Mechanism of Sulfoxidation and CâS Bond Formation Involved in the

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Mechanism of sulfoxidation and C-S bond formation involved in the biosynthesis of ergothioneine catalyzed by Ergothioneine synthase (EgtB) Ge Tian, Hao Su, and Yongjun Liu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b01473 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Mechanism of sulfoxidation and C-S bond formation involved in the biosynthesis of ergothioneine catalyzed by Ergothioneine synthase (EgtB)

Ge Tian, Hao Su, Yongjun Liu* Key Lab of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China (*Corresponding Author: Tel.: +86 53188365576; fax: +86 53188564464. Email address: [email protected])

Abstract: Ergothioneine synthase (EgtB) is a unique non-heme mononuclear iron enzyme that catalyzes the sulfoxidation and C-S bond formation between γ-glutamyl cysteine (γGC) and N-αtrimethyl histidine (TMH) as a pivotal step in the ergothioneine biosynthesis. A controversy has arisen regarding the sequence of sulfoxidation and C-S bond formation in the catalytic cycle. To clarify this issue, the QM/MM approach has been employed to investigate the detailed mechanism of EgtB. Two binding modes of O2 to Fe(II) (“end-on” and “side-on”) have been identified. Within the present computational model, the “end-on” binding mode of O2 is preferred. The open-shell singlet is calculated to be the ground state, whereas the quintet is the most active state. Moreover, the sulfoxidation is prior to the formation C-S bond, and the reaction mainly occurs on the quintet state surface. Due to the electron transfer from the γGC to the ferric superoxide, the sulfur atom of γGC has partial radical characteristic, which facilitates the attack of the distal oxygen atom on the sulfur radical of γGC to form the sulfoxide. The formation of TMH C2 anion, i.e., the abstraction of the proton from imidazole group in TMH by the Fe(IV)-oxo species is the prerequisite for C-S bond formation, which is the rate-limiting step with an energy barrier of 21.7 kcal/mol. Besides, it is also found that although the resulted iron(III)-oxo can easily abstract a proton from Tyr377 to generate a phenolic hydroxyl anion, the subsequent proton transfer from C2 to Tyr377 is calculated to be difficult, thus, Tyr377 is not directly involved in the sulfoxidation and C-S bond formation. Our calculations also reveal that the “side-on” mode is not the catalytic relevant species. This work provides a direct comparison with the previous experimental and theoretical studies, which is helpful for understanding the catalysis of ergothioneine synthase and related enzymes. Keywords: QM/MM; EgtB; nonheme dioxygenase; sulfoxidation; γGC; TMH; ergothioneine 1

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1. Introduction Ergothioneine is a kind of natural thiol-imidazole containing histidine derivative,1 which has a high reduction potential (E0=-0.06 V)2 predominantly because of its thione form, and thereby it is considered as a sort of protectant against oxidative stress in cellular redox homeostasis. Ergothioneine cannot be synthesized in the human body, and it is only absorbed from the dietary sources and enriched in specific human tissues and cells. Since ergothioneine is essential for human body, there has been a longstanding interest in developing the biosynthetic pathway of ergothioneine since the 1960s.3,4 In 2010, Seebeck et al.5 discovered the biosynthetic gene cluster of ergothioneine from Mycobacterium smegmatis, and assigned the five different Egt enyzmes, including EgtA, EgtB6, EgtC7, EgtD and EgtE, which provides a new sulfur transfer strategy for the biosynthesis of sulfur-containing natural products. Among these five Egt enzymes, the sulfoxide synthase EgtB plays a pivotal role in ergothioneine biosynthesis.6 As shown in Scheme 1, EgtB catalyzes the sulfoxidation and C-S bond formation between γ-glutamyl cysteine (γGC) and N-α-trimethyl histidine (TMH) with addition of one oxygen from the molecular dioxygen to generate 2-histidyl-cysteine sulfoxide. EgtB belongs to mononuclear non-heme iron-containing enzymes which activate dioxygen as part of their catalytic mechanism in an array of key biochemical transformations.8-10 Most of these enzymes are characterized by utilization of high-spin Fe(II) active sites and O2 to yield the ferric-superoxide species.

Scheme 1. Overall reaction catalyzed by EgtB. Ovothiol synthase (OvoA) is another sulfoxide synthase, which catalyzes the oxidative insertion of a sulfur atom into the C5-position instead of C2-position on the histidine side chain to generate 5histidyl-cysteine sulfoxide.11 Both OvoA and EgtB are mononuclear non-heme iron enzymes that catalyze the formation of sulfoxide, and follows similar overall processes. However, EgtB and OvoA distinguish themselves by their substrate preference and product C-S bond regioselectivity. OvoA is very selective towards its sulfur donor substrate and only accepts L-cysteine, whereas EgtB requires γ-

2


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glutamyl cysteine as the sulfur donor. Biochemically, the preference for C5 versus C2 oxidation is the main difference between OvoA and EgtB.12, 13

Scheme 2. Two previously proposed mechanisms, A and B. In the previous literatures,11-16 two typical mechanistic models have been proposed to account for the oxidative C-S bond formation in OvoA or EgtB catalysis. In mechanism A11, the sulfoxidation occurs first, whereas in mechanism B the formation of C-S bond is prior to the sulfoxidation13, as shown in Scheme 2. For example, in the OvoA model proposed by Braunshausen et al.11, the Fe(III)-O2•− species firstly attacks the cysteine to generate the sulfoxide by homolytically cleaving the O-O bond, then the sulfurization of histidine occurs in three possible pathways in which the formation of C-S bond may be initiated by a histidyl sp2 radical or histidyl π-radical, or by the direct attack of the cysteine sulfoxide on the histidine (Scheme 2A). In 2012, Bushnell et al. theoretically investigated the ability of different iron-oxygen species to illuminate the catalysis of OvoA and EgtB using cluster model.16 They found that the four-membered ring Fe-O-O-S complex is powerful enough to oxidize the imidazole of histidine to generate a histidyl-derived radical via proton-coupled electron transfer (PCET), and the formation of HisNδ(-H)• radical is preferred to that of HisCδ(−H)• or HisCε(-H)• radical.16 In 2013 Seebeck and his co-worker further performed a series of experiments using different substrates.13 However, the kinetic isotope effect implied that the formation of sp2 radical and imidazyl radical cation seems to be difficult, and the authors suggested another mechanism in which the C-S bond is firstly formed by the attack of thiyl radical on the imidazole ring, followed by rearomatisation and sulfoxidation, as displayed in Scheme 2B.13 In 2015, Goncharenko et al.6 resolved the crystal structure of EgtB and proposed the catalytic 3


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mechanism of EgtB. Specifically, the FeOO•- anion abstracts the proton from the phenolic hydroxyl of Tyr377, meanwhile, the thiyl radical attacks the imidazole ring to form an iminyl radical, which then rearomatizes via a deprotonation process and electron transfer from ligand to iron; finally, the thioether is sulfoxidized by Fe-OOH. In a later experimental study by the same group,17 it was found that the mutation of Tyr377Phe decreases the catalytic activity by 103-fold, and the residue Tyr377 is not a good candidate for mediating the removal of hydrogen/proton from TMH, through KIE (kinetic isotope effect) analysis. In general, the catalytic mechanism of EgtB is still unclear in particular the reaction details at the atomistic level. Steaming from the high resolution crystal structure of EgtB, we started our research work on the catalytic mechanism of EgtB. However, in the process of preparing this article, Liao’ group18 and de Visser’s et al.19 published their works regarding the mechanism of EgtB using QM method and QM/MM approach, respectively. Liao et al. suggested that EgtB is similar to CDO (cysteine dioxygenase) enzyme in the sulfoxidation step, and the sulfoxidation is prior to the C-S bond formation. In particular, the formation of imidazole anion is the prerequisite for C-S bond formation. Nevertheless, de Visser et al. proposed that the Fe(III)-superoxo species firstly reacts via PCET with Tyr377 to create an iron(III)-hydroperoxo intermediate, which prevents the possible thiolate dioxygenation side reaction; then the histidine attacks the S radical to form the C-S bond, and the sulfoxidation is the final step of the catalysis. We noted that de Visser et al. did not address the sulfoxidation step, and the different reactivity of two binding modes of dioxygen (“side-on” and “end-on”) was not compared also. In general, some controversies can be found in these previous studies, as summarized in the following: 1) the sequence of sulfoxidation and C-S bond formation. Liao et al. ruled out the possibility of the formation of C-S bond by the radical attack of γGC on the imidazole ring, whereas de Visser et al. proposed that this step only corresponds to an energy barrier of 14.2 kcal/mol on the quintet surface; 2) the role of Tyr377. de Visser et al. suggested that the residue Tyr377 donates a hydrogen to Fe(III)superoxo in the first step, while Liao et al. proposed that Tyr377 mediates the intramolecular proton transfer of histidine in TMH; 3) the rate-determining step. de Visser et al. revealed that the nucleophilic C-S bond formation is the rate-determining step, but according the results of Liao et al., the hydrogenabstraction step is rate-limiting. Besides, we are also interested in the electronic structures of the reactant iron center, including the radical characteristics and spin properties of key atoms or group, 4


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such as sulfur and Fe-O-O, and the driving force for forming C-S and S=O bonds, as well as why the two research groups gave the different conclusions. In the present work, combined quantum mechanics and molecular mechanics method (QM/MM) has been employed to explore these issues. In recent years, QM/MM approach has been widely and successfully applied in exploring the mechanism of iron-containing enzymes,20 such as α-ketoglutarate (α-KG)-dependent superfamily of enzymes21-23 and dioxygenases24. On the basis of our calculations, the binding modes of dioxygen, the electronic characteristics and geometrical structures of transition states and intermediates, as well as the most possible reaction pathways were illuminated.

2. Computational Methods 2.1 Setup of the System The initial structure of the enzyme was constructed based on the recently reported X-ray structure from Mycobacterium thermoresistibile (PDB code 4X8D, resolution of 1.98 Å).6 In this structure, there are two same domains, which have the identical active sites and binding conformation of substrates (Figure S1b and S1c). Hence, only one chain was employed to construct the reactant models. Figure S2 shows the active site structure from the crystal. In our model, the manganese was changed to iron and the water molecule that initially coordinates with iron was replaced by O2. Three imidazole groups (His51, His134 and His138) coordinate with iron and form a 3-His facial triad, which is a typical feature for many non-heme iron dioxygenases.25-27 The two substrates, γ-glutamyl cysteine (γGC) and N-α-trimethyl histidine (TMH) bind to iron through the S and N atoms. The protonation states of all titratable residues were assigned based on the experimental condition and pKa values calculated by the PROPKA procedure28, and were verified by VMD program29. All glutamate and aspartate residues are deprotonated, whereas all histidine residues are singly protonated at the Nδ atoms. Besides, all arginines and lysines are positively charged. The missing hydrogen atoms were added by the HBUILD module embedded in CHARMM.30 The whole system was solvated with a 36 Å layer of TIP3P type water.31 After stochastically adding nine Na+, the model reached neutralized. The whole system contains 20654 atoms, including 4669 TIP3 water molecules, as shown in Figure S3 (a).

2.2. MD simulations To obtain an equilibrated solvent environment, the systems were subjected to a series of energy 5


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minimizations before the molecular dynamics (MD) simulations. Then, the whole system was subjected to 30 ns MD simulations with stochastic boundary conditions using the CHARMM22/CMAP all-atom force field32. In the MD simulations, the inner active region (within 27 Å of the system) was treated by Newtonion dynamics, and the buffer region (from 27 Å to 31 Å) was treated by Langevin dynamics. As the available force field parameters for iron is not very accurate, to prevent the collapse of the coordinate residues, some researchers often keep the Fe(II)-O2 unit and the ligated residues frozen.33,34 In this work, we adopt the same strategy, i.e., the Fe(II) and its coordinated ligands were kept frozen during the MD simulations. The calculated root-mean-square deviations (RMSDs) of the backbones of the protein are shown in Figure S3(b), which indicate that the system has basically equilibrated after 15 ns simulations. To choose a representative structure for the QM/MM calculations, 14 snapshots were extracted from the trajectories from 17 to 30 ns at an interval of 1 ns, and were optimized by QM/MM method. The superposition of these 14 optimized structures is displayed in Figure S3(c). The RMSDs of all structures were calculated relative to the average one, which ranges from 0.369 to 0.606Å. Since the RMSD of the structure derived from 20 ns corresponds to the smallest one, we supposed this structure as representative.

2.3. QM/MM calculations The whole system was divided into the QM region and MM region. Fe(II), O2, the two substrates TMH and γGC, three ligands in the first-coordination sphere (His51, His134, His138), one water molecule and Tyr377 were selected as the QM region, as depicted in Figure 1. The remaining atoms of the solvated system were assigned to the MM region. During the QM/MM calculations, the region within a distance of 16 Å from Fe center was set as an active region, which includes the QM region and part of MM region.

6


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Figure 1. QM region for QM/MM calculation. Our QM/MM calculations were based on a well-tested procedure that has been proven to be reliable for iron-containing enzymes.20 In this work, the QM/MM calculations were performed by ChemShell35, which combines Turbomole36 for the QM calculation and DL_POLY module37 for the MM calculation. The electronic embedding scheme38 was employed to describe the polarizing effect of the enzyme environment on the QM region. Hydrogen link atoms were applied to treat the QM/MM boundary using the charge-shift model.39 QM/MM optimizations were carried out using the hybrid delocalized internal coordinate (HDLC) optimizer40. To define a contiguous energy profile of the reaction, the potential energy surface (PES) was firstly scanned along the reaction coordinate, and the highest point was chosen as the initial guess for the transition state (TS) geometry, which was further optimized by the partitioned rational function optimization (P-RFO)41 implemented in HDLC code. The last structure in the scanned PES was used to find the next intermediate. All the minima were optimized by using the quasi-Newton limited memory Broyden–Fletcher-Goldfarb-Shanno (L-BFGS) algorithm42,43. In the QM/MM calculations, the QM region was treated by UB3LYP functional in conjugation with the Wachters+f basis set for iron and 6-31G(d,p) basis set (B1 level) for all other atoms. The MM region was described by the CHARMM22/CMAP force field. Considering the spin-state ordering and relative energies of the reactant may depend on the choice of the functionals, we also used other functionals to optimize the reactant, including BP86, BLYP, BVWN and TPSSh. The single-point energy calculations were performed using the larger basis set of 6-311++G(2d,2p), simplified as B2 level, and frequencies were calculated at B1 level to obtain the entropy effect and the Gibbs free energy profiles. In general, the contributions from the entropy effect are quite small (less than 2.7 kcal/mol), as indicated in Table 7


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S1. Given the absence of accurate description of the long-range dispersion interaction in the B3LYP functional, DFT-D3 corrections were carried out. As listed in Table S1, the dispersion effects only make a small change of the relative energies (less than 4.8 kcal/mol). Unless noted otherwise, the energies in the context are corrected by DFT-D3. Furthermore, the bonding nature of the QM region was analyzed by natural bond orbital (NBO) analysis, as implemented in Gaussian 0344. The NPA (natural population atomic) charges were obtained by Turbomole.

3. Results and Discussion 3.1 Binding of dioxygen to iron As the crystal structure lacks O2, the dioxygen was manually added into the active site. There are two possible binding modes for the O2 to Fe, the “side-on” and “end-on” modes. Hence, we have considered different initial binding conformations of O2. In the “end-on” mode, we initially set the dihedrals of D(S-Fe-O1-O2) to different valves, ranging from -180 to 180° at an interval of 20°. After optimization, these structures converged to two typical binding modes of dioxygen, namely as I and II (Figure S4). Since mode I corresponds to an inactive confirmation (the distal oxygen atom O2 is far from the substrate γGC), we only used mode II as the “end-on” reactant model. For the “side-on” mode, different initial binding conformations of dioxygen were also attempted, in which the dihedrals of D(SFe-O1-O2) range from -180 to 180°. When the dihedral was set to the range from -80° to 60°, the optimized structures converged to the “side-on” mode (II), as shown in Figure S5. In the other cases, the optimized structures converged to the “end-on” mode. We noted that the “side-on” mode was not identified by Liao18 and de Visser19. To test the influence of different functionals on the relative stability of the reactant models at different spin states, a series of calculations were performed with BP86, BLYP, B3LYP, BVWN and TPSSh at B1 level. The energy orderings of different spin states are almost consistent (Figure S6). Thus, the widely used density functional B3LYP was employed in the following QM/MM calculations.

3.1.1 Geometries of substrate-Fe-O2 complex In our constructed models, the Fe(II) is coordinated by a 3-His facial triad with a distance about 2.10Å. Although many mononuclear non-heme iron dioxygenases45 feature a 2-His-1-carboxylate 8


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structural motif, there are also some dioxygenases having a 3-His facial triad, such as cysteine dioxygenase46 and salicylate 1,2-dioxygenase47. In addition, de Visser et al. previously suggested that the 3-His structural motif in cysteine dioxygenase is essential for its catalytic activity.48 Due to the open-shell character of the iron and dioxygen, the coupling of the triplet O2 with the Fe(II) may give several possible spin states (septet, quintet, triplet, etc). Without involving the DFT-D3 correction, the septet is calculated to be the ground state, which is in agreement with the calculation results by Liao et al.18, and the open-shell singlet, triplet and quintet are 2.0, 3.4 and 9.0 kcal/mol higher than the septet, respectively. However, taken the DFT-D3 correction into consideration, the open-shell singlet in the “end-on” binding mode is calculated to be the ground state. The change of the spin state ordering is understandable, because the energy gap between the septet and singlet is so small. The optimized structures at different spin states are presented in Figure 2. One can see that both the “side-on” and “end-on” binding modes can be recognized in the ergothioneine synthase, which have also been found in other dioxygenases.24,49

Figure 2. Optimized structures of the reactant models at different spin states: (a) mono-coordinated dioxygen to iron in “end-on” binding mode. Black, purple and red represent the open-shell singlet, triplet and quintet, respectively; (b) di-coordinated dioxygen to iron in “side-on” binding mode. Blue and orange represent the septet and quintet, respectively. The energies (kcal/mol) are calculated at the B3LYP/B2 level, and that of the open-shell singlet is set to zero. All distances are given in Ångstrom (r1=Fe-O1, r2=Fe-O2, r3=O1-O2 and r4=Fe-S). The spin densities (denoted by ρ) of some key atoms are shown in the bottom right. The data in other figures use the same units. End-on mode In the “end-on” binding mode, O2 coordinates with Fe(II) through a single oxygen atom, and only the singlet, triplet and quintet are recognized. The triplet and quintet are 1.0 and 9.4 9


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kcal/mol higher than the open-shell singlet. The spin state ordering coincides with the QM/MM studies of cysteine dioxygenase, and the open-shell singlet is also calculated to be the ground state.50 In addition, the closed-shell singlet is much higher (27.3 kcal/mol) than the open-shell singlet, and it was excluded from our consideration. Unless otherwise noted, the open-shell singlet is simplified as “singlet” in the following context. In the singlet and triplet states (1R and 3R), the proximal oxygen atom (O1) binds tightly to the iron with coordinate distances about 1.90 Å, whereas the distances of distal oxygen (O2) from the iron are about 2.80 Å, as depicted in Figure 2(a). In the quintet state (5R), the distances of Fe-O1 and Fe-O2 are slightly longer than those of the singlet and triplet states, indicating a relative weak coordination of dioxygen in the quintet state. The optimized structural parameters are similar to those in the SDO (salicylate 1,2-dioxygenase) enzyme24b and CDO enzyme50,51. In general, owing to the coordination of proximal oxygen, the O1-O2 bond is greatly weakened. Side-on mode In the “side-on” binding mode, the dioxygen coordinates with Fe(II) by two oxygen atoms. It should be noted that, when we tried to optimize the “end-on” mode at septet spin state, the binding mode always converge to the “side-on” pattern. The septet is only 2.2 kcal/mol higher than the open-shell singlet of the “end-on” binding mode, whereas the quintet state is 11.8 kcal/mol higher than the open-shell singlet. It is noteworthy that the “side-on” mode was not identified in cysteine dioxygenase.50 In the septet and quintet states, the Fe-O1 bond lengths are 2.16 and 2.11 Å, both of them are slightly shorter than the length of Fe-O2 (2.22 Å), as shown in Figure 2(b). The O1-O2 bond lengths are also calculated to be longer than that of the free O2. The angles of ∠Fe-O1-O2 are 77.1 and 75.4° for septet and quintet, respectively. Some other structural parameters are displayed in Table S2. 3.1.2 Electronic structures of substrate-Fe-O2 complex End-on mode The binding of O2 to Fe(II) may couple with the electron transfer between the iron center and O2 or substrate. To understand the electronic structures, spin densities of key atoms and groups (ρ) were calculated. In the optimized singlet and triplet states, spin densities of the ligated dioxygen are -1.05 and 0.95, respectively, which indicate that one unit electron has been transferred from Fe(II) to the dioxygen. As can be seen in Figure 3, the iron center possesses one single unpaired electron in the d-type molecular orbitals (MOs), which is antiferromagnetically (in singlet) or ferromagnetically (in triplet) coupled to the unpaired electron of the dioxygen. The valence electron 10


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orbital surfaces of 1R and 3R are displayed in Figure S7.

Figure 3. Valence electron configurations of γGC-Fe-O2 complex at different spin states. Although the singlet and triplet models correspond to the lower relative energies, the quintet state reactant is calculated to be more energetically favorable for the first step, which will be described in section 3.3. In the related iron-containing enzyme AlkB49c, the reactant in quintet has also been found as the most reactive state. As such, we focus on the natural orbital configuration of quintet state here. In the quintet state, the spin density of dioxygen is -0.36, whereas it is 0.19 for γGC, which indicate that the electron transfer has occurred from γGC to the dioxygen through the Fe(II) center, affording a γGC•-FeII-O2•− radical. Similar cases can be found in homoprotocatechuate 2,3-dioxygenase (HPCD) 49b and homogentisate dioxygenase (HGDO)52. In the homoprotocatechuate 2,3-dioxygenase (HPCD), the spin contribution from the substrate is mainly localized on the ligated oxygen atoms of the substrate due to the covalent bonding between iron and the substrate.49b In the related dioxygenase AlkB49c, the dioxygen binding was also suggested to associate with a charge transfer, which leads to the formation of the Fe(III)-O2•- species that act as a strong nucleophile in the decarboxylation of the pyruvate. In the case of homogentisate dioxygenase (HGDO), the Fe-O2 adduct with the bidentately bound substrate (2AP) has the Fe(III)-superoxo character, while the Fe-O2 complex with the monodentately ligand facilitates the electron transfer from the substrate to the iron center, affording a substrate radical-Fe(II)superoxide.52 In our model, the small spin density of γGC (0.19) indicates a minor radical characteristic of γGC, which is in a good agreement with the results of Liao et al.18. The superoxide radical anion ∗ ↑↓ ∗ ↓ O2•− has the electronic configuration of (the subscript ip and op represent in the Fe-O-O

plane and out of the plane, respectively). The calculated spin densities reflect a dominant electron 11


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∗ ↑↓ ∗ ↑ ∗ ↑ ∗ ↑ ∗ ↑ ∗ ↑ ∗ ↑↓ ∗ ↓ configuration of

for

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5

R. The valence electron orbital

configuration of 5R is depicted in Figure 4, in which the z axis is defined as the axial dz2 orbital of iron. One can see that most of the occupied orbitals are dominated by the metal 3d orbitals, dioxygen 2p and ∗ sulfur 3p orbitals. The lowest orbital is the doubly occupied

, which is an antibonding orbital that ∗ ∗ involves the weak interaction of the iron 3 with the orbitals of dioxygen and γGC. The and

are a little bit higher in energy, and the former represents the weak interaction between 3 and 2 of the O1 along the Fe-O1 bond while the latter is basically the metal 3 . The singly occupied orbital ∗

involves the contributions of iron 3d

, 2 of O1 atom as well as 3 of S atom. Finally, the highest singly occupied ∗ orbital is derived from the weak combination of iron 3 and substrate ∗ TMH amide group. can be defined as the main contribution of γGC. We found that the quintet

state in “end-on” mode is the catalytically active species for EgtB, because the electrons on the substrate and dioxygen are spin unparallel, which facilitates the attack of distal oxygen O2 to the S radical of γGC to form the Fe-O-O-S adduct.

Figure 4. Valence electron orbital diagrams of γGC-Fe-O2 complex (“end-on” model) at quintet. Side-on mode. In the quintet state, the spin densities on Fe, dioxygen and γGC are 2.88, 1.35, and −0.29, respectively, and the complex can be described as γGC•-Fe(III)-O2, which give rise from the 12


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electron transfer from iron to γGC. In contrast to quintet, in septet there is no net electron transfer between the iron and dioxygen, and the spin densities on Fe, dioxygen and γGC are 4.02, 1.53 and 0.15, respectively. Thus, the complex can be described as γGC-Fe(II)-O2. The detailed electron configuration is depicted in Figure 3, and the natural orbital analysis of “side-on” mode is shown in Figure S8. Our results demonstrate that only the quintet (both in “end-on” and “side-on” modes) state of γGCFe-O2 complex shows the radical character of γGC, in other word, it is the coordination of γGC that activates the sulfur atom of the substrate, which is beneficial for the attack by the distal oxygen atom of superoxide radical anion O2•− in the first sulfoxidation step.

3.2 Calculation results of the previously proposed reaction pathways According to the previously proposed reaction mechanism by Seebeck et al.6, the FeOO•- anion first abstracts a proton from residue Tyr377, meanwhile, the thiyl radical attacks the imidazole ring to form an iminyl radical, as illustrated in Figure 5 (path A, from R to A). To verify this concerted pathway, we firstly scanned the potential energy surface (PES) along the reaction coordinate for the first step. However, our scanned results show that the proton abstraction is prior to the formation of C-S bond. Therefore, the synergistic mechanism should be ruled out and the stepwise reaction pathway needs to be considered.

Figure 5. Previously proposed mechanism. When we prepared this article, de Visser et al.19 reported their theoretical study of the reaction mechanism of EgtB, which compared the catalysis of EgtB and cysteine dioxygenase. Their proposed reaction pathway is shown in Figure 5 (Path A′). Firstly, the FeOO•- anion abstracts a hydrogen from Tyr377 with the assistance of a mediated water molecule (not shown in Figure 5 for clarity). Then, the thiyl group attacks the C2 imidazole ring to form C-S bond. Simultaneously, the iron(III) hydroperoxo 13


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loses its proton back to Tyr377. To verify this pathway, we performed a series of repeated calculations using our computational models. Following the proposed mechanism by de Visser, we firstly explored the H-abstraction of Tyr377 by FeOO•- anion. The calculated energy barriers for the first step are 16.7 and 12.2 kcal/mol on triplet and quintet surfaces, which are similar to those in de Visser’s work (17.1 and 6.4 kcal/mol on triplet and quintet surfaces) (Figure S9). The minor difference of energy barriers may be caused by the slight difference of reactant models. Hence, we further compared the active sites of the crystal structure, the optimized model by de Visser, as well as our results, as displayed in Figure S9. It can be seen that the crystal structure superposes well with our model. However, the optimized structure by de Visser shows some minor differences both the binding mode of dioxygen and the relative position of Tyr377 residue. In the active site of the model constructed by de Visser, although the distance between the distal oxygen and OTyr377 is 3.72 Å (it is 2.82 Å in our model), the orientation of phenolic hydroxyl in Tyr377 is suitable for the H-abstraction step mediated by a water molecule. Besides, the dihedral angle of D(S-Fe-O1-O2) in the structure of de Visser’s work is 40.5°, while it is 14.0° in ours (not shown in the figure). As the orientation of FeOO•- is not ideal for H-abstraction and there is no water molecule between the FeOO•- anion and Tyr377 to mediate the H transfer in our calculated model, therefore, in the second step, the formation of C-S bond corresponds to a much high energy barrier of 43.9 kcal/mol on quintet (Figure S9). Although the coordination of γGC activates the sulfur atom of the substrate, the attack of thiyl group on the C2 imidazole ring of TMH to break the aromaticity of the imidazole group is still calculated to be difficult, which has also been confirmed by Liao et al.18 (the C-S bond formation corresponds to an energy barrier of 29.7 kcal/mol from B-Int1 to B-Int2, described in section 3.2). Besides, the Fe-O-O-H group does not return the hydrogen atom back to Tyr377 upon the formation of C-S bond, which should be a result of the inappropriate position between H and Tyr377 (the angle of ∠OTyr377-O2-H is 64.2°). Intermediate B′′ is a high unstable species with a relative energy of 44.0 kcal/mol. It should be noted that, considering the bridging water molecule between Tyr377 and FeOO•- may be significant, a water molecule was manually moved to locate between residue Tyr377 and FeOO•- anion, and the computational results are shown in Figure S10 (a-d). In the H-abstraction step (5TSAW´ and 5

AW´), the energy barrier is 9.9 kcal/mol on the quintet surface, which is close to the result in de

Visser’s work (6.4 kcal/mol). For the C-S bond formation step, we firstly scanned energy profile along 14


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the coordinate, then the involved transition state and intermediate were optimized. During the C-S bond formation (5TSBW´ and 5BW´), the Fe-O-O-H group returns the hydrogen atom back to Tyr377 mediated by the water molecule, which is consistent with de Visser’s results. According to our calculations, the energy barrier for the C-S bond formation is 33.2 kcal/mol, however, it is 14.2 kcal/mol calculated by de Visser et al.,19 as shown in Figure S10(c).To compare our results with that of de Visser’s, we also list the structures (5I1, 5TSCS, and 5I2) calculated by de Visser, as shown in Figure S10 (e-g). From 5I1 to

5

I2, the distance between CE1 and S shortens from 3.81 to 1.98 Å. We also

noted that the dihedral of D(N-Fe-O1-O2) changes from -113.0 to 76.4°, indicating a clear change of the coordination of dioxygen with Fe upon the C-S bond formation, as displayed in Figure S10(i). As such, in de Visser’s work, the much low calculated energy barrier of 14.2 kcal/mol may be partially attributed to the movement of dioxygen. Nevertheless, in our calculation results, the conformation of dioxygen keeps unchanged in the process of C-S bond formation, as shown in the superposition of 5

AW´, 5TSBW´ and 5BW´ (Figure S10 h). Besides, in de Visser’s work, another interesting observation is

that the distances between CE1 and S in 5TSCS and 5I2 are identical (1.98 Å).

3.3 Proposed pathway based our calculations In the previous studies, the sulfurization of histidine was suggested to undergo a C5sp2 radical,11,13 and the formation of the sulfoxide-containing product is a consequence of the reduction of the powerful oxidative Fe-O-O-S intermediate.16 Taken these suggestions into consideration, we designed another reaction pathway. Given that there are two binding modes of dioxygen in the Fe(II)-O2 complex, the detailed reactions will be discussed separately. According to our calculations, the reaction pathway can be illustrated in Figure 6. In the first step, the distal oxygen atom of O2•- radical anion combines with the S• radical of γGC to form a four-membered Fe-O-O-S epoxide IM1; then, the O-O bond cleaves to afford the sulfoxide IM2; subsequently, the iron(IV)-oxo abstracts a proton from C2 of TMH, generating a C2 anion (IM3), which attacks the sulfoxide to form the C-S bond to afford the final product (P). Alternatively, the formation of C2 anion may be mediated by Tyr377 undergoing the intermediates IM3′′ and IM4′′. The energy profiles along the reaction coordinates at different spin states are shown in Figure 7, and the optimized structures of involved species are displayed in Figures 8, 9 and 10. 15


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Considering the calculated results may be influenced by the choice of the initial structures, besides using the snapshot from the MD trajectories at 20ns, other three snapshots at 23, 26 and 29 ns were also employed to explore the first two elementary steps (R→IM1→IM2). With different snapshots, the calculated energy barriers are basically consistent, as shown in Figure S11.

Figure 6. Proposed mechanism for EgtB according to our calculations, denoted as path B. The blue and red arrows in IM2 represent two modes of the H-abstraction of Fe=O, which is either from the C2 of TMH or Tyr377 residue. 3.3.1 Formation of the Fe-O-O-S epoxide Experimental and theoretical examples have demonstrated that the activated O2 by iron(II) can act as a strong nucleophile.53-56 Starting from R, the distal oxygen O2 attacks the S radical of γGC to form a four-membered ring (Fe-O1-O2-S) intermediate IM1. On the quintet surface from 5R to 5IM1, the distance between O2 and S decreases from 3.32 Å (not shown in Figure 2) to 1.67 Å via 2.15 Å in 5TS1 (Figure 8), simultaneously, the O1-O2 bond is greatly weakened with its length increases to 1.47 Å. In 5

IM1, the bond length of O2-S (1.67 Å) shows excellent agreement with that (1.68 Å) in the CDO

enzyme50. As expected, with the formation of O2-S, the coordination of S to Fe is greatly weakened with the distance of Fe-S bond elongated from 2.42 to 2.96 Å. We also note that the four-membered ring (Fe-O1-O2-S) is basically planar where the dihedral angle of D(S-Fe-O1-O2) is only -7.8°. The formation of the Fe-O1-O2-S epoxide corresponds to rather different barriers on the surfaces of singlet, triplet and quintet, which are 22.8, 18.9 and 4.9 kcal/mol relative to 1R, 3R and 5R, respectively (Figure 16


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7). In other words, the quintet reactant 5R is more reactive for the formation of O-S. The free energy barrier is calculated to be 17.0 kcal/mol relative to 1R on quintet surface (Figure S12). It is understandable by analyzing the electronic structures of these spin state reactant models (Figure 3). In 5

∗ R, the singly occupied electron orbital on S ( ) matches well with the singly occupied orbital on

∗ O2 ( ), and both the distal oxygen (O2) and the sulfur atom have the characteristic of radical, which

make the combination of distal O2 and S atom to be quite easy. To shed light on the electron transfer processes along the catalytic reaction, the spin densities of some crucial atoms are also listed in Figure 8. From 5R to 5IM1, the spin density (ρ) on γGC decreases from 0.19 to 0.02, and the electron ∗ ↑↓ ∗ ↑ ∗ ↑ ∗ ↑ ∗ ↑ ∗ ↑↓ ∗ ↑↓ configuration of 5IM1 can be described by

.

Although the open-shell singlet is calculated to be the ground state of the reactant, the formation of S-O may take place on the quintet surface. Figure 7 also shows that the quintet surface is the lowest one among the three spin state energy profiles. Besides, a spin crossing may occur in the first step.

Figure 7. Calculated energy profiles for the suggested mechanism (path B). The energy of open-shell singlet is set to zero. The black, purple and red lines represent the singlet, triplet and quintet surfaces, respectively.

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Figure 8. Optimized structures of the involved species from R to IM2. All distances are given in Å. r1=Fe-O1, r2=Fe-S. Black, blue and red represent singlet, triplet and quintet, respectively. Spin densities of some critical atoms in the quintet state are shown in the bottom right, denoted by ρ. 3.3.2 Formation of the sulfoxide In the second step, the O1-O2 bond cleaves to generate the sulfoxide IM2. The distance of Fe-O1 shortens from 1.98 Å to 1.66 Å upon the formation of 5IM2. Simultaneously, the O1-O2 bond length increases to 3.00 Å, and the length of O2-S shortens to 1.54 Å, indicating the formation of sulfoxide intermediate. The energy barrier of this step is calculated to be 6.0 kcal/mol on the quintet surface (Figure 7), which is very similar to those of the singlet and triplet surfaces (7.7 and 7.6 kcal/mol). Taking the entropy effects into consideration, this elementary step corresponds to a free energy barrier of 5.9 kcal/mol (Figure S12). Besides, the relative energy of 5IM2 is -26.2 kcal/mol at the quintet state, indicating the sulfoxide to be quite stable. In 5IM2, the spin densities of iron and O1 are 3.47 and 0.60, respectively, which are in a good agreement with the calculation results of other non-heme iron(IV)oxo.57 The increase of spin density of O1 from 0.12 to 0.60 should be an indicative of the radical character for Fe(IV)-oxo intermediate. 3.3.3 Formation of C2 anion intermediate The Fe(IV)-oxo species is generally considered as a strong oxidant. In the third step, the Fe(IV)-oxo 18


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species abstracts a proton from C2 of TMH to form the C2 anion intermediate (IM3). This step corresponds a larger energy barrier (21.7 kcal/mol) on the quintet state surface, which is higher than the calculation result by Liao’s group (17.9 kcal/mol using cluster model) 18 and the estimated free energy barrier from the experiment (17.3-17.6 kcal/mol)6. The free energy barrier for the formation of C2 anion is 21.1 kcal/mol in quintet (Figure S12). We attribute the high energy barrier to the unfavorable orientation of the Fe(IV)-oxo species relative the H where the angle of ∠Fe-O1-H is 97.0°, which is not ideal for proton transfer. From 5IM2 to 5IM3, the length of O1-H changes from 2.55 Å to 1.00 Å via 1.17 in 5TS3, and Fe-O1 bond length elongates to 1.82 Å. In addition, the coordination between γGC and Fe gets weak, which can be deduced from the increase of Fe-S distance from 2.51 Å in 5IM2 to 2.82 Å in 5IM3. However, the substrate kinetic isotope effect (KIE) did not support the C2-H bond cleavage to be a rate limiting step.17 The energy profile without including DFT-D3 correction is displayed in Figure S13. Many previously studies56-58 reported that the Fe(IV)-oxo prefers to abstract a H atom rather than a proton from the nearby species. In order to confirm this issue, spin densities and NPA charges were further calculated, as depicted in Table S3 and S4. In 5IM2, the charge and spin density on C2 are 0.57 and 0.07. After the proton abstraction, the NPA value of C2 reduces to 0.03, and the spin density increases a little bit to 0.17. Besides, the spin densities of Fe, S, O1 and O2 are 4.07, −0.43, 0.42 and −0.43 in 5IM3, respectively. These results suggest that the Fe(IV)=O abstracts a proton from TMH, which can also be deduced from the valence electron orbital diagram displayed in Figure S14. Based on our computational model, the cysteine dioxygenase (CDO) pathway has also been investigated on quintet surface, as an alternative pathway from the C-H abstraction (formation of C2 anion intermediate). After the sulfoxidation step, the sulfoxide group first rotates around Ca-S bond, leading to the sulfur atom depart away from the Fe and facilitating the binding of oxygen atom (O2). On the quintet surface, this step corresponds to an energy barrier of 18.3 kcal/mol, which is much higher than that in the CDO enzyme.50 The final step for the formation of sulfinic acid product corresponds to an energy barrier of 10.8 kcal/mol. Thus, the cysteine dioxygenase pathway could be competitive to the C-H abstraction. The detailed information of the involved species in CDO-like mechanism is shown in Figure S15.

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Figure 9. Optimized structures of involved species for the formation of 2-histidyl-cysteine sulfoxide. The distances shown in black, blue and red represent those of the singlet, triplet and quintet, respectively. 3.3.4 Formation of C-S bond In the final step, the C2 anion of TMH attacks the sulfoxide radical to form the product 2-histidylcysteine sulfoxide (P). With the formation of S-C bond, the coordination of γGC with Fe collapses with the distance of Fe-S changing from 2.82 Å in 5IM3 to 3.69 Å in 5P. The spin density on S atom is -0.37 in 5TS4. After the formation of 2-histidyl-cysteine sulfoxide, the spin densities on Fe and O1 are 3.69 ∗ ↑↓ ∗ ↑ ∗ ↑ ∗ ↑ ∗ ↑ and 0.13, respectively. The electron configuration of 5P can be described by

.

The formation of the C-S bond corresponds to energy barriers of 2.6, 8.8 and 3.5 kcal/mol in quintet, triplet and singlet, respectively, which are lower than that in Liao’s work (10.9 kcal/mol in the fifth step). The whole reaction is exothermic by 46.9 kcal/mol on the quintet surface.

20


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Figure 10. Optimized structures of involved species for the hydrogen abstraction step mediated by Tyr377. The H atoms in green and purple come from Tyr377 and TMH, respectively. The data in blue and red represent those of triplet and quintet, respectively. 3.3.5 Does Tyr377 mediate the formation of C2 anion? Considering the optimized structure of intermediate IM2, there are two possible pathways for the formation of C2 anion. In the first pathway, the Fe(IV)=O complex directly abstracts a proton from C2 of TMH, as described in section 3.3.4. In another pathway, the proton-abstraction is mediated by the residue Tyr377, which has been proved to be a good proton donor in fumitremorgin B endoperoxidase (FtmOx1)23. The Tyr377-mediated pathway is illustrated in Figure 6 (from TS3′′ to IM4′′). For comparison, we further performed QM/MM calculations to explore the possibility of this pathway. As the singlet state is not feasible for the reaction (with much higher energy barriers, as described in the previous section), we only optimized the structures at the quintet and triplet states, which are shown in Figure 10. In 5IM2, the phenolic hydroxyl of Tyr377 locates above the Fe(IV)-oxo species with a distance of 3.03 Å, which is ideal for proton-abstraction. From 5TS3′′ to 5IM3′′, the distance between O1 and H1 of Tyr377 shortens from 1.21 Å to 0.97 Å (Figure 10), indicating the formation of Fe(IV)-OH 21


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intermediate. This proton abstraction corresponds to an energy barrier of 9.0 kcal/mol on the quintet surface (Figure 11), which is close to the result of Wang’s work (8.7 kcal/mol, H-abstraction from Tyr228 by Fe(IV)-oxo species in quintet surface).23 IM3′′ is lower than the reactant by ~25.1 kcal/mol, which means IM3′′ to be a stable species.

Figure 11. Calculated energy profiles for the hydrogen abstraction and the formation of C2 anion (from IM2 to IM4′′). After the formation of Fe(IV)-OH complex, the phenolic oxygen anion abstracts a proton from C2 of TMH, and the distance of O-H2 decreases from 2.26 Å in 5IM3′′ to 0.97 Å in 5IM4′′ via 1.18 Å in 5TS4′′, indicating the formation of O-H2 bond. The spin densities on OTyr377 and C2 are 0 and 0.17, respectively. This step corresponds to an energy barrier of 27.5 kcal/mol on the quintet surface (Figure 11), which is 5.8 kcal/mol higher than the direct proton abstraction by Fe(IV)=O complex. Hence, our calculations reveal that although Fe(IV)=O complex can easily abstract a proton from Tyr377, the subsequent proton-abstraction from C2 becomes more difficult, and Tyr377 can not mediate the formation of C2 anion, but it donates a proton to the Fe(IV)-oxo species to generate a relative stable Fe(IV)-OH complex, affecting the formation of C-S bond. 3.3.6 Residue electrostatic analysis In the active site of EgtB, the carboxylate of γGC forms salt bridges with Arg90 (1.82 Å) and Arg87 (1.70 Å), which may have some influences on the calculated reaction barriers, thus, further electrostatic analysis has been performed. The influence of residue i on the energy barrier can be described as ∆Ei-0 = ∆Ei − ∆E0, where ∆Ei is the energy barrier with the charge on residue i set to 0, ∆E0 is the original energy barrier, and ∆Ei-0 is the difference between ∆Ei and ∆E0. In the calculations, the geometrical 22


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structures of the stationary points involved in the reaction were kept. A positive ∆Ei-0 value means that residue i makes contribution to reduce the energy barrier and facilitates the reaction, vice versa. The calculated ∆Ei-0 values are shown in Figure S16, which range from −3.0 kcal mol-1 to 1.6 kcal mol-1 for four steps. These results indicate that Arg90 and Arg87 have different influence on these steps. According to Figure S16b, both residues increase the barrier of the first step and reduce the barrier of the fourth step. However, these ∆Ei-0 values are quite small, indicating the salt bridges between γGC and Arg90 / Arg87 have minor influence on the catalytic reaction. 3.3.7 Calculation results of the “side-on” mode We also explored the possible pathway (Figure S17) by using the “side-on” reactant mode. Firstly, the distal oxygen atom (O2) attacks the S atom of γGC, leading to the formation of intermediate sulfoxide; then, the generated Fe(IV)=O complex abstracts a proton from C2 of TMH to form the imidazole anion; finally, the sulfoxide attacks C2 of TMH, forming the product 2-histidyl-cysteine sulfoxide.

Figure 12. Optimized structures of involved species in the “side-on” reactant mode. The data shown in red and blue represent those of quintet and septet, respectively. In the first step, the distal O2 atom attacks the S atom of γGC to form the sulfoxide intermediate IM1S in which the O2-S bond length is 1.53 Å (Figure 12). This step corresponds to an energy barrier of 27.0 and 30.9 kcal/mol on the quintet and septet surfaces, respectively (Figure 13). The relative high energy barriers may come from the unfavorable electronic structures of the reactant in “side-on” mode 23


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where the dioxygen is not well activated by iron. The following steps are similar to those in the “endon” mode. The formation of C2 anion is also the rate-limiting step with energy barriers of 31.1 and 26.5 kcal/mol on the quintet and septet state surfaces. This energy barrier in “end-on” mode may be caused by the different conformations of sulfoxide intermediates (IM2 vs IM1S). Compared with the sulfoxide intermediate (IM2) in “end-on” mode, the distance of Fe-S bond is about 2.40 Å, whereas it is 2.90 Å in IM1S. Furthermore, in IM2, the distance of O1-H is about 2.50 Å in “end-on” mode, while it is also longer in “side-on” mode (2.70 Å). In a word, the “side-on” mode is not the catalytic relevant species compared with the “end-on” mode.

Figure 13. Calculated energy profiles for the suggested mechanism for EgtB in “side-on” mode. Besides, we also performed calculations according to the pathway that was suggested by Liao et

al.18 (Figure S18) in which the sulfoxidation step is also prior to the formation of C-S bond. In this mechanism, the formation of sulfoxide intermediate also undergoes a four-member ring Fe-O-O-S intermediate, which is similar to our calculation results (section 3.3.1, 3.3.2). However, the subsequent steps are slightly different from our proposal. As shown in Figure S18, Liao et al. proposed that the deprotonated Tyr377 firstly abstracts a proton from the N3 of TMH to form the imidazole anion, which is mediated by Tyr377 and two water molecules, then the C-S bond forms between γGC and TMH; finally, a proton transfer from C1 to N3 on the imidazole ring leads to the formation of the final product (from Int3 to Prod in Figure S18)18. To check the possibility of this pathway, we conducted test calculations using Fe-OH complex (IM3′′). The proton transfer from N3 to Tyr377 anion (from 5TS4L to 5Int4L in Figure S19) is calculated to be quite easy with the assistance of a mediate water molecule, corresponding to an energy barrier of 14.9 kcal/mol on quintet surface. Subsequently, the S radical of γGC attacks to C2 atom of TMH (from 5Int4L to 5Int5L), which couples with one electron transfer to 24


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the iron (spin density of Fe increases from 2.80 to 3.67). The C-S bond formation corresponds to a high barrier of 28.2 kcal/mol. However, according to the calculation results of Liao et al.18 , the C-S bond formation corresponds to a barrier of 10.9 kcal/mol. By checking the geometrical and electronic structures of our results and those of Liao et al., we found that there are two factors may lead to different barriers. On one hand, as shown in Figure S19e in our QM/MM computational models, in the structures of 5Int4L, 5TS5L and 5Int5L, both the coordinate environment of Fe and the substrates keep well, whereas in the optimized structures by Liao et al. (Figure S19f), the two substrates, Fe, Tyr377 underwent a clear conformational changes. On the other hand, in 5Int4L of our model, the three unpaired electron in the d-type molecular orbitals of iron is ferromagnetically coupled to the unpaired electron of the S=O group, while in the 5In4 calculated by Liao et al, the five unpaired electron in the iron center is antiferromagnetically coupled to the unpaired electron of the S=O group. Relatively, the geometries of active site may be the dominant factor to influence the calculation results.

4. Conclusion Started from the crystal structure of the ergothioneine synthase (EgtB), QM/MM calculations have been carried out to explore the catalysis of EgtB. Two binding modes of O2 to Fe(II) have been identified. In the “end-on” mode, the substrate-iron(II)-O2 complex can be described as a hybridization of γGC-iron(III)-O2•-and γGC•-iron(II)-O2•-. While in the “side-on” mode, the reactant can be described as γGC•-iron(III)-O2 and γGC-iron(II)-O2. Although the open-shell singlet is the ground state, the reaction prefers to take place on the quintet surface. It is found that the four-membered ring Fe-O-O-S adduct is firstly formed with an energy barrier of 14.3 kcal/mol on the quintet state surface, and the subsequent sulfoxidation of γGC couples with the collapse of Fe-O-O-S ring, corresponding to an energy barrier of 6.0 kcal/mol; then, the Fe(IV)-oxo adduct abstracts a proton from C2 atom of TMH, leading to the formation of imidazole anion, which is the rate-limiting step with an energy barrier of 21.7 kcal/mol. Besides, we also explored the role of residue Tyr377, and found that the Fe(IV)-oxo species can easily abstracts a proton from residue Tyr377 (9.0 kcal/mol), but the following proton abstraction from C2 by Tyr377 anion is unfeasible. Once the C2 anion forms, the product 2-histidylcysteine sulfoxide can be generated easily. Besides, the reaction pathways suggested by Liao and de Visser have also been calculated using our constructed models. Our calculations reveal that, owing to 25


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the complexity and flexibility of enzymes, especially small ligand molecules (such as O2, H2O, etc) are included in the active site, the calculated barriers usually contain a large energy variation caused by the conformational change. If the connectivity of transition state with the two local minima can not be well confirmed, the calculated barrier may not well reflect the “real” reaction barrier. In addition, the active site structures are highly correlated with the reaction pathways and reactivity of the enzymes. Thus, we must be very careful in constructing the computational model and conducting QM/MM calculations. In a word, the present work may provide more information for understanding the biosynthesis of ergothioneine.

Supporting Information: Relative entropy effects and empirical dispersion corrections; some important parameters of substrate-iron-dioxygen complexes; spin densities of some critical atoms; NPA charges of some critical atoms; overlay structure of the two chains and the active site in the crystal structure; the active region of EgtB; RMSD for protein backbone of the MD simulations; overlap of 14 optimized snapshots; representative structures; spin state orderings with different functionals for the substrate-Fe-O2 complex; the valence electron orbital diagrams of the γGC-Fe-O2 complex in different spin states; superposition of the active sites of the crystal structure with that of de Visser’s work and our study; scanned energy profiles for path A; energy profiles for the first two steps in different snapshots; relative Gibbs free energy profile along the reaction; energy profiles without DFT-D3 corrections; the valence electron orbital diagrams along the reaction on quintet surface; optimized structures involved in the CDO-like mechanism; electrostatic analysis of two residues Arg90 and Arg87; EgtB in “side-on” mode; mechanism proposed by Rongzhen Liao; some important scanned energy profiles along the reaction coordinates; Cartesian coordinates of important species. This information is available free of charge on the ACS Publications website. Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (21773138).

References: 26


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