit not with the efficacy of the wild-type hydroxylation and epoxidation reactions [41]. Although the variable catalytic efficiency of the peroxoferric intermediate itself is not completely understood, as the lifetime of the intermediate cannot be gauged experimentally, calculations by Shaik and colleagues [42] present a significantly unfavourable activation barrier in comparing the peroxo-mediated reaction with the iron-oxo driven one. In addition, these computational models point to the relative proximity of multiple ground states of Compound I alone, offering another paradigm for the multiple reactivities often experimentally observed [43]. Although this may explain the more poorly coupled epoxidation activity of mutant T252A, further work on model substrates and sitedirected mutants must be explored in order to deconvolve kinetic competence from intrinsic chemical reactivity. In addition, solvent polarization and hydrogen-bonding effects at the active centre could dramatically lower the barriers for electrophilic reactivity [44]. C 2003
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The role of proton donation in the reactivity of ferric-peroxo intermediates: oxygenase versus peroxidase reactivity Proton delivery in the P450 mono-oxygenation reaction has been implicated in a number of steps in the reaction scheme [45], and plays a critical role in the evolution of intermediates following reduction of the oxyferrous complex. Unlike the P450s, the peroxidases utilize H2 O2 , instead of dioxygen, to form the hydroperoxo-ferric intermediate, and then catalyse the shift of a proton from proximal to distal oxygen in order to heterolytically cleave the O–O bond. In order to understand the distinction between oxygenase and oxidase activities, the ferric-peroxo complex was made in HRP, through radiolytic reduction of the oxyferrous complex [46], analogous to the work described on P450 above. Both optical and EPR spectra of the peroxo intermediates are in good agreement with cryoradiolytic [16] and stopped-flow [47,48] studies of histidine-ligated haem enzymes (Figure 1). Although the reduced dioxygen intermediate is readily formed in this manner, and firstproton transfer to the distal oxygen still occurs to form the hydroperoxo-ferric intermediate, the enzyme is unable to cleave the O–O bond and form Compound I, unlike the reaction with H2 O2 . Instead, the hydroperoxo species, upon annealing, simply decays to the ferric resting state of the
Figure 1 Optical (A) and EPR (B) spectra of hydroperoxo-ferric HRP
Enzyme Mechanism – A Structural Perspective
enzyme, presumably through the release of the peroxide. Thus despite the commonality of intermediates in these two enzyme systems, the key catalytic control step is the source of protons. In the P450 mono-oxygenase system, these protons are provided by the distal pocket residues and several critical solvent molecules.
Summary The haem-oxygen adducts shown in Scheme 1 not only prove to be critical components in the catalytic cycles of P450 mono-oxygenases, but are also present in a wide number of enzyme families. Unravelling the mechanism of P450 dioxygen activation in individual detail has provided a spectroscopic fingerprint of these intermediates, and the reactivity between them, that can be compared and contrasted with those of other haem enzymes. Structural studies on P450 have revealed themes in dioxygen activation, such as the role of stabilized water, which are likely to be critical in understanding the reactivity of these intermediates in other enzymes. Given the critical nature of proton delivery in controlling the multiple reaction pathways in haem proteins, ˚ ) crystallography the advent of ultra-high resolution (< 1 A will play an important role in future understanding of oxygen metabolism. Clearly the elucidation of the chemical reactions that haem adducts can catalyse, and the role of protons in modulating between these intermediates, is fundamental to the understanding of these important metalloenzyme systems.
This work was supported by a Merit Award grant from the National Institutes of Health (GM31756).
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