How Does Replacement of the Axial Histidine Ligand in Cytochrome c Peroxidase by Nδ-Methyl Histidine Affect Its Properties and Functions? A Computational Study.

Heme peroxidases have important functions in nature related to the detoxification of H2O2. They generally undergo a catalytic cycle where, in the first stage, the iron(III)-heme-H2O2 complex is converted into an iron(IV)-oxo-heme cation radical species called Compound I. Cytochrome c peroxidase Compound I has a unique electronic configuration among heme enzymes where a metal-based biradical is coupled to a protein radical on a nearby Trp residue. Recent work using the engineered Nδ-methyl histidine-ligated cytochrome c peroxidase highlighted changes in spectroscopic and catalytic properties upon axial ligand substitution. To understand the axial ligand effect on structure and reactivity of peroxidases and their axially Nδ-methyl histidine engineered forms, we did a computational study. We created active site cluster models of various sizes as mimics of horseradish peroxidase and cytochrome c peroxidase Compound I. Subsequently, we performed density functional theory studies on the structure and reactivity of these complexes with a model substrate (styrene). Thus, the work shows that the Nδ-methyl histidine group has little effect on the electronic configuration and structure of Compound I and little changes in bond lengths and the same orbital occupation is obtained. However, the Nδ-methyl histidine modification impacts electron transfer processes due to a change in the reduction potential and thereby influences reactivity patterns for oxygen atom transfer. As such, the substitution of the axial histidine by Nδ-methyl histidine in peroxidases slows down oxygen atom transfer to substrates and makes Compound I a weaker oxidant. These studies are in line with experimental work on Nδ-methyl histidine-ligated cytochrome c peroxidases and highlight how the hydrogen bonding network in the second coordination sphere has a major impact on the function and properties of the enzyme.

Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate.

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

Hydrogen Atom Abstraction by High-Valent Fe(OH) versus Mn(OH) Porphyrinoid Complexes: Mechanistic Insights from Experimental and Computational Studies.

High-valent metal-hydroxide species have been implicated as key intermediates in hydroxylation chemistry catalyzed by heme monooxygenases such as the cytochrome P450s. However, in some classes of P450s, a bifurcation from the typical oxygen rebound pathway is observed, wherein the FeIV(OH)(porphyrin) species carries out a net hydrogen atom transfer reaction to form alkene metabolites. In this work, we examine the hydrogen atom transfer (HAT) reactivity of FeIV(OH)(ttppc) (1), ttppc = 5,10,15-tris(2,4,6-triphenyl)-phenyl corrole, toward substituted phenol derivatives. The iron hydroxide complex 1 reacts with a series of para-substituted 2,6-di-tert-butylphenol derivatives (4-X-2,6-DTBP; X = OMe, Me, Et, H, Ac), with second-order rate constants k2 = 3.6(1)-1.21(3) × 104 M-1 s-1 and yielding linear Hammett and Marcus plot correlations. It is concluded that the rate-determining step for O-H cleavage occurs through a concerted HAT mechanism, based on mechanistic analyses that include a KIE = 2.9(1) and DFT calculations. Comparison of the HAT reactivity of 1 to the analogous Mn complex, MnIV(OH)(ttppc), where only the central metal ion is different, indicates a faster HAT reaction and a steeper Hammett slope for 1. The O-H bond dissociation energy (BDE) of the MIII(HO-H) complexes were estimated from a kinetic analysis to be 85 and 89 kcal mol-1 for Mn and Fe, respectively. These estimated BDEs are closely reproduced by DFT calculations and are discussed in the context of how they influence the overall H atom transfer reactivity.

Second-Coordination Sphere Effect on the Reactivity of Vanadium-Peroxo Complexes: A Computational Study.

Vanadium-oxo and vanadium-peroxo complexes are common intermediates in biology and are, for instance, found in the catalytic cycle of vanadium haloperoxidases. In biomimetic chemistry synthetic models have been created that mimic the structural features of the coordination environment of these vanadium-oxo and vanadium-peroxo species. Recently, two novel vanadium-oxo complexes were trapped and characterized with a trigonal bipyramidal ligand design with either a solvent exposed vanadium center or the vanadium inside a cage, designated as the bowl-shaped configuration and the dome-shaped structure, respectively. Density functional theory calculations are reported here on these bowl- and dome-shaped structures where we study the reaction with t-butylhydroperoxide to form the vanadium-peroxo species and its reaction with thioanisole. Although the structural features of the vanadate core are close for both structures, the calculations display a strong second-coordination sphere effect of the ligand architecture on the barrier heights of the reaction with a terminal oxidant even though the rate-determining transition states show little structural differences. A similar observation is seen for the reaction of the two vanadium-peroxo species with thioanisole. Overall, the calculations implicate that vanadium-peroxo is an efficient oxidant of sulfoxidation reactions, although it is not as efficient as analogous iron(IV)-oxo heme and nonheme oxidants that react with substantially lower barriers. The reactivity differences are analyzed with thermochemical cycles and valence bond patterns that explain the differences in chemical properties and identify how the ligands affect the chemical reactivity with substrates.

Reactivity patterns of vanadium(iv/v)-oxo complexes with olefins in the presence of peroxides: a computational study.

Vanadium porphyrin complexes are naturally occurring substances found in crude oil and have been shown to have medicinal properties as well. Little is known on their activities with substrates; therefore, we decided to perform a detailed density functional theory study on the properties and reactivities of vanadium(iv)- and vanadium(v)-oxo complexes with a TPPCl8 or 2,3,7,8,12,13,17,18-octachloro-meso-tetraphenylporphyrinato ligand system. In particular, we investigated the reactivity of [VV(O)(TPPCl8)]+ and [VIV(O)(TPPCl8)] with cyclohexene in the presence of H2O2 or HCO4-. The work shows that vanadium(iv)-oxo and vanadium(v)-oxo are sluggish oxidants by themselves and react with olefins slowly. However, in the presence of hydrogen peroxide, these metal-oxo species can be transformed into a side-on vanadium-peroxo complex, which reacts with substrates more efficiently. Particularly with anionic axial ligands, the side-on vanadium-peroxo and vanadium-oxo complexes produced epoxides from cyclohexene via small barrier heights. In addition to olefin epoxidation, we investigated aliphatic hydroxylation mechanisms by the same oxidants and some oxidants show efficient and viable cyclohexene hydroxylation mechanisms. The work implies that vanadium-oxo and vanadium-peroxo complexes can react with double bonds through epoxidation, and under certain conditions also undergo hydroxylation, but the overall reactivity is highly dependent on the equatorial ligand, the local environment and the presence or absence of anionic axial ligands.

Properties and reactivity of µ-nitrido-bridged dimetal porphyrinoid complexes: how does ruthenium compare to iron?

Methane hydroxylation by metal-oxo oxidants is one of the Holy Grails in biomimetic and biotechnological chemistry. The only enzymes known to perform this reaction in Nature are iron-containing soluble methane monooxygenase and copper-containing particulate methane monooxygenase. Furthermore, few biomimetic iron-containing oxidants have been designed that can hydroxylate methane efficiently. Recent studies reported that µ-nitrido-bridged diiron(IV)-oxo porphyrin and phthalocyanine complexes hydroxylate methane to methanol efficiently. To find out whether the reaction rates are enhanced by replacing iron by ruthenium, we performed a detailed computational study. Our work shows that the µ-nitrido-bridged diruthenium(IV)-oxo reacts with methane via hydrogen atom abstraction barriers that are considerably lower in energy (by about 5 kcal mol‒1) as compared to the analogous diiron(IV)-oxo complex. An analysis of the electronic structure implicates similar spin and charge distributions for the diiron(IV)-oxo and diruthenium(IV)-oxo complexes, but the strength of the O‒H bond formed during the reaction is much stronger for the latter. As such a larger hydrogen atom abstraction driving force for the Ru complex than for the Fe complex is found, which should result in higher reactivity in the oxidation of methane.

Flavonol biosynthesis by nonheme iron dioxygenases: A computational study into the structure and mechanism.

Plants produce flavonol compounds for vital functions regarding plant growth, fruit and flower colouring as well as fruit ripening processes. Several of these biosynthesis steps are stereo- and regioselective and are being carried out by nonheme iron enzymes. Using density functional theory calculations on a large active site model complex of flavanone-3ß-hydroxylase (FHT), we established the mechanism for conversion of naringenin to its dihydroflavonol, which is a key step in the mechanism of flavonol biosynthesis. The reaction starts with dioxygen binding to the iron(II) centre and a reaction with α-ketoglutarate co-substrate gives succinate, an iron(IV)-oxo species and CO2 with large exothermicity and small reaction barriers. The rate-determining reaction step in the mechanism; however, is hydrogen atom abstraction of an aliphatic CH bond by the iron(IV)-oxo species. We identify a large kinetic isotope effect for the replacement of the transferring hydrogen atom by deuterium. In a final step the OH and substrate radicals combine to form the alcohol product with a barrier of several kcal mol-1. We show that the latter is the result of geometric constraints in the active site pocket. Furthermore, the calculations show that a weak tertiary CH bond is shielded from the iron(IV)-oxo species in the substrate binding position and therefore the enzyme is able to activate a stronger CH bond. As such, the flavanone-3ß-hydroxylase enzyme reacts regioselectively with one specific CH bond of naringenin by avoiding activation of weaker bonds through tight substrate and oxidant positioning.

Selective Formation of an FeIV O or an FeIII OOH Intermediate From Iron(II) and H2 O2 : Controlled Heterolytic versus Homolytic Oxygen-Oxygen Bond Cleavage by the Second Coordination Sphere.

We demonstrate that the devised incorporation of an alkylamine group into the second coordination sphere of an FeII complex allows to switch its reactivity with H2 O2 from the usual formation of FeIII species towards the selective generation of an FeIV -oxo intermediate. The FeIV -oxo species was characterized by UV/Vis absorption and Mössbauer spectroscopy. Variable-temperature kinetic analyses point towards a mechanism in which the heterolytic cleavage of the O-O bond is triggered by a proton transfer from the proximal to the distal oxygen atom in the FeII -H2 O2 complex with the assistance of the pendant amine. DFT studies reveal that this heterolytic cleavage is actually initiated by an homolytic O-O cleavage immediately followed by a proton-coupled electron transfer (PCET) that leads to the formation of the FeIV -oxo and release of water through a concerted mechanism.