FULL PAPER DOI: 10.1002/asia.201301608

Metabolism of Halogenated Alkanes by Cytochrome P450 enzymes. Aerobic Oxidation versus Anaerobic Reduction Li Ji,[a] Jing Zhang,[a] Weiping Liu,*[a] and Sam P. de Visser*[b]

Abstract: The cytochromes P450 are a large class of heme-containing enzymes that catalyze a broad range of chemical reactions in biosystems, mainly through oxygen-atom transfer to substrates. A relatively unknown reaction catalyzed by the P450s, but very important for human health, is the activation of halogenated substrates, which may lead to toxicity problems. However, its catalytic mechanism is currently unknown and, therefore, we performed a detailed computational study. To gain insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and re-

ductive P450-mediated activation of tetra- and trichloromethane as halogenated models with density functional theory (DFT) methods. We propose an oxidative halosylation mechanism for CCl4 under aerobic conditions by Compound I of P450, which follows the typical Groves-type rebound mechanism. By contrast, the metabolism of CHCl3 occurs preferentially via an initial hyKeywords: cytochromes · density functional calculations · halogenated substrates · oxidative · reductive

Introduction

(CHCl3) are the two most representative halogenated compounds in chemistry, and are commonly encountered in the environment. A series of studies suggested that the hepatotoxicity produced by CCl4 and CHCl3 is caused by reactive metabolites originating from P450-dependent reaction pathways, namely through the biosynthesis of phosgene (Cl2CO), which is a major product during P450-mediated metabolism of both CHCl3 and CCl4. Phosgene is highly reactive and binds covalently to nucleophilic groups, including proteins, phospholipids, polar heads, and reduced glutathione, and thereby causes irreversible damage to the biosystem.[3] Cytochrome P450 enzymes are the bodys defense system against foreign chemicals and are found in the liver, where they oxygenate substrates. The P450s are heme monoxygenases that utilize molecular oxygen in their catalytic cycle and generally react with substrates via an oxygen atom transfer reaction.[4] For instance, the P450s are known to hydroxylate aliphatic groups, or epoxidize olefin double bonds, but also react via sulfoxidation or dehydrogenation processes. They perform key processes in the human body related to the biodegradation of xenobiotics in the liver, but also to the biosynthesis of, for instance, hormones.[5] The P450s are highly versatile and catalyze a series of important reactions in the body. As the P450s metabolize drugs, there is considerable interest in trying to understand the catalytic mechanism of substrate activation. However, this has been a difficult task, as, for instance, the active species was too shortlived to be detected for a long time and only recently it was

Many widely used chemicals, such as drugs, insecticides, pesticides, herbicides, and organic solvents, contain aliphatic carbonhalogen bonds, which are relatively uncommon in biosystems.[1] How the body deals with these carbonhalogen bond-containing chemicals is still poorly understood. However, it is now well known that the cytochrome P450s (P450s) play a crucial role in the biodegradation of these compounds in the body. P450-catalyzed dehalogenation of carbonhalogen bonds leads to reactive metabolites, but unfortunately, these products often are responsible for toxic effects in the body after exposure to the parent halogenated compound.[2] Carbon tetrachloride (CCl4) and chloroform [a] Dr. L. Ji, J. Zhang, Prof. Dr. W. Liu MOE Key Lab of Environmental Remediation and Ecosystem Health College of Environmental and Resource Sciences Zhejiang University Hangzhou 310058 (China) E-mail: [email protected] [b] Dr. S. P. de Visser Manchester Institute of Biotechnology and School of Chemical Engineering and Analytical Science The University of Manchester 131 Princess Street, Manchester M1 7DN (United Kingdom) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301608.

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drogen-atom abstraction rather than halosylation. Kinetic isotope effect studies should, therefore, be able to distinguish the mechanistic pathways of CCl4 versus CHCl3. We find a novel mechanism that is different from the well accepted P450 substrate activation mechanisms reported previously. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized using Marcus theory equations, which reproduce experimental product distributions.

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characterized spectroscopically.[6] The catalytic action of P450 enzymes has been extensively studied but was mostly focused on aliphatic hydroxylation and olefin epoxidation processes.[7] Little is known on the reactivity of P450 enzymes with halogenated substrates. Over the years, the P450-dependent metabolism of halogenated compounds has been investigated by several groups,[3, 8] and provided interesting yet controversial and confusing experimental results (see Scheme 1). Firstly, the

Weiping Liu, Sam P. de Visser et al.

sor in the catalytic cycle, namely the pentacoordinate ferrous–porphyrin complex, acts as a one-electron reducing agent for some substrates.[10] Previous computational work on P450-catalyzed oxygenation reactions focused on the hydroxylation of CH bonds,[11] epoxidation of C=C bonds,[12] oxidation of aromatic ring systems,[13] and the oxidation of heteroatoms.[14] These studies highlighted a spin-selective reactivity on competing high-spin (HS) and low-spin (LS) states of Cpd I.[9a] Further studies on haloperoxidases also investigated the reactivity of Cpd I models with halides to form hypohalide products.[15] However, the direct reaction of Cpd I with halogenated compounds has never been investigated in detail. As very little is known on the mechanism of oxidative and reductive metabolism of halogenated alkanes by P450 enzymes and the intrinsic features that determine these pathways, this warrants a computational study of which we will present the results here. We have utilized density functional theory (DFT) on a Cpd I model of P450 and investigated the oxidative pathway using CCl4 and CHCl3 as model substrates. Subsequently, we set up models based on the Marcus theory to predict the reaction rates for the reductive pathway between ferrous P450 species and CCl4 and CHCl3.

Results and Discussion Oxidative Reactions Scheme 1. Products observed in P450 activation of CCl4 (a) and CHCl3 (b) under different oxygen concentration conditions.

Figure 1 shows the free energy profiles of the reaction of Cpd I with CCl4 leading to products as well as the geometries of the chlorine abstraction and radical rebound transition states (B3LYP/BSII//BSI level including solvation and dispersion corrections; BSI is LANL2DZ (Fe)-6–31G**ACHTUNGRE(rest); whereas BSII is Wachters + f (Fe)-6-311 + + G**ACHTUNGRE(rest); see the Experimental Section). The initial and ratedetermining step involves a chlorine abstraction by Cpd I via barrier 2, 4TSCl leading to a radical intermediate (2,4IM) consisting of an iron-chlorosyl group and a nearby CCl3 radical, followed by a radical rebound (via barrier 2,4TSreb) to form chlorosylation products (2, 4P). Interestingly, this chlorosylation reaction mechanism follows the typical Groves mechanism of alkane hydroxylation,[16] which is stepwise via a radical intermediate. The free energies of activation of chlorine abstraction are 36.9/34.6 kcal mol1 for the lowspin/high-spin (LS/HS) states without dispersion correction, while inclusion of a dispersion correction lowers the barriers by about 5 kcal mol1 to 31.7/29.2 kcal mol1. This is analogous to observed barrier-lowering effects of dispersion corrections for the H-atom abstraction step in substrate hydroxylation of aliphatic groups by P450 enzymes.[17] The HS transition state 4TSCl is somewhat later along the reaction coordinate than its LS counterpart 2TSCl, as can be seen from a comparison of their C···Cl and Cl···O distances (HS vs. LS: 2.39 vs. 2.26  and 1.86 vs. 1.85 ). Furthermore, the C···Cl and Cl···O distances are much longer than the C···H and H···O distances normally found in hydrogenatom abstraction transition states for alkane hydroxylation

2,4

rate of metabolism of CCl4 to Cl2CO by P450 enzymes increases when the O2 concentration is lowered from 100 % to 5 %. However, a complete lack of Cl2CO products is obtained for oxygen concentration levels below 5 %. Secondly, the electrophilic chlorine was quickly formed from CC14 by P450 at low O2 concentration, while CHCl3 does not yield electrophilic chlorine under aerobic conditions. Thirdly, in vitro and in vivo studies demonstrated that CHCl3 is also metabolized oxidatively to Cl2CO, most likely through a radical pathway. Fourthly, the CCl3C and CHCl2C radicals could be trapped when CCl4 and CHCl3 were metabolized by rat liver microsomes anaerobically. The experimental studies, therefore, implicate differences in the catalytic mechanism of substrate activation by P450 enzymes for CCl4 versus CHCl3 as a substrate. To gain insight into the metabolism of halogenated substrates and the origin of the differences in substrate activation of CCl4 versus CHCl3 as well as the oxygen concentration dependence on the reactivity, we decided to do an in-depth computational study. During the catalytic cycle of P450, a high-valent iron(IV)oxo heme cation radical species known as Compound I (Cpd I) is formed, which is generally accepted as one of the most potent oxidants in nature.[7, 9] However, except for the usual oxidative mechanism, technically P450s also should be able to act as a reducing agent instead, whereby its precur-

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chlorosylation from Figure 1 above. The chlorine abstraction free energy of activation (HS: 35.8 kcal mol1; LS: 48.8 kcal mol1), however, is much higher than the corresponding hydrogen-atom abstraction barrier (HS: 14.5 kcal mol1; LS: 12.3 kcal mol1), and consequently the hydroxylation is kinetically favored. Our prediction is implicit in the preference for hydroxylation over halosylation in CHCl3 catalyzed by Cpd I of P450, and electrophilic chlorine is not expected as a product of P450-mediated oxidative metabolism of CHCl3. The studies presented in Figure 1. Free energy profiles for the quartet (HS) and doublet (LS) chlorosylation of CCl4 by Cpd I of P450, along with optimized geometries with atomic distances in  and angles in degrees for the HS (LS) states. Free Figure 1 and 2 are the first to energies (kcal mol1) obtained at 298.15 K are given relative to the doublet reactant complex 2RC at the report CCl halosylation by UB3LYP/BSII//BSI level including bulk solvation (e = 5.6) and dispersion corrections. P450 Compound I. We find stepwise mechanisms via radical intermediates with a rate-determining initial CCl/CH actiprocesses.[11a,c] Moreover, the O···Cl···C configuration of 2 vation. The free energy of activation for CCl breaking are TSCl is bent (161.58) and contrasts the almost linear chlorhigh and implicate slow reaction processes, which is in ine abstraction of 4TSCl (176.68). Previous work on hydrogen agreement with experimental reports on P450 enzymes.[8] atom abstraction by Cpd I from substrates with aliphatic Moreover, the reaction of Cpd I with chloroform should groups also found O···H···C angles that were close to linearigive dominant hydrogen atom abstraction over halosylation. ty.[11a,c] The imaginary frequencies (HS: i475.9 cm1, LS: The spin density distribution of halosylation shown in the i427.7 cm1) are much smaller than those usually observed Supporting Information demonstrates that the 2,4TSCl transifor H-abstraction processes[11a,c] (typically well above i1000 cm1). tion states for the halosylation mechanism are characterized as ironACHTUNGRE(III) states. Technically, a hydrogen-atom abstraction For the radical rebound step, the dispersion correction by Cpd I can lead to electron transfer into the a2u orbital, lowers the free energies of activation from 12.7/9.7 kcal mol1 to 6.1/3.4 kcal mol1 by about 6.5 kcal mol1 for the thereby giving a p*xz1 p*yz1 a2u2 fSub1 electronic state that can LS/HS state. The complete CCl4 chlorosylation reaction is be described as [FeIV(OH)ACHTUNGRE(Por)—SubC]. Alternatively, elec1 calculated to be slightly exergonic (HS: 2.2 kcal mol , LS: tron transfer fills the p*xz orbital with a second electron and 11.8 kcal mol1). keeps the a2u orbital half-filled to give an ironACHTUNGRE(III)-type radiIn the case of a reaction of Cpd I with CHCl3 as a subcal intermediate: [FeIII(OH)ACHTUNGRE(Por+C)—Sub·]. Previous studies strate, either activation of the CH bond or a CCl bond showed these two electronic states to be close in energy, and can take place, thus leading to competitive hydroxylation environmental perturbations were found to influence the orand chlorosylation. The results of our DFT studies on these dering.[19] Gas-phase studies usually give the iron(IV) pathprocesses are indicated in Figure 2 a and 2 b, respectively. way as the ground state,[9a, 11b,g, 19] but interestingly here the 2,4 The chloroform hydroxylation by Cpd I follows the typical ironACHTUNGRE(III) pathway is well lower in energy. Group spin densities and charges characterize the transition states as 2,4TSClGroves-type hydrogen-atom abstraction/radical rebound mechanism via a rate-determining hydrogen-atom abstracACHTUNGRE(III) electronic structure with a porphyrin cation radical tion barrier 2,4TSH and an iron(IV)-hydroxo radical intermoiety. By contrast, it was reported that the prevalence of 2,4 mediate. Similarly to previous studies on substrate hydroxylTSH(IV) over 2,4TSHACHTUNGRE(III) species is common for most ation by P450 models, the radical rebound has a genuine realkane hydroxylation in gas phase calculations.[11c] In the bound transition state 4TSreb on the HS surface but is barpresent work, we attempted to locate the transition states 2,4 TSCl(IV) by swapping molecular orbitals in 2,4TSClACHTUNGRE(III). rierless on the LS state.[11a] The structures of 4,2TSH show typical features of hydrogen-atom abstraction transition However, despite several attempts we were not able to optistates with an almost linear OHC angle and a large imagmize their geometries, and the electronic configuration reinary frequency (HS: i1365.1 cm1, LS: i1516.8 cm1), which verted to the ironACHTUNGRE(III) states during the SCF convergence. Subsequently, we tried to locate the alternative mechanism implicates that the reaction will proceed with a large kinetic and characterize the iron(IV)-type transition states by runisotope effect.[18] As shown in Figure 2 b, the chlorosylation ning geometry scans starting from the radical intermediate mechanism of CHCl3 closely resembles that for the CCl4

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Figure 2. (a) Free energy profiles for the quartet (HS) and doublet (LS) hydroxylation of CHCl3 by Cpd I of P450, along with optimized geometries with atomic distances in  and angles in degrees for the hydrogen abstraction in the HS (LS) states. (b) Free energy profiles for the quartet (HS) and doublet (LS) chlorosylation of CHCl3 by Cpd I of P450, along with optimized geometries with atomic distances in  and angles in degrees of the chlorine abstraction in the HS (LS) states. Free energies (kcal mol1) obtained at 298.15 K are given relative to the doublet reactant complex 2RC at the UB3LYP/BSII//BSI level including bulk solvation (e = 5.6) and dispersion corrections.

in which different variables were chosen as the “reaction coordinate”. In these geometry scans we did a full geometry optimization at each point along the scan, but with one degree of freedom, that is, the reaction coordinate, fixed. Despite these efforts to find a general feature of the virtual “2,4TSCl(IV)” states, however, the partial geometry optimizations also did not lead to the “2,4TSCl(IV)” states. Therefore, the iron(IV) transition states and radical intermediates are high in energy and will not play a key role in the reaction mechanism. As shown previously in several studies,[20] the hydrogenatom abstraction barrier by iron(IV)-oxo complexes often correlates with the strength of the CH bond that is broken as well as with the strength of the OH bond that is formed. In a similar way, in the halosylation reaction a CCl bond in the substrate is broken and an OCl bond is formed. One would, therefore, expect a correlation between the halosylation barrier TSCl and the strength of the CCl bond that is broken. We thus calculated the bond dissociation energy

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(BDE) of the CCl bond in CCl4 and CHCl3 and found values of 58.4 and 65.7 kcal mol1, respectively, at the B3LYP/aug-cc-pVTZ level of theory. Although the BDE values of the CCl bond in CCl4 and CHCl3 are much lower in energy than the corresponding values of CH bonds for alkanes (typically more than 90 kcal mol1)[11c] this contrasts their relative barrier height for oxygen atom transfer. The overlap effect involving the oxygen and chlorine orbitals, along with the availability of the chlorine lone-pair electron density attacked by the oxygen of Cpd I, may result in marked bond stretching to reach the loose CCl transition state (stretched OCl and CCl lengths as shown in Figure 1 and 2). In such cases, the transition states are of TSClACHTUNGRE(III)-type. Electronically, these transition states match those found for methane hydroxylation by Cpd I, which also proceeds late and on an ironACHTUNGRE(III) potential energy surface.[11a,c] Consequently, the chlorine-transfer transition state provides a different electronic structure compared with that for hydrogenatom transfer processes of most alkanes.

Reductive Reactions As halogenated compounds are highly susceptible to be involved in reductive elimination reactions, we decided to investigate an alternative mechanism of CCl4 and CHCl3 activation by cytochrome P450 enzymes, whereby these substrates react with the iron(II) porphyrin precursor of Cpd I by electron transfer (ET) and thereby split off a haloalkyl radical. During the catalytic cycle of P450, the pentacoordinate ferrous-porphyrin complex is not oxidized under low O2 concentration or anaerobic conditions; thus, this ferrous P450 species could transfer an electron to a substrate and return to the pentacoordinate ferric-porphyrin complex.[5b] The ET may take place either by a stepwise mechanism in which the transfer and bond breaking are successive or by a concerted ET mechanism whereby the bond breakage and the electron transfer happen synchronously,[21] as sketched in Scheme 2.

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ET process (DG*ET) is described in terms of two thermodynamic parameters, namely the free energy of reaction (DG0ET) and the reorganization energy (l), Equation (1). The parameter l consists of two components, namely the inner reorganization energy li, described as a function of the bond dissociation energy (BDE) of the fragmenting bond, and the solvent reorganization energy l0, see the Supporting Information for details of these calculations.

Scheme 2.

Experimental studies have characterized the pentacoordinate ferric-porphyrin complex as a sextet spin ground state, and the one-electron reduced pentacoordinate ferrous-porphyrin complex is known to have a high-spin quintet state.[9a] The high-spin ground states of both species were also confirmed in our current calculations and, therefore, we only consider the high-spin states of ferrous (charge: 1) and ferric (charge: 0) porphyrin complex in the following. We first investigated whether the reduction of CCl4 and CHCl3 by the pentacoordinate ferrous-porphyrin complex is stepwise or concerted. However, geometry optimizations of the radical anions failed to give stable conformations, which imply that the reactions will be concerted. It is clear that the “radical anions” undergo substantial rearrangement to form complexes between the corresponding haloalkyl radicals and halide anions in the gas phase into a radical-anion pair. An optimization in solution (mimicked with a dielectric constant of e = 5.6) led to complete dissociation of the haloalkyl radicals from the halide anions. Firstly, we tested the inner-shell reductive mechanism, whereby one chlorine atom of CCl4 or CHCl3 was presumed to bind to the ferrous center of P450 to form the FeII—ClR species (R = CCl3 or CHCl2), with subsequent CCl bond cleavage to evolve to FeIIICl + R radical by an inner ET process. The present B3LYP/BSI calculations only indicates a binding mode between the chlorine atom and the ferrousporphyrin complex of P450 in the quintet spin state, as shown in Figure 3, in which the (Fe)ClC distance is about 3 , confirming that the ClC bond is broken already. Therefore, the reactant complex between CClR and ground-state porphyrin iron(II) does not exist energetically, and consequently the inner-shell reductive pathway can be ruled out. However, the halogen atoms originating from the reactivity of halogenated alkanes may bind in such a way to inhibit O2 binding.

DGET ¼

ð1Þ

Accordingly, we obtained DG*ET values with the PCM (e = 5.6)-B3LYP/BSI method for the concerted ET mechanism of 5.7 kcal mol1 for CCl4 and 11.9 kcal mol1 for CHCl3, as shown in Figure 4. These free energies of activation are considerably lower in energy than the barriers for chlorine abstraction by Cpd I, whereby values of 29.2/31.7

Figure 4. Free energy profiles (kcal mol1) for electron transfer from the pentacoordinate ferrous-porphyrin complex to the substrates CCl4 (a) and CHCl3 (b).

(HS/LS) and 35.8/48.8 (HS/LS) kcal mol1 for CCl4 and CHCl3, respectively, were obtained (see above). These studies implicate that halogenated hydrocarbons are good electron acceptors and react by electron transfer upon entering the substrate binding pocket of the enzyme. To further assess the electron-accepting ability of CCl4 and CHCl3, we estimated the vertical electron affinities (VEA) using PCMACHTUNGRE(e=5.6)-B3LYP/aug-cc-pVTZ method. Thus, CCl4 and CHCl3 are found to have VEA values of 1.88 eV and 1.26 eV, respectively, which are values that are much larger than those found for alkanes, such as methane (0.54 eV) and propane (0.13 eV). Therefore, VEA maybe a good measurement of the capacity of halogenated hydrocarbons for trapping an electron from the pentacoordinate ferrous-porphyrin complex.

Figure 3. Binding mode of CCl4 and CHCl3 with the ferrous center of the porphyrin complex of P450 in the high-spin quintet ground state at the UB3LYP/BSI level (unit: )

Subsequently, we estimated the free energy barriers of activation of the outer sphere ET processes from the Marcus theory, whereby the Savant model[22] for the concerted ET mechanism was used. The free energy of activation for the

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 2 l DG0ET 1þ 4 l

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O2 concentration conditions. We, therefore, predict a novel mechanism of activation of halogenated alkanes by P450 enzymes that takes place via a reductive dehalogenation with low O2 concentration. A subsequent reaction of the obtained radicals with molecular oxygen forms Cl2CO products and electrophilic chlorine. Figure 5. Free energy profile (kcal mol1) (single-point in PCM, e = 78) along with critical optimized geometries with atomic distances in  for the reactions of CCl3 radical with O2 on doublet spin state and subsequent intramolecular chlorine abstraction leading to Cl2CO and electrophilic chlorine optimized at the UB3LYP/BSII level.

Figure 7 shows the free energy profile in PCM (e = 78) for the decomposition of CCl3OH to Cl2CO. The direct H-atom rearrangement from the OH group to the Cl atom would involve a very large activation barrier of 22.4 kcal mol1. By contrast, micro-solvation with a single water molecule reduces the activation barrier to 9.8 kcal mol1, thus indicating that the ultimate Cl2CO formation apparently can be catalyzed by a single water molecule. So the chief product (CCl3OH) for CHCl3 metabolism under aerobic conditions should rapidly dehydrochlorinate to form Cl2CO, which explains why experimentally only formation of Cl2CO but not of electrophilic chlorine in the oxidative pathway is observed.

Cl2CO and Electrophilic Chlorine Formation Mechanism from the CCl4 Metabolic Pathway In Figure 5 we present the free energy profile as calculated in PCM (e = 78) for the reactions of the CCl3 radical with O2 on the doublet spin state. The reaction is initiated by O2 binding to the carbon center to give a peroxy radical, which is found to be exergonic by 2.7 kcal mol1. Note that the process calculated for the quartet spin pathway is highly endergonic. The overall reaction is highly exergonic (DG = 39.1 kcal mol1), and although that implicates that the reaction can take place at normal temperatures, it actually is hampered by the high barrier for this process. One other feasible mechanism we considered is a radical coupling reaction, whereby the peroxy radical reacts with either a CCl3 radical or the peroxy radical itself to form a CCl3O radical, as described in Equations (2) and (3). Experimental studies showed these reactions to proceed very fast.[23] Our DFT calculations at the BSII level of theory (single-point in PCM, e = 78) show that these two radical coupling reactions are highly exergonic by 33.9 kcal mol1 [Eq. (2)] and 37.2 kcal mol1 [Eq. (3)], respectively. CCl3 O2 C þ CCl3 O2 C ! CCl3 OC þ CCl3 OC þ O2

ð2Þ

CCl3 O2 C þ CCl3 C ! CCl3 OC þ CCl3 OC

ð3Þ

Figure 6. The feasible pathway for the decomposition of the complex of CCl3O2C and ferrous center of P450, along with atomic distances in  of the complex in the sextet (quartet) state optimized at the UB3LYP/BSI level.

The formed CCl3O radical with one very weak CCl bond decomposes readily to form Cl2CO and a chlorine radical with nearly negligible free energy of activation (0.3 kcal mol1 calculated at B3LYP/BSII level; single-point in PCM, e = 78). Another feasible pathway involves an initial complexation of the CCl3O2 radical on the ferrous center of P450. This complex with partly cleavaged OO bond shown in Figure 6 may also produce the CCl3O radical leading to Cl2CO and chlorine radical as products. The above two proposed Cl2CO and electrophilic chlorine formation pathways give a good rationalization of the metabolism rate of CCl4 to Cl2CO by P450 enzymes under low

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Cl2CO Formation Mechanism from the CHCl3 Metabolic Pathway

Figure 7. Free energy profile (kcal mol1) (single-point in PCM, e = 78), along with critical optimized geometries with atomic distances in , for the decomposition of CCl3OH to Cl2CO catalyzed by one water molecule optimized at the UB3LYP/BSII level.

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Conclusions

tions on B3LYP/BSI-optimized structures at the BSII level of theory. Bulk polarity effects were evaluated with the polarizable continuum model (PCM) using chlorobenzene as a solvent (e = 5.6) at the B3LYP/BSI level, except for the Cl2CO and electrophilic chlorine formation pathway, which was done with the PCM model using water (e = 78) at the B3LYP/6-311 + + G** level. The relative free energies for the oxidative reactions shown below are at the B3LYP-D/ BSII//B3LYP/BSI level of theory and include solvation and dispersion corrections, unless pointed out specifically. All calculations were carried out with the Gaussian 09 program package.[30] In previous studies, we extensively benchmarked and calibrated our methodologies and have shown that little changes in free energies of activation are observed when the density functional method is changed;[31] therefore, we focused here on B3LYP with dispersion corrections only.

We present a series of computational studies on the metabolism of CCl4 and CHCl3 by P450 enzymes through both oxidative and reductive pathways. The work investigates for the first time the oxidative mechanism of halosylation of halogenated alkanes by Compound I of P450, which is found to be mechanistically analogous to hydroxylation of alkanes. However, the reductive pathway rationalized using the Marcus theory shows that the electron transfer from the pentacoordinate ferrous-porphyrin complex of P450 to the halogenated alkanes needs much lower barriers than the ones for chlorine abstraction by Compound I of P450 in the oxidative pathway. The P450-dependent metabolism of CCl4 and CHCl3 studied in this work may be extended to most halogenated alkanes with high vertical electron affinity values in the environment: 1) For halogenated alkanes with both halogen and hydrogen atoms, the hydrogen abstraction by Compound I of P450 is favored over chlorine abstraction under aerobic condition, while the reductive dehalogenation is the main pathway in the absence of oxygen, and 2) carbonyl halogen compounds and electrophilic halogen may be formed during P450-mediated metabolism of fully halogenated alkanes at low O2 concentration.

Acknowledgements L.J. gratefully thanks the support from the National Natural Science Foundation of China (Grant No. 21307107). The China National Supercomputing Center in Shenzhen (Shenzhen Cloud Computing Center) is acknowledged for providing computational resource and CPU time (Contract No. S13037).

Experimental Section

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Methods We selected a model of the active site of P450 enzymes that was shown previously to accurately predict reactivities of Cpd I as well as its spectroscopic parameters.[9a, 20c, 24] The model contains iron-protoporphyrin IX, whereby all side chains are replaced by hydrogen atoms and the axial cysteinate ligand is replaced by a thiolate group. This system is overall charge neutral and was investigated in the lowestlying doublet and quartet spin states. The unrestricted hybrid B3LYP density functional[25] was employed combined with the double-z LANL2DZ(Fe)/6-31G** basis set[26] (denoted as BSI) for all geometry optimizations and frequency calculations, except for the Cl2CO and electrophilic chlorine formation mechanism for which the 6-311 + + G** basis set (denoted as BSII) were used. The computed vibrational frequencies were used to quantify the conversion from the electronic energy to the Gibbs free energy at 298.15 K and 101.325 kPa. All local minima are characterized with real frequencies, whereas the transition states had one imaginary frequency for the correct mode. More accurate energies were determined by single-point calculations on the optimized geometries with a basis set which describes iron by the Wachters + f all-electron basis set[27] on iron, and all other atoms by the 6-311 + + G** basis set (denoted as BSII). These methods were previously shown to reproduce experimental rate constants of oxygen atom transfer reactions by iron(IV)-oxo complexes to within 3 kcal mol1.[28] Since B3LYP lacks dispersion interactions,[29] we corrected the energies by single-point B3LYP-D calcula-

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