P-450 Model Research Articles

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study.

Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured Kd values are highly dependent on the redox state of CPR. It is shown that CPRox/CYP2C9 complexes have a much higher dissociation constant than CPR2-/CYP2C9 or CPR4-/CYP2C9 complexes, and a model is presented to account for these results. An Arrhenius analysis of diffusion constants was also carried out, demonstrating that the reduced forms of CPR and CYP2C9 interact differently with the biomimetic ER and may, in addition to protein conformational changes, contribute to the observed NADPH-dependent shift in Kd. Finally, it is also shown that the CPRox/CYP2C9 affinity depends on the nature of the ligand, being higher when a substrate is bound, compared to an inhibitor.

A siloxyl bis-pocket thiolate-tailed Fe(III) porphyrin complex

We report the synthesis and characterization of a bis-pocket iron(III) porphyrin complex that has a covalently attached siloxy thiolate group as the axial ligand. As a new cytochrome P450 model compound, this siloxyl bis-pocket thiolate-tailed iron(III) porphyrin is easily synthesized and is surprisingly stable under air due to the extreme steric protection of the siloxy pockets on both faces of the porphyrin. A single-crystal XRD structure has been determined; the Fe–S bond distance is 2.237 (7) Å with Fe–N bond distances of 2.100 (8) and the Fe is 0.5 Å out of the mean porphyrin plane. The Fe–S bond distance in the siloxy thiolate-tailed iron(III) porphyrin is very similar to that in cytochrome P450 and this structure represents a very rare crystallographically-characterized five-coordinate high spin alkylthiolate-tailed ferric porphyrin. EPR spectrum of this compound showed g values and an E/D ratio very similar to those of cytochrome P450 (CYP450) enzyme, demonstrating the importance of using a very basic thiolate group as the axial ligand in P450 model studies. UV-vis studies of its reduced form with carbon monoxide shows a hyper spectrum, which is characteristic of carbonyl complexes of Fe(II)porphyrin thiolates.

Catalytic activity of biomimetic model of cytochrome P450 in oxidation of dopamine

The introduction of electron-withdrawing group into porphyrin molecule as cytochrome P450 model can tune the energy level and have an effect on the electronic structure. In this work, linking with the strong electron-withdrawing fluorine atoms, a starburst dendritic molecule, 5,10,15,20-tetrakis(pentafluorophenyl)−21H,23H-porphyrin iron (III) chloride (FeTFPP), containing a saddle-shaped porphyrin as the central core and four pentafluorophenyl rings as the peripheral functional groups was successfully synthesized. Subsequently, the macrocyclic conjugate polymer film of FeTFPP was achieved via a low-cost electrochemical method and exploited as an efficient mimetic enzyme. Furthermore, a biomimetic sensor was constructed by the poly(FeTFPP) film and graphene (rGO) sheet (rGO-poly(FeTFPP)) for selective and sensitive detection of dopamine (DA). Here, the FeTFPP polymer performs three functions: electrochemical recognition (owing to the hydrogen bonding between the strongly electronegative fluorine atoms and DA), biomimetic microenvironment (owing to interaction between porphyrin core and DA), electrocatalysis (owing to remarkable catalytic ability of iron (III) ion). Under optimum conditions, the response to DA was linear in the concentration range between 0.05 to 300μM, and the detection limit was 0.023μM. In addition, we applied the rGO-poly(FeTFPP) film to detect DA in real samples and the results implied its feasibility for practical application. As a result, it is believed that the rGO-poly(FeTFPP) film is one of the promising biomimetic catalysts for electrocatalysis and relevant fields.

Molecular cloning and 3D model of first cytochrome P450 from CYP3A subfamily in saltwater crocodile (Crocodylus porosus)

Cytochrome P450s (CYPs) play critical role in oxidative metabolism of numerous xenobiotics and endogenous compounds. The first CYP3A subfamily member in saltwater crocodile has been cloned and modelled for three-dimensional (3D) structure. The full-length cDNA was obtained employing reverse transcription polymerase chain reaction (RT-PCR) strategy and rapid amplification of cDNA ends (RACE). The cDNA sequence of 1659 nucleotides includes 132 nucleotides from 5′ untranslated region (UTR), an open reading frame of 1527 nucleotides encoding 509 amino acids designated as CYP3A163. The alignment of CYP3A163 sequence with CYP3A subfamily across the lineages exhibit the loss of 1 residue in birds and 7 residues in mammals in comparison to reptiles suggesting the adaptation processes during evolution. The amino acid identity of CYP3A163 with Alligator mississippiensis CYP3A77 and Homo sapiens CYP3A4 is 91% and 62% respectively. The 3D structure of CYP3A163 modelled using human CYP3A4 structure as a template with Phyre2 software, represents high similarity with its functionally important motifs and catalytic domain. Both sequence and structure of CYP3A163 display the common and conserved features of CYP3A subfamily. Overall, this study provides primary molecular and structural data of CYP3A163 required to investigate the xenobiotic metabolism in saltwater crocodiles.

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Previous chemical rescue studies identified a putative FeIII-O2− intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.

Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle.

The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, six-coordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme-thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, six-coordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80-90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.

Tunneling Effect That Changes the Reaction Pathway from Epoxidation to Hydroxylation in the Oxidation of Cyclohexene by a Compound I Model of Cytochrome P450.

The rate constants of the C═C epoxidation and the C-H hydroxylation (i.e., allylic C-H bond activation) in the oxidation of cyclohexene by a high-valent iron(IV)-oxo porphyrin π-cation radical complex, [(TMP•+)FeIV(O)(Cl)] (1, TMP = meso-tetramesitylporphyrin dianion), were determined at various temperatures by analyzing the overall rate constants and the products obtained in the cyclohexene oxidation by 1, leading us to conclude that reaction pathway changes from the C═C epoxidation to C-H hydroxylation by decreasing reaction temperature. When cyclohexene was replaced by deuterated cyclohexene (cyclohexene-d10), the epoxidation pathway dominated irrespective of the reaction temperature. The temperature dependence of the rate constant of the C-H hydroxylation pathway in the reactions of cyclohexene and cyclohexene-d10 by 1 suggests that there is a significant tunneling effect on the hydrogen atom abstraction of allylic C-H bonds of cyclohexene by 1, leading us to propose that the tunneling effect is a determining factor for the switchover of the reaction pathway from the C═C epoxidation pathway to the C-H hydroxylation pathway by decreasing reaction temperature. By performing density functional theory (DFT) calculations, the reaction energy barriers of the C═C epoxidation and C-H bond activation reactions by 1 were found to be similar, supporting the notion that small environmental changes, such as the reaction temperature, can flip the preference for one reaction to another.

A Systematic Account on Aromatic Hydroxylation by a Cytochrome P450 Model Compound I: A Low-Pressure Mass Spectrometry and Computational Study.

Cytochrome P450 enzymes are heme-containing mono-oxygenases that mainly react through oxygen-atom transfer. Specific features of substrate and oxidant that determine the reaction rate constant for oxygen atom transfer are still poorly understood and therefore, we did a systematic gas-phase study on reactions by iron(IV)-oxo porphyrin cation radical structures with arenes. We present herein the first results obtained by using Fourier transform-ion cyclotron resonance mass spectrometry and provide rate constants and product distributions for the assayed reactions. Product distributions and kinetic isotope effect studies implicate a rate-determining aromatic hydroxylation reaction that correlates with the ionization energy of the substrate and no evidence of aliphatic hydroxylation products is observed. To further understand the details of the reaction mechanism, a computational study on a model complex was performed. These studies confirm the experimental hypothesis of dominant aromatic over aliphatic hydroxylation and show that the lack of an axial ligand affects the aliphatic pathways. Moreover, a two-parabola valence bond model is used to rationalize the rate constant and identify key properties of the oxidant and substrate that drive the reaction. In particular, the work shows that aromatic hydroxylation rates correlate with the ionization energy of the substrate as well as with the electron affinity of the oxidant.

Valence tautomerism in synthetic models of cytochrome P450

CytP450s have a cysteine-bound heme cofactor that, in its as-isolated resting (oxidized) form, can be conclusively described as a ferric thiolate species. Unlike the native enzyme, most synthetic thiolate-bound ferric porphyrins are unstable in air unless the axial thiolate ligand is sterically protected. Spectroscopic investigations on a series of synthetic mimics of cytP450 indicate that a thiolate-bound ferric porphyrin coexists in organic solutions at room temperature (RT) with a thiyl-radical bound ferrous porphyrin, i.e., its valence tautomer. The ferric thiolate state is favored by greater enthalpy and is air stable. The ferrous thiyl state is favored by entropy, populates at RT, and degrades in air. These ground states can be reversibly interchanged at RT by the addition or removal of water to the apolar medium. It is concluded that hydrogen bonding and local electrostatics protect the resting oxidized cytP450 active site from degradation in air by stabilizing the ferric thiolate ground state in contrast to its synthetic analogs.

Novel nickel(II) and manganese(III) complexes with bidentate Schiff-base ligand: synthesis, spectral, thermogravimetry, electrochemical and electrocatalytical properties

An unsymmetrical bidentate Schiff base ligand, ethane 2-(4-methoxyphenyl)-1-iminosalicylidene, and its novel two mononuclear complexes, Nickel(II) [Ni(II)-2L] and Manganese(III) [Mn(III)Cl-2L] where L represents the ligand, have been synthesized and characterized by various physicochemical methods. The Ni(II) ion is coordinated by two nitrogen and two oxygen atoms with both the bidentate Schiff base ligands in an approximately square planar coordination geometry, while the manganese complex, the Mn(III) ion, is involved in an additional contact with one chloride anion for which the coordination sphere appears as a square pyramidal arrangement. The thermogravimetric analyses of the synthesized compounds revealed three different stages of decomposition for NONO bis-bidentate manganese and nickel complexes. The cyclic voltammetry studies of these complexes in N,N-dimethylformamide showed a redox couple for each one of them, such as Ni(II)/Ni(I) and Mn(III)/Mn(II), which are quasi-reversible. Their catalytic behaviors were tested showing that the nickel complex is an effective electrocatalyst in the reduction of bromocyclopentane. Regarding the manganese complex, it was revealed that it is an efficient catalyst in the activation of molecular dioxygen, currently applied in oxidation reactions of hydrocarbons according to the monooxygenase enzymes as those of cytochrome P450 model.

Physiologically Based Pharmacokinetic (PBPK) Modeling and Simulation Approaches: A Systematic Review of Published Models, Applications, and Model Verification.

Modeling and simulation of drug disposition has emerged as an important tool in drug development, clinical study design and regulatory review, and the number of physiologically based pharmacokinetic (PBPK) modeling related publications and regulatory submissions have risen dramatically in recent years. However, the extent of use of PBPK modeling by researchers, and the public availability of models has not been systematically evaluated. This review evaluates PBPK-related publications to 1) identify the common applications of PBPK modeling; 2) determine ways in which models are developed; 3) establish how model quality is assessed; and 4) provide a list of publically available PBPK models for sensitive P450 and transporter substrates as well as selective inhibitors and inducers. PubMed searches were conducted using the terms "PBPK" and "physiologically based pharmacokinetic model" to collect published models. Only papers on PBPK modeling of pharmaceutical agents in humans published in English between 2008 and May 2015 were reviewed. A total of 366 PBPK-related articles met the search criteria, with the number of articles published per year rising steadily. Published models were most commonly used for drug-drug interaction predictions (28%), followed by interindividual variability and general clinical pharmacokinetic predictions (23%), formulation or absorption modeling (12%), and predicting age-related changes in pharmacokinetics and disposition (10%). In total, 106 models of sensitive substrates, inhibitors, and inducers were identified. An in-depth analysis of the model development and verification revealed a lack of consistency in model development and quality assessment practices, demonstrating a need for development of best-practice guidelines.

Manganese porphyrin functionalized on Fe3O4@nSiO2@MCM-41 magnetic composite: Structural characterization and catalytic activity as cytochrome P450 model

This study reports on the preparation of heterogeneous catalysts with two different pore sizes, the Fe3O4@nSiO2@MCM-41-MnP and Fe3O4@nSiO2@MCM-41(E)-MnP catalysts. Such systems ally the catalytic properties of metalloporphyrins (MeP) with the magnetic properties of magnetite nanoparticles (Fe3O4) in a mesoporous MCM-41 silica matrix. The synthesis of Fe3O4 nanoparticles was followed by the surface coating with a thin silica layer (Fe3O4@nSiO2). Then, an MCM-41-type ordered mesoporous silica structure was grown over the initial particles in the presence of CTAB as surfactant and TEOS as the silica precursor, to yield the Fe3O4@nSiO2@MCM-41 solid. In this step, mesitylene can also be incorporated as a structure expanding agent for the production of larger pores, thus yielding the Fe3O4@nSiO2@MCM-41(E) solids. The resulting composites were further functionalized with aminopropyl groups by the silylating agent APTES. This enabled the covalent immobilization of manganese(III)-5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (Mn(TPF5PP)) onto the composites via a aromatic nucleophilic substitution reaction, to afford the Fe3O4@nSiO2@MCM-41-MnP and Fe3O4@nSiO2@MCM-41(E)-MnP catalysts. Characterization of the catalysts by XRD, UV–Vis, DR UV–vis, FTIR, SEM, TEM, VSM and N2 adsorption/desorption isotherms, aided understanding the catalyst structure and morphology. The catalytic activity of the catalysts in hydrocarbon oxidation ((Z)-cyclooctene and cyclohexane) was evaluated; iodosylbenzene (PhIO) was used as the oxygen donor agent. The catalytic tests demonstrated higher yields of epoxide for Fe3O4@nSiO2@MCM-41(E)-MnP than Fe3O4@nSiO2@MCM-41-MnP catalyst, which can be attributed to the larger pore sizes. Both catalysts were selective for the cyclohexanol product, thus indicating a P450-type biomimetic behavior.

Effect of axial ligand, spin state, and hydrogen bonding on the inner-sphere reorganization energies of functional models of cytochrome P450.

Using a combination of self-assembly and synthesis, bioinspired electrodes having dilute iron porphyrin active sites bound to axial thiolate and imidazole axial ligands are created atop self-assembled monolayers (SAMs). Resonance Raman data indicate that a picket fence architecture results in a high-spin (HS) ground state (GS) in these complexes and a hydrogen-bonding triazole architecture results in a low-spin (LS) ground state. The reorganization energies (λ) of these thiolate- and imidazole-bound iron porphyrin sites for both HS and LS states are experimentally determined. The λ of 5C HS imidazole and thiolate-bound iron porphyrin active sites are 10-16 kJ/mol, which are lower than their 6C LS counterparts. Density functional theory (DFT) calculations reproduce these data and indicate that the presence of significant electronic relaxation from the ligand system lowers the geometric relaxation and results in very low λ in these 5C HS active sites. These calculations indicate that loss of one-half a π bond during redox in a LS thiolate bound active site is responsible for its higher λ relative to a σ-donor ligand-like imidazole. Hydrogen bonding to the axial ligand leads to a significant increase in λ irrespective of the spin state of the iron center. The results suggest that while the hydrogen bonding to the thiolate in the 5C HS thiolate bound active site of cytochrome P450 (cyp450) shifts the potential up, resulting in a negative ΔG, it also increases λ resulting in an overall low barrier for the electron transfer process.

Development and validation of a liquid-chromatography high-resolution tandem mass spectrometry approach for quantification of nine cytochrome P450 (CYP) model substrate metabolites in an in vitro CYP inhibition cocktail.

Knowledge about the cytochrome P450 (CYP) inhibition potential of new drug candidates is important for drug development because of its risk of interactions. For novel psychoactive substances (NPS), corresponding data are not available. For developing a general drug inhibition cocktail assay, a liquid-chromatography high-resolution tandem mass spectrometry multi-analyte approach was developed and validated for quantifying low concentrations of O-diethyl phenacetin for CYP 1A2, 7-hydroxy coumarin for CYP 2A6, 4-hydroxy bupropion for CYP 2B6, N-diethyl amodiaquine for CYP 2C8, 4-hydroxy diclofenac for CYP 2C9, 5-hydroxy omeprazole for CYP 2C19, O-dimethyl dextromethorphan for CYP 2D6, 6-hydroxy chlorzoxazone for CYP 2E1, and 6-beta-hydroxy testosterone for CYP 3A in the incubation mixture in the presence of substrates and inhibitors. The tested matrix effects ranged from 63 to 141 % and the recoveries from 95 to 110 %. Time-saving one-point calibration allowed sufficient quantification, although some of the validation results for 7-hydroxy coumarin, 4-hydroxy bupropion, 4-hydroxy diclofenac, and 6-beta-hydroxy testosterone were outside the acceptance criteria (AC) but without influence of the IC50 calculation. Validation showed also that the approach was sensitive and selective using mass spectral multiplexing. In conclusion, the presented assay was suitable for the quantification of the model substrate metabolites and could be used for the development of a CYP inhibition assay for testing most CYPs and a wide range of drugs of abuse.

Modeling P450 SItes of Metabolism (LB596)

Our group has been working on ways to improve the design and use of P450 modeling technologies. This is important because a large proportion of drugs are metabolized by P450 enzymes, and variants in P450s commonly cause patients to respond differently to medicines. We are pursuing two innovations in this area. First, we have developed P450 models that can predict what atoms are metabolized on molecules based on their structure. Our approach uses training data, molecules where the sites of metabolism are known, to build a predictive model that can accurately predict how new molecules are metabolized by 9 separate P450 isozymes. Knowing which atoms are modified‐‐‐the sites of P450 metabolism‐‐‐enables chemists to modify molecules to improve their metabolic profiles. Second, we developed a strategy for that can make use of region‐resolution site‐of‐metabolism data, instead of exclusively relying on the atom‐resolution data to which prior methods are confined. This unique capability can be leveraged to improve the accuracy of prediction of sites of metabolism on molecule, until now, impossible to predict. Our hope is to extend this technology to model how P450 variants metabolize drugs differently from one another. Effective modeling of metabolism can help overcome the central challenges in modern drug discovery and development: reducing the cost and risk of developing new drugs by ensuring the right medicines are given to the right patients.

English

The alcohol-extraction-water-precipitation fraction of Swertia mussotii Franch. (SME-d) had been proved to be hepatoprotective without toxicity in previous report. In this article, high performance liquid chromatography (HPLC), rat experiment and P450 model tests were employed for studying the pharmacology characteristics of SME-d. The results showed that the contents of sweroside, swertiamarin, mangiferin, gentiopicroside, and isoorientin were 0.24, 3.96, 12.30, 13.53, 16.85 mg/g in SME-d, respectively. SME-d could reduce the CCl4-induced exaltation of alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), total bile acid (TBA) significantly in rat, and the protective activity showed dose-dependent in 0.9 and 1.8 g/kg body weight (BW). The hepatoprotective activity of SME-d was different to positive drug bifendate, which only did on ALT value significantly. Bifendate could inhibit 66.17 ± 2.12% of CYP3A4 activity, while SME-d showed 99.0 ± 0.267% reductions on CYP1A2. The different medicinal characteristics of SME-d to bifendate, which are used widely to cure hepatitis in china, can give more choices for hepatitis. Key words: Swertia mussotii Franch, the alcohol-extraction-water-precipitation fraction, pharmacology characteristic, hepatoprotective activity.

New QSAR Models for Human Cytochromes P450, 1A2, 2D6 and 3A4 Implicated in the Metabolism of Drugs. Relevance of Dataset on Model Development.

Discarding drug-candidates with potential metabolism issues or with significant drug-drug interactions (DDI) is of major importance in drug discovery projects. When a compound is metabolized by catalytic enzymes, especially CYP450 3A4, the risk of DDI is potentially high. [1] An important point in the ADME studies is the characterization of the enzymes involved in the metabolism of drugs. Among these enzymes, the human cytochrome P450 family (CYP), more specifically 1A2, 2C’s, 2D6 and 3A4, are the most intensively studied [2] and QSAR approaches have shown to be valuable for prediction studies. [3] In many of these stud

Mono- and bis-phosphine-ligated H93G myoglobin: Spectral models for ferrous-phosphine and ferrous-CO cytochrome P450

To further investigate the properties of phosphines as structural and functional probes of heme proteins, mono- and bis-phosphine [tris(hydroxymethyl)phosphine, THMP] adducts of H93G myoglobin (Mb) have been prepared by stepwise THMP titrations of exogenous ligand-free ferric and ferrous H93G Mb, respectively. Bubbling with CO or stepwise titration with imidazole (Im) of the bis-THMP-ligated ferrous protein generated a mixed ligand (THMP/CO or THMP/Im, respectively) ferrous complexes. Stable oxyferrous H93G(THMP) Mb was formed at −40°C by bubbling the mono-THMP-Fe(II) protein with O2. A THMP-ligated ferryl H93G Mb moiety has been partially formed upon addition of H2O2 to the ferric mono-THMP adduct. All the species prepared above have been characterized with UV–visible (UV–vis) absorption and magnetic circular dichroism (MCD) spectroscopy in this study. The six-coordinate ferrous bis-phosphine and mono-phosphine/CO complexes of H93G Mb exhibit characteristic spectral features (red-shifted Soret/unique-shaped MCD visible bands and hyperporphyrin spectra, respectively) that only have been seen for the analogous phosphine or CO-complexes of thiolate-ligated heme proteins such as cytochrome P450 (P450) and Caldariomyces fumago chloroperoxidase (CPO). However, such resemblance is not seen in phosphine-ligated ferric H93G Mb even though phosphine-bound ferric P450 and CPO display hyperporphyrin spectra. In fact, bis-THMP-bound ferric H93G Mb exhibits MCD and UV–vis absorption spectra that are similar to those of bis-amine- and bis-thioether-ligated H93G Mb complexes. This study also further demonstrates the utility of the H93G cavity mutant for preparing novel heme iron coordination structures.

(Invited) Synthetic Heme Thiolate Complexes as Precise Model of Cytochrome P450

not Available.

Ruthenium porphyrin and oxidant convert N-nitrosodialkylamines into direct-acting mutagen in the Ames assay

The Ames assay is used for short-term screening of mutagens/carcinogens that induce DNA damage. Most mutagens/carcinogens require enzymatic activation through oxidation by cytochrome P450 in an S-9 mix to exert their mutagenicity. Chemical models for cytochrome P450, consisting of water-soluble or water-insoluble iron porphyrin plus an oxidant, have been used to produce frameshift mutagens from aromatic amines, heterocyclic amines and polyaromatic hydrocarbons. In this study, the mutagenicity of N-nitrosodialkylamines was assayed in the presence of a chemical model, which consists of 5,10,15,20-tetrakis(2,4,6-trimethylphenyl)porphyrinatoruthenium(IV) dichloride (RuMe3) and 2,6-dichloropyridine N-oxide (Cl2pyNO). The chemical model activated symmetrical N-nitrosodialkylamines (alkyl = methyl, ethyl, propyl, butyl), and unsymmetrical N-nitroso-N-methylalkylamine (alkyl = propyl, butyl) in Salmonella typhimurium YG7108. Furthermore, the mutagenicity of N-nitrosodipropylamine (NDP) in S. typhimurium YG7108 was higher than that in S. typhimurium TA1535, suggesting that the mutagenicity derived from NDP using the chemical model was due to DNA alkylation. The results showed that the chemical model can activate N-nitrosodialkylamines to induce base substitution mutations.