Theoretical Insights into the Mechanism and Stereoselectivity of Olefin

Aug 29, 2018 - Engineered P450s can catalyze some non-natural reactions with high efficiency and excellent selectivity, such as the carbine transfer, ...
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Theoretical Insights into the Mechanism and Stereoselectivity of Olefin Cyclopropanation Catalyzed by Two Engineered Cytochrome P450 Enzymes Hao Su,† Guangcai Ma,†,‡ and Yongjun Liu*,† †

Inorg. Chem. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 08/31/18. For personal use only.

Key Lab of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China ‡ College of Geography and Environmental Sciences, Zhejiang Normal University, Jinhua, Zhejiang 321004, China S Supporting Information *

ABSTRACT: Engineered P450s can catalyze some non-natural reactions with high efficiency and excellent selectivity, such as the carbine transfer, nitrene transfer, C−H insertion, and C−H amination, opening alternative routes for sustainable production of chemicals. Recent experiments revealed that two engineered cytochrome P450 enzymes (P450BM3-CIS and P411BM3-CIS) show different efficiencies and stereoselectivities in the olefin cyclopropanation, but key factors that affect the activity remain unclear. In this work, both quantum mechanics (QM) and QM/molecular mechanics (MM) methods were employed to explore the catalytic reactions and selectivity of these two engineered cytochrome P450 enzymes. On the basis of our results, the cyclopropanation of styrene is suggested to mainly occur on the open-shell singlet (OSS) and triplet state surfaces, which contain two elementary steps. The reactive iron(III)−porphyrin carbene (IPC) radical first attacks the terminal alkenyl group of styrene to form a C-radical intermediate, which then undergoes a cyclization reaction affording the cyclopropanation products. Importantly, it is found that the stereoselectivity of cyclopropanations is elucidated only if considering the real protein environment, and the stereoselectivity is determined by multiple factors, such as the relative orientation of IPC to styrene, the binding affinity of the substrate, and the reaction barriers of rate-limiting steps. It is the enzymatic environment that makes the reaction highly stereoselective, which provides useful clues for designing whole-cell catalysts for non-natural chemical reactions.

1. INTRODUCTION

Inspired by the chemo-, regio-, and stereoselectivity of P450s-catalyzed reactions, Arnold’s group reported that the engineered P450BM3 (CYP102A1) enzymes can catalyze the asymmetric olefin cyclopropanation.4 P450BM3 is a long-chain fatty acid hydroxylase from Bacillus megaterium, which has been extensively studied in the past 40 years.5 Mutating some amino acid residues of P450BM3 could change its original catalytic property, making it possible to catalyze the formal carbene transfer from diazoester reagents to olefins with high diastereoand enantioselectivity (Figure 1, right).4 As for the reaction mechanism, it was proposed that the diazoester first reacts with the catalytic center of iron(II)−porphyrin to form a high reactive intermediate iron(III)−porphyrin carbene radical, which is similar to the well-characterized Compound I, and

The impressive catalytic diversity of enzymes such as cytochrome P4501 has extensively spurred chemists to design engineered and artificial biocatalysts to catalyze the unnatural reaction types.2 Natural P450 is an enormous superfamily with more than 1000 sequenced P450 (CYP) genes. P450 monooxygenases usually catalyze the “Compound I”-initiated oxidative R−H activation which involves two sequential steps: (1) hydrogen-abstraction by Compound I to form a radical R·, and (2) hydroxyl rebound to form the R−OH. P450 mono-oxygenases are also capable of catalyzing the epoxidation of olefins, in which the oxygen atom of Compound I shifts directly onto the olefinic group (Figure 1, left). Similar to the P450s-catalyzed epoxidation of olefins, organic synthetic chemists often transfer the isoelectronic carbene into olefins to generate diverse cyclopropanation products;3 however, such a reaction is not readily accessible in organisms.4 © XXXX American Chemical Society

Received: July 5, 2018

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DOI: 10.1021/acs.inorgchem.8b01875 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Left: P450 monooxygenases-catalyzed R−H activation (R−H → R−OH) and epoxidation of olefins; right: engineered P450-catalyzed cyclopropanation of olefins from diazoester reagents via carbene transfer.

little monooxygenation activity and enhanced cyclopropanation activity.4b In intact Escherichia coli cells, both P450BM3-CIS and P411BM3-CIS show better cyclopropanation activity than P450BM3 and P411BM3, and P411BM3-CIS shows about four times more active than P450BM3-CIS and generates the cyclopropanation products with cis enantioselective preference. However, in the absence of reductase domain, the isolated heme domain of P411BM3-CIS (P411BM3‑heme-CIS) shows much lower activity than P411BM3-CIS.9 Besides cyclopropanation, recent studies have reported that the engineered P450 can also catalyze the intramolecular C−H amination, intermolecular aziridination, and imidation of sulfides.10 In addition, Fasan’s group designed the diverse engineered myoglobin catalysts capable of catalyzing the carbene N−H insertion, as well as the outstanding diastereo- and enantioselective olefin cyclopropanation.11 These studies not only enrich the application values of engineered biocatalysts and expand the area of enzyme promiscuity, but also provide a solid basis for designing artificial enzymes for catalyzing the chemical transformations that have no natural counterparts. However, theoretical studies on the engineered P450catalyzed reactions are still very limited.11,12 According to the proposed mechanism by Arnold et al.,4b the cyclopropanation process can be divided into two parts: (1) diazoester activation to form IPC, and (2) carbene transfer to generate cyclopropane products. In 2016, Shaik et al. investigated the iron porphyrin carbenes’ electronic structure, formation, and N−H insertion reaction using DFT with the cluster model.11b Recently, Zhang’s group reported the systematic quantum chemical study using model complexes to explore the effects of binding mode, porphyrin substituent, carbene substituent, and axial ligand on the IPC formation pathways,12a as well as the C−H insertions and cyclopropanations by IPC.12b,c Both Shaik’s11b and Zhang’s12c studies show that the energy differences between electronic states are small, even for the models without axial ligand. In this work, we utilized both quantum mechanics (QM) and QM/molecular mechanics (MM) methods to investigate the carbene transfer process catalyzed by P450BM3-CIS and P411BM3-CIS, aiming at elucidating the detailed cyclopropanation mechanism and nature of stereoselectivity at the atomistic level. On the basis of our calculations, it is found that the stereoselectivity of cyclopropanations is elucidated only if considering the real protein environment.

then the IPC transfers its carbene group to olefin to generate the final cyclopropanation product, and simultaneously the high-valent FeIV returns to its reducing state. Using ethyl diazoacetate (EDA) as the carbene precursor and styrene as olefin to explore the cyclopropanation reaction, Arnold et al. obtained four products, two of which are cis enantiomers, (R, S) and (S,R), and the other two are trans enantiomers, (R,R) and (S,S) (Figure 2). Moreover, experiments have demon-

Figure 2. Enantio- and diastereoselectivity of olefin cyclopropanation catalyzed by engineered P450BM3.

strated that different P450BM3 variants, diazoester reagent and olefin can significantly influence the reaction yields, total turnover numbers (TTNs), and cis/trans ratios of cyclopropanation.4b The formation and reactivity of metalloporphyrin carbenoid complexes, including IPC, have been extensively studied. For example, Wolf et al. studied the styrene cyclopropanation catalyzed by a diverse of synthetic achiral iron(II)−porphyrin complexes and proposed that these species are highly sensitive to the oxygen of air.6a The iron(II)−porphyrin can be rapidly oxidized to form the inactive iron(III)−porphyrin resting state, which completely hampers the carbene transfer process. This means that the cyclopropanation reactions require a stoichiometric amount of reducing agent and anaerobic condition,6 which has been confirmed through the studies on the cyclopropanation activity of various P450BM3 variants in vitro.4 Recently, Arnold and her colleagues reported the whole-cell engineered P450BM3 catalysts, in which the catalytic heme domain was fused into the NADPH-driven P450 reductase domain.4b In addition, the olefin cyclopropanation can also occur in the isolated heme domain (P450BM3‑heme) with the assistance of strong reducing agents over the native NADPH, such as dithionite. During the P450-catalyzed monooxygenation reaction, the axial cysteinate ligation plays an essential role in dioxygen activation and stabilization of the active Compound I intermediate.7 Interestingly, mutation of the axial cysteine into serine has been verified to abolish the monooxygenation activity in P450 2B4.8 Arnold and her coworkers reported the engineered P411BM3 by the axial cysteineto-serine substitution (C400S), and they found that P411BM3 gives significant ferrous CO Soret peak at 411 nm and shows

2. COMPUTATIONAL DETAILS 2.1. Gas Phase QM-only Calculations. To explore the nature of Fe-carbene intermediate and cyclopropanation mechanism, we first performed QM-only calculations on small cluster models by using the Gaussian09 package.13 In the QM calculations, the high reactive Fecarbene intermediate was modeled using Fe-CHCO2Et−porphine. B

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Figure 3. Active site structures of constructed P450BM3-CIS and P411BM3-CIS in complex with iron-porphyrin-carbene (IPC) and styrene. The QM regions are shown in ball and stick models. Similar to the following QM/MM calculations, we constructed two QM models: QM-S and QM-O. In QM-S, the axial cysteine ligand was modeled using a methyl thiolate, whereas the ligated serine in QM-O was represented by a methoxide. Geometry optimizations and frequency calculations were carried out with UB3LYP density functional14 in combination with VTZ basis set for Fe and 631G(d, p) for the other atoms, labeled as BS1, while the energies were further corrected in the gas phase with UB3LYP functionals in conjunction with Wachters+f for Fe and 6-311++G(d, p) for the remaining atoms, labeled as BS2.15,16 To obtain the open-shell singlet (OSS) state, the optimized structure at the triplet state was used as the initial geometry, and then the wave function was optimized to the desired open-shell singlet state, and finally a geometry optimization was performed with this guess. No symmetry restrictions were used in the calculations. 2.2. System Preparation. The initial coordinates of variants P450BM3−CIS and P411BM3−CIS were taken from the X-ray crystal structures (PDB ID: 4H24 and 4H23).4b Compared to the wild type P450BM3, 13 mutations have been introduced into P450BM3−CIS, which includes V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K. The P411BM3− CIS corresponds to P450BM3−CIS with C400S mutation. The active sites of both variants are empty; i.e., they do not contain the substrate styrene and diazoester reagent. To construct the reactant models for cyclopropanation, the FeII−porphyrin complexes in P450BM3−CIS and P411BM3−CIS were modified to high reactive iron(III)− porphyrin-CHCO2Et radical intermediates. Careful inspection on the active sites of both enzymes reveals that, because of the intense steric hindrance from the α-helix (the smallest distance between CHCO2Et and A264 residue of α-helix is only about 2.5 Å), the substrate (styrene) can only enter into the active site on the side adjacent to residue V87. The steric hindrance from the α-helix and the substrate also prohibit the CHCO2Et radical intermediate from freely rotating after the substrate enters into the active site. Then, the styrene was put into the active sites (Figure 3), and L437 and T438 residues fix the orientation of the substrate by steric hindrance. The protonation states of the ionizable residues were determined using the PROPKA module of the PDB 2PQR suite of programs17 in combination with careful visual inspection by VMD software.18 All acidic residues were deprotonated, whereas lysine and arginine residues were protonated. Histidine residues were assigned in singly protonated states according to their local environment and calculated pKa values. All missing hydrogen atoms in crystal structures were added via the HBUILD module as implemented in the CHARMM program package.19 These two systems were solvated with TIP3P water20 spheres of radius of 36 Å, and then 12 Na+ ions were randomly added to neutralize the overall charges of the systems. The hydrogenation, solvation, and neutralization procedures were accompanied by a series of classical energy minimizations to relax the systems.

In the molecular dynamics (MD) procedure, the resulting two solvated systems were slowly heated from 0 to 300 K for 200 ps with a 1 fs time step, and then followed by 200 ps of equilibration at 300 K with a 1 fs time step. After that, 20 ns MD simulations were performed using CHARMM22/CMAP all-atoms force field.21 The Fe-carbene intermediate, styrene substrate, C400 in P450BM3−CIS, and S400 in P411BM3−CIS were kept frozen during the MD simulations. The calculated root-mean-square deviation (RMSD) of the protein structures from the two MD are shown in Figures S1 and S2. One can see that both systems reached plateau after 13 ns. Then, two representative snapshots from the MD trajectories of P450BM3− CIS and P411BM3−CIS were taken as the starting structures of the following QM/MM calculations. 2.3. QM/MM Calculations. QM/MM calculations were performed with the ChemShell program,22 which incorporates the Turbomole23 for the QM region and the DL_POLY24 for MM region. The electronic embedding scheme25 was applied to polarize the QM part by the MM point charges of the force field. No electrostatic cutoffs were introduced for the nonbonding MM and QM/MM interactions. Hydrogen link atoms with the charge shift model26 were used to treat the QM/MM boundary. During the QM/MM geometry optimizations, the QM region was treated by the unrestricted hybrid UB3LYP density functional in combination with VTZ basis set for Fe and 6-31G(d, p) for the other atoms, labeled as BS1. The CHARMM22/CMAP force field was used for the MM treatment. To obtain more accurate energies, the larger basis set BS2 (Wachters +f for Fe and 6-311++G(d, p) for the remaining atoms) were used for single point energy calculations. The obtained energies were further corrected by empirical dispersion correction by using the DFT-D3 program.27 The final QM/MM energies reported in this work are single-point energies at the UB3LYP/BS2/CHHARMM22/CMAP level and corrected by empirical dispersion correction. To distinguish the P450BM3-CIS and P411BM3-CIS systems, we designated the two reactant models as QM/MM-S and QM/MM-O, respectively. The QM region consisted of styrene, Fe-carbene intermediate modeled by the Fe-CHCO2Et−porphine macrocycle without side chains, and the axial ligand side chain (C400 in QM/ MM-S or S400 in QM/MM-O), as shown in Figure 3. The QM region contains 70 atoms, and the total charge of QM region is −1. The remaining atoms of the solvated models were assigned to the MM part. In each model, we defined the active region within 15 Å of Fe atom of Fe-carbene intermediate, including all QM atoms and partial MM atoms. The active regions were fully optimized, whereas the remainder of the solvation model was fixed. On the basis of the scanned potential energy surface (PES) along the reaction coordinates, the transition states were optimized using P-RFO algorithm28 implemented in the HDLC optimizer.29 To obtain the structures of open-shell singlet state, on the basis of the optimized structure of triplet state, one α electron was flipped to β one, and then unrestricted geometry optimization was performed on this structure. C

DOI: 10.1021/acs.inorgchem.8b01875 Inorg. Chem. XXXX, XXX, XXX−XXX

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3. RESULTS AND DISCUSSION 3.1. Structures of Iron-Porphyrin-Carbene Intermediate. The P450-catalyzed mono-oxygenation usually requires a high reactive iron(IV)-oxo porphyrin π cation radical intermediate (Compound I), which can trigger a variety of reactions, such as C/N−H activation, epoxidation of alkenes, and olefination of aldehydes/ketones. According to the mechanistic proposal by Arnold et al.,4a,b in the P450BM3catalyzed cyclopropanation, the isoelectronic carbene transfers from the reactive iron-porphyrin-carbene (IPC) intermediate to olefins. The formation of IPC derived from the reaction of iron(II)-porphyrin with diazoester has been studied by Zhang and Shaik groups.12,11b We first performed QM-only calculations using two cluster models to explore the geometries and electronic structures of IPC. In both models, the porphine macrocycle represents the porphyrin, and the Fe-ligated C400 and S400 are modeled by methyl thiolate and methoxide, respectively. Recently, Shaik and co-workers reported the open-shell singlet as the most stable state.11b The QM-optimized structures of IPC as well as the relative energies of the lowest lying singlet, open-shell singlet, triplet, quintet, and septet spin states are shown in Figure 4. Comparison of the optimized geometries of QM-S

Fe are 1.09 and 1.01, respectively. For the open-shell singlet state, the spin densities on carbene C atom in QM-S and QMO are −0.92 and −0.95, on Fe are 0.96 and 0.91, respectively. Thus, the mutation of the axial cysteine ligand to serine did not affect the spin densities of Fe and carbene C atoms. Given that the quintet and septet states show significant higher energies than triplet, OSS, and singlet, we thus only calculated the triplet, OSS and close-shell singlet PESs for the styrene cyclopropanation process, and the relative energies of involved species are summarized in Table S1. 3.2. Cyclopropanation Mechanism. Although the most stable state of the reactive IPC is the open-shell singlet, however, as can be seen from Table S1, the triplet state of model QM-S and QM-O is only 2.4 and 1.9 kcal/mol higher than those of OSS, and thus both the triplet and OSS may be also the reactive states of IPC. On the basis of our calculations (Table S1), the CSS state refers to about 5 kcal/mol higher than the other two states in both the reactant and transition states, and hence, we can consider the CSS state is not the favorable reactant state. Thus, we mainly focus on the triplet and OSS for the following discussion. The calculated energy barrier of QM-S triplet state is similar to that of Zhang.12c In Zhang’s study, the one-step concerted pathway was suggested for the cyclopropanation reaction of IPC that does not contain the axial ligand. Since the axial ligand has a strong “push effect” on the porphyrin complex as in our model,30 it is reasonable to show differences. The recent study about the C−H amination reaction from Boyd showed that, rather than the previously proposed concerted mechanism in Rh-catalyzed C−H amination, the C−H amination catalyzed by engineered cytochrome P450 is stepwise.32 On the basis of the QM-only calculations, the most plausible styrene cyclopropanation mechanism was proposed, which is shown in Figure 5. The reactive IPC first attacks the terminal alkenyl group of styrene, forming a C-radical intermediate. In this step, the energy

Figure 4. QM-only optimized geometries of IPC intermediates for models QM-S and QM-O. Distances are given in Å, and the relative energies (RE) in kcal/mol. The spin densities (ρ) on Fe and carbene C1 in the closed-shell singlet, OSS, and triplet states are also shown. The energies of QM-S and QM-O in their triplet states are set to zero.

and QM-O shows a minor difference of d(Fe−C) (d1 in Figure 4), but a great difference of d(Fe−O) and d(Fe−S). In both QM-S and QM-O models, OSS, triplet and quintet states show almost identical distances of d(Fe−C), d(Fe−O), and d(Fe−S), whereas d(Fe−C) in the close-shell singlet (CSS) is much shorter than those of the other spin states. Regardless of the axial ligand is methyl thiolate or methoxide, the ground state for IPC intermediate is the OSS state, and the relative energies increase in the order of OSS < triplet < CSS < quintet < septet. Both triplet and OSS states show the iron(III)− porphyrin carbene radical feature. It should be noted that the recent study on Rhodothermus marinus (Rma) cytochrome c, in which axial ligand is a histidine, showed that the OSS state is the most stable state followed by CSS and triplet state.30 This difference should attribute to the different “push effect” from axial ligand.31 In the triplet state, the spin densities on carbene C atoms in QM-S and QM-O are 0.71 and 0.70, and those on

Figure 5. Proposed mechanism of styrene cyclopropanation on the basis of the QM-only calculations at the triplet and open-shell singlet states. D

DOI: 10.1021/acs.inorgchem.8b01875 Inorg. Chem. XXXX, XXX, XXX−XXX

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React_B′, React_C′, and React_D′, respectively. These reactant models were optimized as far as possible to find out the energetically most stable local minima. Figure 6 shows the

barriers of triplet state are almost identical to those of OSS for both models (Table S1). Besides, the four intermediates (Int1), namely, Int1_A, Int1_B, Int1_C, and Int1_D have similar relative energies (Table S1). In the second steps, the formed C-radicals in the Int1 attack the carbene C atom to form four cyclopropanation products with stereoselectivity, and to regenerate the ferrous resting state of IPC. Products P_A and P_D correspond to the trans conformations, while P_B and P_C to cis ones. The optimized geometries of the stationary points of QM-S and QM-O along with the reaction coordinates are summarized in Figures S3−S6, which show very similar structural parameters. The concerted mechanism of cyclopropanation catalyzed by Fe(II)-IPC has also been tested. On the closed-shell singlet state surfaces, the energy of transition states for both QM-S and QM-O is higher than those of the corresponding triplet and OSS states. Thus, in this reaction, the Fe(II) mechanism is not a energetically favorable one. The optimized structures of these stationary points are presented in Figurs S7 and S8. Table S1 summarizes the relative energies of species involved in the styrene cyclopropanation pathway. Although the triplet state reactants have higher energy than the OSS state ones, they correspond to very similar energy barriers for the first step. Besides, models A and D show slight lower barriers than B and C, which may be attributed to the stronger steric hindrance between the phenyl group of styrene and the ethoxycarbonyl group of IPC. Substitution of -SCH3 by -OCH3 has a minor influence on the barriers of the first step and relative energies of Int1; however, it does increase the energy barriers by ∼3 kcal/mol for ring formation (TS2). For better understanding the reaction mechanism, we analyzed the spin densities of key atoms along the reaction coordinates (see Tables S2−S5). From reactant to TS1, the spin density on C1 decreases while those on C2 and C3 increase, indicating an electron transfer from the alkenyl group of styrene to the C1. In TS1 and Int1, some spin density on C3 spread to the benzene ring of styrene via conjugation, which stabilizes the transition state and radical intermediate. In the QM-S model, we also found that the OSS surface cyclopropanation process will lead to CSS products, which is in accordance with the calculated IPC N−H insertion process.11b 3.3. Stereoselectivity of Cyclopropanation. In vitro experimental studies by Arnold’s group revealed that P450BM3CIS catalyzes the selective cyclopropanation with cis:trans about 60:40 under anaerobic conditions, while P411BM3-CIS shows better cis selectivity (cis/trans = 72:28).4b Considering such a small selective difference between P450BM3-CIS and P411BM3-CIS may be quite hard for computational distinguish at the present level, we mainly discuss the factors that affect the stereoselectivity, but do not quantitatively elucidate the selectivity. For both models of QM-S and QM-O, four reaction modes are feasible and show similar barrier heights. These results of QM-only calculations suggest that simple cluster models cannot account for the stereoselectivity, which implies the pivotal importance of the active site environment of engineered P450s. We further performed QM/MM calculations for both P450BM3-CIS and P411BM3-CIS to explain the stereoselectivity of cyclopropanation. On the basis of the different relative orientation between the styrene substrate and carbene group of IPC, we constructed four distinct reactant models for both QM/MM-S and QM/MM-O, which are designated as React_A, React_B, React_C, and React_D, or React_A′,

Figure 6. QM/MM-optimized geometries of four reactants for model QM/MM-S. Distances are given in Å and the relative energies are in kcal/mol. The energy of 3React_A is set to zero.

QM/MM-optimized geometries of four reactant models for QM/MM-S. A comparison of these reactant models reveals that in all cases the QM/MM results show the same spin-state ordering of the QM results. Furthermore, the relative energies for these reactants increase in the order of React_A < React_C < React_D < React_B, and the binding patterns of styrene relative to diazoester would determine the stereoconfiguration (trans and cis) of the products; i.e., one reactant model only corresponds to one cyclopropanation product. React_A and React_D correspond to the trans products, whereas React_B and React_C to the cis products. The energy profiles of the four models at the triplet states are shown in the right panel of Figure 7. One can see that 3React_A corresponds to the lowest energy, followed by 3React_C (3.44 kcal/mol) and 3React_D (9.19 kcal/mol). 3React_B is 12.43 kcal/mol higher than 3 React_A, and thus the possibility of its existence should be the minimum. From the barrier point of view, with respect to their respective reactants, model B shows the lowest barrier of 5.38 kcal/mol, followed by models D (7.53 kcal/mol), A (10.42 kcal/mol), and C (11.18 kcal/mol). Overall, these results indicate that many factors may influence the seteroselectivity of the final products. On one hand, the binding modes of the substrate will affect the cis/trans ratio of the products. On the other hand, the barrier heights of the first steps, which associate with the relative binding modes, will also affect the trans/cis ratio. The calculated results are summarized in Figure 7. The experiment demonstrated that, in vitro the cyclopropanation catalyzed by P450BM3-CIS shows 40% trans and E

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Figure 7. Calculated energy profiles for the four models in QM/MMS at the triplet and OSS states. 3React_A is set to zero of energy.

60% cis selectivity,4b which coincides with the information derived from Figure 7. First, for the most stable state (in OSS), Model A is lower in energy than Model C and Model D by 3.27 and 6.42 kcal/mol, and Model A has similar energy barrier with model C and Model D (9.93 vs 12.20 and 8.81 kcal/mol). Therefore, Model A and Models C/D will lead to a similar amount of products. As for Model B, in the reactant state, Model B is higher than Model A by 12.43 kcal/mol on triplet and 10.47 kcal/mol on the OSS state, which means that this model will afford less reactant. But the much lower energy barrier of Model B (13.2 kcal/mol) makes this model a highly efficient one. So far, our calculation results cannot accurately distinguish the 60:40 ratio selectivity of the P450BM3-CIS. To testify if the protein environment can make the Fe(II) mechanism a favorable one, we also calculated the cyclopropanation at the closed-shell singlet surface. It was found that the reaction follows a concerted mechanism, which corresponds to much higher energy barriers than those of the triplet state (Figures S13 and S14). The experimental studies demonstrated that P411BM3-CIS shows superior 72% cis selectivity in vitro.4b Thus, we further explored the stereoselectivity of P411BM3-CIS (QM/MM-O). The optimized geometries of reactants are shown in Figure 8. Due to the change of the axial ligand, the calculated energies of P411BM3-CIS are different from that of P450BM3-CIS. The calculated results also suggest the open-shell singlet to be the ground state, and the triplet states are only higher than OSS by 1−3 kcal/mol. Thus, we discuss the selectivity of the reaction on the OSS and triplet state surfaces. Figure 9 shows the energy profiles for the QM/MM-O at triplet and OSS states. Comparison of the four reactant models reveals that 3React_B′ and 3React_D′ are thermodynamically more stable than 3 React_A′ and 3React_C′’, but the energetic difference between 3React_B′ and 3React_C′ is very small. Relative to the corresponding reactants, transition states of OSTS1_A′, OS TS1_B′, OS TS1_C′, OS TS1_D′, 3 TS1_A′, 3 TS1_B′, 3 TS1_C′, and 3TS1_D′ correspond to barriers of 13.02, 9.96, 8.81, 19.26,13.53, 9.69, 9.32, and 20.70 kcal/mol, respectively. The transition states of Model B and C on OSS and triplet state show lower energies. Although 3Prod_D′ is more stable than the other products, 3TS1_D′ shows a high barrier that is kinetically unfavorable for obtaining the 3 Prod_D′. Note that the reactant energy of Model A′ is relative higher in energy, and the barrier for the first

Figure 8. Optimized geometries of four reactants for model QM/ MM-O. Distances are given in Å, and the relative energies are given in kcal/mol. The energy of 3React_A′ is set to zero.

Figure 9. Calculated energy profiles at OSS and triplet states for the four models in QM/MM-O. The energy of 3React_A′ is set to zero.

nucleophilic attack increase from 10.41 kcal/mol in Model A to 13.53 kcal/mol in Model A’, thus, Model A’ appears to correspond to an inferior pathway. In contrast, React_B′ and React_C′ have relative lower energies than React_A′, and Model B′ and C′ correspond to lower energy barriers than Model A′ and D′. These results imply that Models B′ and C′ should be responsible for the main products. In general, Model C′ corresponds to the major products. All the optimized geometries of these stationary points are shown in Figure S15− S18. Taking these results into consideration, the cis conformations should be superior in the final products, which is in line with the experiments. Mutation of C400 into F

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Inorganic Chemistry

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S400 not only changes the axial coordination bond but also affects the active site assignment and spin populations on Fe and carbene C atoms of IPC, thereby partially inducing the change of trans and cis selectivity. For QM/MM-O models, the PES of the Fe(II) mechanism at closed-shell singlet state were also calculated (Figures S19 and S20), which show much higher energy barrier than those of triplet and open-shell singlet states. The spin densities of key atoms for QM/MM models are listed in Tables S6−S9. We can see that, in addition to the energy change, the spin distribution of triplet state products also shows large difference compared to those of QM models, which may come from the effect of protein environment. In general, the calculation results provide useful clues for understanding the mechanism of P450BM3-CIS and P411BM3CIS. The selectivity of the cyclopropanation is suggested to originate from the different binding orientations of diazoester and styrene. It should be clarified that the experimentally reported slight difference between the trans and cis selectivity (about three times of difference) for both P450BM3-CIS and P411BM3-CIS together with the potential errors of QM/MM calculations makes it difficult to accurately determine the trans/cis ratio. Actually, our calculation results can only qualitatively elucidate the selective preference for cyclopropanation in P411BM3-CIS. To quantitatively illuminate the stereoselectivity of an enzymatic reaction is usually difficult, because many factors would affect the accuracy of the calculated energies, such as the quantum method to treat the QM region, the force filed parameters to describe the MM atoms, treatment of QM/MM boundary, etc. In addition, the flexibility of proteins may also lead to the fluctuation of calculated energies. Thus, the extensive sampling of conformations is required for obtaining more accurate energies within the selected QM method and MM force field if we aim to address the region- and stereoselectivity of enzymatic reactions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01875. Calculated QM-only relative energies for styrene cyclopropanation at UB3LYP/BSII level; spin densities of key atoms; RMSDs for protein backbone of the MD simulations; optimized structures of stationary points at triplet, OSS, and singlet state for models QM-S and QM-O; QM/MM optimized geometries of stationary points at triplet, OSS, and singlet state for models QM/ MM-S and QM/MM-O. Cartesian coordinates and calculated energies of optimized structures of triplet and open-shell singlet states from QM and QM region of QM/MM calculations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 53188365576; fax: +86 53188564464; e-mail: [email protected]. ORCID

Yongjun Liu: 0000-0002-1686-8272 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21773138, 21707122). REFERENCES

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4. CONCLUSIONS In this work, we utilized both QM and QM/MM calculations to explore the reaction mechanism and stereoselectivity of styrene cyclopropanation catalyzed by the engineered P450BM3 enzymes. QM-only calculations suggest that the ground state of IPC intermediate is the open-shell singlet (OSS) state, which is only slightly lower that the triplet state, regardless whether the axial ligand is methyl thiolate or methoxide, which coincides with the QM/MM results. IPC is a high reactive species, which reacts with styrene substrate to form a C-radical intermediate, which then attacks the C atom of Fe−C to generate the final cyclopropanation products. On the singlet PES, the cyclopropanation proceeds only through one elementary step without undergoing the C-radical intermediate. Actually, the stereoselectivity of cyclopropanation not only depends on the orientation of styrene relative to IPC in the active sites, but also associates with the energy barriers of the rate-limiting step. QM/MM results further reveal that mutation of C400 into S400 facilitates the cis selectivity to a certain extent, which is in agreement with experimental observations. Although theoretical calculations can only qualitatively judge the trans and cis selectivity of olefin cyclopropanation, they are beneficial to help the experimental redesign of engineered P450 enzymes. G

DOI: 10.1021/acs.inorgchem.8b01875 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

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DOI: 10.1021/acs.inorgchem.8b01875 Inorg. Chem. XXXX, XXX, XXX−XXX