J. Phys. Chem. B 2004, 108, 11189-11195
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Theoretical Study on Compound I Formation in Monooxygenation Mechanism by Cytochrome P450 Masayuki Hata,* Yoshinori Hirano,† Tyuji Hoshino, Rie Nishida, and Minoru Tsuda Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba UniVersity, Chiba 263-8522, Japan ReceiVed: January 28, 2004; In Final Form: April 23, 2004
The intermediate structures appearing until compound I formation in the monooxygenation reaction cycle by cytochrome P450 were determined by using the density functional theory. Protons were attached to an O2 molecule binding to the heme iron in a reduced oxy-ferrous state. When two protons were attached to the distal O atom, O-O bond cleavage occurred, and the compound I structure was formed. The structure had an energetic advantage compared to the situation in which one proton each was connected to the proximal and distal O atoms, and the structure would catalyze oxidation of a substrate. The calculated interaction energies between substrate and oxygen ligand of heme were 3-10 kcal/mol and suggests that an interaction between the substrate and the oxygen ligand of heme was maintained through the reaction process from the O2 incorporation to the generation of the compound I and that it contributed the stability of the reaction system.
1. Introduction Cytochrome P450 exists in most living creatures, including animals, plants, and microorganisms, and it plays an extremely important role in metabolism.1-3 The monooxygenation reaction by P450 is initiated by the substrate binding to ferric P450 (Figure 1, stage 1).4 When an electron (first e-) is introduced into substrate-bound P450, the heme iron converts into the reduced form, Fe2+ (Figure 1, stage 2), and then an O2 molecule is incorporated in the heme pocket (Figure 1, stage 3). When another electron (second e-) is introduced (Figure 1, stage 4), the O2 molecule becomes very reactive, and the substrate will be oxygenated by an insertion of one O atom into the R-H bond (Figure 1, stage 5). The detailed mechanism of the substrate monooxygenation reaction in P450 is still unknown2,5 because this reaction proceeds too quick to observed the intermediates. However, the monooxygenation reaction mechanism by cytochrome P450 has been investigated on the basis of the idea that the reaction consists of two sequential processes, the generation of the radical species [FeO]3+ (compound I) [the first step] and monooxygenation of the substrate [the second step]. Several density functional theory (DFT) studies,6-13 including our previous work14 and QM/ MM studies,15,16 elucidated the reaction mechanism of the second step. However, theoretical studies on the first step are much fewer than those on the second step. Harris et al. performed calculations based on the DFT to clarify the reaction process in the first step by using the reduced ferrous dioxygen model P450 heme species.17 Recently, his group elucidated the initial protonation to the reduced oxy-ferrous species of P450eryF using models of proton-transfer system with18 or without19 substrate. In our present work, the stable structures of the first step appearing in the monooxygenation * Corresponding author. E-mail:
[email protected]. Phone: +8143-290-2927. Fax: +81-43-290-2925. † Current Address: Computational Astrophysics Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Figure 1. Monooxygenation reaction cycle of cytochrome P450.
reaction cycle by cytochrome P450 were determined by theoretical calculations considering the substrate-O2 molecule interaction. In the oxygenation reaction by cytochrome P450, it is known that a substrate is introduced into the enzyme before the incorporation of an O2 molecule and that the substrate plays a role of excluding of an H2O molecule bound to the heme iron in the resting state and assisting the attachment of an O2 molecule to the heme iron.5 Since the substrate and the O2 molecule coexist in a heme pocket of the enzyme, it is reasonable to assume that the O2 molecule interacts with the substrate while it is in the pocket. The results of an experiment by Tuckey and Kamin20 supported the notion of interaction between the substrate and the O2 molecule in the heme pocket and also suggested that the introduction of the substrate was favorable for oxy-ferrous P450. 2. Methods 2.1. Construction of the Models for Calculations. A search for stable structures of the reaction intermediates was started from the resting state structure. Poulos et al. determined the three-dimensional crystallographic structure of the substratefree P450cam (resting state) derived from Pseudomonas putida by an X-ray crystallographic structure analysis,21 and they
10.1021/jp0496248 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/17/2004
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Figure 2. Average structure of the oxy-ferrous P450cam during the 310 K MD simulation. Numerals are interatomic distances in units of Å.
registered the data in Protein Data Bank (PDB code 1phc).22 A computational model for DFT calculation was constructed by extracting the heme and its ligands from the PDB data. The protoporphyrin IX in the heme was replaced by porphine and Cys357; the proximal heme ligand was replaced by S--CH3. This computational model was used to determine the structures for the low-spin state, the high-spin state, and the ferrous state (Figure 1). Although a substrate exists in the enzyme in a highspin state and a ferrous state, the computational model of this stage did not contain the substrate because it is far apart from the heme iron (C5 atom of the d-camphor-heme iron: 5-6 Å) as indicated previously.14 In the stage after the incorporation of an O2 molecule (Figure 1, stages 3-5), the substrate, d-camphor, was introduced into the computational model. In advance to the construction of the model in this stage, molecular dynamics (MD) simulation for the oxy-ferrous P450cam23 was executed by using AMBER 4.1 program24 with a hardware accelerator called an MD Engine.25 An O2 molecule was incorporated into the distal side of the heme of the substrate-bound ferric P450cam which was previously obtained by MD simulation, and 300-ps MD simulation was performed in the same manner as that in our previous study.14 The average structure obtained by MD simulation is shown in Figure 2. The distance between the heme iron and the C5 atom of the substrate is 5-6 Å, and the hydrogen bond between the hydroxyl oxygen of Tyr96 and the carbonyl oxygen of the d-camphor is maintained at about 3 Å. These values for the oxy-ferrous P450cam are almost the same as those for the O2-free ferric P450cam obtained in our previous work.14 The C5 atom of the substrate interacts with the O atom of the distal heme ligand (O2): 3.0-3.5 Å. The distance between the exo-H atom connected to the C5 atom of the substrate and the O2 atom is 2.5-2.9 Å (2.75 Å in Figure 2), and that between the endo-H atom connected to the C5 atom and the O2 atom is 3.0-3.5 Å (3.04 Å in Figure 2); i.e., the exo-H atom is always the nearest atom, except for the O1 atom, to the O2 atom. The computational model was constructed from this time-averaged structure of MD simulation, employing the distal heme ligand as it is and replacing the d-camphor with methane through the substitution of H atoms for C4 and C6 atoms. The heme part is the same as that in the former model.
Hata et al. 2.2. Computational Details. The electronic structures of the model compounds were solved by using the DFT method. The basis sets used were 3-21G** for H, Fe, and S atoms and 3-21G for C, N, and O atoms because of program limitations. The exchange functional was Becke’s three-parameter functional,26 and the correlation functional was Lee-Yang-Parr’s formula.27 The structures at the minima on the substrate, the distal ligand of heme, heme iron, and sulfur atom of the proximal ligand on the potential energy hypersurface, were more-precisely fully optimized. Other atoms were fixed to the original positions. Total atomic charge and spin multiplicity depend on the intermediate structure appearing in the monooxygenation reaction by cytochrome P450. These values are described in figure captions on each structure. The computational program used was Gaussian 98.28 Since the active site in a protein is not isolated but enclosed by many molecules or residues, the inclusion of the effect of the dielectric constant seems to be important when attempting to reproduce the environment inside the protein through theoretical approaches. In our previous work,14 geometry optimizations were performed for the stable and the transition states on the reaction path taking the dielectric constant of the surroundings to be 20.29,30 The self-consistent reaction field (SCRF) method using the polarized continuum model (PCM) of Tomasi and coworkers31-40 was used for computations. However, no significant differences were noticed between the results obtained from evaluation using a dielectric constant of 1 and those obtained using a dielectric constant of 20. Hence, dielectric constant of 1 was used in this study. 3. Results The stable structures appearing in the compound I formation step of the monooxygenation reaction cycle by cytochrome P450 were shown in Figures 3-6. The details will be shown in the following subsections. 3.1. Conversion from a Low-Spin State to a High-Spin State. In the resting state, the heme is in a low-spin state with Fe3+, and an H2O molecule is bound to the distal side of heme.5,21 Figure 3, structure a shows the theoretically determined structure of this state. The distance between the distal ligand H2O and the heme iron is 2.11 Å, which is compatible with the value of the X-ray crystallographic structure (2.35 Å).21 The spin density is localized on the heme iron (see Table 1, structure a). When a substrate is introduced into the enzyme, the heme converts from a low-spin state to a high-spin state and, simultaneously, the H2O molecule is detached from the heme41 (Figure 1, stage 1). Figure 3, structure b, shows the structure of the heme in the high-spin state after the detachment of the H2O molecule. The detachment of the H2O molecule induces slight displacement of the heme iron toward Cys357. This displacement was experimentally observed by X-ray crystal structure analysis.41 The spin density is still localized on the heme iron (Table 1, structure b). The sum of potential energies of Figure 3, structure b, and H2O increases by 15.0 kcal/mol compared to that of Figure 3, structure a. Although a substrate exists in the enzyme in this high-spin state, the interaction between the substrate and heme iron can be neglected because of the great distance between them. (The results of previous MD simulation indicated that the distance between the C5 atom of the d-camphor and the heme iron is 5-6 Å.14) Hence, the potential energy in the high-spin state is determined from the sum of energies in Figure 3, structure b, and CH4. 3.2. Introduction of Electrons and Incorporation of an O2 Molecule. The introduction of first e- to substrate-bound P450
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Figure 3. (a) The heme structure in the low-spin state (Fe3+) (the resting state), (b) the high-spin state (Fe3+). Numeral is an interatomic distance (Å). The total charge is 0 e in both structures a and b. The spin multiplicity are the doublet in a and the sextet in b.42
Figure 4. The ferrous heme (Fe2+) and the substrate-bound oxy-ferrous heme. Structure c is the reduced oxy-ferrous heme from Figure 3, structure b. Numerals are interatomic distances (Å). The total charge and the spin multiplicity are -1 e and the quintet state in c,42 -1 e and the triplet state in d, and -2 e and the doublet state in e.
TABLE 1: Spin Density Distributions for Structures a and b
TABLE 2: Spin Density Distributions for Structures c, d, and e
structures groups of atoms H2O Fe Cys (S-CH3) (S of Cys) porphinea
structures
a
b
groups of atoms
c
d
e
-0.00 1.06 0.03 (0.03) -0.09
4.26 0.37 (0.30) 0.37
O1 O2 Fe Cys (S-CH3) (S of Cys) porphinea CH4 (C5 + H1 + H2 + H3 + H4)
4.18 0.20 (0.20) -0.39 -
0.36 0.66 1.04 0.06 (0.06) -0.11 -0.00
0.38 0.63 1.02 -0.00 (-0.00) -1.01 -0.01
a Porphine means the part of the computational model, excluding H2O, Fe, and Cys.
in the high-spin state induces the reduction of the heme iron from Fe3+ to Fe2+ (Figure 1, stage 2). Figure 4, structure c, shows this ferrous heme structure. The spin density of this structure is also localized on the heme iron, and the spin electronic state is a quintet (Table 2, structure c). In addition to this high-spin structure, we tried to determine the structure of the ferrous heme which is in a low-spin state, a spin singlet, as described in some report.42 The potential energy of the low-spin structure (not shown in the figure) was revealed to be 30.7 kcal/mol higher than that of the high-spin state, strongly suggesting that the ferrous heme structure is in a highspin state. Due to the introduction of first e-, the ferrous heme is stabilized by 27.4 kcal/mol, compared to the ferric heme in
a Porphine means the part of the computational model, excluding the O2 molecule, Fe, Cys, and substrate (CH4).
the high-spin state. The potential energy in the ferrous state is estimated from a sum of energies in Figure 4, structure c, and CH4. Subsequently, an O2 molecule is incorporated into the enzyme and bound to the heme (Figure 1, stage 3). Figure 4, structure d, shows the optimized structure of the O2 incorporation. The O atom not directly connected to the heme iron (distal O atom; O2) maintains an interaction with the substrate, and the distance between the O2 atom and the C5 atom of the substrate is 3.14 Å while that between the O2 atom and the exo-H atom of the
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Figure 5. Structures in the attachment of first proton to the distal O atom (f) and the proximal O atom (g). Numerals are interatomic distances (Å). The O1-Fe-C1 in g represents the angle between the O1 atom and the heme plane in degree. The total charge and the spin multiplicity are -1 e and the doublet state in both f and g.
substrate (H1) is 2.04 Å. The bond length between the O atom directly connected to the heme iron (proximal O atom; O1) and the O2 atom is 1.40 Å, and the O1 atom makes a chemical bond with the Fe atom, with a bond length of 1.92 Å. The distance between C5 atom and Fe atom is 5.81 Å. All of these results are consistent with the structure obtained by the MD simulation in Figure 2. According to the spin density distributions, the O2 molecule and the heme iron have large spin density. Due to the incorporation of the O2 molecule, the potential energy decreases by 30.1 kcal/mol. The introduction of second e- increases the reactivity of the O2 molecule bound to the P450 in the oxy-ferrous heme formation (Figure 1, stage 4). Figure 4, structure e, shows the structure in which the reduced oxy-ferrous heme has an interaction with the substrate. Although no significant difference in atomic geometry is seen compared to that of the structure shown in Figure 4, structure d, the spin density distribution is drastically changed; the porphine has a spin density of -0.11 in structure d, whereas it increases to -1.01 in structure e (Table 2). This large β spin density of the porphine is canceled out with the R spin density of the heme iron (+1.02), and then the spin is localized only at the O2 molecule in the total spin density distribution. The potential energy increases by 47.9 kcal/mol through this reduction process, suggesting that there is an increase in the activity of the enzyme. 3.3. Attachment of Two Protons and Generation of the Ultimate Active Species. The introduction of second e- greatly increases the potential energy, and this increase in potential energy causes the oxygenation reaction to proceed very quickly (Figure 1, stage 5). This would be a reason the cleavage of an O-O bond and the transfer of an O atom to the substrate are difficult to observe in experiments. Despite this difficulty, White and Coon proposed the ultimate active species: compound I, which directly generates the monooxygenated products of the P450 catalytic reaction.43 This compound is produced by the attachment of two protons to the distal O atom and by the subsequent detachment of an H2O molecule. It is considered that Lys178,44,45 Arg186,44,45 Asp251,44-49 and Thr25244,48,50 are important for the proton transfer to the distal O atom. Currently, the presence of compound I is believed widely.5 Hence, a proton (H5) was first attached to the distal O atom (O2) in this study. Figure 5, structure f, shows the optimized structure. The connection of the first proton increases the distance between O2 and H1 (2.04 Å f 2.25 Å) compared to that in structure e (Figure 4), which means that there is a decrease in interaction between them. The O1-O2 distance expands from 1.41 to 1.53 Å, and the O1-Fe bond shrinks from 1.94 to 1.83 Å, which
TABLE 3: Spin Density Distributions for Structures f and g structures groups of atoms
f
g
O1 O2 H5 Fe Cys (S-CH3) (S of Cys) porphinea CH4 (C5 + H1 + H2 + H3 + H4)
0.05 -0.01 0.00 1.04 0.05 (0.05) -0.13 -0.00
-0.01 -0.03 0.00 1.07 0.05 (0.05) -0.09 -0.00
a Porphine means the part of the computational model, excluding O1, O2, H5, H6, Fe, Cys, and substrate (CH4).
intimates the separation of the O2 atom to produce compound I. Due to the attachment of the first proton, the potential energy decreases by 443.9 kcal/mol. A structure in which the first proton is attached to the proximal O atom (O1) was also optimized and was shown in Figure 5, structure g. The O2-H1 distance becomes short (2.04 Å f 1.95 Å) compared to that in structure e, and the O1-Fe distance expands (1.94 Å f 1.96 Å), which is contrast to the situation in structure f. The O1-O2 distance expands (1.41 Å f 1.55 Å), which is the same as that shown in Figure 5, structure f. The potential energy decreases by 417.0 kcal/mol from e, while it is higher than that of f by 26.9 kcal/mol. Due to the attachment of the proton, the spin density is localized on the heme iron in both structures f and g (Table 3). Next, a second proton was attached to the reaction active site. Figure 6, structure h, shows the structure in which a second proton is attached to the O2 atom from structure (f). It is notable that a cleavage of the O1-O2 bond spontaneously occurs and compound I is generated with the detachment of the H2O molecule. The detaching O2 atom makes a hydrogen bond of 2.57 Å with the O1 atom through the second proton (H6). The O2 atom also interacts with the substrate because the O2-C5 distance is 3.18 Å and the O2-H1 distance is 2.10 Å. In contrast, the O1 atom that will be inserted into the C5-H1 bond does not interact with the substrate, making distances of 4.63 Å with C5 and 3.83 Å with H1. The O1-Fe bond shrinks to 1.75 Å, which is shorter than that in structure f. Since the spin density of the O1 atom is 0.59, O1 is thought to have the character of free radical (Table 4, structure h). Further calculations were carried out to obtain the structure in which one proton each is attached to the O1 and O2 atoms;
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Figure 6. Structures in the attachment of second proton. (h) Two protons are connected to the distal O atom. (i) Each proton is connected to the distal and the proximal O atoms. Numerals are interatomic distances (Å). The total charge and the spin multiplicity are 0 e and the doublet.
TABLE 4: Spin Density Distributions for Structures h, i, and j structures groups of atoms
h
i
O1 O2 H5 H6 Fe Cys (S-CH3) (S of Cys) porphinea CH4 (C5 + H1 + H2 + H3 + H4)
0.59 0.02 -0.00 -0.00 1.27 -0.67 (-0.66) -0.21 0.00
-0.00 0.00 0.00 -0.00 1.07 0.02 (0.02) -0.09 -0.00
a Porphine means the part of the computational model, excluding O1, O2, H5, H6, Fe, Cys, and substrate (CH4). See the caption of Table 1.
i.e., hydrogen peroxide is generated at the distal side of heme. This structure is shown in Figure 6, structure i. The O1-Fe bond expands to 2.13 Å, while no significant change is seen in the O1-O2 distance (1.54 Å). The O2-C5 distance increases to 3.32 Å. The spin density is localized on the heme iron (Table 4, structure i). In consequence of the attachment of the second proton, the potential energy decreases by 346.5 kcal/mol from structure f to h, by 367.9 kcal/mol from structure g to i, and by 341.0 kcal/ mol from structure f to i. Structure h is more stable than structure i by 5.6 kcal/mol. These large potential energy decrease were seen in the Harris’s result17 though they calculated only the compound I formation reaction without substrate. 3.4. Monooxygenation of the Substrate Caused by the Ultimate Active Species. The detachment of an H2O molecule from the reaction active site of structure h would induce rearrangement of atomic geometry, thereby causing interaction between the O1 atom and the exo-H1 atom, which would initiate monooxygenation of the substrate. As reported in our previous paper,14 the reaction mechanism consists of four elementary process: (1) the extraction of an H atom from the substrate, (2) the rotation of the OH group produced at the distal side of heme, (3) the connection of the OH group to the substrate radical, and (4) the detachment of the oxygenated substrate from the reaction active site. The rate-determining step is the first elementary process, and its activation energy is 15.5 kcal/mol. Further, the potential energy increases by 20.3 kcal/mol owing to the detachment of the H2O molecule.
Figure 7. Comparison of the potential energies for the low- and highspin states in the structures for the resting state and the detached state of the H2O molecule. The abscissa indicates the distance between the heme iron and the H2O molecule (Å), and the ordinate represents potential energy (kcal/mol). Levels a and b correspond to Figure 3, structures a and b. a′ is the spin sextet state in structure a and b" is the spin doublet state in structure b. This energy comparison suggests that the intersystem crossing between the low- and high-spin state occurs during the detachment of H2O molecule from the heme.
4. Discussion 4.1. Mechanism by which an H2O Molecule is Detached from the Resting State. A conversion from the low-spin resting state to the high-spin state with the detachment of the H2O molecule from the heme causes an increase in the potential energy by 15.0 kcal/mol. The mechanism by which H2O becomes detached, however, is still unclear. Figure 7 shows the results of our investigation into this mechanism. In the resting state, the low-spin state (spin doublet) is more stable than the high-spin state (spin sextet) by 3.5 kcal/mol. This is consistent with the experimental findings that the camphor-free P450cam is in the low-spin state.51 In contrast, the low-spin state after detachment of the H2O molecule (structure b" + H2O) is energetically unfavorable by 16.1 kcal/mol compared to the high-spin state after the H2O detachment (structure b + H2O). That is, while the low-spin state is more stable than the highspin state in the resting state, the high-spin state becomes more stable after the H2O detachment. Hence, an intersystem crossing between the low-spin and high-spin states would occur during the detaching process of the H2O molecule from the heme as shown in Figure 7. 4.2. Interaction between the Substrate and the Oxygen Ligand of the Heme. Several DFT studies6-14 and QM/MM studies15,16 on substrate oxidation process in monooxygenation mechanism by cytochrome P450 published elsewhere. However, there are few theoretical studies on the compound I formation step. In this study, the step was elucidated by DFT calculations
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TABLE 5: Stabilization Energy Caused by the Interaction between the Substrate and Distal Heme Liganda stabilization energy (kcal/mol) oxygen molecule bound structure singly protonated structure doubly protonated structure
d-d′ e-e′ f-f′ g-g′ h-h′ i-i′
-6.7 -7.4 -6.7 -10.8 -9.0 -3.4
a d′-i′ indicates the sum of the potential energies between the isolated methane molecule and the substrate-free structures constructed by the removal of the substrate from structures d-i.
using a model compound including substrate because it is considered that the substrate-oxy-heme interaction plays an important role in monooxygenation mechanism by cytochrome P450. Tuckey and Kamin reported that the reduced oxy-P450 energetically preferred to bind the substrate.20 In this subsection, the binding affinity of the substrate to P450 is examined in terms of the energy difference with and without the substrate at the reaction active site. First of all, geometry optimization was performed in the situation in which the substrate is removed from the structure d (Figure 4). The optimized substrate-free structure almost agrees with the substrate-bound structure in Figure 4, structure d, except for the existence of the substrate. An energy comparison between the substrate-bound structure d and the substrate-free structure with the optimized CH4 molecule indicates that the substrate-bound structure is more stable by 6.7 kcal/mol (Table 5d-d′). This result is compatible with the suggestion by Tuckey and Kamin20 and confirms that P450 interacts with the substrate when an O2 molecule is incorporated. Furthermore, the potential energies for substratebound and -free structures were compared for every step up to the generation of the ultimate active species. Table 5 shows a comparison of these energies, which indicates that the substratebound structure is always more stable than is the substrate-free structure and that the energetic advantage is 3-10 kcal/mol in every step. This result suggests that an interaction between the substrate and the oxygen ligand is maintained through the reaction process from the O2 incorporation to the generation of the ultimate active species. However, the interaction between the substrate and the distal heme ligand will be broken in the reaction route in which the ultimate active species of compound I is produced, because an H2O molecule must be detached after structure h. For the reaction route in which compound I is produced, calculations were carried out without the substrate. The changes of the structure and the potential energy after the proton attachment are almost consistent with the computational results by Harris and Loew.17 4.3. Generation of the Ultimate Active Species in the Reaction Process of Monooxygenation. As a consequence of the attachment of two protons to the reduced oxy-ferrous P450 (Figure 4, structure e), the connection of two protons to the distal O atom (O2) gives the route that shows the largest energetic stability and compound I is generated as the ultimate active species. Kamachi and Yoshizawa performed DFT calculations to estimate energetical change in the attachment of two protons using a model including Asp251 and Thr252, which would be important for the proton transfer to the distal O atom.12 The potential energy decreases by first and second proton were ca. 50 and ca. 10 kcal/mol, respectively. Harris performed DFT calculations on the mechanism of initial protonation to the
reduced oxy-ferrous species of cytochrome P450eryF by using a proton-transfer model with substrate.18 The activation energy of the process was 1.31 kcal/mol and the potential energy decrease between reactant and product was 7.9 kcal/mol. Both cases were much smaller than our results. If proton-transfer system is included our model, our values of potential energy decreases might be the same degree as their values. The generation of compound I occurs through the route of a-e f f f h and initiates the monooxygenation, as we reported previously.14 When one proton each is connected to the distal (O2) and proximal (O1) O atom of the reduced oxy-ferrous P450 (Figure 4, structure e), hydrogen peroxide is generated at the distal side of heme through the route of a-e f (f) f i or a-e f g f i. Generation of hydrogen peroxide is, however, frequently observed in experiments with a mutant of P450cam whose ability for oxygenation is low.52 Therefore, the monooxygenation reaction hardly proceeds in the presence of hydrogen peroxide and the enzyme would become inactive. 5. Conclusion The structures of the intermediates appearing until compound I formation in the reaction cycle by cytochrome P450 have been determined by theoretical calculations using the DFT method. When two protons were attached to the distal oxygen atom of oxygen molecule of a reduced oxy-ferrous heme, O-O bond cleavage spontaneously occurred and compound I formed with the detachment of a H2O molecule. The compound I structure was more stable than the structure in which one proton each was connected to oxygen atoms of the oxygen ligand of heme, and would proceeded to oxidation of a substrate.14 The calculated interaction energies between substrate and oxy-heme were 3-10 kcal/mol and suggests that an interaction between the substrate and the oxygen ligand of heme was maintained through the reaction process from the O2 incorporation to the generation of the compound I and that it contributed the stability of the reaction system. The results were compatible with the experiments by Tuckey and Kamin.20 Acknowledgment. The authors thank the Computer Center of the Institute for Molecular Science for the use of Fujitsu VPP5000 computer. The computations were also carried out by DRIA system at Graduate School of Pharmaceutical Sciences, Chiba University. References and Notes (1) Omura, T. In Cytochrome P-450, 2nd ed.; Omura, T., Ishimura, Y., Fujii-Kuriyama, Y., Eds.; Kodansha: Tokyo, 1993; pp 1-15. (2) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. ReV. 1996, 96, 2841-2887. (3) Woggon, W.-D. Top. Curr. Chem. 1997, 184, 39-96. (4) Estabrook, R. W.; Hildebrandt, A. G.; Baron, J.; Netter, K. J.; Leibman, K. Biochem. Biophys. Res. Commun. 1971, 42, 132-139. (5) Ishimura, Y. In Cytochrome P-450, 2nd ed.; Omura, T., Ishimura, Y., Fujii-Kuriyama, Y., Eds.; Kodansha: Tokyo, 1993; pp 80-91. (6) Harris, N.; Cohen, S.; Filatov, M.; Ogliaro, F.; Shaik, S. Angew. Chem., Int. Ed. Engl. 2000, 39, 2003-2007. (7) Ogliaro, F.; Harris, N.; Cohen, S.; Filatov, M.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 8977-8989. (8) Yoshizawa, K.; Kagawa, Y.; Shiota, Y. J. Phys. Chem. B 2000, 104, 12365-12370. (9) de Visser, S. P.; Ogliaro, F.; Harris, N.; Shaik, S. J. Am. Chem. Soc. 2001, 123, 3037-3047. (10) Yoshizawa, K.; Kamachi, T.; Shiota, Y. J. Am. Chem. Soc. 2001, 123, 9806-9816. (11) de Visser, S. P.; Ogliaro, F.; Sharma, P. K.; Shaik, S. J. Am. Chem. Soc. 2002, 124, 11809-11826. (12) Kamachi, T.; Yoshizawa, K. J. Am. Chem. Soc. 2003, 125, 46524661.
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