Conformational Flexibility of Mammalian Cytochrome P450 2B4 in Binding Imidazole Inhibitors with Different Ring Chemistry and Side Chains

Abstract

Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Each of the inhibitors induced type II spectral changes, and IC50 values for enzyme inhibition ranged from 0.1 to 2.4 μm, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated KD values ranging from 0.3 to 4.8 μm and following the same rank order as the IC50 values. Changes in enthalpy at 25 °C ranged from -6.5 to -8.8 kcal mol-1. The largest difference in binding entropy (+5.9 versus -4.1 cal mol-1 K-1) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol-1 K-1), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Accessibility to acrylamide of the only tryptophan (Trp121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4. Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Each of the inhibitors induced type II spectral changes, and IC50 values for enzyme inhibition ranged from 0.1 to 2.4 μm, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated KD values ranging from 0.3 to 4.8 μm and following the same rank order as the IC50 values. Changes in enthalpy at 25 °C ranged from -6.5 to -8.8 kcal mol-1. The largest difference in binding entropy (+5.9 versus -4.1 cal mol-1 K-1) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol-1 K-1), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Accessibility to acrylamide of the only tryptophan (Trp121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4. Cytochromes P450 are a superfamily of enzymes with cysteine thiolate ligation of the catalytic heme center and perform regio- and stereoselective hydroxylation of a wide variety of endogenous and exogenous organic molecules (1Coon M.J. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 1-25Crossref PubMed Scopus (288) Google Scholar). An appreciable number of P450 crystal structures of both bacterial and mammalian origins are now available and show a conserved fold among enzymes with greatly varying primary structures (2Poulos T.L. Johnson E.F. Cytochrome P450: Structure, Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar). A major challenge has been to understand the ability of these proteins to accommodate ligands of varied size and shape. In addition, since the first P450 crystal structure was determined, scientists have continued to struggle to explain how substrates gain access to the deeply buried active site (2Poulos T.L. Johnson E.F. Cytochrome P450: Structure, Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar). However, a recent set of structures has revealed conformational changes in specific regions of the protein, including the F-G loop and helix B′, in response to substrate/inhibitor binding (2Poulos T.L. Johnson E.F. Cytochrome P450: Structure, Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar, 3Poulos T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Crossref PubMed Scopus (1282) Google Scholar, 4Podust L.M. Kim Y. Arase M. Neely B.A. Beck B.J. Bach H. Sherman D.H. Lamb D.C. Kelly S.L. Waterman M.R. J. Biol. Chem. 2003; 278: 12214-12221Abstract PubMed Scopus Google Scholar, H. Ortiz de Montellano P.R. T.L. J. Biol. Chem. PubMed Scopus Google Scholar, Y. J. PubMed Scopus Google Scholar, M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar). and studies from the crystal structures have the flexibility of the access PubMed Scopus Google Scholar, M.J. 2005; PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). The largest conformational observed to in mammalian the wide open binding undergoes a change to 4-(4-chlorophenyl)imidazole 4-CPI, isothermal titration 1-CPI, BEI, 4-CPI, isothermal titration 1-CPI, BEI, of the ligands to in a P450 active site M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). The of the structural between these is in The major structural changes are to the of and C, and and the with at the structural changes are with similar changes observed for bacterial P450 D.C. M. PubMed Scopus Google and H. Ortiz de Montellano P.R. T.L. J. Biol. Chem. PubMed Scopus Google Scholar, Y. J. PubMed Scopus Google Scholar). The CYP2B4 structures the of conformational flexibility available in a P450 The of helix the structural for of and as between helix and P450 cytochrome is to the for M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar, M.R. Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). an explain the of explain enzyme conformational changes in the of and and the loop have been to to and binding in M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar). Here, changes in the of the active site to a in binding ligands of different size in an In the of a of is available to the structural of regio- and of substrates Chem. Toxicol. PubMed Scopus Google Scholar, M. M.R. Johnson E.F. Mol. Pharmacol. PubMed Scopus Google Scholar, T.L. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, H. J. Pharmacol. 2003; PubMed Scopus Google Scholar). of the inferred from these studies are in the site in the closed conformation of the enzyme. structural in the active site and the access the open structure the closed and structures show major structural changes upon binding and ligands the heme for changes to M.R. Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar, J. H. 2003; PubMed Scopus Google Scholar, J. H. PubMed Scopus Google Scholar). the crystal structures reveal of a by the and conformational changes P450 enzymes conformational changes upon binding. is of to structural changes observed by to solution and to study structurally similar for which CYP2B4 was the for In of P450 have been using a wide of spectroscopic and of the including J. Biol. Chem. PubMed Scopus Google PubMed Scopus Google Scholar, M.J. J. Biol. Chem. PubMed Google and catalytic M.J. PubMed Scopus Google using CYP2B4 as of the of is an to and in and in the of P450 enzymes and titration calorimetry is the of to the complete of and in solution Biol. PubMed Scopus Google Scholar, Mol. Biol. Google Scholar, Biol. 2003; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar, M. J. J. Mol. Biol. 2005; Scopus Google Scholar, H. PubMed Scopus Google Scholar). is and in with structural as a major in in a variety of Biol. PubMed Scopus Google Scholar, Mol. Biol. Google Scholar, Biol. 2003; PubMed Scopus Google Scholar). The recent in and have solution to to the H. PubMed Scopus Google Scholar, J. Chem. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). A a complete thermodynamic of the including binding stoichiometry, and changes in enthalpy and has been to study In the of mammalian P450 of the protein that to at the to and to the of have been studies with the bacterial P450 enzymes for a report using to study M. H. PubMed Scopus Google Scholar). In we in with spectroscopic and to the of imidazole inhibitors with CYP2B4 to elucidate the of and to the conformational flexibility of the protein in to the protein in a monomeric Each of the (1-BI), 4-CPI, 4-phenylimidazole (4-PI), and with a perfect 1:1 an with in the thermodynamic the of a versus the of an imidazole versus a major difference in the which is from the plasticity of the the structural flexibility of these inhibitors is The changes in entropy and heat capacity associated with 4-CPI binding to the of the apolar surface area ensuing from a conformational change in the The binding of 4-CPI greatly the of a of helix helix is upon binding to the The thermodynamic with the spectral and IC50 values. that the closed structure of CYP2B4 from the crystal structure only of of the enzyme. BEI, and 4-CPI and 1-CPI as of and from the protein by of the and of a as Johnson E.F. J. Biol. Chem. PubMed Scopus Google was in studies with in the protein as to was with A and and 4.8 at was with the P450 was with and the to The protein was a using a and for The P450 was by difference using an of is the by the to a of and as for CYP2B4 J. PubMed Scopus Google Scholar). at with a different of enzyme. using a at and The and of different with a and an of at and at A μm protein and a 2-fold of in A An was for in the of for the of specific and using the J. PubMed Scopus Google Scholar). A a size of and a of for with J. PubMed Scopus Google Scholar). by using μm a at 25 and in A and was and a was between and following the of a of of the and the same of A and to the of and the by a to the of versus by using and are at a and of was as M. M.R. Johnson E.F. Mol. Pharmacol. PubMed Scopus Google Scholar). A of of of P450 of cytochrome and μm in the of μm with a The and the to a by to the values. and a with a for and using was a and at A protein of μm was in was to the protein to a of to the and the was the in and to using with a was to of from of the to the and with to heat changes to a between the in the and the titration The and binding of the protein in the and for the of the for and to the using a and the and titration A titration the of of with at The titration was at the first of was and the was from the titrations by in the and heat of was from the titration was change in the of the protein titration with The binding to a binding site by to the binding and thermodynamic of the using Calorimetric titrations at and and the change in heat capacity associated with the binding was by the The thermodynamic are from the and are of P450 is in the PubMed Scopus Google Scholar). of the tryptophan in the and of was by acrylamide in A and was °C with different inhibitors at a of to the tryptophan using a with both the and The tryptophan was at and was between and is by the and are the in the and of is the is the of the in the of is the of and is the docking of the inhibitors was using to for the and the to the A.J. J. Chem. Scopus Google Scholar). to the protein using and the for the heme as J. PubMed Scopus Google Scholar). the closed a with a of at and the site was the open a with a of at and the binding at the of the heme was A of that in and the first for the side of the of 4-CPI and 4-PI for similar and the using the at M. J. Mol. PubMed Scopus Google Scholar). of and in study with the was to x-ray and catalytic to Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). titrations of with 4-CPI 1-BI 1-CPI 4-PI and BEI structures are as 4-CPI and 4-PI have a and in The imidazole to the side in 1-CPI, and BEI a from the imidazole ring to the side 4-CPI and 1-CPI from 4-PI and respectively, by the of a 4-CPI and 4-PI from 1-CPI and respectively, by the of an imidazole The flexibility of the inhibitors is in the of BEI and An in BEI and an in 1-BI flexibility between the and imidazole The binding of these inhibitors to induced type II spectral changes, with the at and at of ligation to the heme A of at and versus is for and the to The of these ligands to the of was using as a The IC50 values and the are in The KD values the order 4-CPI < 1-BI < 1-CPI < 4-PI < BEI < 1-PI. The rank order of the IC50 values was that 1-BI a IC50 with 4-CPI A in the was observed upon the of versus by the of the imidazole versus of the with from and spectral titrations The of is and by the following and and by the following and was from the of the in was by a of to the spectral titration in and by the following and and by the following and was from the of the in KD was by a of to the spectral titration in in a of at the and of the the protein monomeric upon binding. to was to in which the enzyme monomeric in solution at the protein in the of inhibitors by the in study was monomeric in A at μm as by the at The enzyme with a of The protein as a upon binding to 1-CPI, and BEI, with an of However, was as a upon 4-CPI with an of was to the of only a with an of was The protein in A an of the protein titrations in A yielded similar with an of for at similar with the at The in the was Calorimetric and of recent in the and of has an for the of thermodynamic as enthalpy and entropy changes with the and number of binding from a the of heat a a complete of the of the spectroscopic Here, by the of enzyme and we to to study The of a which of of to μm at 25 with the of the are for inhibitors 4-CPI 1-BI 1-CPI 4-PI and BEI The a in the heat of binding with was The show the changes for which to the binding site of to binding site to a binding site yielded a with The of are in A 1:1 binding was observed in with the KD values ranging from 0.3 to 4.8 μm following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < same order as the IC50 The was decreased by thermodynamic the was that the 1:1 the active of P450 by difference A of 1-BI with 4-CPI was The values a of KD in the and values in a 25 values of and cal mol-1 observed for 4-CPI and 1-BI the the values with a of -4.1 cal mol-1 observed in the of BEI binding. A of cal mol-1 was obtained for the binding of a imidazole to at J. and J. Biol. Chem. Scholar). However, heat capacity was with of in at BEI was the that the largest and was for heat capacity in with 4-CPI. the in values between the of 4-CPI and BEI with the titrations at and and the change in the heat capacity associated with the binding was from the of the of versus The binding was with a 1:1 the for both 4-CPI and The values and -331 cal mol-1 respectively, for 4-CPI and both the and values a The values with in The the in as a of for 4-CPI and BEI are in and The in as a of of in both in is to and In the the values at the between and and kcal mol-1 for 4-CPI and for The of these values and respectively, for 4-CPI and BEI as from the of versus these the changes and are by the changes and conformational transition the is by changes by both 4-CPI and the observed with only 4-CPI is by the in binding of 4-CPI and BEI binding. The of the change in binding and and binding enthalpy and is for 4-CPI and BEI Accessibility of in observed in the crystal large-scale structural upon 4-CPI binding in the that the heme M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). helix the tryptophan to a to the heme by Accessibility of to the acrylamide was in the and bound of by A the of as a of acrylamide is in The obtained as the of the for the form and for the form of the bound to the inhibitors to the a 2-fold in the of to the is only 4-CPI to the of to the and the closed 4-CPI between in and to form a of these are from the heme in the open structure M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). the structure has been as a for P450 enzymes H. 2005; PubMed Scopus Google was of to the in the closed structure accommodate inhibitors with different chemistry and side and to how these inhibitors in the open A from the of from docking of 4-CPI BEI to both the open and closed structures is in in the closed 4-CPI the same binding in the crystal the docking the heme using the The 4-CPI is in the same as the in the crystal structure the 4-CPI is at an with the heme and the imidazole is to the heme at a of with and at an of with between the of 4-CPI. 1-CPI, and to the same with the and of as in 4-CPI of 1-BI in a similar of the imidazole of the flexibility of the the ring to a and helix BEI docking to the closed structure in of both located to in the However, the imidazole ring the heme in The ring at the and the imidazole the between helix and the to a for heme 4-CPI docking in the open structure in The first in docking by only kcal different The first in the of the at the of the binding between helix and the at from the center of However, in the 4-CPI in the open binding with the imidazole the heme The at a to the heme by as in the closed of 1-CPI, and in and the to the open binding similar to 4-CPI BEI docking to the open structure in the first Here, the first was at the of the binding from the center of the In the the imidazole ring the heme at a of and the was in the open binding The that a binding site is to the the protein in the open conformation structural to BEI, with the obtained from the and spectroscopic the structural of in and catalytic has been by the recent mammalian P450 x-ray structures a closed the structural B′, and have been to a in access to the buried catalytic with studies of and (2Poulos T.L. Johnson E.F. Cytochrome P450: Structure, Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar). However, structures are closed T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Crossref PubMed Scopus (1282) Google Scholar, J. Johnson E.F. Mol. PubMed Scopus Google and binding to change in J. H. PubMed Scopus Google Scholar). In addition, is a between the from crystal structures and the size the of the ligands P450 (2Poulos T.L. Johnson E.F. Cytochrome P450: Structure, Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar, M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar, D.C. M. PubMed Scopus Google Scholar, M.R. Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar, J. H. 2003; PubMed Scopus Google Scholar, J. H. PubMed Scopus Google structural of P450 to binding binding to 4-CPI, CYP2B4 the largest of conformational change in to M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). thermodynamic studies structural in P450 are and is the of to the thermodynamic of binding. However, the major in to the P450 is that protein are to a heat of to the enzyme in the monomeric form to the thermodynamic of ligands is a of the Johnson E.F. J. Biol. Chem. PubMed Scopus Google and monomeric in solution at the μm for only 4-CPI binding induced with 4-CPI binding in the of of the thermodynamic the different of structural observed with of different size in H. Ortiz de Montellano P.R. T.L. J. Biol. Chem. PubMed Scopus Google and J. Mol. Biol. PubMed Scopus Google we a of 1-CPI, and that at a difference in IC50 and spectral KD values. are and in 4-CPI and which are in 1-CPI and 1-PI. The flexibility of these inhibitors is changes in the of protein with 4-CPI and which have a 1-BI and BEI are of The in 1-BI and the in BEI between the imidazole and flexibility to these which in a different in the binding site structurally of these inhibitors has a The thermodynamic signature obtained by a of the of the of these inhibitors and the plasticity of CYP2B4. the difference in changes was for these inhibitors kcal mol-1 at 25 a difference was observed in the and values as in a 1:1 binding of at 25 the values varied from -6.5 to -8.8 kcal mol-1 to for the changes in the values. The of kcal mol-1 for 4-CPI was to kcal mol-1 for 4-PI by a of the The entropy with 4-CPI from the conformational change in the protein both of these inhibitors have the same flexibility and similar values for binding. The binding of 1-BI an kcal mol-1 of with 4-CPI, which for the the that the of to acrylamide was decreased by a different of in the protein with 4-CPI. the observed with 1-CPI, and binding major conformational changes, as to the observed in the protein, the structural among these for BEI the enthalpy is by the at is by and in the protein only 4-CPI binding the complete In the crystal the binding site the that of the a conformational change the of protein Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). The observed among the in study to the imidazole structure and the of the flexibility of BEI to in conformational the binding The largest entropy with a of kcal mol-1 was obtained upon the binding of a imidazole to J. and J. Biol. Chem. Scholar). a conformation of the protein, the binding in the have a crystal structure of a wide open conformation of and J. and J. Biol. Chem. Scholar). are observed between the KD and IC50 values for that the type II spectral changes of M. Chem. Biol. PubMed Scopus Google Scholar, de Montellano P.R. J. Chem. PubMed Scopus Google Scholar, Pharmacol. PubMed Scopus Google Scholar). In 1-BI a spectral with 4-CPI, IC50 values 2-fold of 4-CPI. Steric the from the protein side the to the type II spectral which the of side de Montellano P.R. J. Chem. PubMed Scopus Google Scholar, Pharmacol. PubMed Scopus Google Scholar). in the KD values observed between spectroscopic and titrations for of the However, similar type II spectral changes from the imidazole as a to the heme which a bound the of heat the of a variety of of spectroscopic changes that the between and spectroscopic titrations have been observed in 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of for the spectroscopic titrations yielded similar KD values in the of In addition, of organic to the thermodynamic from for different proteins L.M. J. Biol. Chem. PubMed Scopus Google Scholar, A.J. PubMed Scopus Google Scholar). In the KD values in to the of bound molecules from the CYP2B4 binding However, the of binding for different ligands using solution in H. PubMed Scopus Google Scholar, A.J. PubMed Scopus Google Scholar). 4-CPI and BEI, which for heat capacity solution The from the of changes for is of the thermodynamic for the structural changes in the The for only from of P450 is the of the by J. Chem. Scopus Google that the of apolar to in a in the the study of a of and proteins has that of the apolar surface area in a in the of the Biol. PubMed Scopus Google Scholar, Mol. Biol. Google Scholar, Biol. 2003; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar, M. J. J. Mol. Biol. 2005; Scopus Google Scholar, H. PubMed Scopus Google Scholar, J. Chem. Scopus Google Scholar, Chem. PubMed Google Scholar). The in 4-CPI in change the solution the of values between 4-CPI and The for 4-CPI binding (-604 cal mol-1 K-1), that for BEI binding cal mol-1 K-1), is a of of a of the apolar surface area of upon 4-CPI which is with the structural of B′, and in the crystal structure M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). The as an of to the thermodynamic for 4-CPI binding to from to with an in that is only of the protein at the structural Johnson E.F. J. Biol. Chem. PubMed Scopus Google the from of the surface is The apolar and surface of the open and closed structures of CYP2B4 from the crystal structures by a of the surface J. Chem. Scopus Google Scholar). a in the surface in the closed to the of of the apolar located B′, C, and A apolar surface area of and a surface area of obtained between the open and closed the that the 2-fold obtained upon binding to 4-CPI with BEI from the of apolar surface area from and a conformational In in helix is to the upon 4-CPI the as revealed by a 2-fold in the suggests the of helix to a which is observed in the protein bound to of the these in solution are in with the crystal structures of the from the open to closed conformation helix to a the to a M.R. Johnson E.F. 2003; PubMed Scopus Google Scholar, Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). In the closed is in the with the of heme Johnson E.F. J. Biol. Chem. PubMed Scopus Google Scholar). a buried helix spectroscopic the the of the CYP2B4 structure upon binding to 4-CPI binding to the and the crystal structure an helix J. and J. Biol. Chem. Scholar). 4-CPI is of the ligands to with a a active docking of a of a conformational the to a in the active site the protein flexibility is in the 4-CPI was to the closed structure as conformational the BEI was to the binding in the open the was in an to the closed structure for the conformational to the observed in the inhibition and thermodynamic among 4-CPI, 1-CPI, and the of the docking is from the P450 crystal structures that binding a open closed conformation the chemistry (2Poulos T.L. Johnson E.F. Cytochrome P450: Structure, Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar). The solution thermodynamic in study an to to the thermodynamic between and specific P450 enzymes to the the of specific inhibitors of different P450 enzymes and the of that are to the we that the CYP2B4 have of an open with helix from the heme similar to the open the structure have a binding the same the structure we an open conformation J. and J. Biol. Chem. Scholar). of the challenge of in a monomeric solution studies of and of these enzymes major conformational changes are observed in the x-ray crystal and of and of for access to the and for the studies and for the of P450 with