Mechanism of the Glutathione Persulfide Oxidation Process Catalyzed

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Mechanism of the Glutathione Persulfide Oxidation Process Catalyzed by Ethylmalonic Encephalopathy Protein 1 Beibei Lin, Guangcai Ma, and Yongjun Liu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01417 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Mechanism of the Glutathione Persulfide Oxidation Process Catalyzed by Ethylmalonic Encephalopathy Protein 1 Beibei Lin, Guangcai Ma, Yongjun Liu*

School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China

ABSTRACT: Ethylmalonic encephalopathy protein 1 (ETHE1) is a β-lactamase fold-containing protein, which is related with the increased cellular levels of hydrogen sulfide. ETHE1 is essential for the survival of a range of organisms and catalyzes the oxidation of glutathione persulfide (GSSH). Currently, the catalytic mechanism of ETHE1 still remains unclear, despite a catalytic cycle has been suggested from the crystal structure and a proposal for the mechanistically related cysteine dioxygenase (CDO). In this article, we performed a series of quantum mechanical/molecular mechanical (QM/MM) calculations on the substrate GSSH oxidation by human ETHE1. Our calculation results reveal that the ground state of the iron(II)-superoxo reactant is quintet, which can be described as GSS+•-Fe(II)-O2•, and the most feasible reaction channel was found to starts from the cleavage of dioxygen and a concerted attack of distal oxygen on the sulfur atom of substrate, forming the metal-bound activated oxygen and a sulfite-intermediate. Moreover, the reaction starts from quintet ground state reactant, undergoes triplet intermediate and finally generates the septet product rather than the reaction of CDO which starts from a singlet-quintet crossing. Keywords: Ethylmalonic encephalopathy protein 1(ETHE1); QM/MM; glutathione persulfide; GSSH; oxidation; reaction mechanism.

Corresponding Author: Tel.: +86 53188365576; fax: +86 53188564464. Email address: [email protected] (Y. Liu). 1

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1. Introduction The ethylmalonic encephalopathy protein 1 (ETHE1), also known as sulphur dioxygenase (SDO), is a non-heme iron-dependent oxygenase, which catalyzes the oxygen-dependent oxidation of glutathione persulfide (GSSH), generating glutathione and sulfite, as shown in Scheme 1 (1-3). ETHE1 plays important roles in mitochondrial homeostasis and energy metabolism, and therefore it has been extensively studied in the past decades (4-12). For instance, it has been confirmed that the mutations of the gene encoding for the human ETHE (hETHE1) can lead to Ethylmalonic Encephalopathy (EE) (4), which is an inborn autosomal recessive disorder, effecting on the brain, gastrointestinal tract and peripheral vessels (5-9). Human ETHE1 was also proposed to be related to other diseases, such as acute cardiovascular disorder and myocardial infarction (10, 11). Besides, the role of ETHE1 in Arabidopsis was also investigated: its gene mutation would induce a delay in seed development and embryo arrest by the early heart stage (12). In addition, sequence analysis demonstrates that hETHE1 is a member of the widely distributed metallo-β-lactamase (MBL)-fold family, which is extremely widespread and has a series of biological functions (1, 13, 14).

Scheme 1. Catalytic reaction of hETHE1. The crystal structures of ETHE1 have been described by several research groups (1, 15). The structure of Arabidopsis thaliana ETHE1-like enzyme comprises two central mixed β-sheets, each containing six stands, surrounded on both sides by helices (PDB ID: 2GCU). ETHE1 is much alike the glyoxalase II proteins, however, it does not possess glyoxalase II activity when assayed using the most common glyoxalase II substrates, S-(D)-lactoylglutathione and other glutathione thioesters (3, 16). The crystal structure of hETHE1 (PDB ID: 4CHL) shows significant sequence similarity to ETHE1-like enzyme. Comparison of these two structures indicates similar iron binding mode with nearly identical orientations and metal distances between conserved residues (1). The hETHE1 structure also has striking similarity to isopenicillin N-synthase: employing a facial triad of iron binding residues and involving Fe-S-peptide interactions in mechanisms (3, 17). Despite the physiological substrate of ETHE1 is still unknown, it exhibits activity towards GSSH. In the active site of hETHE1, there exists a channel that is commodious enough to accommodate the substrate molecule of GSSH. 2


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Despite these biochemical discoveries have been obtained, to the best of our knowledge, there is still no relevant theoretical research about the catalytic mechanism of ETHE1. And the structural and electrical characteristic of ETHE1-substrate complex is still not fully understood. Therefore, the present study aims to explore the oxidation mechanism of glutathione persulfide catalyzed by EHTE1 using quantum mechanics/molecular mechanics (QM/MM) calculations (18, 19). In the past decades, the QM/MM method has been extensively applied for exploring the catalytic mechanism of enzymes, including the iron-containing enzyme systems (20-25). Our research group has also studied the catalytic reactions of many enzymes, for examples, the carbapenem synthase and radical SAM enzyme (26, 27). Based on the proposed mechanism of hEHTE1 and CDO (3, 21), three possible reaction pathways are considered here, which start from the two resonance structures, either Fe(III)-superoxo complex or Fe(II)-superoxo complex (1, 3). Spectroscopic studies on Arabidopsis ETHE1 have demonstrated that there is no antiferromagnetically coupled Fe(III)-Fe(II) center and the predominant oxidation state of Fe is Fe(II) (28), however, the spin state of the iron(II)-superoxo complex in hETHE1 is still unclear. To determinate the ground state of the iron(II)-superoxo reactant and the reaction mechanism, the initial structures with different spin states were firstly compared, and then the energy profiles along different spin states and reaction pathways were calculated.

2. Computational methods and details 2.1. Setup of the system, docking and MD Runs The initial structure of the enzyme-substrate complex was constructed on the basis of the crystal structure of hETHE1 at 2.60 Å resolution (PDB ID: 4CHL) (1). The structure of hETHE1 has two chains which represent the dimer in the asymmetric unit, while only one chain was adopted to prepare the initial model and the other one was removed (Figure 1). The structure of GSSH was obtained through the Gaussian View software (29) and Gaussian 03 program package (30) on the basis of the crystal structure of glutathione (GSH, PDB ID: DB00143). Subsequently, the substrate GSSH was docked into the active site of hETHE1 using AutoDock Tools 4.0 (31). In the docking, the three-dimensional grid map was set to 50 Å × 50 Å × 55 Å with a grid spacing of 0.375 Å, centered on the active site of the protein. Then 100 independent docking run was performed, in which the receptor hETHE1 was kept rigid. Docking results were analyzed at the criterion of RMSD (root-mean-square deviation) of 2.0 Å. The representative docking structure was shown in Figure 1c. In addition, O2 was manually added to the iron center, as shown in Figure 3


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1d. Then the obtained structure was further subjected to MD simulation and QM/MM optimization. The protonation states of all charged residues were checked using the PDB2PQR suite of programs in combination with actual reaction condition (32-35). The pKa values of His79, His81, His135 and Asp154 suggest that these residues are unquestionably deprotonated residues. The substrate GSSH was set to be deprotonated at both N and S sites. This notion that the N and S are both deprotonated comes from the complexation behavior of GSH with Platinum(II) where the Pt(II) coordinates to the sulfur and the deprotonated amide nitrogen of the GSH (36). In addition, previous studies also showed that the functional groups of GSH (the amino group of residue, sulfhydryl group of cysteine residue and NH group of the amide bond) would undergo stepwise deprotonation under different pH (37-39). In other word, the Fe ion may act as a soft Lewis acid, which has a relatively high affinity for the deprotonated N ligand of the amide bond. For comparison, we also constructed a model in which the ligated amide of GSSH was set to its neutral state, which will be discussed in the following sections. The missing hydrogen atoms were added by the HBUILD program of the CHARMM package (40, 41). Subsequently, 3990 TIP3P water molecules were placed into the computational model and 8 Na+ were added to neutralize the system (Figure S1). In the MD simulation, the Fe ion, the dioxygen group, the substrate GSSH, and residues His79, His135 and Asp154 were kept fixed. In order to equilibrate the system, a 20 ns molecular dynamics simulation was carried out using the CHARMM22 force field (40-43). The systems were firstly heated from 0 to 298.15 K and then equilibrated at the 298.15 K. Finally, 20 ns MD simulations were carried out.

Figure 1. (a) Crystal structure of ETHE1 (PDB ID: 4CHL), two chains represent the dimer in the asymmetric unit; (b) The structure after the substrate GSSH was docked into ETHE1, ball-and-stick model represents the GSSH; (c) The active site structure after docking, and the arrow points the binding 4


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orientation of the oxygen molecule; (d) Active site structure after the O2 was added into the iron center.

2.2. QM/MM calculations All QM/MM calculations were performed using the Chemshell program, which combines Turbomole for the QM region and DL_POLY for the MM region (44-47). To avoid hyperpolarization of the QM wave function, the electronic embedding scheme was used to account for the polarizing effect of the enzyme environment on the QM region (48). Hydrogen link atoms were used to saturate the valences, and simulate the covalent bonds at the QM/MM interfaces (49, 50). The calculations of QM region were performed by density functional theory (DFT) (51-53) with the unrestricted hybrid density functional UB3LYP (54, 55) at 6-31G(d,p)/Wachters+f (B1) level (56-59), which means the basis set of Wachters+f was used for Fe atom and 6-31G(d,p) for other atoms, and MM region was characterized using the CHARMM22 force field. Since the QM region is negative charged, the basis set of 6-31++G(d,p) which contains diffusion function was also used to optimize the structure of reactant for comparison (60, 61). Geometry optimizations were performed by the hybrid delocalized internal coordinates (HDLC) optimizer (62) and the local minima were searched by the quasi-Newton limited memory Broyden-Fletcher-Goldfarb-Shanno (L-BFGS) method (63, 64). The highest point on the scanned potential energy surface (PES) along the reaction coordinates was determined as transition state, which was further optimized by the partitioned rational function optimization (P-RFO) algorithm (65) following eigenmodes of the Hessian. The intermediate was found by optimizing the last structure from the PES scan, and then it was employed to carry out the next scan to find the consecutive transition state and intermediate. Considering the spin-state ordering and relative energies of the reactant may be influenced by the choice of the functionals, we also used other functionals to calculate the energies of the enzyme-substrate complex (66), including BP86, BLYP, BVWN and TPSSh at B1 level (67-70). To get more accurate energies, the single point energy calculations were performed at 6-311++G(2d,2p)/Wachters+f (B2) level (57, 58). During the QM/MM calculation, the QM atoms and the MM atoms within 15 Å around the Fe ion were allowed to move while the remaining atoms of MM region were kept frozen. In addition, the natural bond orbital (NBO) analysis was also implemented at B2 level to obtain natural population atomic (NPA) charges (71-73).

2.3. The selection of QM region

5


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Figure 2. Created two QM-region models in the QM/MM calculations (models I and II). Iron coordination bonds are shown in black dash lines and possible hydrogen bonds in red dash lines. To determine the ground state of the iron(II)-superoxo reactant (enzyme-substrate complex) and explore the reaction mechanism, we constructed two computational models with different sizes. As shown in Figure 2, model I is a small QM region, which was used to determine the spin ground state of the reactant, and therefore it only contains the essential groups coordinated with the metal, including dioxygen, side chains of His79, Asp154, His135, and part of the substrate GSSH. Model II was used to explore the catalytic reaction pathways, and contains more groups. Compared to Model I, the complete GSSH molecule and His81 were also included in Model II. In total, Model II contains 77 atoms. Thus, the total charges of Model I and Model II were set to -1 and -2, respectively.

2.4. Choice of spin states Since the dioxygen, Fe3+/Fe2+ and GSSH possess a densely orbital manifold, which is only partially filled, some different spin states might exist along with the variation of oxidation states on the iron and its ligands (74). Therefore, our first aim is to determine the ground state of the iron(II)-superoxo reactant. According to the mechanism proposed by Kabil and Banerjee and modified by Pettinati et al, there are two resonant structures (GSS••-Fe(III)-O2• and GSS+•-Fe(II)-O2•) for the initial reactant (1, 3). When the Fe ion is trivalent, the dioxygen may have one single electron, whereas both the substrate GSSH and dioxygen may have a single electron when the Fe ion is divalent, which mean the spin state of the reactant might be singlet, triplet, quintet and septet. To confirm which one is the ground state, geometry optimizations of the lowest-lying singlet, triplet, and quintet spin states were performed on Model I. The relative energies and geometries at the lowest-lying spin state surfaces were calculated and compared.

2.5. Possible reaction pathways 6


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Scheme 2. Possible reaction pathways for the oxidation of GSSH catalyzed by ETHE1. The directions of electron transfer are shown in different colors: red for Path_a, blue for Path_b and green for Path_c. R1 and R2 represent glutamate and glycine groups of GSSH. To get insight into the oxidation process of GSSH, three possible pathways were considered. In Scheme 2, the red, blue and green curved arrows represent the possible direction of electron transfer in the three pathways. Path_a includes two elementary steps from R to IM2a. In the first step, the Oa-Ob bond of Fe(II)-superoxo is firstly broken and the distal oxygen (Ob) forms the new Ob-Sa bond with GSSH, generating IM1a, subsequently, the Fe(II)-Oa bond is broken to form the sulfite intermediate (IM2a). In Path_b, the intermediates (IM1b and IM2b) were borrowed from a proposal of mechanistically related enzymes, isopenicillin N synthase and cysteine dioxygenase (1, 3, 17, 21, 75). Since there is no related spectroscopic study to confirm these intermediates hitherto, it is necessary to verify Path_b through our QM/MM calculation. Path_c represents another possibility that the proximal Oa firstly forms Oa-Sb bond, then the distal Ob attacks the Sa atom to form Ob-Sa bond, leading to the cleavage of dioxygen. Some intermediates in Scheme 2, such as IM2b and IM2c, IM2a, IM3b and IM3c have very similar conformations, however, they are derived from different pathways and have different energies, and therefore are represented by different codes.

3. Results and discussion 3.1. The docking results and MD simulation To construct the enzyme-substrate model, the substrate GSSH was docked into the active site of hETHE1 by using AutoDockTools 4.0 (31). The docking results are shown in Figure 1 and Figure S2. According to 7


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the criterion of RMSD (root-mean-square deviation) 2.0 Å, two typical conformations of the substrate GSSH were found in the active site, as shown in Figure S2. On the basis of the studies of a related cysteine dioxygenase (CDO), the conformation I should represent an unreactive case because the S atom of the substrate GSSH does not coordinate with the Fe ion. In conformation II (Figure S2), both N and S atoms of GSSH have replaced the two water molecules that previously coordinate with the Fe ion. Besides, conformation II corresponds to the minimal binding energy compared to other binding modes. Thus, we chose conformation II as the initial structure for the subsequent MD simulation, as shown in Figure 1(b). One can see that the active site is spacious enough to accommodate the substrate GSSH. The S-S terminal and the N atom (Nsub) are located in the vicinity of the Fe ion. A vacant site remains between the substrate GSSH and residues His79 and His135, which is appropriate for accepting the incoming oxygen molecule. Based on the docking structure, the oxygen molecule was manually positioned to coordinate with the Fe center. The obtained structure was subjected to 20 ns MD simulation. From the calculated root-mean-square deviation (RMSD) of the protein (Figure S3), one can see that the system basically reached equilibrium after 11 ns, which indicates that a 20 ns MD simulation is sufficient to equilibrate the system. To analyze the flexibility and stability of the substrate coordination, the distance between the His81 and the S atom of substrate GSSH was described, as shown in Figure S4. It fluctuates strongly from 2 Å to 5 Å in the beginning, while further reaches equilibrium after 4 ns, which indicates a stable interaction between the substrate and His81 residue. According to the MD trajectory, ten snapshots at 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ns were optimized by QM/MM method. Then an average structure was derived from these ten structures and the RMSDs of these ten structures relative to the average one were calculated to locate a representative reactant model. As shown in Figure S5, these ten optimized structures are superposed well and the green one shows the geometry derived from 20ns. Some key coordinate distances and the average value are listed in Table S1. Both Figure S5 and Table S1 show that the optimized structure from the snapshot at 20ns can be considered as representative, which was used for the following QM/MM calculations. The structure of enzyme-substrate complex was obtained after further QM/MM optimizations. Figure S6 shows the overlay of the optimized geometries of model I (in cyan) and model II (in purple). As mentioned above, Model I refers to the small QM region, which was used to explore the spin state of reactant complex. And model II refers to the big QM region which includes all atoms involved in the chemical process. In general, the optimized structures on the two models are superposed well, which suggests that the Fe atom and its coordinated groups are basically unaffected by the surrounding residues. 8


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3.2. The spin states of enzyme-substrate complex Due to the coupling of Fe with O2, GSSH and the coordinated residues, there are four possible spin states, including the septet, quintet, triplet and singlet states. To determinate which state is the most stable one, we optimized the enzyme-substrate complex in the lowest-lying spin states at B1 level, and their single point energies were calculated at B2 level, which are shown in Figure 3. Although the optimized structures are similar, the relative energy of the quintet is lower than those of singlet (36.9 kcal/mol), triplet (12.9 kcal/mol) and septet (11.4 kcal/mol), respectively, indicating the quintet state of iron(II)-superoxo reactant to be the ground state.

Figure 3. Optimized geometries of the reactant (enzyme-substrate complex) in the lowest-lying spin states. The relative energies (kcal/mol) are given for the singlet (triplet) [quintet] {septet} spin states. Structures depicted here were optimized using model I at B1 level and energies were calculated at B2 level. All the distances are shown in angstroms. The lower-right corner is the overlays of the optimized geometries at different spin states. Table1 Spin density distribution for the GSSH-Fe-O2 complex for the lowest-lying triplet, quintet and septet states at B3LYP/B2 level. species 3

R R 7 R 5

Fe

Oa

Ob

Sa

Sb

Nsub

O2*

GSSH**

1.19 4.08 4.04

0.42 -0.28 0.70

0.47 -0.53 0.69

-0.06 0.24 0.16

-0.01 0.10 0.05

-0.01 0.12 0.11

0.89 -0.81 1.39

-0.08 0.46 0.32

*The total spin density of the dioxygen. **The total spin density of the substrate GSSH, including the Sa, Sb and Nsub atoms.

To understand the electron configuration of various spin states, we further calculated the spin densities and the orbitals. Table 1 shows the spin populations of Fe atom, GSSH and some key atoms. For the lowest-lying quintet state, one can see that the Fe atom and GSSH bear the spin density of 4.08 and 0.46, 9


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respectively, and the dioxygen bears the spin density of -0.81. For the septet state, the Fe atom has the similar spin density (4.04) as in quintet state, but the dioxygen and GSSH have the spin densities of 1.39 and 0.32, respectively. However, for the triplet state, the spin density of Fe atom is only 1.19, and that of the dioxygen is 0.89. On the basis of the data in Table 1, the valence electron configurations of triplet, quintet and septet states are obtained (Scheme 3), which may help us to understand the electron transfer during the catalytic reaction. For example, considering the first step of the reaction involves the formation of O-S bond, the right state of the reactant must be quintet, and the other two configurations (triplet and septet) disfavor the formation of O-S bond. Hence, the reactant complex can be described as GSS+•-Fe(II)-O2•.

Scheme 3. The valence electron configurations for the GSSH-Fe (II)-superoxo complex at the triplet, quintet and septet states. The valence electron orbital diagrams of quintet state are shown in Figure 4, in which the Fe-O bond is defined as the x axis. The metal 3d orbitals interact with the orbitals of neighboring ligands and split into a set of three π∗ obitals ( * * *) and a pair of two σ∗ orbitals (σ∗ σ∗ ). The * orbital represents the antibonding interaction between the metal orbital of Fe and the 2p orbitals of oxygen, whereas the * and * orbitals represent the antibonding interaction between the metal 3d orbitals and 2p orbitals of the substrate. Similarly, the two σ∗ orbitals represent the antibonding interactions between the mental 3d orbitals of Fe and 2p orbitals of substrate. Interestingly, the orbital sequence is unusual as compared to other non-heme metal-oxo calculations (76, 77). It is mainly caused by the different orbital interactions between the mental and the substrate. As for the oxygen orbitals, it was occupied by three electrons with π π . In conclusion, they give an orbital occupation of 10


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π∗ π∗ π∗ σ∗ σ∗ for Fe, and π π for dioxygen and a single spin-parallel electron for GSSH, as shown in Figure 4.

Figure 4. The valence electron orbital diagrams of the GSSH-Fe-O2 complex at the lowest-lying quintet spin state surface. To test the influences of different functionals on the energy of the reactant, a series of calculations were performed with BP86, BLYP, B3LYP, BVWN and TPSSh at B1 level. As shown in Table 2, all functionals predict the quintet to be the ground state, which is separated from other spin states at least 6.4 kcal/mol except for TPSSh functional. The average energy of singlet is 23.9, to which the B3LYP result is the closest. The energy orderings of triplet and septet are inconsistent, i.e., the energy of triplet is lower with BP86, BLYP and BVWN, while the energy of septet is lower with B3LYP and TPSSh. This fluctuation is reasonable by reason of high spin states are generally more stabilized with hybrid density functionals such as B3LYP and TPSSh (21, 66). In any case, all functionals agree that the quintet is the ground state. Therefore, the widely used density functional B3LYP will be chose to optimize the geometries in the following calculations. Table 2 Relative energies of the singlet, triplet, quintet and septet spin states of the enzyme-substrate complex calculated with different functionals at B1 level. All energies are in kcal/mol. Species 1

R

BP86

BLYP

B3LYP

BVWN

TPSSh

23.0

18.7

19.3

17.3

40.1

11


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3

R

6.7

7.2

12.2

6.4

17.2

5

R

0.0

0.0

0.0

0.0

0.0

7

R

7.3

8.0

9.1

7.9

2.1

As we known, the basis set of 6-31G(d,p) has been widely used for geometrical optimization of enzymatic system (78, 79). However, diffuse basis functions are often used for negatively charged system. To explore the influence of basis sets on the structures, we re-optimized enzyme-substrate complex at 6-31++G(d,P) level which includes polarization and diffuse functions. As shown in Figure S7, the key distances of the QM region with the two basis sets are almost identical. And the two optimized structures are high overlapped, which show that the larger basis set of 6-31++G(d,p) does not significantly improve the geometries. Thus, we chose 6-31G(d,p) for geometrical optimization and lager basis set of 6-311++G(2b,2p) for single-point energy calculations.

3.3. Determination of the reaction pathway On the basis of the ground quintet state of the reactant complex (GSS+•-Fe(Ⅱ)-O2•) and the designed reaction pathways in Scheme 2, we performed a series of QM/MM calculations using the larger QM region (model II). In Path_a, the cleavage of Oa-Ob of dioxygen and the formation of Ob-Sa was calculated to be a concerted step, undergoing a coplanar four-member ring (Fe-Oa-Ob-Sa) transition state. In Path_b, the dioxygen bond is kept while the distal oxygen Ob forms O-S bond with Sa atom, generating a twisted four-member ring intermediate (Fe-Oa-Ob-Sa), which then undergoes a transition state and a three-member ring intermediate (Oa-Ob-Sa), finally leading to the cleavage of dioxygen. In Path_c, the Oa atom instead of Ob forms O-S bond, also undergoing a series of three-member ring intermediates and transition states. For Path_a, the optimized geometries of involved species are displayed in Figure 5. In the first step from 5

R to 5IM1a, the distal oxygen Ob attacks the Sa atom of substrate to form Ob-Sa bond, in which the distance

between Ob and Sa decreases from 3.25 Å in 5R to 1.56 Å in 5IM1a via 2.11 Å in TS1a. Simultaneously, the distance of Oa-Ob bond increases from 1.32 Å in 5R via 1.37 Å in 5TS1a to 3.06 Å in 5IM1a (3.06 Å is not shown in Figure 5). In this step, the formation of Ob-Sa bond and the cleavage of dioxygen are concerted. Owing to the cleavage of Oa-Ob bond, the Fe-Oa bond length decreases to 1.64 Å in 5IM1a. In the second step, the Fe-Oa bond is broken to form the sulfite intermediate (5IM2a), in which the length of Fe-Oa increases from 1.64 Å to 2.11 Å. The potential energy profile of this pathway is shown in Figure 6. The first step was calculated to be rate-limiting with an energy barrier of 12.7 kcal/mol, and the second step 12


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corresponds to an energy barrier of 8.1 kcal/mol. The free energy barrier estimated from the experimental kinetic constant is 15.1 kcal/mol (3), which is basically in accordance with the calculated energy barrier. During the whole reaction in Path_a, the Fe atom keeps six coordinated. The changes of coordinated bond distances are displayed in Figure S8. In general, most of the distances are little changed, except the distances Fe-Oa and Fe-Sa which show somewhat fluctuation. For example, Fe-Oa distance is 2.01 Å in 5R, but it changes to1.64 Å in 5IM1a, and retunes to 2.11 Å in 5IM2a.

Figure 5. Optimized quintet spin state geometries for various species in Path_a with model II. All the distances are shown in angstroms. The dot black lines represent the coordination and hydrogen bonds. Some key distances are either listed in the figure directly, or shown at the upper right corner.

Figure 6. Energy profiles of the GSSH oxidation catalyzed by ETHE1 at B2 level in Path_a, Path_b and 13


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Path_c. The energy of the reactant at quintet state was set to zero. In addition, the NPA charges of key atoms are listed in Table S2. Clear changes can be found in Fe, Sa and Oa atoms. For instance, the charge of Fe atom changes from 1.49 in 5R to 1.34 in 5TS1a and 1.45 in 5

TS2a, and finally to 1.38 in 5IM2a. It is understandable because the bonding nature of Fe-O has been

changed during the reaction. Of course, the change of Fe-O bonding nature may result in the change of spin state of the species, and spin-state crossing may occur during the reaction, which will be discussed in the following section. Based on our calculations, the Fe atom is suggested to not only play role in fixing the substrate and the key residues but also act as a Lewis acid. The biggest change is the charge of Sa, which changes from -0.47 in 5R to 1.40 in 5IM2a due to the formation of Ob-Sa bond. Besides, the charge of Oa changes from -0.42 in 5R to -1.00 in 5IM2a. All these changes are consistent with the bond formation and broken processes in Path_a. The charges of O154, N79, N135, and Nsub atoms almost remain unchanged, implying a stable coordination with Fe atom. Path_b contains three elementary steps, including two intermediates IM1b and IM2b. The optimized structures of transition states and intermediates are shown in Figure 7 and the NPA charges of key atoms are listed in Table S3. In the first step, the distal oxygen Ob forms O-S bond with the Sa atom of GSSH while the O-O bond of dioxygen is kept, forming a four-member ring intermediate. The distance between Ob and Sa atoms decreases from 3.25 Å in 5R to 1.68 Å in 5IM1b, and the O-O bond length of dioxygen enlongates from 1.32 Å to 1.48 Å. It is the formation of Ob-Sa bond that leads to the weakness of O-O bond. The energy barrier of this step is 13.7 kcal/mol (Figure 6), which is slightly higher than that of the first step in Path_a. In the second step, the proximal oxygen Oa attacks the Sa atom to form the intermediate (5IM2b), which undergoes a high-energy three-member ring (Oa-Sa-Ob) transition state, corresponding to the energy barrier of 51.9 kcal/mol. By comparing the structures of 5TS2b and 5IM2b, we can see that the formation of the new Oa-Sa bond leads to the partial collapse of Ob-Sa, and the length of Ob-Sa increases from 1.68 Å in 5

IM1b to 2.66Å in 5IM2b. The third step corresponds to the cleavage of dioxygen Oa-Ob, which is

accompanied by the collapses of three-member ring (Oa-Sa-Ob) and the “real” formation of two O-S bonds (Oa-Sa and Ob-Sa), corresponding to an energy barrier of 27.7 kcal/mol. Owing to the collapses of three-member ring (Oa-Sa-Ob) and formation of two O-S bonds, this step is highly exdothermic by 75.2 kcal/mol. Similar to Path_a, Fe atom forms stable coordination with the nearby residues except the Sa atom of GSSH which gradually departs from the Fe atom owing to the formation of Oa-S bond (Figure S9). In conclusion, Path_b corresponds to very high energy barrier and can be ruled out. However, 5IM1b may 14


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transform to 5IM1a through another transition state 5TS2b′ with an energy barrier of 5.7 kcal/mol, as shown in Scheme 2 and Figure 6. In this step, the Oa-Ob bond of dioxygen is easily cleaved owing to the activation by Fe and the formation of Ob-Sa bond. The optimized geometry of 5TS2b′ is shown in Figure S10.

Figure 7. Optimized geometries of various species in Path_b with model II. All the distances are in angstroms. Dot line represents coordination or hydrogen bonding. Some key distances are either listed in the figure directly, or shown at the upper right corner. In Path_c, the reaction starts from the attack of Oa on the Sa atom of GSSH. The optimized structures of the transition states and intermediates are shown in Figure S11 and the NPA charges of key atoms are listed in Table S4. In the first step, the proximal oxygen Oa detaches from the Fe atom and forms O-S bond with the Sa atom of GSSH, generating the intermediate (5IM1c), which contains a three-member ring (Oa-Ob-Sa). The energy barrier of this step is calculated to be 23.9 kcal/mol (Figure 6), which is substantially higher than that in Path_a. The second step involves the formation of Ob-Sa bond. However, owing to the formation of Ob-Sa bond, the Oa-Sa is greatly weakened with its length elongates from 1.67 Å 5IM1c to 2.64 Å 5IM2c. In the third step, the Oa-Ob bond is broken to yield the sulfite intermediate (5IM3c), which corresponds to an energy barrier of 50.0 kcal/mol. Similar to Path_b, Path_c is also computed to be a high-energy pathway and can be ruled out. For comparison, we also constructed another reactant model (Model SI, Figure S12) in which only the S site of GSSH is deprotonated, whereas the NH group is in natural state considering the weak acidity of NH. 15


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Compared to Model II, all the QM atoms in Model SI kept unchanged except for a proton was removed from the NH. Then, to determinate the ground state of Model SI, we re-optimized the enzyme-substrate complex with four spin states, and the corresponding geometries are shown in Figure S13. Similar to Model II, the optimized structures at different spin states are highly overlapped. However, compared to Model II, the residue His81 almost has no hydrogen bonding interaction with the dioxygen molecule. The relative energy of the septet is lower than those of singlet (31.2 kcal/mol), triplet (9.6 kcal/mol) and quintet (6.0 kcal/mol), respectively, indicating the septet state of the reactant (7R′) to be the ground state. Thus, we explored Path_a′ with the septet state. The optimized reactant (R_a′) is displayed in Figure S14. It should be noted that the coordination numbers of Fe ion in 5R and 7R_a′ are different, which may be caused by the different protonation states of NH group. In 5R, the Fe is six-coordination in which both the distal S and N atoms of GSSH coordinate with the Fe ion with lengths of 2.48 Å and 2.13 Å, respectively. However, in R_a′, the distal S and the N atoms of GSSH only weakly interact with the Fe ion, with lengths of 3.81Å and 3.80 Å (not shown in Figure S14), respectively. In addition, both the O atoms of the dioxygen coordinate with the Fe ion with 2.03 Å and 2.13 Å. But any of them seems hard to transfer to the distal S atom owing to the strong interaction between them. The optimized geometries of involved species in Path_a′ are displayed in Figure S15, and the energy profile is shown in Figure S16. These results show that the proximal O is not easy to transfer to the distal S atom, which corresponds to an energy barrier of 23.9 kcal/mol. This result is consistent with the fact that the S ligand is essential for the Fe-O2 reactivity (80). Goldberg and his co-authors have also demonstrated that the binding of sulfur ligand with iron(II) complex are essential for both the reactivity and binding of O2 (80, 81). In other words, the binding of sulfur ligand to iron(II) can increase the reactivity of Fe-O2 species. This notion can be confirmed from the catalytic reaction of the CDO enzyme (21). In general, our calculation results support Mode II as the most reasonable reactant model, which shows similar complexation behavior of the Fe center of CDO. Furthermore, this result also agrees with the proposed mechanism that involves initial complexation of the GSSH cysteinyl-glycine amide nitrogen to the iron (1). We also attempted a proton shuttle mechanism, by which a FeO(H)O species is formed so that the Fe-O bond can cleavage more easily (22, 82). However, no suitable neighboring residue can donate a proton to the proximal O atom of Fe-O2. For example, we tried a mechanism by which the proton transfers from His81 to the proximal O atom of Fe-O2, nevertheless, the scanned energy is always increased, as displayed 16


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in Figure S17. Therefore, the calculated Path_a with Model II is the most possible reaction pathway based on our massive calculations.

3.4. Spin-state crossing and electronic characteristic of Path_a As described above, the most possible reaction pathway (Path_a) has been obtained by our QM/MM calculations. However, the intermediates might be more stable at other spin states because of the spin-crossover. Therefore, we re-calculated the intermediates and transition states at the spin state surfaces of triplet, quintet and septet with model II in Path_a at B2 level. The calculated potential energy profiles are shown in Figure 8. One can see that, the reaction takes place via multistate reactivity patterns on competing triplet, quintet and septet spin-state surfaces. Although the ground state for the reactant is quintet, a spin-state crossing is found upon the formation of the first intermediate (IM1a), i.e., the triplet state (3IM1a) is the ground state of IM1a. This crossing occurs with the cleavage of dioxygen through our geometrical optimization and spin density calculations. 3IM1a is more stable than 5IM1a and 7IM1a by 11.2 and 17.5 kcal/mol. For the second step, the spin-state crossing also occurs upon the formation of the intermediate IM2a. Thus, the optimal reaction pathway starts from 5R to 3IM1a via 5TS1a, then generates 7IM2a via 3

TS2a. The optimized geometries are given in Figure 9. Compared to Figure 5, the optimized structures

seem more reasonable. For example, in 7IM2a, the Sa-Sb bond length has been changed to 4.72 Å with the formation of Oa-S-Ob (SO2), whereas in 5IM2a the Sa-Sb bond length is 2.34 Å.

Figure 8. Energy profiles of the GSSH oxidation catalyzed by ETHE1 at three different spin states. The energy of the reactant at quintet was set to zero.

17


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Figure 9. Optimized geometries for various species in Path_a considering spin crossover. All the distances are in angstroms. Dot lines represent coordination or hydrogen bonding. Some key distances are either listed in the figure directly, or shown at the upper right corner. To further understand the spin-crossover, we now analyze the changes of electronic structures during the reaction, as shown in Figure 10. The NPA charges of key atoms and the spin density population of 5R, 5

TS1a, 3IM1a, 3TS2a and 7IM2a are listed in Table S5 and Table S6, respectively. The valence electron

orbital diagrams of 5R，5TS1a, 3IM1a, 3TS2a and 7IM2a are summarized in Figures S15-S19. The reaction starts with a spin-state crossing from the quintet spin state (5R) to the triplet spin state (3IM1a) with an electron transfer from π to substrate (GSS1), i.e., the β electron on the distal oxygen (Ob) pairs with the

α electron on sulfur (Sa) forming the Sa-Oa bond. This may occur from the formation of transition state (5TS1a). In 5TS1a, the orbital occupation is π∗ π∗ π∗ σ∗ σ∗ , which is different in orbital sequence compared to 5R, as shown in Figure S19 and Figure S18. Indeed, this electron transfer is accordant with the spin density calculations. As shown in Table S6, the spin density on O2 (sum of Oa and Ob) changes from -0.77 in 5R to 0.58 of Oa in 3IM1a, and the spin density on substrate (sum of Sa, Sb and Nsub) changes from 0.46 in 5R to -0.11 in 3IM1a. The intermediate (3IM1a) at triplet spin state is formed with an orbital occupation of π∗ π∗ π∗ σ∗ (Figure S20), in which the σ∗ refers to the antibonding orbital of Oa. With the transfer of the electrons, the Fe ion changes to trivalent in 3IM1a from divalent in 5R. In the second step, the single electron on Oa (σ∗ ) transfers to the substrate (GSS-O21) in 18


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7

IM2a via a transition state (GSS-O1) in 3TS2a. Simultaneously, rearrangement of the orbitals and the

electrons

on

the

Fe

ion

also

occurs,

producing

an

orbital

occupation

of

π∗ π∗ π∗ σ∗ σ∗ GSS-O21 (in 7IM2a) via a triplet state (3TS2a) with an orbital occupation of π∗ π∗ π∗ σ∗ GSS-O1, as shown in Figure S21 and Figure S22. In conclusion, our calculations predict a multistate reactivity pattern in Path_a. If these spin-state crossings are easy to occur, the reaction will proceed along the sequence of 5R, 5TS1a, 3IM1a, 3TS2a and 7IM2a.

Figure 10. Key intermediates and their orbital occupation of the dominant electronic configuration in Path_a considering spin-state crossover.

4. Conclusion In this work, the catalytic mechanism of ethylmalonic encephalopathy protein 1 (ETHE1) has been studied by using QM/MM calculations. The hETHE1-substrate complex was calculated to be the quintet state, which can be best described as GSS+•-Fe(Ⅱ)-O2•. Starting from GSS+•-Fe(Ⅱ)-O2• (5R), three possible reaction pathways have been explored. Our QM/MM calculations support Path_a as the minimum energy pathway. In the first step, the Oa-Ob is cleaved and the distal oxygen forms Ob-Sa bond with the substrate, corresponding to an energy barrier of 12.7 kcal/mol. In the second step, Fe-Oa bond is broken to form the sulfite intermediate with an energy barrier of 8.1 kcal/mol. Calculations on Path_b and Path_c have been identified to be high-energy pathways which will be unable to compete with Path_a. In Path_b, the nucleophilic attack of Ob is stereochemically and energetically feasible, however, the second step involves the cleavage of Fe-Oa to form a three-membered ring intermediate, corresponding to high energy barrier (51.9 kcal/mol). As for Path_c, the first step involves the nucleophilic attack of proximal oxygen (Oa), which is also unlikely to occur with a barrier of 23.9 kcal/mol. Our results are of significance for a better understanding of enzymatic catalysis in general. This is the first QM/MM investigation of the mechanism of HETH1, which might provide meaningful insight into the study of Ethylmalonic encephalopathy. 19


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Associated content Supporting Information Three figures with MD simulations; Fourteen figures with optimized geometries; five figures with valence electron orbital diagrams; Seven tables with NPA charges and spin densities; absolute QM/MM single-point energies and Cartesian coordinates of all the structures.

Author information Corresponding Author: [email protected]. Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21573127, 21373125).

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