Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pKa of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious autooxidation of tyrosine and tryptophan residues in the enzyme’s framework. Here we report the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥ 90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and X-ray absorption spectroscopies, we determine a pKa value for this compound of 11.9. Marcus theory analysis indicates that this elevated pKa results in a >10,000 fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metal-hydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process.

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...

Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate ligated heme proteins that catalyze the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I with variable temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in hydrogen bonding patterns within the “cys-pocket” (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial-thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation.

To provide insight into the iron(IV)hydroxide pKa of histidine ligated heme proteins, we have probed the active site of myoglobin compound II over the pH range of 3.9–9.5, using EXAFS, Mössbauer, and resonance Raman spectroscopies. We find no indication of ferryl protonation over this pH range, allowing us to set an upper limit of 2.7 on the iron(IV)hydroxide pKa in myoglobin. Together with the recent determination of an iron(IV)hydroxide pKa ∼ 12 in the thiolate-ligated heme enzyme cytochrome P450, this result provides insight into Nature’s ability to tune catalytic function through its choice of axial ligand.

Protonated ferryl (or iron(IV)hydroxide) intermediates have been characterized in several thiolate-ligated heme proteins that are known to catalyze C-H bond activation. The basicity of the ferryl intermediates in these species has been proposed to play a critical role in facilitating this chemistry, allowing hydrogen abstraction at reduction potentials below those that would otherwise lead to oxidative degradation of the enzyme. In this contribution, we discuss the events that led to the assignment and characterization of the unusual iron(IV)hydroxide species, highlighting experiments that provided a quantitative measure of the ferryl basicity, the iron(IV)hydroxide pKa. We then turn to the importance of the iron(IV)hydroxide state, presenting a new way of looking at the role of thiolate ligation in these systems.

Addition of anionic donors to the manganese(V)–oxo corrolazine complex MnV(O)(TBP8Cz) has a dramatic influence on oxygen-atom transfer (OAT) reactivity with thioether substrates. The six-coordinate anionic [MnV(O)(TBP8Cz)(X)]− complexes (X = F–, N3–, OCN–) exhibit a ∼5 cm–1 downshift of the Mn–O vibrational mode relative to the parent MnV(O)(TBP8Cz) complex as seen by resonance Raman spectroscopy. Product analysis shows that the oxidation of thioether substrates gives sulfoxide product, consistent with single OAT. A wide range of OAT reactivity is seen for the different axial ligands, with the following trend determined from a comparison of their second-order rate constants for sulfoxidation: five-coordinate ≈ thiocyanate ≈ nitrate < cyanate < azide < fluoride ≪ cyanide. This trend correlates with DFT calculations on the binding of the axial donors to the parent MnV(O)(TBP8Cz) complex. A Hammett study was performed with p-X-C6H4SCH3 derivatives and [MnV(O)(TBP8Cz)(X)]− (X = CN– or F–) as the oxidant, and unusual “V-shaped” Hammett plots were obtained. These results are rationalized based upon a change in mechanism that hinges on the ability of the [MnV(O)(TBP8Cz)(X)]− complexes to function as either an electrophilic or weak nucleophilic oxidant depending upon the nature of the para-X substituents. For comparison, the one-electron-oxidized cationic MnV(O)(TBP8Cz•+) complex yielded a linear Hammett relationship for all substrates (ρ = −1.40), consistent with a straightforward electrophilic mechanism. This study provides new, fundamental insights regarding the influence of axial donors on high-valent MnV(O) porphyrinoid complexes.

Strong electron-donation from the axial thiolate ligand of cytochrome P450 has been proposed to increase the reactivity of compound I with respect to C-H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we have prepared a selenolate-ligated cytochrome P450 compound I intermediate. This isoelectronic perturbation allows for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepared with meta-chloroperbenzoic acid and characterized by UV-visible, Mössbauer and electron paramagnetic resonance spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we find that the selenolate-ligated compound I cleaves C-H bonds more rapidly than the wild-type intermediate.