Active Site Of Cytochrome P450cam Research Articles

Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations

Understanding the mechanisms of ligand binding to enzymes is of paramount importance for the design of new drugs. Here, we report on the use of a novel biased molecular dynamics (MD) methodology to study the mechanism of camphor binding to cytochrome P450cam. Microsecond-long MD simulations allowed us to observe reaction coordinates characterizing ligand diffusion from the active site of cytochrome P450cam to solvent via three egress routes. These atomistic simulations were used to estimate thermodynamic quantities along the reaction coordinates and indicate diverse binding configurations. The results suggest that the diffusion of camphor along the pathway near the substrate recognition site (SRS) is thermodynamically preferred. In addition, we show that the diffusion near the SRS is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The conformational change accompanying this switch is characterized by the retraction of the F and G helices and the disorder of the B′ helix. These results are corroborated by experimental studies and provide detailed insight into ligand binding and conformational behavior of the cytochrome family. The presented methodology is general and can be applied to other ligand-protein systems.

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.

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)

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)

1P-121 チトクロムP450camの基質結合反応における活性部位近傍Asp297の役割(ヘム蛋白質,第46回日本生物物理学会年会)

1P-121 チトクロムP450camの基質結合反応における活性部位近傍Asp297の役割(ヘム蛋白質,第46回日本生物物理学会年会)

Role of Threonine 101 on the Stability of the Heme Active Site of Cytochrome P450cam: Multiwavelength Circular Dichroism Studies

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.

Substrate binding in the active site of cytochrome P450cam

We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT interaction energy is well reproduced by QM/MM calculations, which allows for application of QM/MM to the catalytic cycle of cytochrome P450s. The substrate is distorted considerably due to the presence of the protein environment, which however does not have a large impact on the strong hydrogen bonding interactions.

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants.

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0-2.8 M in the presence of camphor and 0-1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20-60 degrees C in the presence of the substrate and 20-47 degrees C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.

Protein side-chain motion and hydration in proton-transfer pathways. Results for cytochrome p450cam.

Proton-transfer reactions form an integral part of bioenergetics and enzymatic catalysis. The identification of proton-conducting pathways inside a protein is a key to understanding the mechanisms of biomolecular proton transfer. Proton pathways are modeled here as hydrogen bonded networks of proton-conducting groups, including proton-exchanging groups of amino acid side chains and bound water molecules. We focus on the identification of potential proton-conducting pathways inside a protein of known structure. However, consideration of the static structure alone is often not sufficient to detect suitable proton-transfer paths, leading, for example, from the protein surface to the active site buried inside the protein. We include dynamic fluctuations of amino acid side chains and water molecules into our analysis. To illustrate the method, proton transfer into the active site of cytochrome P450cam is studied. The cooperative rotation of amino acids and motion of water molecules are found to connect the protein surface to the molecular oxygen. Our observations emphasize the intrinsic dynamical nature of proton pathways where critical connections in the network may be transiently provided by mobile groups.

How do substrates enter and products exit the buried active site of cytochrome P450cam? 2. Steered molecular dynamics and adiabatic mapping of substrate pathways

Three possible channels by which substrates and products can exit from the buried active site of cytochrome P450cam have been identified by means of random expulsion molecular dynamics simulations. In the investigation described here, we computed estimates of the relative probabilities of ligand passage through the three channels using steered molecular dynamics and adiabatic mapping. For comparison, the same techniques are also applied to investigate substrate egress from cytochrome P450-BM3. The channel in cytochrome P450cam, for which there is the most supporting evidence from experiments (which we name pathway 2a), is computed to be the most probable ligand exit channel. It has the smallest computed unbinding work and force. For this channel, the ligand exits between the F/G loop and the B′ helix. Two mechanistically distinct, but energetically similar routes through this channel were observed, showing that multiple pathways along one channel are possible. The probability of ligand exit via the next most probable channel (pathway 3), which is located between the I helix and the F and G helices, is estimated to be less than 1/10 of the probability of exit along pathway 2a. Low-frequency modes of the protein extracted from an essential dynamics analysis of a 1 ns duration molecular dynamics simulation of cytochrome P450cam with camphor bound, support the opening of pathway 2a on a longer timescale. On longer timescales, it is therefore expected that this pathway becomes more dominant than estimated from the present computations.

How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 Å) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 Å) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.

Piperonyl butoxide-mediated inhibition of cytochrome P450-catalysed insecticide metabolism: a rational approach

The inhibitory effect of piperonyl butoxide (PBO) on cytochrome P450 was studied by theoretical methods. Binding conformations of PBO were obtained by the recently developed low-mode conformational search within the active site of cytochrome P450cam. Increased activity of PBO relative to other methylendioxyphenyl inhibitors was rationalized by its decreased conformational mobility and the steric block created by its long side-chain in the substrate access channel of the enzyme. © 1999 Society of Chemical Industry

Inhibitors of cytochrome P450 catalyzed insecticide metabolism: A rational approach

The inhibition of cytochrome P450 mediated oxidative metabolism affected by methylenedioxyphenyl synergists was studied by theoretical methods. Binding conformations of safrole-based inhibitors [piperonyl butoxide (PBO), safrole, and izosafrole] were obtained by the recently developed low-mode conformational search within the active site of cytochrome P450cam. Increased activity of PBO was rationalized by the steric block created by its long side chain in the substrate access channel of the enzyme. Stability of the Fe(II)–carbene complex was confirmed by quantum chemical calculations. In addition to the effect of back-donation, the role of the carbene orientation was explored. Molecular dynamics (MD) simulations revealed that hydrogen bonding between the carbene oxygen and Thr252-OH might stabilize the Fe(II) complex. Changes in the coordination of axial thiolate during carbene complexation were found to be crucial for the inhibitory action. Decreasing redox potential of the enzyme is caused by the increasing thiol character of the proposed drifting of the axial ligand stabilized by NHS hydrogen bonds with Gly359 and Leu358. ©1999 John Wiley & Sons, Inc. Int J Quant Chem 73: 123-135, 1999

Cytochrome P-450 Catalyzed Insecticide Metabolism. Prediction of Regio- and Stereoselectivity in the Primer Metabolism of Carbofuran: A Theoretical Study

The molecular mechanism of carbofuran metabolism was investigated by molecular modeling using the energy-minimized active site of cytochrome P-450cam. A feasible binding conformation of carbofuran ...

Active sites of the cytochrome p450cam (CYP101) F87W and F87A mutants. Evidence for significant structural reorganization without alteration of catalytic regiospecificity.

Ferricyanide oxidation of the aryl-iron complexes formed by the reaction of cytochrome P450 enzymes with arylhydrazines causes in situ migration of the aryl group from the iron to the porphyrin nitrogen atoms. The regiochemistry of this migration, defined by the ratio of the four possible N-arylprotoporphyrin IX isomers, provides a method for mapping the topologies of cytochrome P450 active sites. The method has been validated by using it to examine the active site of cytochrome P450cam (CYP101), for which a crystal structure is available. In agreement with the crystal structure, reaction with phenylhydrazine gives a 5:25:70 ratio of the NA:NC:ND (subscript indicates pyrrole ring) N-phenylprotoporphyrin IX isomers. Naphthylhydrazine, however, yields exclusively the NC regioisomer and 4-(phenyl)phenylhydrazine the NA:NC:ND isomers in a 14:40:46 ratio. These isomer ratio differences are readily explained by topological differences between the upper and lower reaches of the active site. Having validated the aryl-iron shift as a topological probe, we used it to investigate the structural changes caused by mutation of Phe-87, a residue that provides the ceiling over pyrrole ring D in the crystal structure of cytochrome P450cam. Mutation of Phe-87 to a tryptophan causes no detectable change in the regiochemistry of camphor hydroxylation and only minor changes in the N-aryl isomer ratios. However, mutation of Phe-87 to an alanine, which was expected to open up the region above pyrrole ring D, severely decreased the proportion of the ND in favor of the NA isomer. Less rather than more space is therefore available over pyrrole ring D in the F87A mutant despite the fact that the regiochemistry of camphor hydroxylation remains unchanged. These results provide evidence for significant structural reorganization in the upper regions of the substrate binding site without alteration of the camphor hydroxylation regiospecificity in the F87A mutant.

Active site topologies of bacterial cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3). In situ rearrangement of their phenyl-iron complexes.

The reactions of cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3) with phenyldiazene result in the formation of phenyl-iron complexes with absorption maxima at 474-478 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 decreases the 474-478 nm absorbance and shifts the phenyl group from the iron to the porphyrin nitrogens. Acidification and extraction of the prosthetic group from each of the ferricyanide-treated enzymes yields a different mixture of the four possible N-phenylprotoporphyrin IX regioisomers. The ratios of the regioisomers with the phenyl ring on pyrrole rings B, A, C, and D (in order of elution from the high performance liquid chromatography column) are, respectively: cytochrome P450cam, 0:0:1:4; P450terp, 0:0:0:1; and P450BM-3, 2:10:2:1. The isomer ratio for recombinant cytochrome P450BM-3 without the cytochrome P450 reductase domain (2:9:2:1) shows that the reductase domain does not detectably perturb the active site topology of cytochrome P450BM-3. Potassium ions modulate the intensity of the spectrum of the phenyl-iron complex of cytochrome P450cam, but do not alter the N-phenyl isomer ratio. Computer graphics analysis of the crystal structure of the cytochrome P450cam phenyl-iron complex indicates that the active site of cytochrome P450cam is open above pyrrole ring D and, to a small extent, pyrrole ring C, in complete agreement with the observed N-phenylprotoporphyrin IX regioisomer pattern. The regioisomer ratios indicate that the active site of cytochrome P450terp is only open above pyrrole ring D, whereas that of cytochrome P450BM-3 is open to some extent above all the pyrrole rings but particularly above pyrrole ring A. The bacterial enzymes thus have topologies distinct from each other and from those of the mammalian enzymes so far investigated, which have active sites that are open to a comparable extent above pyrrole rings A and D.

Solvation of the active site of cytochrome P450-cam

Energetically favorable water binding sites in the substrate pocket of cytochrome P450-cam have been predicted by a molecular mechanics method. Binding sites corresponding to all the experimentally observed water sites in this region of the enzyme were located. The calculations also indicate the presence of two further water binding sites. One of these is located in a hydrophobic region of the protein where a water molecule would not bind tightly to the substrate-free enzyme. However, in the substrate-bound enzyme, a water molecule in this region could donate a hydrogen bond of optimum geometry to the carbonyl oxygen atom of the camphor substrate and could therefore contribute to the correct positioning of the camphor substrate for 5-exo-hydroxylation. These calculations also suggest that a steric analogue of camphor, containing an alkyl group which could prevent a water molecule from binding in this region, might inhibit cytochrome P450-cam by forming a more stable enzyme-ligand complex than camphor itself.

Investigations of the resonance Raman excitation profiles of cytochrome P450cam

We have measured the resonance Raman excitation profiles (REP’s) of several vibrational modes associated with the heme active site of cytochrome P450cam. The important Fe–S axial ligand mode (351 cm−1) of the substrate bound (high-spin ferric) complex is found to have structure in its blue shifted REP. Inverse transform techniques allow the line shape of the resonant charge transfer absorption to be reconstructed directly from the REP data. The observed splitting (3200 cm−1) is associated with an inequivalence in the resonant S→Fe charge transfer excitations. The position of the high-energy component (∼323 nm) is found to be in excellent agreement with z-polarized single crystal measurements, while the low-energy component (∼360 nm) is not clearly observed in the single crystal analysis. The ‘‘spin-marker’’ band ν3 is found to have a REP that is significantly red shifted with respect to the theoretical predictions. A variety of perturbations including non-Condon and multiple state coupling, as well as state dependent damping, are unable to account for the observed red shift. Studies of other ferric heme protein systems, including substrate free cytochrome P450cam (low-spin) and aquomet myoglobin (high-spin) reveal ‘‘normal’’ behavior of their REP’s. The electron–nuclear coupling strengths are directly extracted from the measured absolute cross sections in these cases.

Cloning and sequencing of chloroperoxidase cDNA.

An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA. A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322. The complete primary sequence of chloroperoxidase has been derived. We have also determined about 73% of the peptide sequence by amino acid sequencing. The DNA sequence data matches all of the available known peptide sequences. The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons. A putative signal peptide of 21 amino acids is proposed from DNA sequence data. The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences. Three cysteine residues are found in the protein sequence. A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam. No other heme protein homologues can be detected. We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group. A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E. coli or yeast.