The Experts below are selected from a list of 36408 Experts worldwide ranked by ideXlab platform
Alexander I. Archakov - One of the best experts on this subject based on the ideXlab platform.
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Electrochemical sensor systems based on one dimensional (1D) nanostructures for analysis of bioaffinity interactions
Biochemistry (moscow) Supplement Series B: Biomedical Chemistry, 2012Co-Authors: Victoria V. Shumyantseva, Elena V. Suprun, Tatiana V. Bulko, Alexander I. ArchakovAbstract:Modification of screen printed graphite electrodes (SPE) with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) enhances such analytical characteristics of the sensor as effective surface area, electrocatalytic properties and kinetics of heterogeneous electron transfer. The reason for such improvement may be associated with the synergistic effect of AuNPs and PbNWs. Nanowires ensembles on the electrode surface were employed for the detection of cardiac myoglobin in human blood, Cytochrome P450 2B4, Cytochrome c. Composite materials based on nanoparticles with different dimensions (three dimensional (3D) gold nanoparticles and one dimensional (1D) Pb nanowires) create a platform for electrochemical analysis of proteins with low detection limits.
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Stoichiometry of electrocatalytic cycle of Cytochrome P450 2B4.
Journal of inorganic biochemistry, 2008Co-Authors: Yuri O. Rudakov, Victoria V. Shumyantseva, Elena V. Suprun, Tatiana V. Bulko, N. F. Samenkova, G. P. Kuznetsova, Alexander I. ArchakovAbstract:Abstract Stoichiometry of the electrocatalytical cycle of Cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized Cytochrome P450 2B4 were used as the operating electrode (at the potential E 0′ = −450 mV) and electrodes, modified with Cytochrome c ( E 0′ = −50 mV) or Prussian Blue ( E 0′ = 0), as measuring electrodes (for H 2 O 2 ) and Clark-type electrode (for O 2 ). Benzphetamine N -demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.
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Direct electron transfer of Cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles.
Analytical chemistry, 2004Co-Authors: Victoria V. Shumyantseva, Alexander I. Archakov, Yuri D. Ivanov, Nikitas Bistolas, Frieder W. Scheller, Ulla WollenbergerAbstract:A method for construction of biosensors with membranous Cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated Cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between −0.292 and −0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of p...
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Fluorescent assay for riboflavin binding to Cytochrome P450 2B4.
Journal of inorganic biochemistry, 2004Co-Authors: Victoria V. Shumyantseva, Tatiana V. Bulko, N. A. Petushkova, N. F. Samenkova, G. P. Kuznetsova, Alexander I. ArchakovAbstract:Abstract The interactions between the hemoprotein Cytochrome P450 2B4 (CYP 2B4) and riboflavin – a low molecular weight component of the flavoprotein NADPH-dependent Cytochrome P450 reductase – were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by Cytochrome P450 2B4 was used to probe the ligand–enzyme binding ( λ ex =385 nm, λ em =520 nm). Fluorescence titration experiments showed formation of a complex between Cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K d =8.8 ± 1 μM. The fluorescence intensity of riboflavin was decreased with increasing the Cytochrome P450 2B4 concentration, indicating the transfer of resonance excitation energy from riboflavin (energy donor) to the Cytochrome P450 2B4 heme (energy acceptor). The data obtained are suggestive of the existence of riboflavin binding site(s) on the hemeprotein molecule.
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Study of interaction of Cytochrome P450 2B4 with riboflavin by fluorescence spectrometry
Biofizika, 2004Co-Authors: N. A. Petushkova, Alexander I. ArchakovAbstract:Fluorescence quenching of riboflavin by Cytochrome P450 2B4 was used to probe the ligand--enzyme binding interaction ((lambda ex = 385 nm, lambda em = 520 nm). Riboflavin is a component of a flavoprotein NADPH dependent Cytochrome P450 reductase, an essential electron carrier during Cytochrome P450 catalysis. Fluorescence titration measurements revealed that Cytochrome P450 2B4 and riboflavin formed a complex with an apparent Kd = 8.8 +/- 1 microM. The fluorescence intensity of riboflavin decreased upon the addition of Cytochrome P450 2B4, which may be caused by the resonance excitation energy transfer from the fluorescent donor riboflavin to the Cytochrome P450 2B4 heme acceptor. These data suggest that there may exist specific sites of binding of riboflavin with the protein globule of Cytochrome P450 2B4.
James R Halpert - One of the best experts on this subject based on the ideXlab platform.
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effect of detergent binding on Cytochrome P450 2B4 structure as analyzed by x ray crystallography and deuterium exchange mass spectrometry
Biophysical Chemistry, 2016Co-Authors: Manish B. Shah, Qinghai Zhang, David C Stout, Hyun-hee Jang, Ross P Wilderman, David Lee, James R HalpertAbstract:Abstract Multiple crystal structures of CYP2B4 have demonstrated the binding of the detergent 5-cyclohexyl-1-pentyl-β-D-maltoside (CYMAL-5) in a peripheral pocket located adjacent to the active site. To explore the consequences of detergent binding, X-ray crystal structures of the peripheral pocket mutant CYP2B4 F202W were solved in the presence of hexaethylene glycol monooctyl ether (C8E6) and CYMAL-5. The structure in the presence of CYMAL-5 illustrated a closed conformation indistinguishable from the previously solved wild-type. In contrast, the F202W structure in the presence of C8E6 revealed a detergent molecule that coordinated the heme-iron and extended to the protein surface through the substrate access channel 2f. Despite the overall structural similarity of these detergent complexes, remarkable differences were observed in the A, A′, and H helices, the F-G cassette, the C-D and β4 loop region. Hydrogen-deuterium exchange mass spectrometry (DXMS) was employed to probe these differences and to test the effect of detergents in solution. The presence of either detergent increased the H/D exchange rate across the plastic regions, and the results obtained by DXMS in solution were consistent in general with the relevant structural snapshots. The study provides insight into effect of detergent binding and the interpretation of associated conformational dynamics of CYP2B4.
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the role of Cytochrome P450 2b6 and 2B4 substrate access channel residues predicted based on crystal structures of the amlodipine complexes
Archives of Biochemistry and Biophysics, 2014Co-Authors: Hyun-hee Jang, Dmitri R. Davydov, Gayoung Lee, Chulho Yun, James R HalpertAbstract:Recent X-ray crystal structures of human Cytochrome P450 2B6 and rabbit Cytochrome P450 2B4 in complex with amlodipine showed two bound ligand molecules, one in the active site and one in the substrate access channel. Based on the X-ray crystal structures, we investigated the interactions of P450 2B4 and 2B6 with amlodipine using absorbance spectroscopy, and determined the steady-state kinetics of 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin oxidation by some access channel mutants to evaluate the functional role of these residues in substrate turnover. The results of absorbance titrations are consistent with a simple mechanism with two parallel binding events that result in the formation of the enzyme complex with two molecules of amlodipine. Using this model we were able to resolve two separate ligand-binding events, which are characterized by two distinct KD values in each enzyme. The access channel mutants R73K in P450 2B6 and R73K, V216W, L219W, and F220W in P450 2B4 showed a significant decrease in kcat/KM with the both substrates. Overall, the results suggest that P450 2B4 and 2B6 form an enzyme complex with two molecules of amlodipine in solution, and R73, V216, L219 and F220 in P450 2B4 may play an important role in substrate metabolism.
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Potent mechanism-based inactivation of Cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of Cytochrome P450 catalysis.
Biochemistry, 2013Co-Authors: Haoming Zhang, Manish B. Shah, Qinghai Zhang, C. David Stout, James R Halpert, Maryam Foroozesh, Jiawang Liu, Yoichi Osawa, Paul F. HollenbergAbstract:The mechanism-based inactivation of Cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min–1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, resul...
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X-ray crystal structure of the Cytochrome P450 2B4 active site mutant F297A in complex with clopidogrel: insights into compensatory rearrangements of the binding pocket.
Archives of biochemistry and biophysics, 2013Co-Authors: Manish B. Shah, Qinghai Zhang, C. David Stout, Hyun-hee Jang, James R HalpertAbstract:Prior X-ray crystal structures of Cytochrome P450 2B4 revealed the pivotal role of rearrangement of the side chains of residues F206 and F297 in the active site in accommodating various inhibitors or substrates. To explore the role of these residues, 2B4 F206A and F297A were created by site-directed mutagenesis and characterized functionally. The structure of F297A with clopidogrel demonstrated the reorientation of the ligand such that the methyl ester group is oriented toward the heme, whereas the thiophene moiety now extends to the additional void in the F297A mutant. Most interestingly, movement of the I helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A-virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the real F297A mutant or a virtual mutant with the I-helix re-positioned oriented clopidogrel preferentially with either the methyl ester or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate and interact with the substrate in a distinct orientation while maintaining the overall closed protein conformation.
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Conformational Adaptation of Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4 Revealed Upon Binding Multiple Amlodipine Molecules
Biochemistry, 2012Co-Authors: Manish B. Shah, P. Ross Wilderman, Qinghai Zhang, C. David Stout, Jaime Pascual, James R HalpertAbstract:Structures of human Cytochrome P450 2B6 and rabbit Cytochrome P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine have been determined by X-ray crystallography. The presence of two drug molecules suggests clear substrate access channels in each P450. According to a previously established nomenclature, amlodipine molecules were trapped in access pathway 2f in P450 2B6 and in pathway 2a or 2f in P450 2B4. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described solvent channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B', F, F', and G' and part of helix G is substantially more open in the amlodipine complexes than in the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F', and B' and the β4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states not observed with previous P450 2B structures and provide clear evidence of the substrate access channels in two drug-metabolizing P450s. In addition, the structures exhibit the versatility that can be exploited via in silico studies with other P450 2B6 ligands as large as raloxifene and itraconazole.
Lucy Waskell - One of the best experts on this subject based on the ideXlab platform.
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Structure of Cytochrome P450 2B4 with an acetate ligand and an active site hydrogen bond network similar to oxyferrous P450cam.
Journal of inorganic biochemistry, 2018Co-Authors: Yuting Yang, Jennifer L. Meagher, Jeanne A. Stuckey, Lucy WaskellAbstract:Superposition of the active site of acetate-bound P4502B4 and oxyferrous P450cam. Bond lengths between the heme iron, the sixth ligand, and the hydroxyl of the conserved threonine are shown. Note that different threonine rotamers form the hydrogen bonds to the acetate and oxygen.
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Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs
Angewandte Chemie (International ed. in English), 2018Co-Authors: Carlo Barnaba, Lucy Waskell, Bikash R. Sahoo, Thirupathi Ravula, Ilce Gabriela Medina-meza, Gattadahalli M. Anantharamaiah, Ayyalusamy RamamoorthyAbstract:Although membrane environment is known to boost drug metabolism by mammalian Cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal Cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.
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Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.
Biochemistry, 2016Co-Authors: Roman Davydov, Brian M. Hoffman, Muralidharan Shanmugam, William A. Gunderson, Naw May Pearl, Lucy WaskellAbstract:Crystallographic studies have shown that the F429H mutation of Cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole 14N but has relatively little effect on the 1H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal ...
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Insights into the role of substrates on the interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR.
Scientific reports, 2015Co-Authors: Meng Zhang, Shivani Ahuja, Lucy Waskell, Rui Huang, Stephanie Valerie Le Clair, Ayyalusamy RamamoorthyAbstract:Mammalian Cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to Cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound Cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.
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Insights into the Role of Substrates on the Interaction between Cytochrome b_5 and Cytochrome P450 2B4 by NMR
Scientific Reports, 2015Co-Authors: Meng Zhang, Shivani Ahuja, Lucy Waskell, Rui Huang, Stephanie Valerie Le Clair, Ayyalusamy RamamoorthyAbstract:Mammalian Cytochrome b _5 (cyt b _5) is a membrane-bound protein capable of donating an electron to Cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b _5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b _5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b _5 and full-length substrate-free/substrate-bound Cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b _5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b _5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b _5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b _5-P450 complexation process.
Victoria V. Shumyantseva - One of the best experts on this subject based on the ideXlab platform.
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Electrochemical sensor systems based on one dimensional (1D) nanostructures for analysis of bioaffinity interactions
Biochemistry (moscow) Supplement Series B: Biomedical Chemistry, 2012Co-Authors: Victoria V. Shumyantseva, Elena V. Suprun, Tatiana V. Bulko, Alexander I. ArchakovAbstract:Modification of screen printed graphite electrodes (SPE) with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) enhances such analytical characteristics of the sensor as effective surface area, electrocatalytic properties and kinetics of heterogeneous electron transfer. The reason for such improvement may be associated with the synergistic effect of AuNPs and PbNWs. Nanowires ensembles on the electrode surface were employed for the detection of cardiac myoglobin in human blood, Cytochrome P450 2B4, Cytochrome c. Composite materials based on nanoparticles with different dimensions (three dimensional (3D) gold nanoparticles and one dimensional (1D) Pb nanowires) create a platform for electrochemical analysis of proteins with low detection limits.
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Stoichiometry of electrocatalytic cycle of Cytochrome P450 2B4.
Journal of inorganic biochemistry, 2008Co-Authors: Yuri O. Rudakov, Victoria V. Shumyantseva, Elena V. Suprun, Tatiana V. Bulko, N. F. Samenkova, G. P. Kuznetsova, Alexander I. ArchakovAbstract:Abstract Stoichiometry of the electrocatalytical cycle of Cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized Cytochrome P450 2B4 were used as the operating electrode (at the potential E 0′ = −450 mV) and electrodes, modified with Cytochrome c ( E 0′ = −50 mV) or Prussian Blue ( E 0′ = 0), as measuring electrodes (for H 2 O 2 ) and Clark-type electrode (for O 2 ). Benzphetamine N -demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.
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Nanoelectrochemistry of Cytochrome P450s: Direct electron transfer and electrocatalysis
Biochemistry (moscow) Supplement Series B: Biomedical Chemistry, 2007Co-Authors: Victoria V. Shumyantseva, Tatiana V. Bulko, N. F. Samenkova, G. P. Kuznetsova, I. I. Karuzina, Yu. O. Rudakov, A. V. Lisitsa, A I ArchakovAbstract:Direct electron transfer has been demonstrated between Cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14α-demethylase (CYP51MT) and screen printed graphite electrodes, modified by gold nanoparticles and didodecyldimethyl ammonium bromide (DDAB). The proposed method for preparation of enzymatic nanostructured electrodes may be used for electrodetection of this hemoprotein provided that 2–200 pmol P450 per electrode has been adsorbed.
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Nanoelectrochemistry of Cytochrome P450s: Direct electron transfer and electrocatalysis
Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2007Co-Authors: Victoria V. Shumyantseva, Tatiana V. Bulko, N. F. Samenkova, G. P. Kuznetsova, I. I. Karuzina, Yu. O. Rudakov, A. V. Lisitsa, A. I. ArchakovAbstract:Direct electron transfer has been demonstrated between Cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14α-demethylase (CYP51MT) and screen printed graphite electrodes, modified by gold nanoparticles and didodecyldimethyl ammonium bromide (DDAB). The proposed method for preparation of enzymatic nanostructured electrodes may be used for electrodetection of this hemoprotein provided that 2–200 pmol P450 per electrode has been adsorbed. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, and amperometry.
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Direct electron transfer of Cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles.
Analytical chemistry, 2004Co-Authors: Victoria V. Shumyantseva, Alexander I. Archakov, Yuri D. Ivanov, Nikitas Bistolas, Frieder W. Scheller, Ulla WollenbergerAbstract:A method for construction of biosensors with membranous Cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated Cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between −0.292 and −0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of p...
Haoming Zhang - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional characterization of a Cytochrome P450 2B4 F429H mutant with an axial thiolate-histidine hydrogen bond.
Biochemistry, 2014Co-Authors: Yuting Yang, Haoming Zhang, Naw May Pearl, Dandamudi Usharani, Michael Tarasev, Freeborn Rwere, Jennifer L. Meagher, Cuthbert SunAbstract:The structural basis of the regulation of microsomal Cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of Cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the eN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine–thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I–IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.
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Potent mechanism-based inactivation of Cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of Cytochrome P450 catalysis.
Biochemistry, 2013Co-Authors: Haoming Zhang, Manish B. Shah, Qinghai Zhang, C. David Stout, James R Halpert, Maryam Foroozesh, Jiawang Liu, Yoichi Osawa, Paul F. HollenbergAbstract:The mechanism-based inactivation of Cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min–1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, resul...
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Structural Analysis of Mammalian Cytochrome P450 2B4 Covalently Bound to the Mechanism-Based Inactivator Tert-Butylphenylacetylene: Insight Into Partial Enzymatic Activity
Biochemistry, 2011Co-Authors: Haoming Zhang, P. Ross Wilderman, Tong Liu, Arthur G. Roberts, Qinghai Zhang, Virgil L. Woods, C. David Stout, Hsia Lien Lin, Paul F. HollenbergAbstract:A combined structural and computational analysis of rabbit Cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.
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Defining the Structural Consequences of Mechanism-Based Inactivation of Mammalian Cytochrome P450 2B4 Using Resonance Raman Spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Piotr J. Mak, Haoming Zhang, Paul F. Hollenberg, James R. KincaidAbstract:In view of the potent oxidizing strength of Cytochrome P450 intermediates, it is not surprising that certain substrates can give rise to reactive species capable of attacking the heme or critical distal-pocket protein residues to irreversibly modify the enzyme in a process known as mechanism-based (MB) inactivation, a result that can have serious physiological consequences leading to adverse drug−drug interactions and toxicity. While methods exist to document the attachment of these substrate fragments, it is more difficult to gain insight into the structural basis for the altered functional properties of these modified enzymes. In response to this pressing need to better understand MB inhibition, we here report the first application of resonance Raman spectroscopy to study the inactivation of a truncated form of mammalian CYP2B4 by the acetylenic inhibitor 4-(tert-butyl)phenylacetylene, whose activated form is known to attach to the distal-pocket T302 residue of CYP2B4.
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tert butylphenylacetylene is a potent mechanism based inactivator of Cytochrome P450 2B4 inhibition of Cytochrome P450 catalysis by steric hindrance
Molecular Pharmacology, 2009Co-Authors: Haoming Zhang, Djemel Hamdane, Hsia Lien Lin, Vyvyca J Walker, Paul F. HollenbergAbstract:We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for Cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.
