P450 Heme Research Articles

The Dissociation Process of NADP+ from NADPH-Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation.

NADPH-cytochrome P450 reductase (CPR) plays a vital role as a redox partner for mammalian cytochrome P450 enzymes (P450s), facilitating the transfer of two electrons from NADPH to the P450 heme center in a sequential manner. Previous experimental studies revealed substantial domain movements of CPR, transitioning between closed and open states during the electron transfer (ET) cycle. These transitions are essential and are influenced by the binding of NADPH or the release of NADP+. However, the intricate molecular mechanisms governing the CPR-mediated ET cycle have largely remained elusive. This study employed molecular dynamics (MD) simulation techniques to explore the dissociation of NADP+ from CPR, a crucial step preceding the initial ET from CPR to a P450. Alongside the binding structure of NADP+ observed in a crystal structure (pose I), our MD simulations identified an alternative binding structure (pose II). Although pose II exhibits slightly lower stability than pose I, it can be formed through an approximate 210° counterclockwise rotation of the adenine group, with a free energy barrier of only 2.76 kcal/mol. The simulation results further suggest that NADP+ dissociation involves a tentative formation of pose II from pose I before complete dissociation, and that the binding of NADP+ to CPR is primarily governed by nonbonded interactions within the adenosine binding pocket.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Association between the oxidative stress gene polymorphism and chronic obstructive pulmonary disease risk: a meta-analysis

BackgroundThe association between the oxidative stress gene polymorphism and chronic obstructive pulmonary disease (COPD) risk has been extensively studied but the results have been controversial. This study aimed to investigate the overall association between the oxidative stress gene including glutathione S-transferase (GST), epoxide hydrolase exon (EPHX), superoxide dismutase (SOD), catalase (CAT), cytochrome P450 system (CYP) and heme oxygenase (HO-1) polymorphism and the risk of COPD.MethodsWe searched the PubMed and EMBASE database to identify studies that investigated the association between the oxidative stress gene polymorphism and risk of COPD. The relevant data were extracted and statistical analyses were performed using the Revman 5.4 and STATA 12 software. Dominant genetic model, recessive model, co-dominant model, heterozygote model, and allele model were analyzed. Venice criteria and publication bias were conducted to access the credibility and reliability.ResultsIn total, 63 publications including 14,733 patients and 50,570 controls were included in the meta-analysis.15 genetic variants of 6 genes were analyzed, and 7 SNPs in GSTP1, CAT, CYP, SOD were first analyses until now. In our study, EPHX T113C C allele, GSTM1 null, GSTT1 null, GSTP1 A313G G and C341T T allele, CYP1A1 MspI C allele, SOD3 A213G G allele and L type in Ho-1 showed increased COPD risk, especially in Asians. T allele in CAT C262T and C allele in SOD2 Val 9 Ala were associated with decreased COPD risk. To avoid high heterogeneity and publications bias, subgroups analysis was performed in accord with HWE and ethnicity. Publication bias was assessed by Begg’s funnel plots and Egger’s test, and no publication bias were found for recessive models. 4 variants were identified with strong levels of epidemiological evidence of associations with the COPD risk.ConclusionsOur results confirm that oxidative stress gene polymorphism was associated with COPD risk. These finding can improve human understanding of this disease gene molecular level and enable early intervention and prevention of COPD. Well-designed studies with large sample sizes are essential to clarify the association of these significant variants with the susceptibility to COPD.

Selective steroidogenic cytochrome P450 haem iron ligation by steroid-derived isonitriles

Alkyl isonitriles, R—NC, have previously been shown to ligate the heme (haem) iron of cytochromes P450 in both accessible oxidation states (ferrous, Fe2+, and ferric, Fe3+). Herein, the preparation of four steroid-derived isonitriles and their interactions with several P450s, including the steroidogenic CYP17A1 and CYP106A2, as well as the more promiscuous drug metabolizers CYP3A4 and CYP2D6, is described. It was found that successful ligation of the heme iron by the isonitrile functionality for a given P450 depends on both the position and stereochemistry of the isonitrile on the steroid skeleton. Spectral studies indicate that isonitrile ligation of the ferric heme is stable upon reduction to the ferrous form, with reoxidation resulting in the original complex. A crystallographic structure of CYP17A1 with an isonitrile derived from pregnanalone further confirmed the interaction and identified the absolute stereochemistry of the bound species.

Cytochrome P460 Cofactor Maturation Proceeds via Peroxide-Dependent Post-translational Modification.

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide. They bear specialized "heme P460" cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link-deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, this proenzyme undergoes maturation to active enzyme with spectroscopic and catalytic properties that match wild-type cyt P460. This maturation reactivity requires no chaperones─it is intrinsic to the protein. This behavior extends to the broader cytochrome c'β superfamily. Accumulated data reveal key contributions from the secondary coordination sphere that enable selective, complete maturation. Spectroscopic data support the intermediacy of a ferryl species along the maturation pathway.

A one-pot Pd- and P450-catalyzed chemoenzymatic synthesis of a library of oxyfunctionalized biaryl alkanoic acids leveraging a substrate anchoring approach

A one-pot chemoenzymatic approach was developed by combining Palladium-catalysis with selective cytochrome P450 enzyme oxyfunctionalization. Various iodophenyl alkanoic acids could be coupled with alkylphenyl boronic acids to generate a series of alkyl substituted biarylalkanoic acids in overall high yield. The identity of the products could be confirmed by various analytical and chromatographic techniques. Addition of an engineered cytochrome P450 heme domain mutant with peroxygenase activity upon completion of the chemical reaction resulted in the selective oxyfunctionalization of those compounds, primarily at the benzylic position. Moreover, in order to increase the biocatalytic product conversion, a reversible substrate engineering approach was developed. This involves the coupling of a bulky amino acid such as L- phenylalanine or tryptophan, to the carboxylic acid moiety. The approach resulted in a 14 to 49% overall biocatalytic product conversion increase associated with a change in regioselectivity of hydroxylation towards less favored positions.

Toxicity prediction using target, interactome, and pathway profiles as descriptors

In silico methods are essential to the safety evaluation of chemicals. Computational risk assessment offers several approaches, with data science and knowledge-based methods becoming an increasingly important sub-group. One of the substantial attributes of data science is that it allows using existing data to find correlations, build strong hypotheses, and create new, valuable knowledge that may help to reduce the number of resource intensive experiments.In choosing a suitable method for toxicity prediction, the available data and desired toxicity endpoint are two essential factors to consider. The complexity of the endpoint can impact the success rate of the in silico models. For highly complex endpoints such as hepatotoxicity, it can be beneficial to decipher the toxic event from a more systemic point of view.We propose a data science-based modelling pipeline that uses compounds` connections to tissue-specific biological targets, interactome, and biological pathways as descriptors of compounds. Models trained on different combinations of the collected, compound-target, compound-interactor, and compound-pathway profiles, were used to predict the hepatotoxicity of drug-like compounds. Several tree-based models were trained, utilizing separate and combined target, interactome and pathway level variables. The model using combined descriptors of all levels and the random forest algorithm was further optimized. Descriptor importance for model performance was addressed and examined for a biological explanation to define which targets or pathways can have a crucial role in toxicity. Descriptors connected to cytochromes P450 enzymes, heme degradation and biological oxidation received high weights. Furthermore, the involvement of other, less discussed processes in connection with toxicity, such as the involvement of RHO GTPase effectors in hepatotoxicity, were marked as fundamental. The optimized combined model using only the selected descriptors yielded the best performance with an accuracy of 0.766.The same dataset using classical Morgan fingerprints for compound representation yielded models with similar performance measures, as well as the combination of systems biology-based descriptors and Morgan fingerprints. Consequently, adding the structural information of compounds did not enhance the predictive value of the models. The developed systems biology-based pipeline comprises a valuable tool in predicting toxicity, while providing novel insights about the possible mechanisms of the unwanted events.

Improved Bioproduction of the Nylon 12 Monomer by Combining the Directed Evolution of P450 and Enhancing Heme Synthesis.

The nylon 12 (PA12) monomer ω-aminododecanoic acid (ω-AmDDA) could be synthesized from lauric acid (DDA) through multi-enzyme cascade transformation using engineered E. coli, with the P450 catalyzing terminal hydroxylation of DDA as a rate-limiting enzyme. Its activity is jointly determined by the heme domain and the reductase domain. To obtain a P450 mutant with higher activity, directed evolution was conducted using a colorimetric high-throughput screening (HTS) system with DDA as the real substrate. After two rounds of directed evolution, a positive double-site mutant (R14R/D629G) with 90.3% higher activity was obtained. Molecular docking analysis, kinetic parameter determination and protein electrophoresis suggested the improved soluble expression of P450 resulting from the synonymous mutation near the N-terminus and the shortened distance of the electron transfer between FMN and FAD caused by D629G mutation as the major reasons for activity improvement. The significantly increased kcat and unchanged Km provided further evidence for the increase in electron transfer efficiency. Considering the important role of heme in P450, its supply was strengthened by the metabolic engineering of the heme synthesis pathway. By combining P450-directed evolution and enhancing heme synthesis, 2.02 ± 0.03 g/L of ω-AmDDA was produced from 10 mM DDA, with a yield of 93.6%.

Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460.

Product selectivity of ammonia oxidation by ammonia-oxidizing bacteria (AOB) is tightly controlled by metalloenzymes. Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO). The non-metabolic enzyme cytochrome (cyt) P460 also oxidizes NH2OH, but instead produces nitrous oxide (N2O). While both enzymes use a heme P460 cofactor, they selectively oxidize NH2OH to different products. Previously reported structures of Nitrosomonas sp. AL212 cyt P460 show that a capping phenylalanine residue rotates upon ligand binding, suggesting that this Phe may influence substrate and/or product binding. Here, we show via substitutions of the capping Phe in Nitrosomonas europaea cyt P460 that the bulky phenyl side-chain promotes the heme-lysine cross-link forming reaction operative in maturing the cofactor. Additionally, the Phe side-chain plays an important role in modulating product selectivity between N2O and NO during NH2OH oxidation under aerobic conditions. A picture emerges where the sterics and electrostatics of the side-chain in this capping position control the kinetics of N2O formation and NO binding affinity. This demonstrates how the outer coordination sphere of cyt P460 is tuned not only for selective NH2OH oxidation, but also for the autocatalytic cross-link forming reaction that imbues activity to an otherwise inactive protein.

The protein conformational basis of isoflavone biosynthesis

Isoflavonoids play important roles in plant defense and also exhibit a range of mammalian health-promoting activities. Their biosynthesis is initiated by two enzymes with unusual catalytic activities; 2-hydroxyisoflavanone synthase (2-HIS), a membrane-bound cytochrome P450 catalyzing a coupled aryl-ring migration and hydroxylation, and 2-hydroxyisoflavanone dehydratase (2-HID), a member of a large carboxylesterase family that paradoxically catalyzes dehydration of 2-hydroxyisoflavanones to isoflavone. Here we report the crystal structures of 2-HIS from Medicago truncatula and 2-HID from Pueraria lobata. The 2-HIS structure reveals a unique cytochrome P450 conformation and heme and substrate binding mode that facilitate the coupled aryl-ring migration and hydroxylation reactions. The 2-HID structure reveals the active site architecture and putative catalytic residues for the dual dehydratase and carboxylesterase activities. Mutagenesis studies revealed key residues involved in substrate binding and specificity. Understanding the structural basis of isoflavone biosynthesis will facilitate the engineering of new bioactive isoflavonoids.

Differences in Enantioselective Hydroxylation of 2,2',3,6-Tetrachlorobiphenyl (CB45) and 2,2',3,4',6-Pentachlorobiphenyl (CB91) by Human and Rat CYP2B Subfamilies.

Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2',3,6-tetrachlorobiphenyl (CB45) and 2,2',3,4',6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4'-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.

Comparing the Impact of NIR, Visible and UV Light on ROS Upregulation via Photoacceptors of Mitochondrial Complexes in Normal, Immune and Cancer Cells.

The effect of UV/visible/NIR light (380/450/530/650/808/1064 nm) on ROS generation, mitochondrial activity and viability is experimentally compared in human neuroblastoma cancer cells. The absorption of photons by mitochondrial photoacceptors in Complexes I, III and IV is in detail investigated by sequential blocking with selective pharmaceutical blockers. Complex I absorbs UV/blue light by heme P450, resulting in a very high rate (14 times) of ROS generation leading to cell death. Complex III absorbs green light, by cytochromes b, c1 and c, and possesses less ability for ROS production (seven times), so that only irradiation lower than 10 mW cm-2 causes an increase in cell viability. Complex IV is well-known as the primary photoacceptor for red/NIR light. Light of 650/808 nm at 10-100 mW cm-2 generates a physiological ROS level about 20% of a basal concentration, which enhance mitochondrial activity and cell survival, while 1064 nm light does not show any distinguished effects. Further, ROS generation induced by low-intensity red/NIR light is compared in neurons, immune and cancer cells. Red light seems to more rapidly stimulate ROS production, mitochondrial activity and cell survival than 808 nm. At the same time, different cell lines demonstrate slightly various rates of ROS generation, peculiar to their cellular physiology.

Protein/protein interactions in the human cytochrome P450 system

Human cytochrome P450 enzymes are heme‐containing monooxygenases that can act on diverse substrates including drugs, steroids, fatty acids, and vitamins. Most of these are microsomal enzymes that require electrons from NADPH‐cytochrome P450 reductase for catalysis. Specifically, the reductase FMN transfers electrons to the P450 heme, but little is known about the details of this transient interaction. Herein the reductase FMN domain was fused to various human P450 enzymes to promote their interaction, and the effects were measured on P450 ligand binding and catalysis occurring in the P450 active site some 15‐20 Å away from the FMN domain/P450 interaction. These FMN domain‐P450 fusion proteins were generated for both steroidogenic (CYP17A1 and CYP21A2) and drug‐metabolizing (CYP3A4, CYP2A6, and CYP2D6) P450 enzymes and compared with the respective isolated P450 enzyme. Fusion of the FMN domain does not appear to change the mode in which ligands bind in the P450 active site. Compounds that bound in the P450 active site either by displacing the heme water (type I interactions) or by directly binding the heme iron (type II interactions) in the isolated P450 enzyme, also did so in the corresponding FMN domain‐P450 fusion enzyme. However, among drug‐metabolizing enzymes fusion of the FMN domain can 1) alter the percentage of the P450 population that binds a particular ligand and 2) increase or decrease the ligand binding affinity, in a P450 and ligand‐specific way. The FMN domain appeared to have significantly less effects on steroidogenic enzymes, regardless of the ligand. Similar catalytic studies are being performed to relate these observed ligand binding trends to substrate metabolism. These current results demonstrate that the effect of the FMN domain is both isoform and ligand dependent, revealing an intricate communication between the P450 surface where the FMN domain binds and the buried P450 active site where substrates and inhibitors bind. This variability suggests that different P450 enzymes interact with the same reductase FMN domain in distinct ways. Since electron delivery is required for catalysis, this knowledge could potentially be exploited to selectively modulate the function of individual P450 enzymes to treat disease.

Deranged hembiosynthetic pathway in gasoline dispensers in Nigeria: Implications for risk of myeloproliferative disorders and chemoprevention

Objectives: There is increasing exposure to petrochemicals, including benzene, particularly in the low and medium-income countries. Benzene is a component of many petrochemicals and a ubiquitous environmental pollutant. Phenol is one of its principal metabolites and serves as a biomarker of exposure to benzene. The mechanism of its toxicity is incompletely elucidated. Benzene’s interaction with key micronutrients; copper (Cu), iron (Fe), and zinc (Zn) in the haemopoietic system has only been poorly explored, particularly in the developing countries where their status is variable and uncertain, with attendant intense exposure to petrochemicals. Material and Methods: Two groups of 50 gasoline dispensers (GDs) and 50 non-occupationally exposed participants were selected from Oye Local Government Area, Nigeria. The duration of occupational exposure was 2–10 years. Serum levels of Cu, Fe, and Zn were determined using flame atomic absorption spectrophotometry while heme and phenol were determined by standard spectrophotometry. Results: Phenol was significantly higher in GDs (P = 0.000), compared to controls (P < 0.05). The micronutrients, Cu, Fe, and Zn were all significantly decreased in GDs compared to controls (P = 0.000 in all cases). Phenol and Fe demonstrated significant inverse correlation (r = −0.557, P = 0.00), while heme and Zn also exhibited inverse correlation respectively to phenol (r = −0.38, P = 0.01; r = −0.37, P = 0.01). Conclusion: These data suggest intense perturbation of the haemopoietic system in GDs; likely from altered xenobiotic metabolism requiring heme in cytochrome P450; cell cycle dysregulation, where Zn is pivotal, p53 suppression also dependent on Zn and oxidative stress all converging in haemopoietic dysregulation. Importantly, depression of these micronutrients implies potentiation of myelotoxicity and risk of myeloproliferation, probably arising from alterations in transcription, differentiation errors, genome instability, and derangement in cell signal transduction moderated by Zn; accentuating risk of myeloproliferation; suggesting a role for these micronutrients in chemoprevention. Understanding these events may be important in risk assessment, policy formulation, regulatory measures and chemoprevention in GDs and the general population.

Protein/Protein Interactions in the Human Cytochrome P450 System

Cytochrome P450 enzymes are the major superfamily of enzymes involved in xenobiotic metabolism and steroid biosynthesis. In vivo, human P450 enzymes are embedded in the lipid membrane and require a membrane-bound redox partner protein for catalysis. This study focuses on P450 enzyme interactions with their redox partner, cytochrome P450 reductase. This protein is the required redox partner for 50 of the 57 human P450 enzymes. It accepts electrons from NADPH via its FAD domain, transfers them to its FMN domain, and then supplies those electrons to the P450 heme to support P450 catalysis. The FMN domain binds directly to the proximal face of P450 enzymes, but this interaction is transient and little is known about the details of the interactions of the FMN domain and different human P450 enzymes. Thus, this study sought to better define the structural interactions and the functional implications of the reductase FMN domain binding to multiple human P450 enzymes, including P450 enzymes responsible for both steroidogenic and xenobiotic metabolism. To facilitate interactions of the reductase FMN domain and the different P450 enzymes, artificial fusion proteins were generated. The fusion proteins were successfully expressed in E. coli and purified. Binding studies and substrate metabolism assays with typical ligands were used to compare the functions of these fusion proteins with the respective isolated P450 enzyme. These fusions are also being used to generate X-ray structures to reveal the specific interactions between the reductase FMN domain and each different P450 enzyme. While the P450 proximal surface is thought to be the general binding surface for the reductase FMN domain, we expect to observe variations in the specific FMN domain/P450 interactions that may correlate with functional differences between the enzymes.

Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1.

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR). As the POR concentration is limiting when compared with P450 and HO-1, they must effectively compete for POR to function. In addition to these functionally required protein-protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, 'How are the HO-1 and P450 systems organized in the endoplasmic reticulum?' Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins. The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner. Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR. The POR•CYP1A1 complex was readily disrupted by the addition of HO-1. CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller. This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1. Based on the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

Human Cytochrome P450 2C9 and its Polymorphic Modifications: Electroanalysis, Catalytic Properties and Approaches to the Regulation of Enzymatic Activity

The electrochemical properties of cytochrome P450 2C9 (CYP2C9) and polymorphic modifications P450 2C9*2 (CYP2C9*2) and P450 2C9*3 (CYP2C9*3) were studied. To analyze the comparative catalytic activity, the enzymes were immobilized on electrodes modified with a membrane-like synthetic surfactant (didodecyldimethylammonium bromide (SPE/DDAB). Peaks of reduction and oxidation of the heme iron, which confirms the adequate choice of the type of modified electrode, characterize cyclic voltammograms of cytochromes P450 under anaerobic conditions. The midpoint potential, Emid, of cytochrome P450 2C9 was -0.318 ± 0.01 V, and Emid =-0.324 ± 0.01 V, and Emid = -0.318 ± 0.03 V for allelic variant 2C9*2 and allelic variant 2C9*3, respectively. In the presence of substrate diclofenac under aerobic conditions, cytochrome P450 2C9 and its polymorphic modifications P450 2C9*2 and P450 2C9*3 exhibit catalytic properties registered by increasing the catalytic current. The amplitude ratio of diclofenac/oxygen currents ((IDF/IO2, catalytic index) is 1.80 for cytochrome P450 2C9, 1.33 and 2.18 for isoforms 2C9 *2 and 2C9*3, respectively. Antioxidant medications mexidol and taurine (at a concentration of 98 μM and 100 μM, respectively) stimulated aerobic reduction of cytochromes P450 heme iron. In the presence of mexidol, an increase in the catalytic activity of cytochrome P450 2C9 in relation to diclofenac by 1.7 times is registered. Taurine, vitamin-like compound with antioxidant properties also stimulates the metabolism of diclofenac by 1.5 times.

Heme P460: A (Cross) Link to Nitric Oxide.

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2-) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2-. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c-heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea, which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH2OH and were both originally reported to produce NO2-. Each can inspire means to effect controlled release of chemical energy.Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2- was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O2, thus producing NO2- abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2- is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2- enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen "defixation".Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH2OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NH2OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-links. The heme-Lys cross-link of cyt P460 enforces the relative position of the cofactor and second-sphere residues. Moreover, the cross-link prevents the dissociation of the axial histidine residue, which stops catalysis, emphasizing the importance of this unique post-translational modification.

The nitrosoamphetamine metabolite is accommodated in the active site of human hemoglobin: Spectroscopy and crystal structure

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group. We report the 2.15 Å resolution X-ray crystal structure of the AmphNO adduct of adult human hemoglobin as the Hb [α-FeIII(H2O)][β-FeII(AmphNO)] derivative. We show that the binding of AmphNO to the β subunit is enabled by an E helix movement and stabilization of ligand binding by H-bonding with the distal His63 residue. We also observe an AmphNHOH group in the Xe2 pocket in close proximity to the α heme site in this derivative. Additionally, UV–vis spectroscopy was used to characterize this and related wt and mutant Mb adducts. Importantly, our X-ray crystal structure of this Hb-nitrosoamphetamine complex represents the first crystal structure of a wild-type heme protein adduct of any amphetamine metabolite. Our results provide a framework for further studies of AmphNHOH/AmphNO interactions with Hb and Mb as viable processes that potentially contribute to the overall biological inorganic chemistry of amphetamine drugs.