Mechanistic Insights into the Directing Effect of Thr303 in Ethanol

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Mechanistic Insights into the Directing Effect of Thr303 in Ethanol Oxidation by Cytochrome P450 2E1 Qianqian Lu, Jinshuai Song, Peng Wu, Chunsen Li, and Walter Thiel ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b00907 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Catalysis

Mechanistic Insights into the Directing Effect of Thr303 in Ethanol Oxidation by Cytochrome P450 2E1 Qianqian Lu,a,b Jinshuai Song,a,b Peng Wu,a,c Chunsen Li*a,b and Walter Thiel*d a.

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of

Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China. b.

Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, Xiamen, Fujian

361005, China. c. University

of Chinese Academy of Sciences, Beijing 100049, China.

d. Max-Planck-Institut

für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr,

Germany.

ABSTRACT There is a long-standing mechanistic consensus that alcohol oxidation by Cytochrome P450 enzymes is triggered by hydrogen abstraction from the α-C-H bond of the alcohol. Through combined MD simulations and QM/MM calculations we demonstrate that this is not the case in P450 2E1-mediated ethanol oxidation. We show that while the O-H bond is stronger than the α-C-H bonds in alcohols, the intrinsic reactivity of O-H and α-C-H bonds is comparable for hydrogen abstraction, due to the strong electrostatic interaction between the ethanol hydroxyl group and the Fe=O moiety. Thus, the binding of ethanol to the Fe=O moiety in the P450 2E1 pocket is of particular importance to the reaction mechanism. We further show that the Thr303 residue plays a crucial role in confining the ethanol substrate in the active site of P450 2E1 and thereby steering the initial hydrogen abstraction from the O-H bond of ethanol. Because of the highly endothermic O-H bond cleavage, the subsequent hydrogen abstraction of α-C-H bond is the overall rate-determining step for ethanol oxidation. These mechanistic findings are in agreement with available experimental data (e.g., kinetic isotope experiments, electron spin resonance analysis). Our work sheds light on the puzzling mechanism of ethanol oxidation in P450 2E1 by identifying the directing effect of Thr303 on substrate orientation, which complements its role as proton-shuttle mediator during the formation of compound I.

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Keywords: P450, alcohol oxidation, hydrogen abstraction, quantum mechanics/molecular mechanics, molecular dynamics

1. Introduction Cytochrome P450 enzymes, capable of activating molecular oxygen, are important biocatalysts for a wide range of reactions, such as hydroxylation, epoxidation, sulfoxidation and N-dealkylation.1-5 Cytochrome P450 2E1 (CYP2E1) is one of the most intriguing human P450 enzymes due to its predominant role in drug metabolism and human health.6-10 CYP2E1 has a broad substrate specificity,11,12 including short chain alcohols and aldehydes, bicyclic and monocyclic compounds (e.g., benzene and caffeine), and some long chain fatty acids such as arachidonic acid. The ability of CYP2E1 to metabolize ethanol was not discovered until 1968, when Lieber and DeCarli characterized a P450-dependent microsomal ethanol oxidizing system (MEOS) accounting for chronic alcohol consumption.13,14 Since MEOS represents the second most important pathway for ethanol metabolism15 and ethanol-induced oxidant stress has a great impact on the process of alcoholic liver injuries,16-20 the high activity of CYP2E1 in ethanol metabolism has drawn intense attention. HA_O Mechanism H Hydrogen Abstraction of O-H

CH3CH2O O

Hydrogen Abstraction of  -C-H

Fe

O Fe(IV)

H3C

SH

CH3CH2OH

Rebound

gem-diol Mechanism

HO

CH OH

Fe

Dehydration

SH

SH

H

Hydrogen Abstraction of  -C-H

CH3CHOH

CH3CHO H2O Fe SH

O Fe Hydrogen Abstraction of O-H SH

HA_C Mechanism

Scheme 1. Proposed mechanisms for P450-mediated ethanol oxidation. Conceptually, three mechanisms have been proposed for P450-mediated ethanol oxidation based on the iron(IV)-oxoporphyrin π-cation radical, so-called compound I (Cpd I). As shown in Scheme 1, the HA_O and HA_C mechanisms are dual hydrogen abstraction pathways, which differ in the sequence of O-H bond cleavage and α-C-H bond cleavage. As a branch of the HA_C mechanism, the gem-diol mechanism includes the rebound of the Fe-OH/substrate-radical and the dehydration of the gem-diol intermediate. Given that α-C-H bond cleavage was suggested to be 2


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rate-determining,21,22 the gem-diol or HA_C mechanisms are commonly accepted as the plausible pathways for alcohol oxidation by P450 enzymes. However, in 2017, Iwahashi et al. detected ethanol-derived oxygen-centered radicals in a biomimetic ethanol oxidation reaction mediated by the (TPP)•+Fe(IV)=O complex,23 which indicates that the O-H bond of ethanol is broken first during ethanol oxidation. Besides, an early quantum mechanical (QM) study reported by Shaik et al. also revealed the feasibility of the cleavage of ethanol O-H bond.24 While QM model calculations can be used to explore the intrinsic mechanism of ethanol oxidation, a truncated active-site model may not account for the actual reaction in CYP2E1. Besides, the ethanol-binding machinery in the active site of CYP2E1 seems complicated in view of the large variation of the CYP2E1 binding pocket. To unveil the mechanism of ethanol oxidation in CYP2E1, we conducted combined molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations.25,26 As we shown herein, the Thr303 residue is crucial to keep the ethanol substrate near the Fe=O moiety in CYP2E1 pocket. Furthermore, the strong hydrogen-bonding interaction between the side chain hydroxyl of Thr303 and the hydroxyl of ethanol confines the orientation of ethanol and thereby facilitates the cleavage of the ethanol O-H bond during ethanol oxidation.

2. Computational Details 2.1 System Preparation and Molecular Dynamics Simulations All calculations during system setup were performed with the CHARMM program.27 To date, there are six crystal structures of CYP2E1 complex in the Protein Data Bank.28-30 The initial protein coordinates of CYP2E1 were assigned based on the chain A of crystal structure 3E6I.30 The missing residue Met138 was added by superimposing structure 3E6I on another X-ray structure of CYP2E1 (PDB ID 3T3Z),28 while other missing residues on the protein surface were ignored. The Fe=O species was constructed by adding an oxygen atom at a distance of 1.65 Å above the iron atom of the heme moiety. The ethanol substrate was manually modelled into the active site of CYP2E1 based on a docking investigation using Chimera.31 Hydrogen atoms of the protein residues were added by the HBUILD module32 of the CHARMM program. The protonation states of titratable residues were determined by the PROPKA program33,34 and visual inspection of their corresponding hydrogen-bonding networks.35 3


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The substrate-bound enzyme was solvated by using the VMD software36 to construct a water layer of 13 Å. Six Cl

―

ions were added stochastically to neutralize this system. In order to

equilibrate the inner water layer, the following steps were executed: (i) optimizing the inner 7 Å layer of solvent by 1000 steps of steepest descent (SD) and 1000 steps of adopted basis Newton-Raphson (ABNR) while keeping the protein backbone and the outer 6 Å layer of water fixed, (ii) heating the protein from 0 to 300 K for 15ps, (iii) equilibrating the inner 7 Å layer water at 300 K for 15 ps with a 1 fs time step, (iv) repeating the above three steps until the requisite water molecules for solvation were less than 50. After the solvation of the protein, the whole system was subjected to MD simulations (up to 50 ns) while keeping the Fe=O moiety, the Cl ― ions and the outer 6 Å of the water layer fixed. 2.2 QM/MM Calculations All QM/MM calculations were performed with the ChemShell program.37 The entire system was separated into a QM and a MM region, with the QM region containing ethanol and propionate-truncated Cpd I. During the QM/MM geometry optimizations, the MM atoms located within 8 Å from the QM region were included in the active region. The QM region was calculated with Turbomole38 while the MM region was modeled with the CHARMM22 force field using the DL_POLY module.39 The electronic embedding scheme40 was applied to account for the polarizing effect of the enzyme environment on the QM part. Hydrogen link atoms41 with the charge-shift model42 were employed to treat the QM/MM boundary. The unrestricted B3LYP functional43,44 combined with a mixed basis set (Def2-TZVP for iron and Def2-SVP45 for the other atoms, denoted as B1) was used for geometry optimization, as B3LYP is known to perform well for P450 systems.46,47 Transition states were optimized by a relaxed potential energy surface (PES) scan followed by a full transition state optimization via the dimer method as implemented in the DL-FIND optimizer.48 Frequencies were calculated at the UB3LYP/B1 level of theory to characterize each stationary point as a local minimum or a transition state and to obtain the zero-point energy (ZPE). Single-point calculations were conducted at the UB3LYP/Def2-TZVPP level (B2).45 QM/MM energies reported in this work were obtained by adding the ZPE to the single-point energies obtained at the UB3LYP/B2 level of theory. Both the doublet and quartet states of Cpd I were investigated in this work. The calculated results show that intermediates and transition states on the doublet and quartet potential energy 4


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surfaces have similar energies, in accordance with the two-state reactivity.49,50 Considering that the species in the doublet and quartet states feature the same interactions between the QM region and surrounding protein residues, we just present the results of the doublet surface in the main text. The calculated results of the quartet surface are summarized in Supporting Information, for the sake of brevity.

3. Results and Discussion Figure 1a displays the active cavity of CYP2E1. The dome region is constituted by five phenylalanine residues (Phe106, Phe116, Phe207, Phe298 and Phe478), while the wall region is mainly formed by residues Ile115, Ala299, Leu363, Val364 and Leu368. Thr303 is the only polar residue adjacent to the active site of CYP2E1. The available crystal structures of CYP2E1 do not contain water molecules in the active site,28-30 in line with the hydrophobic character of both the CYP2E1 pocket and the bound inhibitors (e.g., methyl pyrazole, indazole, pilocarpine). Based on the structural features of the active cavity of CYP2E1, we first conducted MD simulations to investigate the ethanol-binding machinery.

pose-A pose-B

(a)

(b)

Figure 1. (a) Active cavity of CYP2E1 (PDB ID: 3E6I). (b) Molecular docking of ethanol substrate into the active site of CYP2E1. 3.1 MD results of CYP2E1 with ethanol In 2003, Harris et al. studied the interactions of CYP2E1 with various substrates, such as chlorzoxazone, p-nitrophenol, acetominophen, and methoxyflurane.51 The docking of these substrates to the oxyferryl moiety of CYP2E1 suggested that the lowest-energy configurations 5


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involve intimate interactions between the substrates and oxyferryl.51 For example, the docking configuration of p-nitrophenol included a hydrogen-bonding interaction between the hydroxyl group of p-nitrophenol and the oxyferryl.51 By contrast, the present docking study with ethanol favors a configuration in which ethanol closely interacts with the Thr303 residue. In this configuration, the hydroxyl group of ethanol engages in two hydrogen-bonding interactions, one with the side chain hydroxyl of Thr303 and the other one with the backbone carbonyl of Ala299 (Figure S1). The difference from the previous docking results51 can be attributed to the different sizes of the alcohols (ethanol vs. p-nitrophenol). Interestingly, when Thr303 is mutated into Ala, the docking favors a configuration in which the hydroxyl of ethanol interacts with the oxyferryl (Figure S1). The present docking results thus indicate that the ethanol substrate may engage in hydrogen-bonding interactions with the oxyferryl (pose-A, Figure 1b) or the side chain hydroxyl of Thr303 (pose-B, Figure 1b) in the active cavity of CYP2E1. Therefore, we carried out two MD simulations starting from pose-A and pose-B, respectively. We note that after the solvation procedure, the relatively large ethanol binding pocket of CYP2E1 contains three water molecules (Figure S2): whether these water molecules stay in the active site of CYP2E1 during the MD simulations depends on the orientation of ethanol, as discussed below. In the 22 ns MD simulation starting from pose-A (Figure 2a), ethanol tumbles freely in the CYP2E1 pocket. There are two dominant orientations: either the hydroxyl group of ethanol gets close to Fe=O moiety and forms a hydrogen-bonding interaction with the Fe=O end (scenario a, Figure S3), or ethanol turns the hydroxyl group upward and directs its α-CH2 group towards the oxo moiety (scenario b, Figure S3). These two orientations of ethanol are associated with different solvation patterns within the binding pocket: in the former case (scenario a), 1-2 water molecules are moving freely in the active cavity, while in the latter case (scenario b), water molecules are likely to engage in a water network with the hydroxyl group of ethanol and the backbone carbonyl of protein residues (Figure S3).52 However, in scenario b, the water network involving ethanol is quite unstable and breaks down quickly during the MD simulations. Moreover, the dome region of the CYP2E1 pocket is gradually distorted due to the repeated flips of ethanol, and consequently ethanol moves away and remains far from the Fe=O moiety after 21 ns of dynamics (Figure 2a).53 6


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ACS Catalysis

H3C

C

H

O

H1

H2 O Fe

(a)

THR303 O

H

H3C O H1

H C

H2 O Fe

(b)

Figure 2. Variation of the H…O=Fe distances during the MD simulations for pose-A (Figure 2a) and pose-B (Figure 2b). H1 refers to the hydroxyl hydrogen of ethanol, and H2 refers to one α-CH2 hydrogen of ethanol. In the 50 ns MD simulation starting from pose-B, the ethanol substrate does not maintain its initial location (Figure 1b), but rather slides into the middle between Thr303 and the Fe=O moiety after 5 ns. In this conformation, the hydroxyl group of ethanol engages in hydrogen-bonding interactions with both the side chain hydroxyl of Thr303 and the oxygen of the Fe=O moiety (Figure 2b). The Thr303 residue does not hold ethanol tightly during the early stages of the simulation, as reflected by occasional fluctations of the distance between ethanol and Fe=O moiety. This is because Thr303 sometimes flips its hydroxyl group to form a hydrogen-bonding interaction with the carbonyl oxygen of the Ala299 residue. However, in the course of the simulation, the interaction between Thr303 and ethanol is enhanced while the distance between Thr303 and Ala299 increases. During the last 10 ns of the dynamics, the ethanol substrate is stuck tightly and unable to slide away from the Fe=O moiety. Water molecules are absent in the ethanol binding pocket for almost the entire simulation (pose-B) because Thr303 prevents the access of water molecules to the hydroxyl group of ethanol. 7


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The two MD simulations demonstrate that the Thr303 residue is essential to place ethanol near the Fe=O moiety of CYP2E1 pocket, and the large pocket of CYP2E1 can be readily distorted by flips of the substrate. By comparing the earlier 6 ns trajectories (see Figure S5), we find that the Thr303-ethanol hydrogen-bonding interaction in the second MD simulation (pose-B) derives from a continuous contact between ethanol and Thr303 that weakens the original Thr303-Ala299 interaction and frees Thr303 to form an H-bond with ethanol. A diminished interaction between threonine and nearby alanine or glycine residues was previously also reported in P450BM3 and P450cam.54,55 Furthermore, these MD results also reveal that the water molecules in the active site of CYP2E1 are too flexible to form a stable water network, consistent with the absence of hydrophilic residues in the distal pocket of CYP2E1. Considering that the MD simulation from pose-B results in a converged configuration with ethanol stuck between the Fe=O moiety and the Thr303 residue, pose-B represents an appropriate model for exploring the actual mechanism of ethanol oxidation in CYP2E1 enzyme. Nevertheless, the MD simulation from pose-A provides a large variety of ethanol conformations, and thus serves as a good basis for studying the mechanism of ethanol oxidation without the influence of the Thr303 residue. The comparison of QM/MM calculations in the absence and presence of Thr303 will clarify how Thr303 affects the mechanism of ethanol oxidation. Therefore, configurations from both MD simulations were used as starting points for QM/MM calculations.

3.2 QM/MM results 3.2.1 Ethanol oxidation in the absence of the Thr303 effect The QM/MM results of this subsection are meant to clarify the intrinsic reactivity of ethanol O-H and α-C-H bonds in the hydrogen abstraction process. For this purpose, we extracted two starting structures from the early stages of the MD simulation from pose-A to investigate ethanol oxidation mechanisms trigged by either ethanol O-H cleavage or α-C-H cleavage. Figure 3 shows the energy profiles of the HA_O and gem-diol mechanisms (in the S=1/2 spin state of Cpd I), and Figure 4 displays the QM/MM optimized structures of critical intermediates. HA_O mechanism The HA_O mechanism starts from complex A1_RC, in which the hydroxyl group of ethanol points to the oxo of Cpd I. The distance between ethanol and Cpd I is 1.81 Å and the Fe-O-H angle is 124.9º (Figure 4). These structural features are similar to the reactant complex 8


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ACS Catalysis

of the QM study reported by Shaik et al.24 The hydrogen abstraction of the ethanol O-H bond occurs via transition state A1_TS1 and generates HO-Fe(IV)/ethoxyl radical intermediate A1_IM1. Thereafter the second hydrogen abstraction will occur on the α-C-H bond of the ethoxyl radical. Intriguingly, A1_IM1 first isomerizes to intermediate A1_IM2 which is more stable than A1_IM1 by 6.9 kcal/mol. Since a simple Fe-OH bond rotation should not change the energy much,56 the stabilization is expected to be caused by the protein enviroment. Indeed, as shown in Figure 4, the structure of A1_IM2 features a hydrogen-bonding interaction between the Fe-OH moiety and the carbonyl oxygen of Ala299 (1.76 Å, Figure 4). Thus, the thermodynamical stability of the Fe-OH/ethoxyl radical is governed by the surrounding active-site residues. The second hydrogen abstraction transition state A1_TS2 occurs with a small barrier of 1.8 kcal/mol to deliver the acetaldehyde product. Gem-diol mechanism For the gem-diol mechanism, the starting complex is A2_RC, in which ethanol engages in an extended hydrogen-bonding network ranging from the backbone carbonyl of the Gly300 residue to the protein surface (Figure 4). Due to the hydrogen-bonding network, A2_RC has different Fe-O1-H2 and O1-H2-C angles compared with the QM optimized structure.24 The first hydrogen abstraction via transition state A2_TS1 has a barrier of 15.5 kcal/mol. From the resulting Fe-OH intermediate A2_IM1, either the second hydrogen abstraction (HA_C mechanism) or the OH-rebound (gem-diol mechanism) might occur. However, the second hydrogen abstraction is impossible, since the hydroxyl group of ethanol constrained by water network cannot be abstracted by the Fe-OH moiety. By contrast, the rebound process is barrierless and highly exothermic (Figure S6), indicating that the production of gem-diol intermediate A2_IM2 is favorable not only kinetically but also thermodynamically.57 Subsequent dehydration via A2_TS2 requires an energy barrier of 41.7 kcal/mol, but this step could be greatly promoted by water acting as proton shuttle.58

9


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(a)

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CH3 H2C O

H

O1-H1-O2 = 171.5 O

O

HC

CH3 H

Fe

O

SH

A1_RC 0.0

A1_IM1 +12.0

A1_IM2 +5.1

O

A1_TS2 +6.9

CH3 O C H2 H O

SH

Fe

A1_TS1: hydrogen abstraction of OH

H

O C

H

SH

O1-H2-C = 169.0 1.26 Fe-O1-H2 = 132.8

H2

CH3

1.48 O1 H

O

Fe

Fe

Fe

SH

SH

-62.0

A1_TS2: hydrogen abstraction of -C-H of oxygen-centered radical

A1_PC (b)

O1

Fe

C

Fe

OH O

1.10

SH

CH3 H2C O H

CH3 H2C

H1

1.33

H

Fe

A1_TS1 +16.0

Fe-O1-H1 = 117.9

O2

OH H3C C H H

C H2

O

1.22

1.41

Fe SH

A2_TS1 +15.5

A2_RC

H

O O

H3C C H

S

Fe

A2_IM1 -10.4

SH

H

H O H

A2_TS2 -21.1

OH H3C CH2

A2_TS1: hydrogen abstraction of -C-H

H

O C

1.34 1.16

Fe SH

OH H3C CH

SH

O Fe

H

SH

OH Fe

1.65

CH3 H

O

Fe-O1-H2 = 135.8

O1 Fe

OH H3C CH

Fe

0.0

O1-H2-C = 164.5

-62.8 A2_IM2

-65.7 A2_PC

A2_TS2: gem-diol dehydration

Figure 3. (a) Energy profiles for ethanol oxidation by the HA_O mechanism. (b) Energy profiles for ethanol oxidation by the gem-diol mechanism. Also shown are key geometry data of transition states (distances in angstroms and angles in degrees). Energies are given in kcal/mol. The results refer to pose-A (no direct involvement of Thr303). Comparision between the HA_O and gem-diol mechanisms According to the calculated results, the first hydrogen abstraction is the rate-determining step for both mechanisms. Besides, the energies of A1_TS1 and A2_TS1 are similar, in analogy to previous QM results.24 Therefore, the ethanol O-H and α-C-H bonds have comparable reactivities for hydrogen abstraction, regardless of the higher bond-dissociation energy (BDE) of the O-H bond compared with C-H bonds in alcohols.59,60 To unveil the origin of this phenomenon, we performed a distortion/interaction analysis61 of the hydrogen abstraction transition states A1_TS1 and A2_TS1. 10


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ACS Catalysis

O2

1.76

1.81 H1 O1 Fe

2.18

Fe-O1-H1 = 124.9 O1-H1-O2 = 170.2

A1_RC

1.69

2.03

1.81

H2

A1_IM2

1.77

1.78

1.98

1.69

C

1.76 2.32 1.99

2.70

3.83

O1 Fe

Fe-O1-H2 = 163.3 O1-H2-C = 123.4

A2_RC

A2_IM2

Figure 4. QM/MM optimized structures of key intermediates in the HA_O and gem-diol mechanisms. Distances and angles are given in Ångstroms and degrees, respectively. The results refer to pose-A (no direct involvement of Thr303). Distortion/interaction analysis of A1_TS1 and A2_TS1 In this approach, the activation barrier (ΔEǂ) is decomposed into a deformation energy (ΔEdef) and an interaction energy (ΔEint). The deformation energy (ΔEdef) is the energy required to distort the geometries of ethanol and Cpd I upon going from the reactant to the transition state, and the interaction energy (ΔEint) is the difference between activation barrier (ΔEǂ) and deformation energy (ΔEdef). The energies of A1_TS1 and A2_TS1 in Scheme 2 are higher than those in Figure 3 due to the exclusion of ZPE. As shown in Scheme 2, the distortion energy of ethanol is larger than that of Cpd I for both A1_TS1 and A2_TS1, indicating that the barrier of hydrogen abstraction mainly comes from the deformation of the ethanol substrate. Additionally, the deformation energy of ethanol in A1_TS1 is remarkably higher than that in A2_TS1. This can be explained by the later transition state of 11


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A1_TS1 where the O-H bond of ethanol (1.33 Å) is much more elongated than the α-C-H bond of A2_TS1 (1.22 Å). Nevertheless, there is a large interaction energy in transition state A1_TS1 (-23.6 kcal/mol, Scheme 2) which decreases the barrier of A1_TS1 from 42.5 kcal/mol to 18.9 kcal/mol. This is in sharp contrast to A2_TS1, in which the ethanol-Cpd I interaction increases the barrier by 5.4 kcal/mol. Considering that the Mulliken charge on the abstracted hydrogen of A1_TS1 is more positive than that of A2_TS1 (0.27 versus 0.14), A1_TS1 will have a stronger electrostatic interaction between the polar hydroxyl group of ethanol and the oxo group of Cpd I, which will counteract the influence of the larger O-H bond strength on hydrogen abstraction. CH3 H2C

O

O

OH

Fe

Fe

+

SH

SH

ethanol (RC)

CpdI (RC)

A1_RC

CH3 H2C O

H

Edist(CpdI)

E

Edist(ethanol)

O O

+

Fe

Fe A1_TS1

CH3CH2OH

CH3CH2O H

SH

SH CpdI (TS)

ethanol (TS)

Eint = E - Edist(CpdI) - Edist(ethanol)

Transition States

E

Edist(CpdI)

Edist(ethanol)

Eint

A1_TS1

18.9

3.0

39.5

-23.6

A2_TS1

18.5

5.1

8.0

5.4

Scheme 2. Distortion/interaction analysis of transition states A1_TS1 and A2_TS1. The energies (kcal/mol) were calculated at UB3LYP/ Def2-TZVPP level of theory.

3.2.2 Ethanol oxidation in the presence of the Thr303 effect The MD simulations from pose-B provide a favorable substrate-enzyme conformation where the ethanol substrate is held tightly by the Thr303 residue and the Fe=O moiety. Since Thr303 constrains ethanol within the active site of CYP2E1, the QM/MM calculations starting from such MD snapshots are expected to correspond to the actual mechanism of ethanol oxidation by CYP2E1. Figure 5a shows the energy profiles of the HA_C and HA_O mechanisms from reactant

12


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B_RC, and Figure 5b displays the QM/MM optimized configurations of selected intermediates and transition states. (a)

T303 CH3

CH3

C H

C

O H

O H

H T303

H O

T303

Fe

CH3

C

SH

H

H3C C

O H

O

H O

H H

B_TS3 21.0

Fe

T303 CH3

CH3

O

H

C H

CH3 H

O H

H

O

17.1

B_IM1 +15.0 CH3 O H

H3C C

H H

O

H

O Fe

H3C

H

C O

H

SH

H H

-50.3 B_PC

O Fe

Fe

H

SH

H

C

H

B_TS2

C

T303

H

Fe

SH

0.0 B_RC

H O

O C

O

T303

C

H

O

B_TS1 +16.0

-50.3 B_PC

CH3

CH3

C

H

SH

SH

HA_C mechanism

HA_O mechanism

(b)

1.85

1.91

O2 H1

3.28

1.73

1.79

O1 Fe

Fe-O1-H1 = 133.1 O1-H1-O2 = 171.5

B_RC

B_IM1

1.90

1.83

1.23 C

C

H2

2.16

O1 1.50

O1

Fe

H2 1.22

1.37

Fe

Fe-O1-H2 = 125.6 O1-H2-C = 170.4

Fe-O1-H2 = 119.4 O1-H2-C = 141.3

B_TS3

B_TS2

13


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Figure 5. (a) Energy profiles (kcal/mol) of the HA_O and HA_C mechanisms for ethanol oxidation starting from B_RC. (b) The optimized structures of B_RC, B_IM1, B_TS2 and B_TS3. The results refer to pose-B (with involvement of Thr303). The optimized structure of B_RC shows that ethanol is closer to the Fe=O moiety compared with A1_RC (1.73 Å versus 1.81 Å).62 The Fe-O-H angle of 133.1° resembles the favorable Fe-O-H angles (between 110°-130°) in hydrogen abstraction transition states of the C-H bond. Hence, the side chain hydroxyl of Thr303 not only fixes the ethanol substrate, but also cooperates with the Fe=O moiety to orient the ethanol O-H bond in a position suitable for hydrogen abstraction. The hydrogen abstraction of ethanol O-H bond via B_TS1 has a barrier of 16.0 kcal/mol. This is in accord with the barrier of O-H cleavage without the influence of Thr303 (Figure 3). We were unable to find any low-energy Fe-OH intermediate similar to A1_IM2. Inspection of B_IM1 shows that the conformation of Ala299 is different from that in A1_IM1. In B_IM1, the carbonyl group of Ala299 is far away from the active site and cannot form a hydrogen-bonding interaction with Fe-OH to stabilize the product of O-H cleavage (Figure 5b). This indicates that the switch from the Thr303-Ala299 to the Thr303-ethanol interaction changes the orientations of the surrounding residues, thus destabilizing the HO-Fe(IV)/ethoxyl radical intermediate. Therefore, the second hydrogen abstraction of the α-C-H bond via B_TS2 becomes the rate-determining step in the HA_O mechanism. The energy profile of the HA_C mechanism is shown on the left side of Figure 5a (starting from B_RC and going towards the left). In this case, the hydrogen abstraction passes through the transition state B_TS3 and then directly yields the acetaldehyde product B_PC, with concomitant hydrogen abstraction of the O-H bond that occurs spontaneously without an intervening intermediate. Hence, the hydrogen abstraction with α-C-H bond cleavage is the rate-determining step of the HA_C mechanism. According to these QM/MM results, the cleavage of the ethanol α-C-H bond is rate-determining in CYP2E1 for both the HA_C and HA_O mechanisms. The transition state of α-C-H bond cleavage B_TS2 is lower in energy than B_TS3 by 3.9 kcal/mol, indicating that the preferred mechanism for ethanol oxidation by CYP2E1 is the HA_O mechanism. The difference between B_TS2 and B_TS3 is not due to steric effects but to hydrogen-bonding interactions with Thr303. 14


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In B_TS2, the α-C-H bond of the ethoxyl radical is ideally oriented for hydrogen abstraction (Fe-O1-H2 =125.6°, O1-H2-C = 170.4°, Figure 5b), because the ethoxyl radical is only weakly coordinated by Thr303 and the Fe-OH moiety in B_IM1. Whereas in B_TS3, the O1-H2-C angle of 141.3° is greatly distorted due to the strong hydrogen-bonding interaction betweeen Thr303 and ethanol in B_RC. 3.2.3 Summary: mechanisms of ethanol oxidation in CYP2E1 In the case of ethanol oxidation without the influence of Thr303, the computed QM/MM barriers (Figure 3) are similar for hydrogen abstraction from the ethanol O-H and α-C-H bonds. This indicates that the bond dissociation energy is not the only determinant of hydrogen abstraction reactivity although it greatly affects the reaction energy, since the cleavage of the ethanol O-H bond is more endothermic and thermodynamically much less favorable than the cleavage of the α-C-H bond (Figure 3). Thus, judging from the computed intrinsic barriers, it is uncertain whether the first hydrogen abstraction is the rate-determining step in the HA_O mechanism. This is consistent with the experimental observation that the rate-determining step of alcohol oxidation is dependent on the form of cytochrome P450.21 We also note that the presence of the Fe-OH/ethoxyl radical intermediate in the HA_O mechanism is in accord with the detection of ethanol-derived oxygen-centered radicals in biomimetic ethanol oxidation.23 Considering the actual ethanol oxidation in CYP2E1 under the influence of Thr303, the MD simulations suggest that the Thr303 residue keeps the ethanol substrate near the Fe=O moiety of the CYP2E1 pocket, in an orientation well suited for hydrogen abstraction. According to the QM/MM results, the HA_O mechanism (Figure 5) is favored in CYP2E1-mediated ethanol oxidation. The transition state for the second hydrogen abstraction involving α-C-H bond cleavage is the highest-energy point on the HA_O pathway for ethanol oxidation, in agreement with experimental observations that the cleavage of ethanol α-C-H bond is isotopically sensitive.22 While there is no mutagenesis study of ethanol oxidation reaction in CYP2E1, Thr303 was previously shown to be critical for the reactivity and the selectivity in CYP2E1-mediated reactions. For example, Fukuda et al. reported that the mutation of Thr303 to valine will reduce the reactivity of CYP2E1 in fatty acid hydroxylation63,64 Hollenberg and co-workers found that the T303A mutation in CYP2E1 will affect the isothiocyanates metabolism by changing the orientation of the inactivator.65 Furthermore, the influence of protein residues on the alcohol oxidation mechanism 15


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was recently explored in the oxidation of (S)-α-hydroxymyristic acid by CYP152.66 Finally, it is intriguing to see that a few other P450 enzymes that oxidize ethanol also contain a conserved threonine residue in the active cavity, such as Thr309 of CYP3A4, Thr319 of CYP1A2 and Thr302 of CYP2B4. Therefore, it appears that the conserved threonine residue in P450 is not only of significance to dioxygen activation,67 but also has a great impact on the substrate-binding machinery in the active cavity.

4. Conclusions We report MD simulations of CYP2E1 complemented by QM/MM calculations that clarify the ethanol-binding machinery in CYP2E1 as well as the mechanism of ethanol oxidation. The Thr303 residue is found to be decisive for confining and orienting the ethanol substrate in the active cavity of CYP2E1. To identify its crucial influence, we examined the HA_O and HA_C mechanisms for two cases, i.e., ethanol oxidation with and without the involvement of Thr303. In the absence of the Thr303 effect, the HA_O and HA_C mechanisms are competitive, since the thermodynamic preference for C-H cleavage is largely compensated by stronger electrostatic interactions between the ethanol O-H group and the Fe=O moiety in the transition state for O-H cleavage. However, upon the involvement of Thr303, hydrogen-bonding interactions of ethanol with Thr303 and the Fe=O moiety orient the ethanol O-H bond such that it is well positioned for hydrogen abstraction. The preferred mechanism for ethanol oxidation in CYP2E1 is thus found to proceed via an initial hydrogen abstraction of the ethanol O-H bond followed by a subsequent hydrogen abstraction of the ethanol α-C-H bond (with a slightly higher transition state). Our computational results rationalize much of the available experimental observations on alcohol oxidation by P450 enzymes and imply that the threonine residue may generally affect the reactivity of small substrates in the active site of P450 enzymes.

Corresponding Authors [email protected]; [email protected]

Conflicts of Interest 16


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ACS Catalysis

There are no conflicts of interest to declare.

Supporting Information Supporting ﬁgures, spin density distribution, QM/MM and ZPE energies, and Cartesian coordinates of all computed species in doublet state.

Acknowledgements We thank the NSFC (21573237), the Hundred-Talent Program of the Chinese Academy of Sciences, the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB20000000), and the China Postdoctoral Science Foundation (2016LH00019, 2016M600508) for financial support.

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Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Structure and Chemistry of Cytochrome P450. Chem. Rev. 2005, 105, 2253-2277.

5 Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Heme-Containing Oxygenases. Chem. Rev. 1996, 96, 2841-2887. 6 Manikandan, P.; Nagini, S. Cytochrome P450 Structure, Function and Clinical Significance: A Review. Curr. Drug Targets 2018, 19, 38-54. 7 Lewis, D. F.; Ito, Y. Human P450s Involved in Drug Metabolism and the Use of Structural Modelling for Understanding Substrate Selectivity and Binding Affinity. Xenobiotica 2009, 39, 625-635.

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Injury. Arch. Toxicol. 2009, 83, 519-548. 19 Lu, Y.; Cederbaum, A. I. CYP2E1 and Oxidative Liver Injury by Alcohol. Free Radical Bio. Med. 2008, 44, 723-738. 20 Dey, A.; Cederbaum, A. I. Alcohol and Oxidative Liver Injury. Hepatology 2006, 43, 63-74. 18


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21 Vaz, A. D. N.; Coon, M. J. On the Mechanism of Action of Cytochrome P450: Evaluation of Hydrogen Abstraction in Oxygen-Dependent Alcohol Oxidation. Biochemistry 1994, 33, 6442-6449. 22 Ekstrӧm, G.; Norsten, C.; Cronholm, T.; Ingelman-Sundberg, M. Cytochrome P450 Dependent Ethanol Oxidation. Kinetic Isotope Effects and Absence of Stereoselectivity. Biochemistry 1987, 26, 7348-7354. 23 Nishizaki, D.; Iwahashi, H. Oxygen-Centered Radicals Formed in the Reaction Mixtures Containing Chloroiron Tetraphenylporphyrin, lodosylbenzene, and Ethanol. Inorg. Chem. 2017, 56, 13166-13173. 24 Wang, Y.; Yang, C. L.; Wang, H. M.; Han, K. L.; Shaik, S. A New Mechanism for Ethanol Oxidation Mediated by Cytochrome P450 2E1: Bulk Polarity of the Active Site Makes a Difference. ChemBioChem 2007, 8, 277-281. 25 Shaik, S.; Kumar, D.; de Visser, S. P.; Altun, A.; Thiel, W. Theoretical Perspective on the Structure and Mechanism of Cytochrome P450 Enzymes. Chem. Rev. 2005, 105, 2279-2328. 26 Shaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. P450 Enzymes: Their Structure, Reactivity, and Selectivity-Modeled by QM/MM Calculations. Chem. Rev. 2010, 110, 949-1017. 27 Brooks, B. R.; Brooks, C. L.; Mackerell, A. D.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; Caflisch, A.; Caves, L.; Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.; Gao, J.; Hodoscek, M.; Im, W.; Kuczera, K.; Lazaridis, T.; Ma, J.; Ovchinnikov, V.; Paci, E.; Pastor, R. W.; Post, C. B.; Pu, J. Z.; Schaefer, M.; Tidor, B.; Venable, R. M.; Woodcock, H. L.; Wu, X.; Yang, W.; York, D. M.; Karplus, M. CHARMM: The Biomolecular Simulation Program. J. Comput. Chem. 2009, 30, 1545-1614. 28 DeVore, N. M.; Meneely, K. M.; Bart, A. G.; Stephens, E. S.; Battaile, K. P.; Scott, E. E. Structural Comparison of Cytochromes P450 2A6, 2A13, and 2E1 with Pilocarpine. FEBS J. 2012, 279, 1621-1631.

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29 Porubsky, P. R.; Battaile, K. P.; Scott, E. E. Human Cytochrome P450 2E1 Structures with Fatty Acid Analogs Reveal a Previously Unobserved Binding Mode. J. Biol. Chem. 2010, 285, 22282-22290. 30 Porubsky, P. R.; Meneely, K. M.; Scott, E. E. Structures of Human Cytochrome P450 2E1 Insights into the Binding of Inhibitors and Both Small Molecular Weight and Fatty Acid Substrates. J. Biol. Chem. 2008, 283, 33698-33707. 31 Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera—A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25, 1605-1612. 32 Brünger, A. T.; Karplus, M. Polar Hydrogen Positions in Proteins: Empirical Energy Placement and Neutron Diffraction Comparison. Proteins: Struct. Funct. Genet. 1988, 4, 148-156. 33 Søndergaard, C. R.; Olsson, M. H.; Rostkowski, M.; Jensen, J. H. Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of pKa Values. J. Chem. Theory Comput. 2011, 7, 2284-2295. 34 Olsson, M. H.; Søndergaard, C. R.; Rostkowski, M.; Jensen, J. H. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. J. Chem. Theory Comput. 2011, 7, 525-537. 35 In this work, we used the deprotonated form of the Glu302 residue, because the salt-bridge between Glu302 and Arg309 is crucial to stabilize the active cavity of CYP2E1. In MD simulations with protonated Glu302, the ethanol substrate is unstable and escapes from the active cavity of CYP2E1 after 12 ns, due to the deformation of CYP2E1 pocket. 36 Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graph. 1996, 14, 33-38. 37 Sherwood, P.; de Vries, A. H.; Guest, M. F.; Schreckenbach, G.; Catlow, C. R. A.; French, S. A.; Sokol, A. A.; Bromley, S. T.; Thiel,W.; Turner, A. J.; Billeter, S.; Terstegen, F.; Thiel, S.; Kendrick, J.; Rogers, S. C.; Casci, J.; Watson, M.; King, F.; Karlsen, E.; Sjøvoll, M.; Fahmi, A.; Schäfer, A.; Lennartz, C. QUASI: A General Purpose Implementation of the QM/MM Approach and Its Application to Problems in Catalysis. J. Mol. Struct: THEOCHEM 2003, 632, 1-28. 20


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38 Ahlrichs, R.; Bär, M.; Häser, M.; Horn, H.; Kölmel, C. Electronic Structure Calculations on Workstation Computers: The Program System Turbomole. Chem. Phys. Lett. 1989, 162, 165-169. 39 Smith, W.; Yong, C. W.; Rodger, P. M. DL_POLY: Application to Molecular Simulation. Mol. Simulat. 2002, 28, 385-471. 40 Bakowies, D.; Thiel, W. Hybrid Models for Combined Quantum Mechanical and Molecular Mechanical Approaches. J. Phys. Chem. 1996, 100, 10580-10594. 41 de Vries, A. H.; Sherwood, P.; Collins, S. J.; Rigby, A. M.; Rigutto, M.; Kramer, G. J. Zeolite Structure and Reactivity by Combined Quantum-Chemical-Classical Calculations. J. Phys. Chem. B 1999, 103, 6133-6141. 42 Senn, H. M.; Thiel, W. QM/MM Methods for Biomolecular Systems. Angew. Chem., Int. Ed. 2009, 48, 1198-1229. 43 Miehlich, B.; Savin, A.; Stoll, H.; Preuss, H. Results Obtained with the Correlation Energy Density Functionals of Becke and Lee, Yang and Parr. Chem. Phys. Lett. 1989, 157, 200-206. 44 Lee, C. T.; Yang, W. T.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron Density. Phys. Rev. B: Condens. Matter Mater. Phys 1988, 37, 785-789. 45 Weigend, F.; Ahlrichs, R. Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys. Chem. Chem. Phys. 2005, 7, 3297-3305. 46 Chen, H.; Song, J. S.; Lai, W. Z.; Wu, W.; Shaik, S. Multiple Low-Lying States for Compound I of P450cam and Chloroperoxidase Revealed from Multireference Ab Initio QM/MM Calculations. J. Chem. Theory Comput. 2010, 6, 940-953. 47 Altun, A.; Kumar, D.; Neese, F; Thiel, W. Multireference Ab Initio Quantum Mechanics/Molecular Mechanics Study on Intermediates in the Catalytic Cycle of Cytochrome P450cam. J. Phys. Chem. A 2008, 112, 12904-12910. 48 Kästner, J.; Carr, J. M.; Keal, T. W.; Thiel, W.; Wander, A.; Sherwood, P. DL-FIND: An Open-Source Geometry Optimizer for Atomistic Simulations. J. Phys. Chem. A 2009, 113, 11856-11865. 21


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49 Schröder, D.; Shaik, S.; Schwarz, H. Two-State Reactivity as a New Concept in Organometallic Chemistry. Acc. Chem. Res. 2000, 33, 139-145. 50 Shaik, S.; Hirao H.; Kumar, D. Reactivity of High-Valent Iron-Oxo Species in Enzymes and Synthetic Reagents: A Tale of Many States. Acc. Chem. Res. 2007, 40, 532-542. 51 Park, J. Y.; Harris, D. Construction and Assessment of Models of CYP2E1: Predictions of Metabolism from Docking, Molecular Dynamics, and Density Functional Theoretical Calculations. J. Med. Chem. 2003, 46, 1645-1660. 52 For scenario a, the scattered water molecules leave the binding pocket to form hydrogen-bonding interactions with the backbone carbonyl groups of protein residues or other waters in the distal pocket of CYP2E1 upon QM/MM optimization. For scenario b, the water network is still present in the binding pocket after the QM/MM optimizations due to the presence of hydrogen-bonding interactions. This is the reason behind the different solvation patterns of the ethanol binding pocket in A1_RC and A2_RC (Figure 4). 53 For the deformation of the dome region, see Figure S4 for details. 54 Clark, J. P.; Miles, C. S.; Mowat, C. G.; Walkinshaw, M. D.; Reid, G. A.; Daff, S. N.; Chapman, S. K. The role of Thr268 and Phe393 in cytochrome P450BM3. J. Inorg. Biochem. 2006, 100, 1075-1090. 55 Tripathi, S.; Li, H.; Poulos, T. Structural Basis for Effector Control and Redox Partner Recognition in Cytochrome P450. Science 2013, 340, 1227-1230. 56 Balcells, D.; Sauer, E. L. O.; Raynaud, C.; Brudvig, G. W.; Crabtree, R. H.; Eisenstein, O. Manganese Catalysts for C-H Activation: An Experimental/Theoretical Study Identiﬁes the Stereoelectronic Factor That Controls the Switch between Hydroxylation and Desaturation Pathways. J. Am. Chem. Soc. 2010, 132, 7605-7616. 57 Similar results of rebound step was previously reported by Shaik et al. Please refer to: Harris, N.; Cohen, S.; Filatov, M.; Ogliaro, F.; Shaik, S. Two-State Reactivity in the Rebound Step of Alkane Hydroxylation by Cytochrome P450: Origins of Free Radicals with Finite Lifetimes. Angew. Chem., Int. Ed. 2000, 39, 2003-2007. Ogliaro, F.; Harris, N.; Cohen, S.; Filatov, M.; de Visser, S. P.; Shaik, S. A Model “Rebound” Mechanism of 22


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Hydroxylation by Cytochrome P450: Stepwise and Effectively Concerted Pathways, and Their Reactivity Patterns. J. Am. Chem. Soc. 2000, 122, 8977-8989. 58 On the basis of DFT calculations, the barrier of dehydration is 21.5 kcal/mol when two water molecules serve as proton shuttles (Figure S7). Thus, it is expected that dehydration should be much easier in the enzyme enviroment. 59 Blanksby, S. J.; Ellison, G. B. Bond Dissociation Energies of Organic Molecules. Acc. Chem. Res. 2003, 36, 255-263. 60 Ervin, K. M.; DeTuri, V. F. Anchoring the Gas-Phase Acidity Scale. J. Phys. Chem. A 2002, 106, 9947-9956. 61 Bickelhaupt,

F.

M.;

Houk,

K.

N.

Analyzing

Reaction

Rates

with

the

Distortion/Interaction-Activation Strain Model. Angew. Chem., Int. Ed. 2017, 56, 10070-10086. 62 Two other snapshots were extracted from the MD simulations of pose-B, and the optimized structures are similar to B_RC (Figure S9). 63 Fukuda, T.; Imai, Y.; Komori, M.; Nakamura, M.; Kusunose, E.; Satouchi, K.; Kusunose, M. Different Mechanisms of Regioselection of Fatty Acid Hydroxylation by Laurate (ω-1)-Hydroxylating P450s, P450 2C2 and P450 2E1. J. Biochem. 1994, 115, 338-344. 64 Fukuda, T.; Imai, Y.; Komori, M.; Nakamura, M.; Kusunose, E.; Satouchi, K.; Kusunose, M. Replacement of Thr303 of P450 2E1 with Serine Modifies the Regioselectivity of Its Fatty Acid Hydroxylase Activity. J. Biochem. 1993, 113, 7-12. 65 Moreno, R. L.; Goosen, T.; Kent, U. M.; Chung, F. L.; Hollenberg, P. F. Differential Effects of Naturally Occurring Isothiocyanates on the Activities of Cytochrome P450 2E1 and the Mutant P450 2E1 T303A. Arch. Biochem. Biophys. 2001, 391, 99-110. 66 Onoda, H.; Shoji, O.; Suzuki, K.; Sugimoto, H.; Shiro, Y.; Watanabe, Y. α-Oxidative Decarboxylation of Fatty Acids Catalysed by Cytochrome P450 Peroxygenases Yielding Shorter-Alkyl-Chain Fatty Acids. Catal. Sci. Technol. 2018, 8, 434-442. 67 For representive reports about the effect of Thr252 for the O2 activation of Cytochrome P450cam, see: Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.; Benson, D. E.; Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. The Catalytic Pathway of Cytochrome P450cam at Atomic Resolution. Science 2000, 287, 1615-1622. 23


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Kamachi T.; Yoshizawa, K. A. Theoretical Study on the Mechanism of Camphor Hydroxylation by Compound I of Cytochrome P450. J. Am. Chem. Soc. 2003, 125, 4652-4661. Zheng, J.; Wang, D.; Thiel, W.; Shaik, S. QM/MM Study of Mechanisms for Compound I Formation in the Catalytic Cycle of Cytochrome P450cam. J. Am. Chem. Soc. 2006, 128, 13204-13215.

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Table of Contents Thr303

Thr303

QM/MM calculations

Ethanol

Acetaldehyde

25


Mechanistic Insights into the Directing Effect of Thr303 in Ethanol

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