Alicja Franke

Drug Metabolism by Cytochrome P450 Enzymes: What Distinguishes the Pathways Leading to Substrate Hydroxylation Over Desaturation?

Cytochrome P450 enzymes are highly versatile biological catalysts in our body that react with a broad range of substrates. Key functions in the liver include the metabolism of drugs and xenobiotics. One particular metabolic pathway that is poorly understood relates to the P450 activation of aliphatic groups leading to either hydroxylation or desaturation pathways. A DFT and QM/MM study has been carried out on the factors that determine the regioselectivity of aliphatic hydroxylation over desaturation of compounds by P450 isozymes. The calculations establish multistate reactivity patterns, whereby the product distributions differ on each of the spin-state surfaces; hence spin-selective product formation was found. The electronic and thermochemical factors that determine the bifurcation pathways were analysed and a model that predicts the regioselectivity of aliphatic hydroxylation over desaturation pathways was established from valence bond and molecular orbital theories. Thus, the difference in energy of the OH versus the OC bond formed and the π-conjugation energy determines the degree of desaturation products. In addition, environmental effects of the substrate binding pocket that affect the regioselectivities were identified. These studies imply that bioengineering P450 isozymes for desaturation reactions will have to include modifications in the substrate binding pocket to restrict the hydroxylation rebound reaction.

Direct Comparison of the Reactivity of Model Complexes for Compounds 0, I, and II in Oxygenation, Hydrogen‐Abstraction, and Hydride‐Transfer Processes

The iron(III) meso-tetramesitylporphyrin complex is a good biomimetic to study the catalytic reactions of cytochrome P450. All of the three most discussed reactive intermediates concerning P450 catalysis (namely, Cpd 0, Cpd I, and Cpd II) can be selectively produced, identified, and stabilized for many minutes in solution at low temperature by choosing appropriate reaction conditions. In this way, their reactivity against various substrates was determined by utilizing low-temperature rapid-scan UV/Vis spectroscopy. Since all reactive intermediates are derived from a single model complex, the results of these kinetic measurements provide for the first time a full comparability of the determined rate constants for the three intermediates. The rate constants reveal a significant dependence of the reactivity on the type of reaction (e.g., oxygenation, hydrogen abstraction, or hydride transfer), which closely correlates with the chemical nature of Cpds 0, I, and II. The detailed knowledge of the reactivity of these intermediates provides a valuable tool to evaluate their particular role in biological systems.

Combined Experimental and Theoretical Study on the Reactivity of Compounds I and II in Horseradish Peroxidase Biomimetics

For the exploration of the intrinsic reactivity of two key active species in the catalytic cycle of horseradish peroxidase (HRP), Compound I (HRP-I) and Compound II (HRP-II), we generated in situ [Fe(IV) O(TMP(+.) )(2-MeIm)](+) and [Fe(IV) O(TMP)(2-MeIm)](0) (TMP=5,10,15,20-tetramesitylporphyrin; 2-MeIm=2-methylimidazole) as biomimetics for HRP-I and HRP-II, respectively. Their catalytic activities in epoxidation, hydrogen abstraction, and heteroatom oxidation reactions were studied in acetonitrile at -15 °C by utilizing rapid-scan UV/Vis spectroscopy. Comparison of the second-order rate constants measured for the direct reactions of the HRP-I and HRP-II mimics with the selected substrates clearly confirmed the outstanding oxidizing capability of the HRP-I mimic, which is significantly higher than that of HRP-II. The experimental study was supported by computational modeling (DFT calculations) of the oxidation mechanism of the selected substrates with the involvement of quartet and doublet HRP-I mimics ((2,4) Cpd I) and the closed-shell triplet spin HRP-II model ((3) Cpd II) as oxidizing species. The significantly lower activation barriers calculated for the oxidation systems involving (2,4) Cpd I than those found for (3) Cpd II are in line with the much higher oxidizing efficiency of the HRP-I mimic proven in the experimental part of the study. In addition, the DFT calculations show that all three reaction types catalyzed by HRP-I occur on the doublet spin surface in an effectively concerted manner, whereas these reactions may proceed in a stepwise mechanism with the HRP-II mimic as oxidant. However, the high desaturation or oxygen rebound barriers during CH bond activation processes by the HRP-II mimic predict a sufficient lifetime for the substrate radical formed through hydrogen abstraction. Thus, the theoretical calculations suggest that the dissociation of the substrate radical may be a more favorable pathway than desaturation or oxygen rebound processes. Importantly, depending on the electronic nature of the oxidizing species, that is, (2,4) Cpd I or (3) Cpd II, an interesting region-selective conversion phenomenon between sulfoxidation and H-atom abstraction was revealed in the course of the oxidation reaction of dimethylsulfide. The combined experimental and theoretical study on the elucidation of the intrinsic reactivity patterns of the HRP-I and HRP-II mimics provides a valuable tool for evaluating the particular role of the HRP active species in biological systems.

Axial Ligand and Spin-State Influence on the Formation and Reactivity of Hydroperoxo–Iron(III) Porphyrin Complexes

The present study focuses on the formation and reactivity of hydroperoxo-iron(III) porphyrin complexes formed in the [Fe(III)(tpfpp)X]/H(2)O(2)/HOO(-) system (TPFPP=5,10,15,20-tetrakis(pentafluorophenyl)-21H,23H-porphyrin; X=Cl(-) or CF(3) SO(3)(-)) in acetonitrile under basic conditions at -15 °C. Depending on the selected reaction conditions and the active form of the catalyst, the formation of high-spin [Fe(III)(tpfpp)(OOH)] and low-spin [Fe(III)(tpfpp)(OH)(OOH)] could be observed with the application of a low-temperature rapid-scan UV/Vis spectroscopic technique. Axial ligation and the spin state of the iron(III) center control the mode of O-O bond cleavage in the corresponding hydroperoxo porphyrin species. A mechanistic changeover from homo- to heterolytic O-O bond cleavage is observed for high- [Fe(III)(tpfpp)(OOH)] and low-spin [Fe(III)(tpfpp)(OH)(OOH)] complexes, respectively. In contrast to other iron(III) hydroperoxo complexes with electron-rich porphyrin ligands, electron-deficient [Fe(III)(tpfpp)(OH)(OOH)] was stable under relatively mild conditions and could therefore be investigated directly in the oxygenation reactions of selected organic substrates. The very low reactivity of [Fe(III)(tpfpp)(OH)(OOH)] towards organic substrates implied that the ferric hydroperoxo intermediate must be a very sluggish oxidant compared with the iron(IV)-oxo porphyrin π-cation radical intermediate in the catalytic oxygenation reactions of cytochrome P450.

Factors that affect the nature of the final oxidation products in "peroxo-shunt" reactions of iron-porphyrin complexes.

The present study focuses on the oxidation of the water-soluble and water-insoluble iron(III)-porphyrin complexes [Fe(III)(TMPS)] and [Fe(III)(TMP)] (TMPS = meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphyrinato, TMP = meso-tetrakis(2,4,6-trimethylphenyl)porphyrinato), respectively, by meta-chloroperoxybenzoic acid (m-CPBA) in aqueous methanol and aqueous acetonitrile solutions of varying acidity. With the application of a low-temperature rapid-scan UV/Vis spectroscopic technique, the complete spectral changes that accompany the formation and decomposition of the primary product of O-O bond cleavage in the acylperoxoiron(III)-porphyrin intermediate [(P)Fe(III)-OOX] (P = porphyrin) were successfully recorded and characterized. The results clearly indicate that the O-O bond in m-CPBA is heterolytically cleaved by the studied iron(III)-porphyrin complexes independent of the acidity of the reaction medium. The existence of two different oxidation products under acidic and basic conditions is suggested not to be the result of a mechanistic changeover in the mode of O-O bond cleavage on going from low to high pH values, but rather the effect of environmental changes on the actual product of the O-O bond cleavage in [(P)Fe(III)-OOX]. The oxoiron(IV)-porphyrin cation radical formed as a primary oxidation product over the entire pH range can undergo a one- or two-electron reduction depending on the selected reaction conditions. The present study provides valuable information for the interpretation and improved understanding of results obtained in product-analysis experiments.

Temperature and pressure effects on C-H abstraction reactions involving compound I and II mimics in aqueous solution.

The presented results cover a comparative mechanistic study on the reactivity of compound (Cpd) I and II mimics of a water-soluble iron(III) porphyrin, [meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III), Fe(III)(TMPS). The acidity of the aqueous medium strongly controls the chemical nature and stability of the high-valent iron(IV) oxo species. Reactivity studies were performed at pH 5 and 10, where the Cpd I and II mimics are stabilized as the sole oxidizing species, respectively. The contributions of ΔH(‡) and ΔS(‡) to the free energy of activation (ΔG(‡)) for the oxidation of 4-methoxybenzaldehyde (4-MB-ald), 4-methoxybenzyl alcohol (4-MB-alc), and 1-phenylethanol (1-PhEtOH) by the Cpd I and II mimics were determined. The relatively large contribution of the ΔH(‡) term in comparison to the -TΔS(‡) term to ΔG(‡) for reactions involving the Cpd II mimic indicates that the oxidation of selected substrates by this oxidizing species is clearly an enthalpy-controlled process. In contrast, different results were found for reactions with application of the Cpd I mimic. Depending on the nature of the substrate, the reaction at room temperature can be entropy-controlled, as found for the oxidation of 4-MB-alc, or enthalpy-controlled, as found for 1-PhEtOH. Importantly, for the first time, activation volumes (ΔV(‡)) for the oxidation of selected substrates by both reactive intermediates could be determined. Positive values of ΔV(‡) were found for reactions with the Cpd II mimic and slightly negative ones for reactions with the Cpd II mimic. The results are discussed in the context of the oxidation mechanism conducted by the Cpd I and II mimics.

Mechanistic Insight into Peroxo‐Shunt Formation of Biomimetic Models for Compound II, Their Reactivity toward Organic Substrates, and the Influence of N‐Methylimidazole Axial Ligation

High-valent iron-oxo species have been invoked as reactive intermediates in catalytic cycles of heme and nonheme enzymes. The studies presented herein are devoted to the formation of compound II model complexes, with the application of a water soluble (TMPS)Fe(III)(OH) porphyrin ([meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III) hydroxide) and hydrogen peroxide as oxidant, and their reactivity toward selected organic substrates. The kinetics of the reaction of H2O2 with (TMPS)Fe(III)(OH) was studied as a function of temperature and pressure. The negative values of the activation entropy and activation volume for the formation of (TMPS)Fe(IV)=O(OH) point to the overall associative nature of the process. A pH-dependence study on the formation of (TMPS)Fe(IV)=O(OH) revealed a very high reactivity of OOH(-) toward (TMPS)Fe(III)(OH) in comparison to H2O2. The influence of N-methylimidazole (N-MeIm) ligation on both the formation of iron(IV)-oxo species and their oxidising properties in the reactions with 4-methoxybenzyl alcohol or 4-methoxybenzaldehyde, was investigated in detail. Combined experimental and theoretical studies revealed that among the studied complexes, (TMPS)Fe(III)(H2O)(N-MeIm) is highly reactive toward H2O2 to form the iron(IV)-oxo species, (TMPS)Fe(IV)=O(N-MeIm). The latter species can also be formed in the reaction of (TMPS)Fe(III)(N-MeIm)2 with H2O2 or in the direct reaction of (TMPS)Fe(IV)=O(OH) with N-MeIm. Interestingly, the kinetic studies involving substrate oxidation by (TMPS)Fe(IV)=O(OH) and (TMPS)Fe(IV)=O(N-MeIm) do not display a pronounced effect of the N-MeIm axial ligand on the reactivity of the compound II mimic in comparison to the OH(-) substituted analogue. Similarly, DFT computations revealed that the presence of an axial ligand (OH(-) or N-MeIm) in the trans position to the oxo group in the iron(IV)-oxo species does not significantly affect the activation barriers calculated for C-H dehydrogenation of the selected organic substrates.

Spectroscopic and Mechanistic Studies on Oxidation Reactions Catalyzed by the Functional Model SR Complex for Cytochrome P450: Influence of Oxidant, Substrate, and Solvent

Kinetic and mechanistic studies on the formation of an oxoiron(IV) porphyrin cation radical bearing a thiolate group as proximal ligand are reported. The SR complex, a functional enzyme mimic of P450, was oxidized in peroxo-shunt reactions under different experimental conditions with variation of solvent, temperature, and identity and excess of oxidant in the presence of different organic substrates. Through the application of a low-temperature rapid-scan stopped-flow technique, the reactive intermediates in the SR catalytic cycle, such as the initially formed SR acylperoxoiron(III) complex and the SR high-valent iron(IV) porphyrin cation radical complex [(SR(*+))Fe(IV)=O], were successfully identified and kinetically characterized. The oxidation of the SR complex under catalytic conditions provided direct spectroscopic information on the reactivity of [(SR(.+))Fe(IV)=O] towards the oxidation of selected organic substrates. Because the catalytically active species is a synthetic oxoiron(IV) porphyrin cation radical bearing a thiolate proximal group, the effect of the strong electron donor ligand on the formation and reactivity/stability of the SR high-valent iron species is addressed and discussed in the light of the reactivity pattern observed in substrate oxygenation reactions catalyzed by native P450 enzyme systems.

Mechanistic studies on the reactions of cyanide with a water-soluble Fe(III) porphyrin and their effect on the binding of NO.

The reaction of the water-soluble Fe(III)(TMPS) porphyrin with CN(-) in basic solution leads to the stepwise formation of Fe(III)(TMPS)(CN)(H(2)O) and Fe(III)(TMPS)(CN)(2). The kinetics of the reaction of CN(-) with Fe(III)(TMPS)(CN)(H(2)O) was studied as a function of temperature and pressure. The positive value of the activation volume for the formation of Fe(III)(TMPS)(CN)(2) is consistent with the operation of a dissociatively activated mechanism and confirms the six-coordinate nature of the monocyano complex. A good agreement between the rate constants at pH 8 and 9 for the formation of the dicyano complex implies the presence of water in the axial position trans to coordinated cyanide in the monocyano complex and eliminates the existence of Fe(III)(TMPS)(CN)(OH) under the selected reaction conditions. Both Fe(III)(TMPS)(CN)(H(2)O) and Fe(III)(TMPS)(CN)(2) bind nitric oxide (NO) to form the same nitrosyl complex, namely, Fe(II)(TMPS)(CN)(NO(+)). Kinetic studies indicate that nitrosylation of Fe(III)(TMPS)(CN)(2) follows a limiting dissociative mechanism that is supported by the independence of the observed rate constant on [NO] at an appropriately high excess of NO, and the positive values of both the activation parameters ΔS(‡) and ΔV(‡) found for the reaction under such conditions. The relatively small first-order rate constant for NO binding, namely, (1.54 ± 0.01) × 10(-2) s(-1), correlates with the rate constant for CN(-) release from the Fe(III)(TMPS)(CN)(2) complex, namely, (1.3 ± 0.2) × 10(-2) s(-1) at 20 °C, and supports the proposed nitrosylation mechanism.

A complete volume profile for the reversible binding of camphor to cytochrome P450cam

The effect of pressure on the kinetics and thermodynamics of the reversible binding of camphor to cytochrome P450cam was studied as a function of the K+ concentration. The determination of the reaction and activation volumes enabled the construction of the first complete volume profile for the reversible binding of camphor to P450cam. Although the volume profiles constructed for the reactions conducted at low and high K+ concentrations are rather similar, and both show a drastic volume increase on going from the reactant to the transition state and a relatively small volume change on going from the transition to the product state, the position of the transition state is largely affected by the K+ concentration in solution. Similarly, the activation volume determined for the dissociation of camphor is influenced by the presence of K+, which reflects changes in the ease of water entering the active site of camphor-bound P450cam that depends on the K+ concentration. Careful analysis of the components that contribute to the observed volume changes allowed the estimation of the total number of water molecules expelled to the bulk solvent during the binding of camphor to P450cam and the subsequent spin transition. The results are discussed in reference to other studies reported in the literature that deal with the kinetics and thermodynamics of the binding of camphor to P450cam under various reaction conditions.