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Degradation mechanism of biphenyl and 4-4′-dichlorobiphenyl cis-dihydroxylation by

non-heme 2,3 dioxygenases BphA: A QM/MM approach

Ledong Zhu, Jie Zhou, Ruiming Zhang, Xiaowen Tang, Junjie Wang, Yanwei Li,
Qingzhu Zhang, Wenxing Wang

PII: S0045-6535(20)30035-7
DOI: https://doi.org/10.1016/j.chemosphere.2020.125844
Reference: CHEM 125844

To appear in: ECSN

Received Date: 4 November 2019

Revised Date: 19 December 2019
Accepted Date: 3 January 2020

Please cite this article as: Zhu, L., Zhou, J., Zhang, R., Tang, X., Wang, J., Li, Y., Zhang, Q., Wang,
W., Degradation mechanism of biphenyl and 4-4′-dichlorobiphenyl cis-dihydroxylation by non-heme
2,3 dioxygenases BphA: A QM/MM approach, Chemosphere (2020), doi: https://doi.org/10.1016/
j.chemosphere.2020.125844.

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Credit Author Statement

Ledong Zhu: Conceptualization, Methodology, Software, Formal analysis, Writing -

Original Draft

Jie Zhou: Conceptualization, Formal analysis, Data Curation, Writing - Original

Draft

Ruiming Zhang: Software

Xiaowen Tang: Software

Junjie Wang: Software

Yanwei Li: Writing - Review & Editing

Qingzhu Zhang: Writing - Review & Editing, Resources, Funding acquisition,

Project administration

Wenxing Wang: Supervision

 1 Degradation Mechanism of Biphenyl and
2 4-4’-dichlorobiphenyl cis-dihydroxylation by Non-Heme
3 2,3 Dioxygenases BphA: a QM/MM Approach

1
5 Ledong Zhu, 1Jie Zhou, 1Ruiming Zhang, 2Xiaowen Tang, 1Junjie Wang,
1
6 Yanwei Li, 1Qingzhu Zhang*, 1Wenxing Wang
1
7 Environment Research Institute, Shandong University, Qingdao 266237,

8 P.R. China
2
9 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou

10 510006, P. R. China

11

12

13

14

15

16 Keywords

17 Rieske-type enzyme; Non-heme iron enzymes; Quantum

18 mechanics/molecular mechanics; Biphenyl 2,3-dioxygenase

19 ___________________________________________________________
*
20 Corresponding authors. E-mail: zqz@sdu.edu.cn,

21 Phone: 86-532-5863 1971

1
 1 Abstract

2 Biphenyl 2,3-dioxygenase (BphA), a Rieske-type and first enzyme in the aerobic

3 degradation process, plays a key role in the metabolizing process of

4 biphenyl/polychlorinated biphenyl aromatic pollutants in the environment. To

5 understand the catalytic mechanism of biphenyl 2,3-dioxygenase, the conversions

6 leading to the cis-diols are investigated by means of quantum mechanics/molecular

7 mechanics (QM/MM) method. A hydroperoxo-iron(III) species is involved in the

8 enzyme-catalyzed reaction. Herein, we explored the direct reaction mechanism of

9 hydroperoxo-iron(III) species with biphenyl and 4-4’-dichlorobiphenyl. The reaction

10 process involves an epoxide intermediate, it could develop into a carbocation

11 intermediate, and ultimately evolve into a cis-diol product. The important roles of

12 several residues during the dioxygenation process were highlighted. This study may

13 provide theoretical support for further directed mutations and enzymatic engineering

14 of BphA, as well as promote the development of degrading environmentally persistent

15 biphenyl/polychlorinated biphenyl aromatic contaminants.

16

17 1. Introduction

18 Biphenyl is an aromatic hydrocarbon having the central C-C bond connecting two

19 benzene rings. The π-conjugation structure of the two twisted phenyl planes gives it

20 strong stability (Johansson and Olsen, 2008; Chakraborty and Das, 2016). Biphenyl is

21 an important organic raw material widely used in medicine, pesticides, dyes, liquid

22 crystal materials and other fields. Although the use of biphenyl has decreased, it is a

2
 1 non-negligible pollutant and widespread existence in the environment. Great effort

2 has been made to explore its toxicological effects and environmental fate

3 (Chakraborty and Das, 2016). Many in vivo and in vitro experiments revealed the

4 toxicity of biphenyl to humans. Chronic exposure to biphenyl could cause damage to

5 the nervous system and digestive system (Moody et al., 2002; Pieper, 2005;

6 Hyeon-Yeong et al., 2015; Ma et al., 2017). Biphenyl is the precursor of

7 polychlorinated biphenyls (PCBs), a kind of organic compound formed by the

8 substitution of hydrogen atoms on biphenyl rings by chlorine. PCBs are one of the 12

9 persistent organic pollutants (POPs) in the Stockholm Convention, which have 209

10 congeners. PCBs have been widely used in the fields of industry such as plasticizers,

11 heat transfer fluid, fire retardants, and sealant (Xing et al., 2009; Li et al., 2018; Wang

12 et al., 2018; Fiedler et al., 2019). PCBs were banned three decades ago; however, they

13 are still found in the environment worldwide. Due to the lipophilic and chemical

14 stability properties, PCBs can migrate from a long distance with the earth's water-gas

15 circulation and are even found at the Antarctic and Arctic (Ge et al., 2013; Wang et al.,

16 2018). Many studies revealed that the atmospheric fine particles (PM) could enter the

17 human body through respiration and cause significant adverse effects on human

18 health (Manigrasso and Avino, 2012; Heo, 2017; Zhang et al., 2017). As one of the

19 PM components, PCBs have received special attention for years because of their

20 toxicity, persistency, bioaccumulation and carcinogenicity (Zhao et al., 2017; Zhu et

21 al., 2018b; Satsangi and Agarwal, 2019). PCBs are widely existent in the atmospheric

22 environment in many regions including Africa, Asia and Polar Regions (Cabrerizo et

3
 1 al., 2017; Trinh and Chang, 2018; Zhang et al., 2018). The global atmospheric passive

2 sampling (GAPS) study showed high concentrations of PCBs detected in urban areas

3 or near urban areas (Pozo et al., 2008). Atmospheric PCBs can enrich in the soil

4 through dry and wet deposition mechanisms and then migrate to various organisms in

5 the soil environment. They are further amplified through the food chain, leading to an

6 increased threat to human health (Fan and Sloan, 2008; Günindi et al., 2011).

7 Consequently, the environmental remediation of PCBs has become a major global

8 problem (Shao et al., 2011). The understanding of the biological degradation

9 mechanism of PCBs is significant for the bioremediation of PCBs in environment

10 contaminant management. Due to the relatively high aqueous solubility, the low

11 chlorinated PCBs are important for assessing the transport and the environmental fate

12 of PCBs. Herein, in this study, the biological degradation mechanism of PCBs was

13 studied by using a primary congener of PCBs, 4-4’-dichlorobiphenyl, as the model

14 substrate.

15

16 Biological degradation is an environment-friendly and cost-effective strategy that

17 plays a fundamental role in the carbon cycle and the removal of POPs from the

18 environment (Wang et al., 2018). Furukawa, Suenaga, Seeger, Pieper, and their

19 coworkers proposed that there are four enzymes involved in initiating the degradation

20 process of biphenyl and PCBs (Hikaru et al., 2002; Seeger and Pieper, 2010). They

21 are Biphenyl 2,3-dioxygenase (BphA), cis-2,3-Dihydro-2,3-dihydroxybiphenyl

22 dehydrogenase (BphB), 2,3-Dihydroxybiphenyl 1,2- dioxygenase (BphC), and

4
 1 2-Hydroxy-6-phenyl-6-oxohexa-2,4-dienoate hydrolase (BphD), as shown in Scheme

2 1 (Hikaru et al., 2002; Kensuke et al., 2004). BphA is the first enzyme in the aerobic

3 degradation of biphenyl and PCBs. BphA is a multicomponent Rieske-type enzyme

4 comprising: (1) a terminal oxygenase component (NDO), it contains one mononuclear

5 iron center and a Rieske [2Fe-2S] cluster, (2) a ferredoxin, and (3) a ferredoxin

6 reductase (Arnett et al., 2000; Furusawa et al., 2004; Kumar et al., 2011; Zhao et al.,

7 2018; Agulló et al., 2019). In addition, the terminal oxygenase consists of two

8 subunits (BphA1 and BphA2), in which the α-subunit (BphA1) is the central catalytic

9 domain. Although it does not involve any reactive active site and is absent in the

10 reaction with the substrate directly, the β-subunit (BphA2) maintains the correct

11 conformation of the α-subunit (Pham et al., 2012; Agulló et al., 2019). The ferredoxin

12 and the ferredoxin reductase transfer two electrons from nicotinamide adenine

13 nucleotide (NADH) to the NDO component. The electrons pass through the

14 mononuclear iron catalytic center and consume one molecule of oxygen under the

15 action of NDO to complete the oxygenation with the substrate. Based on different

16 experimental observations and computational results, as shown in Scheme 2, a

17 possible enzyme-catalyzed reaction pathway for biphenyl and PCBs dioxygenation

18 can be proposed (Karlsson et al., 2003; Bassan et al., 2004; Kovaleva and Lipscomb,

19 2008).

20

21 Biodegradation of biphenyl and PCBs by microbes has been studied intensively in the

22 past. Many studies focus on the aerobic bacteria capable of degrading biphenyl and

5
 1 PCBs, including P. pnomenusa B356, B. xenovorans LB400, and Rhodococcus RHA1.

2 The strain LB400 is widely used for the oxidative degradation of PCBs (Kitagawa et

3 al., 2001; Pham et al., 2012; Bhattacharya and Khare, 2017; Agulló et al., 2019). To

4 obtain a more efficient enzyme, Sonali Dhindwal et al. replace seven residues in

5 BphAELB400 and mutate it to BphAEII9. The variant of BphAELB400, BphAEII9, is

6 obtained by replacing the seven residues in region III (Thr335, Phe336, Asn337,

7 Asn338, Ile339, Arg340, and Ile341) of BphAELB400 with the corresponding residues

8 (Gly333, Ile334, Asn335, Thr336, Ile337, Arg338, and Thr339) of BphAEB356.

9 Experiments have revealed that BphAEII9 is more effective for PCBs degradation than

10 BphAELB400 or BphAEB356 (Dhindwal et al., 2016).

11

12 In this study, the combined quantum mechanics/molecular mechanics (QM/MM)

13 method was employed to explore the catalytic process of biphenyl by the BphA

14 enzyme at the atomic level. The QM/MM method has increasingly become a powerful

15 tool to complement experimental enzyme chemistry. To study the degradation ability

16 of BphA to biphenyl and PCBs, we selected biphenyl and 4-4’-dichlorobiphenyl for

17 simulation studies. In addition, the details of the transition states and short-lived

18 intermediates are provided, of which the information is inaccessible by the

19 experimental enzyme chemistry. To reveal the effect of the residues surrounding the

20 active site involved in the epoxide formation, the electrostatic analyses are

21 investigated, it may become a valuable tool to complement experimental enzyme

22 chemistry studies.

6
 1

2 2. Calculation Methods

3 2.1 Setup of the System and Molecular Dynamic Simulations

4 The original crystal structure of BphA was obtained from the Protein Data Bank

5 (http://www.rcsb.org): BphAEII9 (PDB ID: 5AEW). Since there is no crystal structure

6 of biphenyl and hydroperoxo-iron(III) complexes or PCBs and hydroperoxo-iron(III)

7 complexes, the substrate in the BphAEII9 was further modified with the aid of the

8 Discovery Studio 2.5 program. We used the Swiss-Pdb Viewer

9 (https://spdbv.vital-it.ch/) to check the protein structure and repaired nine absent

10 amino acid side chain atoms. Then, the molecular docking was performed to simulate

11 the binding of biphenyl- hydroperoxo-iron(III) complexes and

12 PCBs-hydroperoxo-iron(III) complexes using CDOCKER. With the aid of

13 CHARMM27 force field, the HBUILD facility in the CHARMM package

14 complements the missing hydrogen atoms in the crystal structure (Brooks et al., 2010a;

15 Brooks et al., 2010b). The protonation states of ionizable residues were determined by

16 the pKa values obtained from PROPKA3.1 (Olsson et al., 2011). The enzyme

17 complexes were initially placed in a cubic water box with a size of 35 Å × 35 Å × 35

18 Å. The water box then modified to a water sphere (TIP3P model) with a diameter of

19 70 Å (Jorgensen et al., 1983). Consequently, a total of 10681 water molecules and

20 10624 water molecules were included in the biphenyl-hydroperoxo-iron(III)

21 complexes system and PCBs-hydroperoxo-iron(III) complexes system, respectively.

22 The two systems were sufficiently minimized with a combination of Steepest Descent

7
 1 and Adopted Basis Newton-Raphson minimization after neutralized with twelve

2 sodium ions at random positions, respectively. The two systems were equilibrated

3 within 50 ps, followed by a 20 ns (0.001 ps × 20,000,000 steps) molecular dynamic

4 simulation with the canonical ensemble (NVT, 298.15 K). The Langevin dynamics

5 method and leap-frog algorithm attached in the CHARMM package were applied

6 during the reaction fitting. The obtained root-mean-square deviation (RMSD) of the

7 two systems were displayed in Fig. S1.

9 2.2 QM/MM Methodology

10 The QM/MM calculations were performed by using ChemShell 3.6 platform

11 (SHERWOOD et al., 2003), which integrates Turbomole (Ahlrichs et al., 1989) and

12 DL-POLY (Smith and Forester, 1996) programs. The hybrid delocalized internal

13 coordinate (HDLC) optimizer with the quasi-Newton limited memory

14 Broyden−Fletcher−Goldfarb−Shanno (L-BFGS) method (Billeter et al., 2000) and the

15 Hessian eigenmode-following algorithm (P-RFO) (Banerjee et al., 1985) were

16 employed for the geometry optimization and the optimization of the transition states,

17 respectively.

18

19 The electrostatic embedding method (And and Thiel, 1996) was applied to account for

20 the polarizing effect of the QM density in the QM region. The boundary between the

21 QM region and the MM region is realized by the connection atom method combined

22 with the charge shift model (Jr et al., 2002; Brooks et al., 2010b). All of the

8
 1 calculations for geometry optimization and single-point energy calculation were

2 performed using the unrestricted hybrid B3LYP-D3 exchange–correlation functional

3 method (Grimme et al., 2010). The geometry optimizations were conducted with the

4 double-zeta LANL2DZ (Los Alamos National Laboratory 2 double ζ) basis set on

5 iron and 6-31G** basis set on other atoms. Moreover, the final energy calculations for

6 the optimized configuration were combinate with the triple-zeta LACV3P+* basis set

7 on iron and 6-311++G** basis set on other atoms. The B3LYP-D3

8 exchange–correlation functional method has been successfully applied on iron

9 compounds with a reasonable deviation from experimental energy (Rydberg et al.,

10 2014). The LANL2DZ basis sets are widely implemented to describe transition metal

11 containing systems (Yang et al., 2009). Besides, Multiwfn 3.7 was employed to

12 perform the orbital composition analysis, natural bond orbital (NBO) analysis and

13 quantitative molecular surface analysis (Lu and Chen, 2011, 2012a, b). We can reveal

14 the molecular mechanism of the degradation of biphenyl and 4-4’-dichlorobiphenyl

15 by BphA enzyme through calculation. The QM regions contained substrates, a ferric

16 hydroperoxo intermediate ([FeIII-OOH]2+) and three residues coordinating with iron.

17 In addition, residues close to the active sites were classified to the QM region.

18 Therefore, residues Gln226, His233, His239, Asp388, [FeIII-OOH]2+, and the substrate

19 (biphenyl and 4-4’-dichlorobiphenyl) were considered to be the QM region. The

20 number of atoms in the QM region of both systems is 62. The former calculations

21 showed that the barriers of rate-determining steps for Rieske non-heme iron

22 dioxygenases in the quartet-spin and doublet-spin states were higher than in the

9
 1 sextet-spin state (Bassan et al., 2004). Therefore, the reactions were only carried out

2 in the sextet-spin state in this study. For the convenience of description, the two

3 systems involving two substrates are named as BP (biphenyl) and Cl-BP

4 (4-4’-dichlorobiphenyl). The reactant, transition state, intermediate and product are

5 labeled as R1, TS1, IM1and P1 in the BP system, R2, TS2, IM2 and P2 in the Cl-BP

6 system.

8 3. Results and Discussion

9 3.1 Molecular Docking and Dynamics

10 In Scheme 2, there are two electrons involved in the dioxygen activation in NDO. The

11 FeII of the non-heme iron complex and the Rieske cluster are oxidized. During the

12 dioxygen activation, the peroxide directly attacks the substrate. The intermediate in

13 the reaction pathway is a hydroperoxo-iron(III) species. This conclusion has been

14 verified by previous experiments and calculations (Wolfe and Lipscomb, 2003;

15 Bassan et al., 2004). We used Discovery Studio 2.5 program to obtain the structure of

16 hydroperoxo-iron(III)-substrate complex. As the enzyme was characterized as a dimer

17 in the X-ray diffraction (XRD) study (Dhindwal et al., 2016), therefore, two chains

18 (protomer K and L) were included in the subsequent MD simulations. The MD

19 simulations were performed for 20 ns, and a final 2000 conformations were collected

20 for the two systems. After 10 ns, the dynamic trajectories of the two systems tend to

21 be stable. The RMSD value of the BP reaction system is 1.0 Å, and the RMSD value

22 of the Cl-BP reaction system is 1.7 Å. We obtained two thousand snapshots from the

10
 1 MD simulations, and used cluster analysis tools to divide the two thousand of

2 conformations into several categories. Similar structures in the trajectories of

3 molecular dynamics were combined and TTClust was employed to cluster each group.

4 Hierarchical clustering method was implemented to group similar conformations and

5 backbone RMSD was used to obtain a distance matrix (Tubiana et al., 2018; Ono et

6 al., 2019). To understand the structural changes of the enzymes in the MD simulations,

7 the molecular dynamics of BP and Cl-BP were analyzed by TTClust. During the

8 dynamic simulation, the corresponding RMSD of the backbone for the two systems

9 are within 0.07Å (Fig. 1(a1) and Fig. 1(b1)). It indicates the two systems are stable

10 with only minor fluctuations. In the simulation process, the structure of

11 enzyme-substrate complex was maintained consistently. The dendrogram (Fig. 1(a2)

12 and Fig. 1(b2)) facilitate choosing an appropriate clusterization level (here adjusted to

13 yield 7 clusters of BP and 3 clusters of Cl-BP) and could describe the relationship

14 between the various conformations in each group. For example, in the BP system, the

15 RMSD of the other six groups of the structures are 4.63 Å, 4.62 Å, 5.00 Å, 5.02 Å,

16 4.96 Å and 5.17 Å compared to the first group of protein structures. Therefore, it is

17 considered that the active region and the substrate have a stable spatial structure.

18 Among the seven groups of structures (Fig. 1(a3)), Group 6 has the most significant

19 proportion – 604 structures. This indicates that Group 6 has relatively stable structures

20 in the BP system. Group 1 has only 99 configurations, indicating that the structures

21 are unstable during the binding of the protein to the substrate. Fig. 1(a4) shows the 2D

22 projection plot of the relative distances between clusters based on the RMSD between

11
 1 representative frames. The minimum and maximum distances are 0.36 and 0.40, and

2 the minimum and maximum spreads (average RMSD within clusters) are 0.39 Å and

3 0.52 Å. According to the results of cluster analysis, we could have a more reliable

4 basis on selecting the molecular structure to perform further calculations. Therefore,

5 one snapshot was selected from Group 6 to study the catalytic mechanism of biphenyl

6 2,3-dioxygenase.

8 3.2 The dioxygen-NDO complex

9 According to the side-on binding of dioxygen observed in the crystal structures of

10 naphthalene dioxygenase, the structure of the non-heme Fe-O2 complex contains one

11 iron(II) atom, one electron, one molecule of oxygen and one proton ([FeIII-OOH]2+).

12 Whether the hydroperoxo-iron(III) species can undergo cis-dihydroxylation in BphA

13 enzyme has not been studied in detail. Here, we proposed a mechanism for the

14 cis-dihydroxylation of the BP and the Cl-BP systems as shown in Scheme 3. Bassan

15 and co-workers proposed that the FeIII was attacked by hydroperoxo ligand in two

16 manners, end-on or side-on manner. However, the high-spin state (S = 5/2) has more

17 favorable energy than the low-spin state (S = 3/2) (Bassan et al., 2004).

18

19 In this study, we focus on the hydroperoxo ligand binding to the iron(III) in the

20 side-on manner, and the spin state is sextet-spin (S = 5/2). In the side-on binding mode,

21 the FeIII is six-coordinated by His233, His239, Asp388, and hydroperoxo ligand. The

22 FeIII iron center maintains five unpaired electrons in the d orbitals in sextet-spin. To

12
 1 understand the electronic structures more comprehensively, spin densities of crucial

2 atoms were calculated as displayed in Fig. 2. Furthermore, to gain insight into the

3 sextet-spin reactive state, the specific electron configuration is calculated as depicted

4 in Scheme 4, and the natural orbital analysis of the side-on binding mode is performed

5 as shown in Fig. 3 and Fig. S2. The spin populations of Fe atom and two oxygen

6 atoms in hydroperoxide were displayed in Table 1. For both complexes species in the

7 two systems, iron presents a typical spin density of ferric iron, about 4. For example,

8 for the BP system (Fig. 3), the calculated spin densities reflect that the iron 3d orbitals

9 could interact with the neighboring ligands and split into a set of three π* orbitals

10 (π*z2, π*x2- y2, π*xz) and a pair of two σ* orbitals (σ*yz, σ*xy). The lowest orbital is the

11 π*xz orbital representing the weak interaction of the iron dxz orbital with the orbitals of

12 surrounding ligands. The π*x2- y2 and π*z2 orbitals illustrate the antibonding interaction

13 between the 2p orbitals of hydroperoxide and the Fe(III) 3d orbitals. Similarly, the

14 antibonding interactions between the 2p orbitals of the cation intermediate and the

15 Fe(III) 3d orbitals are represented by the two σ* orbitals. In conclusion, the orbital

16 occupations are π*1xzπ*1x2-y2π*1xzσ*1xyσ*1yz for iron and π2uπ2g for hydroperoxide and

17 a single spin-parallel electron for BP. In addition, the carbon of the arene cation

18 intermediate should be sp2 hybrid carbon, which we can also reveal through the

19 natural orbital analysis.

20

21 3.3 Reaction Mechanism and Potential barriers

22 We conducted further calculations to delineate the catalytic mechanism of biphenyl

13
 1 2,3-dioxygenase. Herein, the chemical transformations leading to the cis-diols are

2 based on the experimental X-ray structure. It should be noted that the valence state of

3 FeIII iron before and after the reaction process remains unchanged, inferring that iron

4 is not involved in any redox reaction. The optimized key bond lengths of the involved

5 species are shown in Table 2. Starting from R, the cleavage of the O1-O2 bond of

6 hydroperoxide and the formation of the new C1-O2 and C2-O2 bonds were revealed as

7 a concerted step, which generates an epoxide intermediate. The distance between O1

8 and O2 increases from 1.44 Å in R1 to 2.97 Å in IM11 via 1.95 Å in TS11.

9 Simultaneously, the length of the C1-O2 bond decreases from 3.26 Å in R1 to 1.43 Å

10 in IM11 via 2.50 Å in TS11, and the length of the C2-O2 bond decreases from 3.28 Å

11 in R1 to 1.43 Å in IM11 via 2.30 Å in TS11 (Fig. 2(a)). The spin density computed on

12 the iron with the epoxide intermediate is 4.21, with the remaining spin density related

13 to the five unpaired d-electrons delocalized on the ligands. The distance of C2-O2

14 increases from 1.43 to 2.40 Å, which indicates the C2-O2 bond is broken. And a

15 carbocation intermediate (IM21) is formed in the second step. The third step is the

16 interaction between the cationic intermediates and the hydroxide. In the final product,

17 the distance of C2-O1 bond is 1.48 Å, and the newly formed product cis-diol is bound

18 to the metal.

19

20 We used Multiwfn 3.7 (Lu and Chen, 2012a) to analyze the wave function to elucidate

21 the molecular interaction and the characteristic of the carbocation group. The color

22 mapped isosurface graphs of electrostatic potential (ESP) were drawn by VMD 1.9.3

14
 1 program (Humphrey et al., 1996). ESP on the molecular vdW surface is important for

2 cognizing and predicting molecular interaction (Murray and Politzer, 2011; Manzetti

3 and Lu, 2013). A deep exploration of ESP allows identifying important interaction

4 between substrate and BphA. The ESP-mapped vdW surface of carbocation

5 intermediate of the BP system is displayed in Fig. 4(a). The surface area in different

6 ESP ranges is sketched as shown in Fig. 4(b). The majority of the vdW surface of BP

7 has moderate ESP value (i.e. within −30 to +30 kcal/mol) which mainly pertains to

8 the biphenyl. The ESP value over the biphenyl carbons is low negative (i.e. < −18

9 kcal/mol), indicating that the rich π-cloud of the carbons. In addition, a small segment

10 of the vdW surface has a significant negative value of ESP (i.e. < −30 kcal/mol),

11 which is attributed to the surface close to the oxygens of the carboxyl group. The

12 value of ESP around the arene cation is −17.32 kcal/mol, which is a direct

13 consequence of the unpaired electrons of the cation intermediate. Methyl group and

14 hydrogens are the main factors leading to the proportional surface area with positive

15 ESP values. The global minima and maxima of ESP on the surface are −75.50 and

16 +70.53 kcal/mol, corresponding to the hydrogen and the oxygen in the carboxyl group,

17 respectively.

18

19 The potential energy profiles of the two systems are displayed in Fig. 5. The first step

20 of epoxide formation is revealed to be the rate-determining process, and the energy

21 barriers of the BP and the Cl-BP systems are 17.6 kcal/mol and 19.8 kcal/mol,

22 respectively. The experimentally measured free energy barrier (using biphenyl as the

15
 1 substrate) is 17.7 kcal/mol (Gómez-Gil et al., 2007), which agrees well with the

2 computational energy barrier. The following step is the formation of carbocation

3 species with the energy barriers of −6.8 kcal/mol and −4.6 kcal/mol for the BP and the

4 Cl-BP systems, respectively. In the final step, bonding of the hydroxo ligand to the

5 carbocation intermediate to produce the diol occurs with low energy barriers of −23.1

6 kcal/mol and −20.7 kcal/mol for the BP and the Cl-BP systems, respectively. For the

7 Cl-BP system, the calculated barrier of each step is slightly higher than that of the BP

8 system. The energy calculation results imply that compared with the Cl-BP system,

9 the BP system has more advantages in energy and more likely to occur. The trend of

10 the calculated potential barriers of the two systems is consistent with the Bassan’s

11 work (Bassan et al., 2004).

12

13 3.4 Individual Residue Influence

14 To explore the electrostatic influences on epoxide formation, we calculated the

15 electrostatic interaction energies of thirteen residues according to previous

16 experimental crystal structure study (Dhindwal et al., 2016) and further manually

17 inspection. In addition, the calculation of the electrostatic residue analysis could

18 further explain the significance of designing new engineer BphAE. Therefore, we

19 chose to investigate the seven unique residues (Gly335, Ile336, Asn337, Thr 338,

20 Ile339, Arg340, and Thr341) in BphAEII9 that are different in the BphAELB400. The

21 remaining six residues (Phe227, Asp230, Val287, His233, Leu333, and Phe378) are

22 within 5 Å of the reaction site and are considered important sites that have a

16
 1 significant effect on the reaction. The electrostatic influence of an amino acid i

2 surrounding the reactive center can be represented as follows :

3
∆Ei−0 = ∆Ei −∆E0

4 Where, ΔEi-0 is the changes of potential energy, ∆Ei is the potential energy with

5 charges on residue i set to 0, and ∆E0 is the original value of the potential barrier. In

6 the process of calculations, the geometric structures of the stationary points remained

7 unchanged. A negative ∆Ei-0 value indicates that the ith residue can increase the

8 potential barrier and suppress the enzyme reaction. In contrast, a positive ∆Ei-0 value

9 means that the ith residue can decrease the potential barrier and promote the enzyme

10 reaction (Zhu et al., 2018a). The ∆Ei-0 values of thirteen residues on the BP and the

11 Cl-BP systems are displayed in Fig. 6(a) and the structures of each residue are shown

12 in Fig. 6(b).

13

14 For the BP system, residue Arg340 significantly suppresses the epoxide formation

15 process by increasing the reaction barrier by about 4.1  kcal/mol. In addition, residues

16 Asp230, Gly335, Asn337, Thr338, and Ile339 facilitate the process through

17 decreasing the reaction barrier by about 2.4 kcal/mol, 2.3 kcal/mol, 3.2 kcal/mol, 1.6

18 kcal/mol and 3.9 kcal/mol, respectively. The remaining seven residues exhibit weaker

19 contribution (−1 kcal/mol ∆Ei-0 1 kcal/mol) on the BP system. Phe227 and His323

20 are located near the binding site of the reaction, reducing the active pocket of the

21 enzyme reaction. The binding reaction of the hydroperoxo-iron(III) ligand with the

22 substrate is suppressed. Among them, there is a hydrogen bonding between His323

17
 1 and the substrate, and there is an edge-to-face π-π interaction between Phe227 and the

2 substrate. The significant hydrogen bond between Arg340 and Phe227 makes Phe227

3 more stable, which indirectly hinders the hydroperoxo-iron(III) ligand to the substrate.

4 Asp230 is located around the distal ring of the active site. The hydrogen bond

5 between Asp230 and the substrate helps stabilize the substrate and makes the reaction

6 easier to occur. Asn338 in BphAELB400 is replaced with Thr338 in BphAEII9. Due to

7 the different side-chain lengths of Asn and Thr, the side chains of Arg340 are located

8 at different positions of BphAELB400 and BphAEII9. The weakening of a significant

9 hydrogen bond between Arg340 and Phe378 affected the spatial position of Phe378 in

10 BphAEII9. The Phe378 is close to the reaction active site; its position affects the

11 docking of the substrate with the hydroperoxo-iron(III) ligand and the interaction

12 between them. Similarly, the Gly335, Asn337, and Ile339, located around the distal

13 ring, affect the reaction through interactions with amino acids near the reaction active

14 site.

15

16 For the Cl-BP system, Phe227, Ile336, and Arg340 suppress the epoxide formation

17 process through increasing the reaction barrier by about 1.3 kcal/mol, 2.7  kcal/mol,

18 and 3.1 kcal/mol, respectively. Asn337, Ile339, and Phe378 promote the process by

19 decreasing the reaction barrier by about 2.3 kcal/mol, 2.5 kcal/mol, and 1.1 kcal/mol,

20 respectively. The other seven residues exhibit weaker contribution (−1 kcal/mol ∆Ei-0

21 1 kcal/mol) on the Cl-BP system. Due to the enhanced hydrogen bond between the

22 chlorine atom at the distal ring and the hydrogen atom in Ile336, the hydrogen bond in

18
 1 the reactant is stronger than the transition state. Therefore, compared with the BP

2 system, the inhibition effect of Ile336 on the Cl-BP system is more obvious. Six

3 residues are highlighted, for both the BP system (Asp230, Gly335, Asn337, Thr338,

4 Ile339, and Arg340) and the Cl-BP system (Phe227, Ile336, Asn337, Ile339, Phe378,

5 and Arg340). The results of electrostatic influence analysis can provide candidates for

6 future mutation chemistry studies.

8 3.5 Environmental Implications

9 The development of methods for analyzing and predicting the biotransformation

10 processes of POPs have significantly promoted the understanding of their

11 environmental fates and impacts on human health (Katsoyiannis and Samara, 2005;

12 Zhang et al., 2016). In this study, we performed computational calculations to reveal

13 the crucial information of the cis-dihydroxylation mechanisms of biphenyl and PCBs

14 that are hardly accessed by experimental studies. The computed energetics and

15 electrostatic interaction energies can provide guidelines for the development of

16 eco-friendly and low energy consumption biodegradation techniques. In particular, the

17 calculation results in this study provide candidates for future directed mutations and

18 enzymatic engineering of BphA.

19

20 4. Conclusions

21 To elucidate the catalytic mechanism of biphenyl 2,3-dioxygenase, we implemented

22 QM/MM method to investigate the chemical transformations leading to the cis-diols.

19
 1 The dioxygenation process of the two substrates consists of three elementary reactions.

2 The rate-determining step is epoxide formation, in which the cleavage of the O1-O2

3 bond of hydroperoxide and the formation of the new C1-O2 and C2-O2 bonds were

4 considered to be a concerted step. The distance of C2-O2 bond increases gradually and

5 then brokes to form the carbocation intermediate in the second step. The final step is

6 the interaction between the hydroxo ligand and the carbocation, which generates the

7 cis-diol product with a shallow barrier. During the process, the hydroperoxo-iron(III)

8 species is considered to be sextet-spin (S=5/2). The calculation highlighted the

9 important roles of six residues for both the BP system (Asp230, Gly335, Asn337,

10 Thr338, Ile339, and Arg340) and the Cl-BP system (Phe227, Ile336, Asn337, Ile339,

11 Phe378, and Arg340) in the dioxygenation process. This study may provide

12 theoretical support for further directed mutations and enzymatic engineering of BphA.

13 Furthermore, it is promising for future research to combine computational

14 calculations with experimental study to achieve degrading pollutants in a more

15 effective and eco-friendly manner.

16

17 Acknowledgments

18 The work was financially supported by (National Natural Science Foundation of

19 China, project Nos. 21577082) and Taishan Scholars (No. ts201712003).

20 Declarations of interest: none

21

22 Supporting Information Available

20
1 Root-mean-square deviations (RMSD) of the backbone for molecular dynamic

2 simulation involved in the BP system and the Cl-BP system (Fig. S1). The valence

3 electron orbital diagrams of R1, IM11, and P1 in the BP system, R2, IM12, IM22 and P2

4 in the Cl-BP system (Fig. S2).

21
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 1 Atmospheric Environment 173, 38-45.
2
3
4
5
6
7
8
9
10
11

26
 1 Table 1a. Spin distributions for [FeIII-OOH]2+ in biphenyl 2,3-dioxygenase (in the BP
2 system).
Spin density
species Fe O1 O2
1
R 4.18 0.37 0.06
TS11 3.95 0.65 0.52
IM11 4.21 0.03 0.41
TS21 4.23 0.41 0.45
IM21 4.01 0.22 0.38
TS31 4.19 0.46 0.38
P1 4.18 0.37 0.06
3
4
5
6
7
8

9 Table 1b. Spin distributions for [FeIII-OOH]2+ in biphenyl 2,3-dioxygenase (in the

10 Cl-BP system).
Spin density
species Fe O1 O2
2
R 4.01 0.36 0.09
TS12 4.02 0.35 0.39
IM12 4.03 0.04 0.43
TS22 3.95 0.65 0.49
IM22 4.03 0.35 0.45
TS32 4.03 0.43 0.21
P2 3.91 0.45 0.05
11
12
13
14
15
16
17
18
19
20
21
22
23
24
27
 1 Table 2. Selected key bond distances (in Å) in the reactants, transition states,
2 intermediates, and products involved in the BP and the Cl-BP systems during the
3 whole reactions at the B3LYP-D3/6-31G(d,p)/ LANL2DZ //CHARMM36 level.
d(Fe-O1) d(Fe-O2) d(O2-O1) d(O1-C2) d(O2-C1) d(O2-C2)
1
R 2.30 1.88 1.44 3.33 3.26 3.28
TS11 1.99 1.90 1.95 3.38 2.50 2.30
IM11 1.84 3.15 2.97 3.52 1.43 1.43
TS21 1.82 2.33 2.64 3.44 1.42 2.07
IM21 1.85 1.90 2.71 3.38 1.44 2.40
TS31 1.90 1.95 2.34 2.90 1.50 2.44
P1 2.81 2.02 2.24 1.48 1.43 2.35
R2 2.26 1.87 1.46 3.03 3.06 2.99
TS12 1.90 1.81 2.37 3.05 2.38 2.76
IM12 1.79 4.00 2.83 3.73 1.43 1.45
TS22 1.75 1.93 2.34 3.50 1.72 2.72
IM22 1.85 2.03 2.42 3.39 1.43 2.38
TS32 1.87 2.29 2.07 2.84 1.44 2.40
P2 3.01 1.99 2.17 1.49 1.43 2.26
4
5
6
7
8
9
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
25
26
27
28
29

28
 1 Figure Captions

2 Scheme 1. Pathway of aerobic PCB metabolism by biphenyl-oxidizing bacteria,

3 including BphA, BphB, BphC, and BphD enzymes.

4 Scheme 2. Catalytic cycle of biphenyl 2,3-dioxygenase, where a hydroperoxo-iron(III)

5 species is invoked as intermediate.

6 Scheme 3. Proposed mechanisms for the cis-dihydroxylation of the BP and the Cl-BP

7 systems with biphenyl 2,3-dioxygenase.

8 Scheme 4. Valence Electron Configurations for the hydroperoxo-iron(III) complex at

9 the Quintet, and the Sextet States.

10 Figure 1. TTClust was applied to the analysis of molecular dynamics simulations

11 (20ns) of the two systems. The distance matrix saved as a heatmap to allow a quick

12 overview of the number of possible clusters of the BP system (a1) and the Cl-BP

13 system (b1). Clustering dendrogram of seven clusters of the BP system (a2) and three

14 clusters of the Cl-BP system (b2). Clustering of the BP system (a3) and the Cl-BP

15 system (b3) along the trajectory with a timeline (top) and a distribution of clusters

16 (bottom). 2D projection plot of the relative distances between clusters based on the

17 RMSD between representative frames of the BP system (a4) and the Cl-BP system

18 (b4). The minimum and maximum distances are indicated, as well as the minimum

19 and maximum spreads (average RMSD within clusters).

29
 1 Figure 2. The three-dimensional structures of R, TS1, IM1, TS2, IM2, TS3, and P

2 involved in the dihydroxylation process of the (a) BP system and the (b) Cl-BP

3 system. Relevant spin populations, relative energies and bond lengths (Å) are shown.

4 Figure 3. Valence electron orbital diagrams of the Biphenyl-hydroperoxo-iron(III)

5 complex at the lowest-lying sextet spin-state surface of IM21.

6 Figure 4. (a) ESP mapped molecular vdW surface of the BP system. The unit is in

7 kcal/mol. Significant surface local minima and maxima of ESP are represented as

8 orange and cyan spheres and labeled by blue and red texts, respectively. (b) The

9 surface area in each ESP range on the vdW surface of the BP system.

10 Figure 5. Energy diagram of the concerted mechanism leading to the cis-diol in

11 biphenyl 2,3-dioxygenase of the (a) BP system and the (b) Cl-BP system.

12 Figure 6. (a) ∆Ei-0 values of 13 individual residues toward the rate-determining step

13 involved in the dihydroxylation process of the BP system and the Cl-BP system. (b)

14 The structures of each residue toward the rate-determining step involved in the BP

15 system and the Cl-BP system.

16

17

18

19

20

30
 1

3 Scheme 1
4
5
6
7
8
9

10
11

12 Scheme 2
13
14
15
16
17
18
19
20
21

31
1
2

3 Scheme 3

6 Scheme 4

32
1
2
3 Figure 1.
33
1
2
3 Figure 2(a).
4

34
1
2
3 Figure 2(b).
4

35
1
2
3 Figure 3.
4
5

36
 1
2
3 Figure 4(a).
4
5
6
7
8

9
10 Figure 4(b).
37
 1

2
3
4 Figure 5(a).
5
6
7
8
9
10

11
12
13 Figure 5(b).
14

38
1
2
3 Figure 6(a).
4
5

6
7
8 Figure 6(b).
39
The research highlights in this paper:

1. The degradation mechanism of biphenyl and PCBs were revealed by MD and

QM/MM methods.

2. A hydroperoxo-iron(III) species is involved in the enzyme-catalyzed reaction.

3. Electrostatic influence analysis can provide targets for future mutation studies.
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: