Catalytic Mechanism for 2,3-Dihydroxybiphenyl Ring Cleavage by

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Catalytic Mechanism for 2,3-Dihydroxybiphenyl Ring-Cleavage by NonHeme Extradiol Dioxygenases Bphc: Insights from QM/MM Analysis Junjie Wang, Jinfeng Chen, Xiaowen Tang, Yanwei Li, Ruiming Zhang, Ledong Zhu, Yanhui Sun, Qingzhu Zhang, and Wenxing Wang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11008 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 19, 2019

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Catalytic Mechanism for 2,3-Dihydroxybiphenyl Ring-Cleavage by Non-Heme Extradiol Dioxygenases BphC: Insights from QM/MM Analysis 1Junjie

Wang, 1Jinfeng Chen, 2Xiaowen Tang, 1Yanwei Li, 1Ruiming

Zhang, 1Ledong Zhu, 3Yanhui Sun, 1Qingzhu Zhang*, 1Wenxing Wang 1Environment

Research Institute, Shandong University,

Qingdao 266237, P. R. China 2School

of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China

3College

of Environment and Safety Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China

Keywords Non-heme, Extradiol dioxygenases BphC, Quantum mechanics/molecular mechanics, 2,3-Dihydroxybiphenyl, Reactive oxygen species ___________________________________________________________ *Corresponding

authors. E-mail: [email protected],

Fax: 86-531-8836 1990 1


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Abstract An extradiol-cleaving catecholic dioxygenase, BphC, plays important roles in catabolism of biphenyl/polychlorinated biphenyls (PCBs) aromatic contaminants in the environment. To better elucidate the biodegradable pathway, a theoretical investigation of the ring-open degradation for 2,3-dihydroxybiphenyl (DHBP) was performed with the aid of quantum mechanical/molecular mechanical (QM/MM) calculations. A quintet state of the DHBP-iron-dioxygen group adducts was found to be reactive state with a substrate radical-FeII-superoxo (DHBP• ↑-FeII-O2• ‾↓) character. The HOO• species was the reactive oxygen species responsible for subsequent attack of DHBP. Among the whole reaction energy profile, the first step proton-coupled electron transfer was determined to be the rate-determining step with a potential energy barrier of 17.2 kcal/mol, which is close to the experimental value (14.7 kcal/mol). Importantly, the residue His194 shows distinct roles in the catalytic cycle, where it acts as an acid-base catalyst to deprotonate the hydroxyl group of DHBP at the early stage, then stabilizes the negative charge on the dioxygen group and, at the final stage, promotes the semialdehyde product formation as a proton donor.

2



1. Introduction

Polychlorinated biphenyls (PCBs), typical persistent organic pollutants (POPs) on the Stockholm Convention, are subject to long-range atmospheric transport, recalcitrant degradation and bioaccumulation in aquatic and terrestrial ecosystems.1-3 Despite that the production and usage of PCBs have been banned, PCBs are globally dispersed in the ecosystem by historical emission and are detected in human blood due to the incomplete degradation and the remaining mass remained in use.4-14 PCB degradation has attracted much concern and interest because of the refractory degradation, bioaccumulation in food chain and threats to human health. Among various PCBs degradation techniques, environmental biodegradation with the ability to accelerate chemical transformations has been reported to be effective in precious experimental studies.15-19 Jim A., Bedard, Furukawa, Pieper and co-workers have proposed the pathway of aerobic PCBs degradation by biphenyl-oxidizing bacteria as shown in Figure 1.15,

20-22

As biphenyl is the primary substrate for these PCBs/biphenyl

degradation bacteria, the gene clusters responsible for the PCB/biphenyl aerobic degradation are named as bph. As presented in Figure 1, four enzymes, BphA, BphB, BphC and BphD, can degrade PCBs to 2-hydroxypenta-2,4-dienoic acid and chlorobenzoic acid.21,

23

Among bph genes products, the 2,3-dihydroxybiphenyl

dioxygenase (BphC) is an indispensable and extremely important enzyme participating 3


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in the PCBs degradation process. BphC obtained from Pseudomonas sp. Strain KKS102 is a non-heme iron-dependent oxygenase, which could catalyze the extradiol cleavage of catechol ring of 2,3-dihydroxybiphenyl (DHBP).17-18,

24

The BphC enzyme plays

critical roles in the biodegradation of PCBs/biphenyl and in the catabolism of aromatic contaminants in the environment, therefore, it has been extensively investigated in the past decades.25-33 Study has shown that biphenyl can undergo halogenation, belonging to the precursors of PCBs.34 And, the mechanism of ring-open of PCBs is homologous to that of biphenyl.29 In addition, several experimental evidences show that higher chlorinated PCB congeners can undergo reductive dehalogenation by anaerobic bacteria, resulting in lower chlorinated PCB or biphenyl, while they would be degraded by biphenyl-oxidizing aerobic bacteria.15, 35 Consequently, this work was mainly focus on the catalytic mechanism of DHBP ring-cleavage. The Pseudomonas sp. Strain KKS102 can degrade a wide range of PCBs, which have been regarded to be refractory to biological degradation33, therefore, detailed characterization for the representative biphenyl would be extended to other PCBs in the natural environment. The crystal structure of the DHBP-BphC enzyme complexes shows that an FeΙΙ is coordinated by the so-called 2-His-1-carboxylate facial triad (His145, His209 and Glu260), the DHBP and one vacant site.29 The crystal structure of the DHBP-BphC enzyme-NO was solved and the oxygen-binding site of BphC was identified in the study by Nobuyuki and co-workers29, which was a breakthrough as NO were acted as a probe for the oxygen-binding site in BphC enzyme. And NO has been acted extensively as a probe for the oxygen-binding site in previous experiment.36 In addition, a catalytic 4



mechanism was proposed for oxygen-dependent metabolic process in their study as shown in Scheme 1.29 The first step of the catalytic process involves in the DHBP binding to the iron in the active-site in a monoanionic form through the displacement of two coordinated water molecules, where the OαH of DHBP is found to be deprotonated in the crystal structure. Subsequently, the dioxygen molecule binds to the vacant iron coordination site. In the following step, the O2 binding results in the complete deprotonation of the OβH group of the DHBP, where the His194 acts as an acid-base catalyst. Simultaneously, an electron transfer from the DHBP to dioxygen occurs, resulting in the formation of a substrate radical-FeΙΙ-superoxo. At this stage, the negative charge of the superoxide can be stabilized by the protonated His194. Then the dioxygen attacks the DHBP to form the FeΙΙ-alkylperoxo intermediate. After that, the O-O bond cleavage and the C-C bond fission through the Criegee rearrangement generate a lactone intermediate. Finally, ring cleavage of the catechol proceeds through a seven-membered lactone ring, generating the 2-hydroxy-6-oxo-6-phenylhexa-2,4dienoic acid (HOPDA). Several experimental investigations have proposed the proposal of ring-cleavage mechanism for BphC15, 24, 29, 37-39. FeΙΙ-O(H)O species is the reactive oxygen species attacking the substrate as reported in a recent study by Dong and co-workers25 and FeΙΙ is the predominant oxidation state evidenced by spectroscopic and X-ray studies.29, 37, 40 To date, little is known regarding the spin state of the DHBPBphC enzyme-dioxygen group complex, the role of His194, the rate-limiting step, the reactive oxygen species, the detailed valence-state change of iron and the molecular orbitals of iron in the degradation process of DHBP by BphC enzyme. 5


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The

present

work

was

carried

out

by

combining

quantum

mechanics/molecular mechanics (QM/MM) method to characterize the catalytic process of DHBP by BphC enzyme at the atomic level and complement the experimental enzyme chemistry. On the basis of the previously proposed mechanism of the DHBP degradation by BphC, additional possible reaction pathways are considered in this study. The spin densities and relative energies of the initial configuration with three spin states (triplet - S = 1, quintet - S = 2 and septet - S = 3) were first compared to determinate the ground state of the DHBP-BphC enzymedioxygen group. Subsequently, the energy profiles along the ground state were computed. Spin density, valence-state change and relative energy analysis of reactant, transition states, intermediates and product were also calculated to provide foundation information for understanding the catalytic mechanism for the DHBP ring-cleavage by non-heme extradiol dioxygenases BphC, which provides new insights into the degradation mechanism of PCBs.

2. Calculation Methods 2.1 Molecular Dynamic Simulation

The initial model for the simulation was built on the basis of the X-ray crystal structure of the 2,3-dihydroxybiphenyl dioxygenase in complex with 2,3dihydroxybiphenyl (BphC-DHBP) (PDB code: 1KW6, resolution: 1.45 Å). The protonation states of ionizable residues were manually determined through visual inspection based on the pKa values with the aid of the PROPKA procedure41. And the 6



force filed parameterization for the substrate DHBP was parameterized by using SwissParam and the partial charges parameters were further optimized with the aid of Gaussian09.42 And force field parametrizations for the iron and dioxygen group were added manually to the topology file. Hydrogen atoms of BphC enzyme were added with the HBUILD module of the CHARMM package using CHARMM36 force field.43-46 The entire enzyme was simulated in a cubic water box (TIP3P model47) with a size of 35 × 35 × 35 molecules and a water sphere with a radius of 36.12 Å was selected by deleting superfluous water molecules of the cube for subsequent simulation. Nine sodium ions were included to neutralize the enzyme-water system via randomly substitution of the solvent water molecules. The entire enzyme system was first relaxed through energy minimizations, and then was heated from 0 K to 298.15 K in 50 ps (1 fs/step) and equilibrated thermally within 1 ns (1 fs/step) to reach absolutely thermodynamic equilibrium state. Finally, a 20 ns stochastic boundary molecular dynamics (SBMD) simulation was performed at 298.15 K. The Langevin temperature coupling method and the leap-frog algorithm were employed in the simultation.43 2.2 QM/MM Calculations

All QM/MM calculations were performed with the aid of ChemShell48 platform that combines Turbomole49 and DL-POLY50. Turbomole was used for the QM region and DL-POLY with the CHARMM36 forced field was employed to analyze the MM region. Charge shift model51 and electrostatic embedding method52-54 were applied to avoid the hyperpolarization of QM wave function. The density functional theory 7


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(DFT) with the hybrid density functional B3LYP at a double-ζ Def-SV(P) basis set (B1)55 level was used in the calculations for the QM region. Energies were computed by single-point calculations with the aid of a larger Def-TZVP basis set (B2).56 In addition, Multiwfn57-58 were used to perform the natural bond orbital (NBO) analysis and orbital composition analysis. Important residues related to the formation or the cleavage of bonds were selected in the QM region and residues correlated to the distributions of electron density were also considered. Overall, the DHBP, iron, dioxygen group, residues His145, His194, His209 and Glu260, a total of 69 atoms (shown in Figure S1), were included in the QM region of BphC. All the atoms within 15 Å of the Fe ion, a total of 1590 atoms, were allowed to move and the remaining atoms were fixed during the QM/MM calculation. Among the Chemshell package, the hybrid delocalized internal coordinate (HDLC) optimizer was introduced for the geometry optimization, which employed the quasi-Newton limited memory Broyden−Fletcher−Goldfarb−Shanno (LBFGS) method for energy minimization searching.59-60 And the transition state structure was determined by scanning the potential energy profile from reactant to intermediate. The partitioned rational function optimization (P-RFO) algorithm was employed for the optimization of transition state, where a Hessian eigenmode-following algorithm (PRFO) was used for the reaction core and the L-BFGS algorithm was used for the environment, respectively.59, 61

3. Results and Discussion 8



After the SBMD simulation, one snapshot (20 ns) from the trajectory was selected as the initial model for the QM/MM simulation and the corresponding rootmean-square deviation (RMSD) of the enzyme skeleton was inspected to verify the reliability of the molecular dynamics method (Figure S2). The RMSD trajectory is stable after 13 ns at RMSD value of 1.68 Å. 3.1 Spin states of DHBP-Fe-O2 adducts

Three possible spin states (triplet, quintet and septet) would occur when the ground-state dioxygen group (triplet-oxygen, 3O2) enters and turns into the surrounding ligand of the FeΙΙ coordination centre. These three spin states were all considered to determine the ground state of the initial reactant model. The single-point energies and spin densities of the triplet, quintet and septet states of iron, dioxygen group, DHBP and key atoms optimized at the B2 level are shown in Table 1. In addition, energies computed for the reactant with BLYP, B3LYP, M06-2X, PBE0 and TPSSH methods at the B1 level are presented in Table 2. To better understand the electron transfer in the reactant, the valence electron configurations and the shaded surface map with projection of localized orbital locator (LOL) for the triplet, quintet, and septet states were calculated as displayed in Scheme 2 and Figure 2, respectively. As shown in Table 1, spin density and single-point data reveal that the septet state is the energetically stable state, which is also evidenced by the obtained energies shown in Table 2. The spin densities of iron, dioxygen group and substrate are 9


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3.83, 1.91 and 0.1 respectively, illustrating the dioxygen group is in the ground state (triplet-oxygen, 3O2) and all the unpaired electrons in FeII and O2 have parallel spin (DHBP-FeII-O2 in Scheme 2). For the triplet spin state, the iron, dioxygen group and DHBP bear spin densities of 0.96, 0.98 and 0.08, respectively, and the electron transfer occurs between the iron and dioxygen group, where the reactant complex can be described as substrate-FeIII-superoxo (DHBP-FeIII-O2• ‾ in Scheme 2). And it can also imply that the triplet state is a low-spin S=1/2 FeIII ferromagnetically coupled to a superoxo radical. However, the low-spin triplet state is energetically extremely unstable (18.9 kcal/mol). In the quintet spin state, the spin densities are 4.04, -0.38, 0.21 for iron, dioxygen group and DHBP, respectively, and the reactant complex have a substrate radical-FeII-superoxo (DHBP• ↑-FeII-O2• ‾↓) character as shown in Scheme 2. The above characterizations for three spin states are also evidenced by the shade surface map as shown in Figure 2. The localized orbital locator (LOL) distribution characteristics of the iron are related to the electrons distribution in d orbital, and the LOL can emerge spherically symmetric when the electron configuration is a half-filled or full d subshell. The LOL distributions of the iron, as shown in Figure 2, in the quintet and septet states with six electrons on the 3d orbital have an approximate ellipse shape. However, the LOL distribution of the iron presents an irregular quadrilateral shape in the triplet state even though there are five electrons on the 3d orbital. It is inferred that the quintet and septet states have the FeII character and the triplet state is a low-spin state with the FeIII coordination centre. To test the influences of a variety of functionals on the energy of the 10



triplet, quintet and septet state of reactant, a series of energy calculations with BLYP, B3LYP, M06-2X, PBE0 and TPSSH methods at the B1 level were performed in Table 2. It can be indicated that the average energies of the triplet and septet state are 13.6 and -6.5 kcal/mol respectively. And the energies of triplet and septet state with B3LYP are 10.5 and -5.5 kcal/mol respectively, where the B3LYP result is the closest. In addition, the energy orderings of the triplet and septet state are consistent where all the energies of the triplet state are higher than that of septet state. Therefore, the widely used density functional B3LYP would be suitable for the whole reaction and it would be selected to optimize the geometries in the subsequent reactions. As shown in Figure 3, the distances between O1 and O2 are 1.29 Å, 1.26 Å and 1.21 Å in the triplet, quintet and septet states, respectively. In the septet states, 1.21 Å is a typical value for triplet-oxygen, 3O2. The distance between O1 and FeII in the septet state is 2.91 Å and no electron transfer occurs between the dioxygen group and FeII centre as illustrated in Figure 2. In addition, almost all functionals in Table 2 indicate that the septet state is most energetically stable. Consequently, the septet state is the initial “ground” state for the system, but not for the reactant model. In the triplet state, the distance of the O2-C2 bond is 3.54 Å, which is much longer than that in the quintet state (3.17 Å), and the distal oxygen O2 orientates far away from the C2 in the DHBP (Figure 3a). These above characterizations are not beneficial to the subsequently attacking the C2 by the O2 on. Particularly, the energy of the triplet state is 18.9 kcal/mol greater than that of the quintet state. Therefore, the triplet state can not become the ground state for the reactant model. In the quintet state, the distance of the O1-O2 is 1.26 11


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Å and the spin densities of the DHBP and the dioxygen group are 0.2 and -0.44, respectively, illustrating that the transfer from the DHBP to the dioxygen group is not fully accomplishment but the quintet state have been ready for the subsequent catalytic reaction. The dioxygen group has a superoxide character and the opposite spin between the substrate radical and the superoxide (DHBP• ↑-FeII-O2• ‾↓) can be the reactive state for the subsequent catalytic reaction. The above results are similar to many previous theoretical studies of the non-heme iron-containing extrodiol dioxygenases.24-25, 27-28, 6269

To better understand the further insight into the quintet reactive state, molecular

orbital diagram was obtained (Figure 4), which indicates that the iron 3d orbitals could interact with surrounding ligands and split into a pair of two π* orbitals ( π *xz, π *yz) and a pair of two σ* orbitals (σ *x2 - y2, σ *z2). As shown in Figure 4, the π *yz and π *xz of the 3d orbitals on FeII represent the antibonding combinations with the 2p orbital of dioxygen and DHBP, whereas the dxy orbital is not changed since there is not any antibonding interaction between the iron and DHBP. Similarly, the σ *x2 - y2 orbital represents the antibonding interaction between the 3d orbital on FeII and the 2p orbital on DHBP by head-on overlapping. And the σ *z2 orbital represents the antibonding interaction between the 3d orbital of FeII and 2p orbital of the substrate DHBP. In conclusion, the orbital occupations are 𝑑xy 2π *xz 1π *yz 1σ *x2 - y2 1 σ *z2 1 , πu2πg1 and one single spin-parallel electron for the iron, dioxygen and DHBP, respectively.

3.2 Determination of the oxygen reactive species 12



As the quintet state is the ground reactive state, we therefore proposed the subsequent reaction pathways on the basis of the quintet state as shown in Scheme 3. The calculated energy profiles in the preceding steps of the DHBP ring-open are displayed in Figure 5. Simulation of the distal oxygen O2 attacking the DHBP C2 is performed without consideration for His194 and yields the alkylperoxo bridge intermediate (Figure 5a). However, the potential energy barriers is 29.8 kcal/mol, which is much greater than the experimental value (14.7 kcal/mol18), and this process is energetically unfavorable. When considering the residue His194, an alternative pathway is possible to form the alkylperoxo bridge intermediate. In the initial step from 51

to IM1, a proton H1 donated from the DHBP is accepted by the His194, where the

distance between H1 and Nα decreases from 1.70 Å to 1.13 Å via 1.20 Å in TS1 with a potential energy barrier 17.2 kcal/mol (Table 3). In addition, potential energy comparison between the B3LYP and B3LYP-D3 methods was also considered, 17.2 kcal/mol and 15.8 kcal/mol respectively, for non-bonded interactions in the Figure S3. And the difference could be ignored, consequently the subsequent calculations would be in the B3LYP method. As illustrated in Figure S4, during the above hydrogen abstraction process, a proton-coupled electron transfer (PCET) step through iron occurs, where the spin densities of the DHBP and the dioxygen group changed from 0.2 and 0.44 to 0.4 and -0.72 (as shown in Table S1), respectively, and the spin population of iron changed from 4.04 to 4.19 via 2.92 in TS1. The hydrogen abstraction process is energetically favored because of the concerted proton-coupled electron transfer event.25, 13


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29, 38, 70-76

Then, the proton H1 transfers to the proximal oxygen O1, promoted by the

conformation rotation of the protonated His194 orientating towards the proximal O1 (as shown in Figure S5). In these two steps that His194 acts as an acid-base catalyst in the proton transfer and then the protonated His194 can stabilize a negative charge on the superoxide radical located in the hydrophobic O2-binding cavity. For the step of transferring the H1 to the proximal O1, we supposed the Fe-O(H)O was the reactive oxygen species for the subsequent attack of the distal oxygen O2 on the C2 (Figure 5b), where the TS2´ and IM2´ energies are 12.2 kcal/mol and 5.9 kcal/mol, respectively. However, the transition state from IM2´ to IM3 was not found. Therefore, an alternative pathway is considered and the corresponding energies of TS2 and IM2 are 3.3 kcal/mol and -4.6 kcal/mol, respectively, which are much lower than that of TS2´ and IM2´ and the transition state (TS3) from IM2 to IM3 are found. It is an intriguing discovery that the attack of the distal oxygen O2 on the C2 and the transfer of the proton H1 to proximal oxygen O1 are a concerted step (IM1 to IM2), during which the distance between O2 and C2 decreases from 4.70 Å to 2.37 Å via 2.76 Å in TS2. In addition, the distance (IM1 to IM2) between O1 and Fe increased from 2.12 Å to 2.38 Å, which might assist the attack of the oxygen species on the DHBP. Consequently, instead of the Fe-O(H)O species, the HOO• is determined to be the reactive oxygen species for subsequent reactions. In the reaction pathways involving the HOO• reactive species, the O1-O2 bond cleaving occurs to form a substrate epoxide radical intermediate (IM3) with a potential energy barrier of 8.9 kcal/mol. The distances of O1-O2 bond and O2-C1 bond 14



change from 1.44 Å to 2.82 Å and 2.37 Å to 1.44 Å via 2.20 Å and 2.21 Å in TS3, respectively. In addition, the distance between the iron and O1 decreases from 2.38 Å to 1.78 Å, suggesting that after the O1-O2 bond cleaving, the hydroxyl group (O1H1) becomes the ligand of iron. As shown in Table S1, in the IM3, the spin densities of the iron, dioxygen group and DHBP are 2.89, 0.11 and 0.92, respectively, where the iron bearing the spin population of 2.89 is oxidized to FeIII. And the spins on the DHBP and dioxygen group can also verify the substrate epoxide radical characteristic in IM3. Therefore, the formation of IM3 promotes the subsequent catalytic reaction of DHBP ring open. 3.3 Reaction mechanism for the DHBP ring-open and semialdehyde formation

The energy profiles along DHBP ring-open and semialdehyde product formation are shown in Figure 6. The C1-C2 bond is cleaved following the formation of IM3, resulting in a seven-membered ring (lactone radical) intermediate (IM4 in Scheme 3) with a potential energy barrier of 1.1 kcal/mol (Figure 6). The distance between C1C2 bond increases from 1.54 Å to 2.54 Å via 1.65 Å in TS4. Thus, the Criegee rearrangement reaction was found via a stepwise cleavage (IM2 to IM4), where the O1O2 bond cleavage gave an epoxide radical intermediate (IM3) and the C1-C2 bond cleavage resulted in a lactone radical intermediate (IM4). Due to the lactone radical intermediate, the hydroxyl group, O1H1, would attack the C2 of DHBP quiet easily to generate the IM5. Along IM4 to IM5, the spin densities of iron, dioxygen group and DHBP changed from 3.91, 0.18 and -0.24 to 3.79, 0.02 and 0.03 respectively, 15


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suggesting that the iron center has now been re-reduced to its initial FeII oxidation state due to the departure of the hydroxyl group (O1H1) on FeIII and the radical intermediate promoted the attack of O1 on the C2. And the distances of Fe-O1 bond and O1-C2 bond change from 1.87 Å to 2.28 Å and 3.11 Å to 1.42 Å via 1.90 Å and 2.39 Å in TS5, respectively. DHBP ring-open occurs subsequently to generate a semialdehyde intermediate (IM6), accompanying a transfer of the proton H1 from the hydroxyl group (O1H1) to the residue His194. The distances of O2-C2 bond and H1-Nα bond change from 1.46 Å to 2.65 Å and 1.73 Å to 1.09 Å via 1.89 Å and 1.52 Å in TS6, respectively. In the final step, the proton H1 transferring from His194 to the hydroxyl group (OβH1) occurs to generate the 1-oxo-1-phenylhexa-3-hydroyl-3,5-dienoate product (P in Figure 6). The distance between H1-Nα bond increases from 1.09 Å to 1.83 Å via 1.19 Å in TS7. And His194 acts as a proton donor in this proton transfer step. Among the entire catalytic reaction (Figure 5 and Figure 6), the first step of the protoncoupled electron transfer (51 to IM1) is found to be the rate-determining step with a potential energy barrier of 17.2 kcal/mol, instead of the O1-O2 bond cleavage (IM2 to IM3) with a barrier of 8.9 kcal/mol.

4. Conclusion In this work, we implanted the QM/MM calculations to investigate the reaction mechanism of ring-open degradation of DHBP by the BphC enzyme. The septet state was found to be the initial state when the dioxygen group entered and then the quintet was found to be the ground reactive state for the subsequent catalytic reactions. The attack of dioxygen group on the DHBP consists of two steps. The first 16



step is a proton-coupled electron transfer from DHBP to His194, where the His194 acts as an acid-base catalyst and the process is found to be the rate-determining step with a potential energy barrier of 17.2 kcal/mol. In the second step, the proton is transferred from the protonated His194 to the proximal oxygen of the superoxide ligand and the HOO• species is determined to be the reactive oxygen species for subsequent attack. Criegee rearrangement occurs stepwise that the O1-O2 bond cleavage results in an epoxide radical intermediate and the C1-C2 bond cleavage generates a seven-membered lactone radical intermediate. Then the lactone radical intermediate is attacked by the hydroxyl group bound to the FeIII. Subsequently the DHBP ring-open occurs through O2-C2 bond cleavage accompanying the proton transfer from the hydroxyl group (O1H1) to His194 to form the semialdehyde intermediate. Finally, the proton is transferred from His194 to DHBP yielding the semialdehyde product, where the His194 plays a role as a proton donor. Overall, this mechanistic insight sheds light on the DHBP ring cleavage degradation process by the extradiol dioxygenases, and can facilitate designing more effective chlorinated biphenyl removal approaches.

Acknowledgments

The work was financially supported by (National Natural Science Foundation of China, project Nos. 21337001 and 21577082) and Taishan Scholars (No. ts201712003) and National Major Science and Technology Program for Water 17


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Pollution Control and Treatment (No. 2017ZX07202-002).

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Cycloaddition with Alkenylisoxazoles. The Journal of organic chemistry 2018.

Table 1. Spin Densities and relative energies of the DHBP-BphC complex for the triplet, quintet, and septet states computed at the B3LYP/B2 Level. The “t” refers to the total spin density of the dioxygen group, and the “Sub” represents the total spin density of the substrate DHBP.

spin density species

Fe

O1

O2

O2t

Oβ

Oα

Sub

ΔE(kcal/mol)

31

0.96

0.39

0.59

0.98

0

0.05

0.08

18.9

51

4.04

-0.06

-0.38

-0.44

0.03

0.14

0.21

3.5

71

3.83

0.96

0.95

1.91

0.01

0.07

0.1

0

23


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Table 2. The relative energies of the triplet, quintet, and septet spin states of the DHBPBphC complex among different functionals calculated at the B1 level. All energies are shown in kcal/mol. species

BLYP

B3LYP

M062X

PBE0

TPSSH

31

1.8

10.5

35.9

16.7

3.5

51

0.0

0.0

0.0

0.0

0.0

71

0.03

-5.5

-12.6

-8.1

-6.4

24



Page 26 of 45

Table 3. Key bond distances and potential energy barriers of the DHBP-BphC complex in the quintet state calculated at B1 level. Distance/Å Energy

Fe-O1

O1-O2

O1-C2

O2-C1

O2-C2

C1-C2

H1-Nα

0

2.01

1.26

3.30

3.86

3.17

1.42

1.70

TS1

17.2

2.11

1.28

3.22

3.85

3.18

1.41

1.20

TS1´

29.8

1.98

1.33

2.52

2.59

1.77

1.49

1.63

IM1

2.1

2.12

1.29

3.42

4.70

3.74

1.41

1.13

IM1´

25.5

1.83

1.45

2.36

2.38

1.45

1.53

1.61

TS2

3.3

2.31

1.38

2.78

2.76

2.14

1.46

1.65

TS2´

12.2

2.27

1.31

3.21

3.87

3.27

1.40

1.33

IM2

-4.6

2.38

1.44

2.39

2.37

1.50

1.53

1.71

IM2´

5.9

2.40

1.31

3.24

3.82

3.24

1.40

1.39

TS3

8.9

1.84

2.20

2.70

2.21

1.37

1.55

1.95

IM3

-2.0

1.78

2.82

2.85

1.44

1.42

1.54

1.87

TS4

1.1

1.80

2.92

2.81

1.43

1.40

1.65

1.94

IM4

-29.7

1.87

3.17

3.11

1.35

1.37

2.54

1.98

TS5

-26.2

1.90

2.99

2.39

1.38

1.37

2.31

1.88

IM5

-56.8

2.28

2.29

1.42

1.35

1.46

2.44

1.73

TS6

-52.3

2.23

2.48

1.35

1.28

1.89

2.83

1.52

IM6

-62.0

2.17

3.15

1.29

1.24

2.65

3.40

1.09

TS7

-59.4

2.13

3.29

1.27

1.23

2.74

3.36

1.19

P

-67.4

2.06

3.46

1.27

1.23

2.84

3.32

1.83

species 51

25


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Figure Captions

Scheme 1. Reaction mechanism for the DHBP extradiol ring cleavage by BphC enzyme proposed by Nobuyuki Sato and co-workers. The “R” means the benzene ring. Scheme 2. Valence electron configurations for the dioxygen group-iron-DHBP complex at the triplet, quintet, and septet states. The “R” represents the benzene ring. The brown arrow, white arrow and black arrow refer to the electrons in the DHBP, dioxygen group and iron respectively. Scheme 3. Proposed mechanism for the ring-open catalytic pathway of the DHBP by BphC enzyme. The “R” represents the benzene ring. Figure 1. Pathway of aerobic polychlorinated biphenyl metabolism by biphenyloxidizing bacteria, including BphA, BphB, BphC and BphD enzymes. The HOPDA refers to 2-hydroxy-6-oxo-6-(chloro)phenylhexa-2,4-dienoic acid. Figure 2. Shaded surface map with projection of localized orbital locator (LOL) in a plane of the iron and dioxygen group for triplet, quintet and septet respectively. The covalent regions have high LOL value, the electron depletion regions between valence shell and inner shell was represented by the blue circle. The purple arrow refers to a lone pair. Figure 3. Optimized structures and key bond distances for the reactant of the triplet, quintet and septet states. A is the superposition of the optimized reactant in the triplet (iron and dioxygen in yellow), quintet (dioxygen in red and iron in green) and septet (dioxygen in orange and iron in green) states. B is detailed geometry of the quintet state. 26



All of the distances are shown in Å. And “R” represents the benzene ring. Figure 4. Molecule orbital for the reactant of quintet state. The brown arrow, white arrow and black arrow refer to the electrons in the DHBP, dioxygen group and iron respectively. Figure 5. Energy profiles of the attack of dioxygen group on the DHBP. a reveals that the directly attack of dioxygen group on the DHBP is very sluggish and difficult to occur. b is demonstrated that the HOO•, not the Fe-O(H)O species, is determined to be the reactive oxygen species with 8.9 kcal/mol lower than the formation of Fe-O(H)O species. And “R” represents the benzene ring. Figure 6. Energy profiles of the formation of semialdehyde product complex. The relative energies (kcal/mol) are calculated at the B3LYP/B2 level. The “R” represents the benzene ring.

Scheme 1

27


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Scheme 2

28



Scheme 3

29


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Figure 1

30



Figure 2

31


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Figure 3

Figure 4

32



a

33


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b Figure 5

Figure 6

34



Reaction mechanism for the DHBP extradiol ring cleavage by BphC enzyme proposed by Nobuyuki Sato and co-workers. The “R” means the benzene ring. 707x738mm (96 x 96 DPI)


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Valence electron configurations for the dioxygen group-iron-DHBP complex at the triplet, quintet, and septet states. The “R” represents the benzene ring. The brown arrow, white arrow and black arrow refer to the electrons in the DHBP, dioxygen group and iron respectively. 762x370mm (96 x 96 DPI)



Proposed mechanism for the ring-open catalytic pathway of the DHBP by BphC enzyme. The “R” represents the benzene ring. 1448x646mm (96 x 96 DPI)


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Pathway of aerobic polychlorinated biphenyl metabolism by biphenyl-oxidizing bacteria, including BphA, BphB, BphC and BphD enzymes. The HOPDA refers to 2-hydroxy-6-oxo-6-(chloro)phenylhexa-2,4-dienoic acid. 1025x304mm (96 x 96 DPI)



Shaded surface map with projection of localized orbital locator (LOL) in a plane of the iron and dioxygen group for triplet, quintet and septet respectively. The covalent regions have high LOL value, the electron depletion regions between valence shell and inner shell was represented by the blue circle. The purple arrow refers to a lone pair. 123x46mm (220 x 220 DPI)


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Optimized structures and key bond distances for the reactant of the triplet, quintet and septet states. A is the superposition of the optimized reactant in the triplet (iron and dioxygen in yellow), quintet (dioxygen in red and iron in green) and septet (dioxygen in orange and iron in green) states. B is detailed geometry of the quintet state. All of the distances are shown in Å. And “R” represents the benzene ring. 146x73mm (220 x 220 DPI)



Molecule orbital for the reactant of quintet state. The brown arrow, white arrow and black arrow refer to the electrons in the DHBP, dioxygen group and iron respectively. 588x282mm (96 x 96 DPI)


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Energy profiles of the attack of dioxygen group on the DHBP. a reveals that the directly attack of dioxygen group on the DHBP is very sluggish and difficult to occur. b is demonstrated that the HOO•, not the FeO(H)O species, is determined to be the reactive oxygen species with 8.9 kcal/mol lower than the formation of Fe-O(H)O species. And “R” represents the benzene ring. 386x186mm (96 x 96 DPI)



Energy profiles of the attack of dioxygen group on the DHBP. a reveals that the directly attack of dioxygen group on the DHBP is very sluggish and difficult to occur. b is demonstrated that the HOO•, not the FeO(H)O species, is determined to be the reactive oxygen species with 8.9 kcal/mol lower than the formation of Fe-O(H)O species. And “R” represents the benzene ring. 549x218mm (96 x 96 DPI)


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Energy profiles of the formation of semialdehyde product complex. The relative energies (kcal/mol) are calculated at the B3LYP/B2 level. The “R” represents the benzene ring. 561x373mm (96 x 96 DPI)


Catalytic Mechanism for 2,3-Dihydroxybiphenyl Ring Cleavage by

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