Ascorbate Peroxidase Compound II Is an Iron(IV) Oxo Species.

The protonation state of the iron(IV) oxo (or ferryl) form of ascorbate peroxidase compound II (APX-II) is a subject of debate. It has been reported that this intermediate is best described as an iron(IV) hydroxide species. Neutron diffraction data obtained from putative APX-II crystals indicate a protonated oxygenic ligand at 1.88 Å from the heme iron. This finding, if correct, would be unprecedented. A basic iron(IV) oxo species has yet to be spectroscopically observed in a histidine-ligated heme enzyme. The importance of ferryl basicity lies in its connection to our fundamental understanding of C-H bond activation. Basic ferryl species have been proposed to facilitate the oxidation of inert C-H bonds, reactions that are unknown for histidine-ligated hemes enzymes. To provide further insight into the protonation status of APX-II, we examined the intermediate using a combination of Mössbauer and X-ray absorption spectroscopies. Our data indicate that APX-II is an iron(IV) oxo species with an Fe-O bond distance of 1.68 Å, a K-edge pre-edge absorption of 18 units, and Mössbauer parameters of ΔEq = 1.65 mm/s and δ = 0.03 mm/s.

Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex.

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.

Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity.

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.

A new look at the role of thiolate ligation in cytochrome P450.

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.

Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme.

We report on the protonation state of Helicobacter pylori catalase compound II. UV/visible, Mössbauer, and X-ray absorption spectroscopies have been used to examine the intermediate from pH 5 to 14. We have determined that HPC-II exists in an iron(IV) hydroxide state up to pH 11. Above this pH, the iron(IV) hydroxide complex transitions to a new species (pKa = 13.1) with Mössbauer parameters that are indicative of an iron(IV)-oxo intermediate. Recently, we discussed a role for an elevated compound II pKa in diminishing the compound I reduction potential. This has the effect of shifting the thermodynamic landscape toward the two-electron chemistry that is critical for catalase function. In catalase, a diminished potential would increase the selectivity for peroxide disproportionation over off-pathway one-electron chemistry, reducing the buildup of the inactive compound II state and reducing the need for energetically expensive electron donor molecules.

Significantly shorter Fe-S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase.

Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse 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 using 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 the 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.

Oxygen-atom transfer reactivity of axially ligated Mn(V)-oxo complexes: evidence for enhanced electrophilic and nucleophilic pathways.

Addition of anionic donors to the manganese(V)-oxo corrolazine complex Mn(V)(O)(TBP8Cz) has a dramatic influence on oxygen-atom transfer (OAT) reactivity with thioether substrates. The six-coordinate anionic [Mn(V)(O)(TBP8Cz)(X)](-) complexes (X = F(-), N3(-), OCN(-)) exhibit a ∼5 cm(-1) downshift of the Mn-O vibrational mode relative to the parent Mn(V)(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 Mn(V)(O)(TBP8Cz) complex. A Hammett study was performed with p-X-C6H4SCH3 derivatives and [Mn(V)(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 [Mn(V)(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 Mn(V)(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 Mn(V)(O) porphyrinoid complexes.

Setting an upper limit on the myoglobin iron(IV)hydroxide pK(a): insight into axial ligand tuning in heme protein catalysis.

To provide insight into the iron(IV)hydroxide pK(a) 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 pK(a) in myoglobin. Together with the recent determination of an iron(IV)hydroxide pK(a) ∼ 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.

Iron(IV)hydroxide pK(a) and the role of thiolate ligation in C-H bond activation by cytochrome P450.

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 pK(a) (where K(a) is the acid dissociation constant) 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 auto-oxidation of tyrosine and tryptophan residues in the enzyme's framework. Here, we report on 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 pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

Reactive intermediates in cytochrome p450 catalysis.

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 (P450ST). 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.

O(2)-evolving chlorite dismutase as a tool for studying O(2)-utilizing enzymes.

The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas's modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O(2) at concentrations far exceeding 2 mM. This method was used to define the O(2) concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme's Mn(IV)/Fe(III) cofactor forms from a Mn(II)/Fe(II) complex and O(2) via a Mn(IV)/Fe(IV) intermediate, at effective O(2) concentrations as high as ~10 mM. With a more soluble receptor, myoglobin, an O(2) adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C-H-bond-cleaving Fe(IV)-oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo-Fe(2)(III/III) complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe(2)(III/IV) intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O(2) evolution permits a >5 mM "pulse" of O(2) to be generated in <1 ms at the easily accessible Cld concentration of 50 µM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O(2) addition steps.