Cytochrome P450cam Research Articles

1SE0920 チトクロムP450cam触媒サイクルの最初のステップにおける水分子排出機構について(1SE 生体反応のダイナミクスに基づく生物機能解明の新展開,第48回日本生物物理学会年会)

1SE0920 チトクロムP450cam触媒サイクルの最初のステップにおける水分子排出機構について(1SE 生体反応のダイナミクスに基づく生物機能解明の新展開,第48回日本生物物理学会年会)

2P113 Characterization of hisdine-tagged cytochrome P450cam and its synthesis in a cell-free system(The 48th Annual Meeting of the Biophysical Society of Japan)

2P113 Characterization of hisdine-tagged cytochrome P450cam and its synthesis in a cell-free system(The 48th Annual Meeting of the Biophysical Society of Japan)

3P113 Substrate binding excludes water cluster from active site of cytochrome P450cam - mutation analysis of water expelling system(Heme proteins,The 48th Annual Meeting of the Biophysical Society of Japan)

3P113 Substrate binding excludes water cluster from active site of cytochrome P450cam - mutation analysis of water expelling system(Heme proteins,The 48th Annual Meeting of the Biophysical Society of Japan)

Tuning the substrate specificity by engineering the active site of cytochrome P450cam: A rational approach

Rational design of the active site of cytochrome P450cam has been carried out to catalyse oxygenation of various potentially important chemical reactions. The modeling studies showed that the distal pocket of the heme consisting of the Y96, T101, F87 and L244 residues could be suitably mutated to change the substrate specificity of the enzyme. We found that the mutant enzymes could catalyse oxygenation of indole to produce indigo. While Y96F was found to be several times better as a catalyst for conversion of indole to indigo, the double mutant Y96F/L244A showed the highest NADH oxidation rate as well as yield of indigo. The oxidative catalysis using H(2)O(2) as the oxygen source was found to produce a higher purity of indigo, and lesser or no formation of indirubin was detected. The enzymatic oxygenation of aromatic hydrocarbons such as coumarin and analogues was also found to be enhanced on mutation of Y96 and L244 residues in the enzyme. The studies also showed that mutation of suitable residues can alter the regio-selectivity of hydroxylation of the aromatic hydrocarbons.

Compound I Reactivity Defines Alkene Oxidation Selectivity in Cytochrome P450cam

Prediction of the chemoselectivity of drug oxidation by the human cytochrome P450 enzymes will aid in the avoidance of adverse drug reactions. The chemoselectivity of alkene oxidation is an important problem to address, as it can result in the formation of epoxides, which can have toxic effects. In this paper the epoxidation and hydroxylation of cyclohexene and propene by the bacterial P450(cam) isoform are modeled with hybrid quantum mechanical/molecular mechanical (QM/MM) methods. Snapshots for QM/MM modeling are chosen from molecular dynamics trajectories, to sample the different conformations of the enzyme-substrate complex. The energy barriers obtained for these processes are in qualitative agreement with experimental work, supporting the use of QM/MM methods in the study of selectivity for this class of enzyme. This work highlights the complexity involved in modeling these systems with QM/MM and the importance in the selection of starting geometries.

Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-009-0608-3) contains supplementary material, which is available to authorized users.

Spectroscopic studies of the oxidation of ferric CYP153A6 by peracids: Insights into P450 higher oxidation states

Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-20309; J. Inorg. Biochem. 100 (2006) 2034-2044; J. Biol. Inorg. Chem. 13 (2008) 599-611] spectrally characterized compound I (ferryl iron plus a porphyrin pi-cation radical (Fe(IV)O/Por(.+))), Cpd ES, and Cpd II (Fe(IV)O/Tyr() or Fe(IV)O). We now report that reactions of CYP153A6 with peracids yield all these intermediates, with kinetic profiles allowing better resolution of all forms at pH 8.0 compared to similar reactions with WT P450cam. Properties of the reactions of these higher oxidation state intermediates were determined in double-mixing experiments in which intermediates are pre-formed and ascorbate is then added. Reactions of heptane-bound CYP153A6 (pH 7.4) with mCPBA resulted in conversion of P450 to the low-spin ferric form, presumably as heptanol was formed, suggesting that CYP 153A6 is a potential biocatalyst that can use peracids with no added NAD(P)H or reducing systems for bioremediation and other industrial applications.

Metal Ion Facilitated Dissociation of Heme from b-Type Heme Proteins

Addition of Ni(2+), Cu(2+), or Zn(2+) (10-40 equiv) to metMb in sodium bicarbonate buffer (25 degrees C) at alkaline pH (7.8-9.5) results in a time-dependent (2-6 h) change in the electronic absorption spectrum of the protein that is consistent with dissociation of the heme from the active site and that can be largely reversed by addition of EDTA. Similar treatment of cytochrome b(5), indoleamine 2,3-dioxygenase, and cytochrome P450(cam) (in the presence or absence of camphor) produces a similar spectroscopic response. Elution of metMb treated with Ni(2+) in this manner over an anion exchange column in buffer containing Ni(2+) affords apo-myoglobin without exposure to acidic pH or organic solvents as usually required. Bovine liver catalase, in which the heme groups are remote from the surface of the protein, and horseradish peroxidase, which has four disulfide bonds and just three histidyl residues, exhibit a much smaller spectroscopic response. We propose that formation of carbamino groups by reaction of bicarbonate with protein amino groups promotes both protein solubility and the interaction of the protein with metal ions, thereby avoiding precipitation while destabilizing the interaction of heme with the protein. From these observations, bicarbonate buffers may be of value in the study of nonmembrane proteins of limited solubility.

A General Boundary Potential for Hybrid QM/MM Simulations of Solvated Biomolecular Systems

We present a general boundary potential for the efficient and accurate evaluation of electrostatic interactions in hybrid quantum mechanical/molecular mechanical (QM/MM) approaches called solvated macromolecule boundary potential (SMBP), which is designed for QM/MM calculations with any kind of QM method. The SMBP targets QM/MM single-point energy calculations and geometry optimizations. In the SMBP scheme, the outer solvent and macromolecule region is described by a boundary potential obtained with the use of Poisson-Boltzmann calculations (treating the bulk solvent as a dielectric continuum). In the QM calculations, the SMBP is represented by virtual point charges on a surface enclosing the explicitly treated inner region. These charges and their interactions with the QM density are determined through a self-consistent reaction field procedure. The accuracy of the SMBP is evaluated on three diverse test systems: the intramolecular proton transfer of glycine in water, the hydroxylation reaction in p-hydroxybenzoate hydroxylase, and the spin state energy splittings in the pentacoordinated ferric complex of cytochrome P450cam. In the case of solvated glycine, application of the SMBP turns out to be problematic since analogous QM/MM/SMBP and full QM/MM geometry optimizations lead to different close-lying local minima. In both enzymes, the SMBP performs very well and closely reproduces the results from full QM/MM optimizations of these more rigid test systems. Starting from optimized QM/MM/SMBP structures along a reaction path, one can apply the previously implemented generalized solvent boundary potential (GSBP) to sample over MM phase space in QM/MM free energy calculations within the framework of free energy perturbation theory. This reduces the overall computational costs of sampling by 1 order of magnitude while maintaining good accuracy. The combined use of SMBP and GSBP thus allows for efficient QM/MM free energy studies of enzymes.

Finite-temperature effects in enzymatic reactions — Insights from QM/MM free-energy simulations

We report potential-energy and free-energy data for three enzymatic reactions: carbon–halogen bond formation in fluorinase, hydrogen abstraction from camphor in cytochrome P450cam, and chorismate-to-prephenate Claisen rearrangement in chorismate mutase. The results were obtained by combined quantum mechanics/molecular mechanics (QM/MM) optimizations and two types of QM/MM free-energy simulations (free-energy perturbation and umbrella sampling) using semi-empirical or density-functional QM methods. Based on these results and our previously published free-energy data on electrophilic substitution in para-hydroxybenzoate hydroxylase, we discuss the importance of finite-temperature effects in the chemical step of enzyme reactions. We find that the entropic contribution to the activation barrier is generally rather small, usually of the order of 5 kJ mol–1 or less, consistent with the notion that enzymes bind and pre-organize the reactants in the active site. A somewhat larger entropic contribution is encountered in the case of chorismate mutase where the pericyclic transition state is intrinsically more rigid than the chorismate reactant (also in the enzyme). The present results suggest that barriers from QM/MM geometry optimization may often be close to free-energy barriers for the chemical step in enzymatic reactions.

QM/MM Study of the Second Proton Transfer in the Catalytic Cycle of the D251N Mutant of Cytochrome P450cam

Protonation of Compound 0 in the catalytic cycle of cytochrome P450cam may lead to the formation of either the reactive Compound I (coupling) or the ferric resting state (uncoupling). In this work, we investigate the effect of the D251N mutation on the coupling and uncoupling reaction by combined quantum mechanics/molecular mechanics (QM/MM) calculations. The mutated Asn251 residue has two possible orientations, i.e. directed toward the active site (no flip) or away from the active site (flip), with the latter one being preferred in classical molecular dynamics (MD) simulations. The possible proton transfer mechanisms in the coupling and uncoupling reaction were studied for three models of the D251N mutant, i.e. no flip (model I), flip (model II), and flip with an extra water (model III). According to the QM/MM calculations, the uncoupling reaction is always less favorable than the coupling reaction. The coupling reaction in the D251N mutant follows the same mechanism as in the wild-type enzyme, with initial O-O cleavage followed by proton transfer. The barrier for the initial step is similar in all D251N models, but the proton transfer is most facile in model III. The hydroxide anion formed in model III is not reprotonated easily by neighboring residues, while proton delivery from bulk solvent seems possible via a water network that remains intact during 2 ns classical MD simulation. The computational results are consistent with the experimental findings that the coupling reaction dominates the consumption of dioxygen in the D251N mutant, but with lower activity than in the wild-type enzyme.

Tricistronic overexpression of cytochrome P450 cam , putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system

The catalytic turnover of cytochrome P450( cam ) from Pseudomonas putida requires two auxiliary reduction partners, putidaredoxin (Pd) and putidaredoxin reductase (PdR). We report the functional expression in Escherichia coli of tricistronic constructs consisting of P450( cam ) encoded by the first cistron and the auxiliary proteins, Pd and PdR by the second and the third. Transformed bacterial whole cells efficiently oxidized (1R)-(+)-camphor to 5-exo-hydroxycamphor and, interestingly, limonene to (-)-perillyl alcohol. These bioengineered E. coli cells possess a heterologous self-sufficient P450 catalytic system that may have advantages in terms of low cost and high yield for the production of fine chemicals.

Redox-Dependent Dynamics in Cytochrome P450cam

Local protein backbone dynamics of the camphor hydroxylase cytochrome P450(cam) (CYP101) depend upon the oxidation and ligation state of the heme iron. (1)H-(15)N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H-D exchange) and (15)N relaxation measurements, and these results are compared with previously published results obtained by H-D exchange mass spectrometry. In general, the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B' helix and beta3 and beta5 sheets) and binding of putidaredoxin (C and L helices), the iron-sulfur protein that acts as the effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and the relevance of the observed effects to the enzyme mechanism is discussed.

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

Structural and Dynamic Implications of an Effector-induced Backbone Amide cis–trans Isomerization in Cytochrome P450 cam

Experimental evidence has been provided for a functionally relevant cis–trans isomerization of the Ile88–Pro89 peptide bond in cytochrome P450 cam (CYP101). The isomerization is proposed to be a key element of the structural reorganization leading to the catalytically competent form of CYP101 upon binding of the effector protein putidaredoxin (Pdx). A detailed comparison of the results of molecular dynamics simulations on the cis and trans conformations of substrate- and carbonmonoxy-bound ferrous CYP101 with sequence-specific Pdx-induced structural perturbations identified by nuclear magnetic resonance is presented, providing insight into the structural and dynamic consequences of the isomerization. The mechanical coupling between the Pdx binding site on the proximal face of CYP101 and the site of isomerization is described.

How is the Reactivity of Cytochrome P450cam Affected by Thr252X Mutation? A QM/MM Study for X = Serine, Valine, Alanine, Glycine

Proton transfer reactions play a vital role in the catalytic cycle of cytochrome P450cam and are responsible for the formation of the iron-oxo species called Compound I (Cpd I) that is supposed to be the active oxidant. Depending on the course of the proton transfer, protonation of the last observable intermediate (ferric hydroperoxo complex, Cpd 0) can lead to either the formation of Cpd I (coupling reaction) or the ferric resting state (uncoupling reaction). The ratio of these two processes is drastically affected by mutation of the Thr252 residue. In this work, we study the effect of Thr252X (X = serine, valine, alanine, glycine) mutations on the formation of Cpd I by means of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations and classical simulations. In the wild-type enzyme, the coupling reaction is favored since its rate-limiting barrier is 13 kcal/mol lower than that for uncoupling. This difference is reduced to 7 kcal/mol in the serine mutant. In the case of valine, alanine, and glycine mutants, an additional water molecule enters the active site and lowers the activation energy of the uncoupling reaction significantly. With the additional water molecule, coupling and uncoupling have similar barriers in the valine mutant, and the uncoupling reaction becomes favored in the alanine and glycine mutants. These findings agree very well with experimental results and thus confirm the assumption that uncontrolled proton delivery by solvent water networks is responsible for the uncoupling reaction. The present study provides a detailed mechanistic understanding of the role of the Thr252 residue.

A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Site

Cytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor. The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side chains interact with Arg, His, and/or Asp residues. To understand the role of the heme-7-propionate side chain, we prepared reconstituted P450cam with an artificial one-legged heme which has a methyl group at the position of the 7-propionate. Removal of 7-propionate dramatically decreases the d-camphor affinity by 3 orders of magnitude relative to that of the wild-type enzyme, and spectroscopic data indicate that 74% of the ferric P450cam exhibits a low-spin state owing to water molecule occupancy in the substrate-binding site under the normal assay conditions. Thus, the monooxygenase activity of the reconstituted protein is remarkably low due to the decrease in the rate of the first electron transfer from reduced putidaredoxin, whereas 87% of oxidized NADH was utilized to produce 5-hydroxy-d-camphor without any significant uncoupling reactions. X-ray structural analysis of the reconstituted enzyme reveals a novel water array extending from the substrate-binding site to bulk solvent through the position occupied by 7-propionate. This water array appears without causing any major changes in the protein structure with the notable exception of conformational changes occurring at Asp297 and Gln322 residues. We propose that the 7-propionate forms a barrier against entry of bulk water molecules and therefore in combination with Asp297, Arg299, and Gln322 plays an essential role in the process of elimination of the substrate-binding site water cluster which occurs upon d-camphor binding.

1P-079 急速混合凍結EPR法によるシトクロムP450cam反応中間体の捕捉と動的解析(ヘム蛋白質,第47回日本生物物理学会年会)

1P-079 急速混合凍結EPR法によるシトクロムP450cam反応中間体の捕捉と動的解析(ヘム蛋白質,第47回日本生物物理学会年会)

3P-075 基質カンファー結合によるチトクロムP450camの構造変化(ヘム蛋白質,第47回日本生物物理学会年会)

3P-075 基質カンファー結合によるチトクロムP450camの構造変化(ヘム蛋白質,第47回日本生物物理学会年会)

3P-076 チトクロムP450camの活性部位からの水クラスター排出機構(ヘム蛋白質,第47回日本生物物理学会年会)

3P-076 チトクロムP450camの活性部位からの水クラスター排出機構(ヘム蛋白質,第47回日本生物物理学会年会)