P450 Compound Research Articles

Oxygen‐18 Labeling Reveals a Mixed Fe−O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α‐Demethylation

AbstractThe 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2−) or Compound I (perferryl oxygen, FeO3+) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18O incorporation into formic acid from 18O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18O (one atom) into formic acid, consistent with a major but not exclusive FeO2− mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X‐ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Å away from the heme iron atom. Experiments with human P450 51A1 and H218O yielded primarily one 18O atom but 14 % of the formic acid product with two 18O atoms, indicative of a minor contribution of a Compound I mechanism. LC–MS evidence for a Compound 0‐derived Baeyer–Villiger reaction product (a 14α‐formyl ester) was also found.

Oxygen-18 Labeling Reveals a Mixed Fe-O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α-Demethylation.

The 14α-demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three-step reaction terminates in a C-C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2 - ) or Compound I (perferryl oxygen, FeO3+ ) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18 O incorporation into formic acid from 18 O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18 O (one atom) into formic acid, consistent with a major but not exclusive FeO2 - mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X-ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Å away from the heme iron atom. Experiments with human P450 51A1 and H2 18 O yielded primarily one 18 O atom but 14 % of the formic acid product with two 18 O atoms, indicative of a minor contribution of a Compound I mechanism. LC-MS evidence for a Compound 0-derived Baeyer-Villiger reaction product (a 14α-formyl ester) was also found.

Insights into Cytochrome P450 Enzyme Catalyzed Defluorination of Aromatic Fluorides.

Density functional calculations establish a novel mechanism of aromatic defluorination by P450 Compound I. This is achieved via either an initial epoxide intermediate or through a 1,2-fluorine shift in an electrophilic intermediate, which highlights that the P450s can defluorinate fluoroarenes. However, in the absence of a proton donor a strong Fe-F bond can be obtained as shown from the calculations.

Insights into Cytochrome P450 Enzyme Catalyzed Defluorination of Aromatic Fluorides

The biodegradability of many fluorinated compounds is limited due to the robustness of the C‐F bond. Recently, experimental studies suggested the potential involvement of cytochrome P450 enzymes in facilitating aromatic defluorination, raising questions where this can be applied in biocatalysis. Our study offers an in‐depth computational examination into the oxidative defluorination process of a drug molecule, mediated by cytochrome P450 Compound I. We explored a large number of potential mechanisms, and identify two competitive low‐energy pathways that are initiated with an electrophilic attack on the aromatic ring, and followed by either a 1,2‐fluorine shift or a ring‐closure to form an epoxide intermediate. Both of these intermediates are shown to react rapidly through defluorination assisted by a solvent proton. Interestingly, defluorination in the vicinity of a heme group may generate a stable iron(III)‐fluoride complex, potentially leading to enzyme inactivation.

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study.

Bisphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)propane) is used as a precursor in the synthesis of polycarbonate and epoxy plastics; however, its availability in the environment is causing toxicity as an endocrine-disrupting chemical. Metabolism of BPA and their analogues (substitutes) is generally performed by liver cytochrome P450 enzymes and often leads to a mixture of products, and some of those are toxic. To understand the product distributions of P450 activation of BPA, we have performed a computational study into the mechanisms and reactivities using large model structures of a human P450 isozyme (P450 2C9) with BPA bound. Density functional theory (DFT) calculations on mechanisms of BPA activation by a P450 compound I model were investigated, leading to a number of possible products. The substrate-binding pocket is tight, and as a consequence, aliphatic hydroxylation is not feasible as the methyl substituents of BPA cannot reach compound I well due to constraints of the substrate-binding pocket. Instead, we find low-energy pathways that are initiated with phenol hydrogen atom abstraction followed by OH rebound to the phenolic ortho- or para-position. The barriers of para-rebound are well lower in energy than those for ortho-rebound, and consequently, our P450 2C9 model predicts dominant hydroxycumyl alcohol products. The reactions proceed through two-state reactivity on competing doublet and quartet spin state surfaces. The calculations show fast and efficient substrate activation on a doublet spin state surface with a rate-determining electrophilic addition step, while the quartet spin state surface has multiple high-energy barriers that can also lead to various side products including C4-aromatic hydroxylation. This work shows that product formation is more feasible on the low spin state, while the physicochemical properties of the substrate govern barrier heights of the rate-determining step of the reaction. Finally, the importance of the second-coordination sphere is highlighted that determines the product distributions and guides the bifurcation pathways.

Substrate sulfoxidation by a biomimetic cytochrome P450 Compound I mimic: How do porphyrin and phthalocyanine equatorial ligands compare?

Substrate sulfoxidation by a biomimetic cytochrome P450 Compound I mimic: How do porphyrin and phthalocyanine equatorial ligands compare?

A three states model for hydrogen abstraction reactions with the cytochrome P450 compound I is revisited. Isolobal and isospin analogy among Fe(IV)=O, O = O and O

Radical abstraction reactions by the cytochrome P450 compound I are investigated on the basis of the one quartet two doublet model consisted of three configurations; 4{2[↑• +Por]–[↑• 3Fe(IV)=O •↑]} (4CpdI; S = 3/2), 2{2[↓• +Por]–[↑• 3Fe(IV)=O •↑]} (2CpdIa; S = 1/2) and 2{1[Por(↑↓)]–2[↑• Fe(V)=O]} (2CpdIb; S = 1/2) where Por denotes porphyrin and axial cysteine anion (–SR) is neglected. The dπ-pπ bond of 2CpdIb was highly spin-polarized in the transition-state region of hydrogen abstraction reactions, providing the doublet configuration; 2{1[Por(↑↓)]–2[↑↑•• Fe(IV)–O •↓]} (2CpdIb,oxyl). The oxyl-radical character of 2CpdIb,oxyl plays an important role for hydrogen radical abstraction from alkanes. The spin populations on the iron atom at the transition structures by available density functional theory (DFT) results were about 2.0 for 2CpdIb,oxyl, whereas they were about 1.0 for 4CpdI and 2CpdIa. On the other hand, the spin density population on the carbon atom of alkyl radical were negative and positive in sign for 2CpdIb,oxyl and 4CpdI, respectively, in consistent with their spin structures. In order to elucidate possible reaction pathways by the cytochrome P450 compound I, relative stabilities between 4CpdI and 2CpdIb were examined on the basis of available DFT and beyond DFT results. The refined DFT results obtained by the beyond DFT elucidated one of the possible reaction pathways for hydrogen abstractions; 2CpdIa → 2Cpdb → 2CpdIb,oxyl → 2(TS) → (rebound). Implications of the computational results are also discussed in relation to water oxidation in artificial catalysts.

In silico toxicity prediction, synthesis, characterization, antimicrobial and antioxidant activity of different di substituted chalcones

Right now the amalgamation of novel arrangement of 4'- chloro-3'- nitro-phenyl-(3-keto-1,2diene-1-flurobenzene), 4'- chloro-3'nitro-phenyl-(3-keto-1,2diene-1-chlorobenzene),4'- chloro-3'- nitro-phenyl-(3-keto-1,2diene-1-thiophene),4'- chloro-3'nitro-phenyl-(3-keto-1,2diene-1-pyridine), 4'- chloro-3'nitro-phenyl-(3-keto-1,2diene-1-furon).The target mixes were integrated by the Claisen-schimidt buildup utilizing corrosive and base as a response impetus. insilico enhancement strategies by utilization of various information base like molinspiration for atomic properties, bioactivity, ochem for organic information, pre ADME for pharmacokinetic properties, preToxicity for harmfulness profile of the mixes, swiss ADME for pharmacokinetic property, Swiss lethality for natural danger of the mixes, and Osiris(datawarrior) for poisonous quality forecast for dynamic lead particles ID. From the outcome mixes indented as dynamic leads they used to assess distinctive organic action. The mixes additionally finish the amees assessment for the most part for mutagenecity discovery that mixes shows mutagencity towards the test living being they likewise safe to hinder cytochrome P450 compound subunits like cyp1A2, cyp2C9, cyp2C19 which are situated in endoplasmic reticulum. Alpha beta unsaturated chalcones have fantastic cell reinforcement and antimicrobial action so the orchestrated mixes were screened for their antimicrobial by utilizing five bacterial strains and three parasitic strains and cancer prevention agent action by DPPH technique. From hostile to oxidant results compound 3a, 3b, 3c show critical action the measure esteems are closer to standard medication esteem. Antimicrobial movement results the compound 3a, 3c, 3e indicated noteworthy action against Bacillus subtilis, E Coli, Salmonella tyhphi, Pseudomonas aeruginosa though the compound 3b, 3d demonstrated moderate activitycompared to standard medication vale. Compound 3b, 3d demonstrated fantastic antifungal action against Candida albicans, Aspergillusniger, Alternariaalternata. The mixes 3a, 3c, 3e show moderate antifungal movement when contrasted with standard worth.

Cross-linking of aromatic phenolate groups by cytochrome P450 enzymes: a model for the biosynthesis of vancomycin by OxyB.

The cytochromes P450 are a versatile class of enzymes involved in many chemical reactions in biosystems and as such they take part in biodegradation as well as biosynthesis pathways in many organisms. These enzymes use molecular oxygen on a heme centre and often react as mono-oxygenases. Lesser known reactions catalyzed by the P450s include desaturation pathways and ring-closure reactions. In this work we study the aromatic cross-linking of glycopeptide units as, for instance, performed by the P450 isozyme OxyB as part of vancomycin biosynthesis. A series of density functional theory studies are reported on a large active site cluster model of 258 atoms containing the heme with its coordinated ligands, a representative substrate and its interacting protein residues. We show that the catalytic cycle intermediates Compound I and Compound II of P450 can rapidly and successively abstract a phenolic hydrogen atom from adjacent peptide groups to give a biradical intermediate with small reaction barriers. The latter can form the ether cross-link between the two aromatic residues, which is the rate-determining step in the reaction mechanism and involves a simultaneous proton transfer from the ipso-position to the ketone. A thermochemical analysis reveals that weak phenolic O-H bonds lead to hydrogen atom abstraction easily by Compound I and Compound II, enabling a selective aromatic cross-linking reaction.

Reduction Potentials of P450 Compounds I and II: Insight into the Thermodynamics of C-H Bond Activation.

We present a mixed experimental/theoretical determination of the bond strengths and redox potentials that define the ground-state thermodynamics for C-H bond activation in cytochrome P450 catalysis. Using redox titrations with [Ir(IV)Cl6]2-, we have determined the compound II/ferric (or Fe(IV)OH/Fe(III)OH2) couple and its associated D(O-H)Ferric bond strength in CYP158. Knowledge of this potential as well as the compound II/ferric (or Fe(IV)O/Fe(III)OH) reduction potential in horseradish peroxidase and the two-electron compound I/ferric (or Fe(IV)O(Por•)/Fe(III)OH2(Por)) reduction potential in aromatic peroxidase has allowed us to gauge the accuracy of theoretically determined bond strengths. Using the restricted open shell (ROS) method as proposed by Wright and co-workers, we have obtained O-H bond strengths and associated redox potentials for charge-neutral H-atom reductions of these iron(IV)-hydroxo and -oxo porphyrin species that are within 1 kcal/mol of experimentally determined values, suggesting that the ROS method may provide accurate values for the P450-II O-H bond strength and P450-I reduction potential. The efforts detailed here indicate that the ground-state thermodynamics of C-H bond activation in P450 are best described as follows: E0'Comp-I = 1.22 V (at pH 7, vs NHE) with D(O-H)Comp-II = 95 kcal/mol and E0'Comp-II = 0.99 V (at pH 7, vs NHE) with D(O-H)Ferric = 90 kcal/mol.

Hole Hopping through Tryptophan in Cytochrome P450.

Electron-transfer kinetics have been measured in four conjugates of cytochrome P450 with surface-bound Ru-photosensitizers. The conjugates are constructed with enzymes from Bacillus megaterium (CYP102A1) and Sulfolobus acidocaldarius (CYP119). A W96 residue lies in the path between Ru and the heme in CYP102A1, whereas H76 is present at the analogous location in CYP119. Two additional conjugates have been prepared with (CYP102A1)W96H and (CYP119)H76W mutant enzymes. Heme oxidation by photochemically generated Ru3+ leads to P450 compound II formation when a tryptophan residue is in the path between Ru and the heme; no heme oxidation is observed when histidine occupies this position. The data indicate that heme oxidation proceeds via two-step tunneling through a tryptophan radical intermediate. In contrast, heme reduction by photochemically generated Ru+ proceeds in a single electron tunneling step with closely similar rate constants for all four conjugates.

Enzymatic catalysis: New functional twists for P450s.

Two papers provide insight into the reactivity of cytochrome P450s. A direct link between electron donation and reactivity has been shown with a selenocysteine-ligated P450 compound I, whereas a serine-ligated P450 (P411) has been engineered to catalyse an intermolecular C–H amination via nitrene transfer.

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.

Oxygen Atom Transfer Using an Iron(IV)-Oxo Embedded in a Tetracyclic N-Heterocyclic Carbene System: How Does the Reactivity Compare to Cytochrome P450 Compound I?

N-Heterocyclic carbenes (NHC) are commonly featured as ligands in transition metal catalysis. Recently, a cyclic system containing four NHC groups with a central iron atom was synthesized and its iron(IV)-oxo species, [FeIV (O)(cNHC4 )]2+ , was characterized. This tetracyclic NHC ligand system may give the iron(IV)-oxo species unique catalytic properties as compared to traditional non-heme and heme iron ligand systems. Therefore, we performed a computational study on the structure and reactivity of the [FeIV (O)(cNHC4 )]2+ complex in substrate hydroxylation and epoxidation reactions. The reactivity patterns are compared with cytochrome P450 Compound I and non-heme iron(IV)-oxo models and it is shown that the [FeIV (O)(cNHC4 )]2+ system is an effective oxidant with oxidative power analogous to P450 Compound I. Unfortunately, in polar solvents, a solvent molecule will bind to the sixth ligand position and decrease the catalytic activity of the oxidant. A molecular orbital and valence bond analysis provides insight into the origin of the reactivity differences and makes predictions of how to further exploit these systems in chemical catalysis.

Will 1,2-dihydro-1,2-azaborine-based drugs resist metabolism by cytochrome P450 compound I?

1,2-dihydro-1,2-azaborine is a structural and electronic analogue of benzene which is able to occupy benzene-binding pockets in T4 lysozyme and has been proposed as suitable arene-mimicking group for biological and pharmaceutical applications. Its applicability in a biological context requires it to be able to resist modification by xenobiotic-degrading enzymes like the P450 cytochromes. Quantum chemical computations described in this work show that 1,2-dihydro-1,2-azaborine is much more prone to modification by these enzymes than benzene, unless steric crowding of the ring prevents it from reaching the active site, or otherwise only allows reaction at the less reactive C4-position. This novel heterocyclic compound is therefore expected to be of limited usefulness as an aryl bioisostere.

Evaluating the Disposition of a Mixed Aldehyde Oxidase/Cytochrome P450 Substrate in Rats with Attenuated P450 Activity.

Marketed drugs cleared by aldehyde oxidase (AO) are few, with no known clinically relevant pharmacokinetic drug interactions associated with AO inhibition, whereas cytochrome P450 (P450) inhibition or induction mediates a number of clinical drug interactions. Little attention has been given to the consequences of coadministering a P450 inhibitor with a compound metabolized by both AO and P450. Upon discovering that VU0409106 (1) was metabolized by AO (to M1) and P450 enzymes (to M4-M6), we sought to evaluate the in vivo disposition of 1 and its metabolites in rats with attenuated P450 activity. Male rats were orally pretreated with the pan-P450 inactivator, 1-aminobenzotriazole (ABT), before an i.p. dose of 1. Interestingly, the plasma area under the curve (AUC) of M1 was increased 15-fold in ABT-treated rats, indicating a metabolic shunt toward AO resulted from the drug interaction condition. The AUC of 1 also increased 7.8-fold. Accordingly, plasma clearance of 1 decreased from 53.5 to 15.3 ml/min per kilogram in ABT-pretreated rats receiving an i.v. dose of 1. Consistent with these data, M1 formation in hepatic S9 increased with NADPH-exclusion to eliminate P450 activity (50% over reactions containing NADPH). These studies reflect possible consequences of a drug interaction between P450 inhibitors and compounds cleared by both AO and P450 enzymes. Notably, increased exposure to an AO metabolite may hold clinical relevance for active metabolites or those mediating toxicity at elevated concentrations. The recent rise in clinical drug candidates metabolized by AO underscores the importance of these findings and the need for clinical studies to fully understand these risks.

Origin of the Enhanced Reactivity of μ-Nitrido-Bridged Diiron(IV)-Oxo Porphyrinoid Complexes over Cytochrome P450 Compound I

μ-Nitrido-bridged diiron porphyrins and phthalocyanines are known to be efficient oxidants that are able to oxidize some of the strongest C–H bonds in nature, such as the one in methane. The origin of their catalytic efficiency is poorly understood, and in order to gain insight into the structural and electronic features of this chemical system, we performed a detailed and systematic study into their chemical properties and reactivities using density functional theory. Our work shows that μ-nitrido-bridged diiron porphyrins and phthalocyanines are highly active catalytic oxidants, which react with methane with very low reaction barriers and a rate-determining hydrogen-atom-abstraction step. Furthermore, the μ-nitrido-bridged diiron porphyrin and phthalocyanine complexes react with a free energy of activation that is more than 10 kcal mol–1 lower in energy than that found for cytochrome P450 Compound I, which is known to be one of the most efficient C–H hydroxylating agents in Nature. We have analyzed the ...

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 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.

Characterizing the Intermediates Compound I and II in the Cytochrome P450 Catalytic Cycle with Nonlinear X-ray Spectroscopy: A Simulation Study.

Cytochrome P450 enzymes are an important family of biocatalysts that oxidize chemically inert CH bonds. There are many unresolved questions regarding the catalytic reaction intermediates, in particular P450 Compound I (Cpd-I) and II (Cpd-II). By using simple molecular models, we simulate various X-ray spectroscopy signals, including X-ray absorption near-edge structure (XANES), resonant inelastic X-ray scattering (RIXS), and stimulated X-ray Raman spectroscopy (SXRS) of the low- and high-spin states of Cpd-I and II. Characteristic peak patterns are presented and connected to the corresponding electronic structures. These X-ray spectroscopy techniques are complementary to more conventional infrared and optical spectroscopy and they help to elucidate the evolving electronic structures of transient species along the reaction path.

Decarboxylation of fatty acids to terminal alkenes by cytochrome P450 compound I.

OleT(JE), a cytochrome P450, catalyzes the conversion of fatty acids to terminal alkenes using hydrogen peroxide as a cosubstrate. Analytical studies with an eicosanoic acid substrate show that the enzyme predominantly generates nonadecene and that carbon dioxide is the one carbon coproduct of the reaction. The addition of hydrogen peroxide to a deuterated substrate-enzyme (E-S) complex results in the transient formation of an iron(IV) oxo π cation radical (Compound I) intermediate which is spectroscopically indistinguishable from those that perform oxygen insertion chemistries. A kinetic isotope effect for Compound I decay suggests that it abstracts a substrate hydrogen atom to initiate fatty acid decarboxylation. Together, these results indicate that the initial mechanism for alkene formation, which does not result from oxygen rebound, is similar to that widely suggested for P450 monooxygenation reactions.