Theoretical Study of the Mechanism of the Nonheme Iron Enzyme

Article pubs.acs.org/IC

Theoretical Study of the Mechanism of the Nonheme Iron Enzyme EgtB Wen-Jie Wei,† Per E. M. Siegbahn,‡ and Rong-Zhen Liao*,† †

Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education, Hubei Key Laboratory of Bioinorganic Chemistry and Materia Medica, Hubei Key Laboratory of Materials Chemistry and Service Failure, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China ‡ Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-10691 Stockholm, Sweden S Supporting Information *

ABSTRACT: EgtB is a nonheme iron enzyme catalyzing the C−S bond formation between γ-glutamyl cysteine (γGC) and N-αtrimethyl histidine (TMH) in the ergothioneine biosynthesis. Density functional calculations were performed to elucidate and delineate the reaction mechanism of this enzyme. Two different mechanisms were considered, depending on whether the sulfoxidation or the S−C bond formation takes place first. The calculations suggest that the S−O bond formation occurs first between the thiolate and the ferric superoxide, followed by homolytic O−O bond cleavage, very similar to the case of cysteine dioxygenase. Subsequently, proton transfer from a second-shell residue Tyr377 to the newly generated iron−oxo moiety takes place, which is followed by proton transfer from the TMH imidazole to Tyr377, facilitated by two crystallographically observed water molecules. Next, the S−C bond is formed between γGC and TMH, followed by proton transfer from the imidazole CH moiety to Tyr377, which was calculated to be the rate-limiting step for the whole reaction, with a barrier of 17.9 kcal/mol in the quintet state. The calculated barrier for the rate-limiting step agrees quite well with experimental kinetic data. Finally, this proton is transferred back to the imidazole nitrogen to form the product. The alternative thiyl radical attack mechanism has a very high barrier, being 25.8 kcal/mol, ruling out this possibility. isopenicillin synthase (IPNS),24 EgtB,8 and the ovothiol biosynthesis enzyme OvoA.25 EgtB and OvoA catalyze similar reactions using different substrates.25−28 OvoA preferentially uses histidine and cysteine as the substrates to generate a C−S bond at the imidazole Cδ position. Consequently, these two enzymes show different regioselectivity for C−S bond formation. The X-ray crystal structure of EgtB from Mycobacterium thermoresistibile has been solved in three different forms, namely, the apo form, in complex with iron and the TMH substrate, and in complex with manganese and the two natural substrates γGC and TMH at 1.98 Å resolution, with the active site for the third one shown in Figure 1.14 In the active site, the metal is hexacoordinated and ligated by three histidines (His51, His134, and His138), the two substrates (via a sulfide of γGC and an imidazole nitrogen of TMH), and a water molecule. This water molecule has been proposed to be displaced by an oxygen molecule prior to substrate oxidation.14 A second-shell residue, Tyr377 forms a hydrogen bond with the water molecule. In addition, two positively charged residues, Arg90 and Arg87, form hydrogen bonds with the substrate γGC. Furthermore, several additional water molecules form a hydrogen bonding network interacting with the two substrates.

1. INTRODUCTION Ergothioneine, first isolated from the ergot fungus Claviceps purpurea by Tanret in 1909,1 is a thiolhistidine betaine derivative involved in detoxifying heavy metal ions and scavenging reactive oxidative species generated from oxidative stress. Ergothioneine is synthesized only by certain fungi and the Actinomycetales bacteria. By using an ergothioneinespecific transporter, humans absorb ergothioneine from food and enrich it in many parts of our body.2−7 The biosynthetic genes of ergothioneine in Mycobacterium smegmatis were identified in 2010, including five enzymes, namely, EgtA, EgtB, EgtC, EgtD, and EgtE.8 EgtB catalyzes the critical C−S bond formation at the imidazole Cε position between γglutamyl cysteine(γGC) and N-α-trimethyl histidine (TMH) using O2 as the oxidant (Scheme 1). EgtB belongs to the mononuclear nonheme iron dioxygenase family. For the typical dioxygenases, the first-shell ligand framework is composed of two histidine residues and one carboxylate, being either glutamate or aspartate.9−11 However, there exist several dioxygenases with distinct ligand binding motifs.12 For example, quercetin dioxygenase consists of three histidines and a glutamate,13 while EgtB, cysteine dioxygenase, and the acetylacetone-cleaving enzyme Dke1 possesses a threehistidine structural motif.14−16 There are several mononuclear nonheme iron enzymes known to catalyze C−S bond formation,17−22 namely, cysteine dioxygenase (CDO),23 © 2017 American Chemical Society

Received: January 6, 2017 Published: March 9, 2017 3589

DOI: 10.1021/acs.inorgchem.6b03177 Inorg. Chem. 2017, 56, 3589−3599

Article

Inorganic Chemistry Scheme 1. Reaction Catalyzed by EgtB

is followed by O−O bond cleavage to generate a sulfoxide FeIVO intermediate.25 Then the sulfoxide attacks the imidazole Cε with its hydrogen transferred to the oxo moiety, producing the final product. In the alternative mechanism B, the thiyl radical, which is generated by intramolecular electron transfer from the thiolate to the ferric superoxide, attacks the imidazole Cε first, with the hydrogen transferred to the peroxide moiety facilitated by Tyr377.28 This leads to the formation of a thioester, which then undergoes sulfoxidation to generate the final product. In the present work, a quantum chemical cluster approach was used to investigate the reaction mechanism of EgtB. With a model of the active site designed on the basis of the crystal structure (PDB entry 4X8D),14 density functional theory (DFT) was used to calculate the potential energy profiles for different reaction pathways. This kind of approach has been successfully applied to the study of various classes of enzymes,29−33 including a number of nonheme mononuclear iron dixoygenases,34−48 heme enzymes,49−59 and diiron enzymes.60−63

Figure 1. X-ray structure of the active site of EgtB from Mycobacterium thermoresistibile complex with γ-glutamyl cysteine and N-α-trimethyl histidine (coordinates taken from PDB entry 4X8D). Mn is replaced by Fe in the active site.14

The reaction mechanism of EgtB and OvoA remains elusive, and two different mechanistic scenarios have been proposed, depending on whether S−O or S−C bond formation takes place first (Scheme 2).14,25−28 During the reaction, an oxygen molecule first binds to the ferrous ion, leading to the formation of a ferric superoxide complex. Then the reaction diverges into two different mechanisms. In mechanism A, the ferric superoxide attacks the thiolate to form an S−O bond, which

2. COMPUTATIONAL DETAILS A model of the active site of EgtB was built based on the X-ray structure of the wild-type EgtB in complex with the two substrates (PDB ID 4X8D).14 A comparison of this structure with the other available crystal structures has been made (Figure S1 in the Supporting Information), and they show very high structural consistence. The

Scheme 2. Suggested Two Different Mechanisms for EgtB

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Inorganic Chemistry model (Figure 2) consists of an iron ion along with its first ligands, His51, His134, His138, the two substrates, and an oxygen molecule. In

polarization effects of the protein environment on the active site model were considered by performing single-point calculations on the optimized structures using the SMD68 solvation model method. The dielectric constant was chosen to be 4, which is widely applied for the modeling of the enzyme surroundings. Previous studies on several different classes of enzymes suggested that the solvation effects decrease very quickly when the size of the active model reaches 150− 200 atoms, and the choice of the dielectric constant becomes unimportant.69−72 Analytic frequency calculations were carried out at the same level of theory as the geometry optimizations to obtain zero-point energies (ZPE) and to further verify the nature of the various stationary points. Several atoms (labeled in the red circle in Figure 2) at the periphery of the active site model were kept fixed to their X-ray positions during geometry optimization, in order to mimic the steric constraints imposed by the protein matrix. With acetylene hydratase as an example,73 we have shown that the coordinate error of the starting Xray structure, roughly estimated by varying constraints, has a quite small effect on the calculated potential energy profile, especially when the resolution of the structure used is better than 2.0 Å (1.98 Å for EgtB in this case).

3. RESULTS AND DISCUSSION 3.1. Mechanism A: Sulfoxidation Followed by S−C Bond Formation. We first consider the mechanism in which the sulfoxidation takes place first, and this pathway is reminiscent of the nonheme iron enzyme cysteine dioxygenase.23,74 The structure of the active site with the two substrates and the O2 molecule bound (React) was optimized and is shown in Figure 2 (for the core part of the full model, see Figure 3). The optimized structure agrees quite well with the experimental crystal structure, and the RMSD for the metal− ligand distances is 0.13 Å (Table S1). During O2 coordination to the ferrous ion, one electron is transferred from the metal to the O2 moiety to generate a ferric−superoxide complex in which the O2 moiety preferentially binds to the iron in an endon fashion.34−47,74−77 Electron transfer from the substrate instead of the iron to the oxygen molecule has also been found by Roy and Kästner in the nonheme iron enzyme salicylate dioxygenase.78 The metal and the superoxide centers can interact in either a ferromagnetic or an antiferromagnetic fashion. This leads to in total six possible electronic states considering that the ferric ion can be in a low-spin (1α), an intermediate-spin (3α), or a high-spin (5α) state. The calculated energies for these electronic structures are shown in Table 1. The septet was calculated to be the most stable one, followed by the quintet (+1.4 kcal/mol relative to the septet), in which the high-spin ferric ion (spin density of 4.10) antiferromagnetically interacts with the superoxide (spin

Figure 2. Optimized structure of the active site model of EgtB. Atoms marked with red circles were fixed at their X-ray structure positions during the geometry optimizations. addition, five important second-shell residues, Asp48, Gln55, Arg87, Arg90, and Tyr377, were also included. Furthermore, seven crystallographically observed water molecules were added in the model. As shown in Figure 2, one water molecule (W1) forms two hydrogen bonds with Arg87 and TMH while another water molecule (W2) is hydrogen bonded to Asp48, Arg90, and γGC. The remaining five water molecules form a hydrogen bonding network between TMH and Tyr377, and they play an important role in the proton transfer during the reaction (vide infra). Hydrogen atoms were added manually, and the amino acids were typically truncated at their αcarbon atoms and saturated with hydrogen atoms, except for Arg87 and Arg90, which were represented by N-methyl-guanidine. The full model of the TMH substrate was used, while the γGC substrate was truncated at its α-carbon. The model is thus composed of 191 atoms and has a total charge of +1. All density functional calculations were carried out using the Gaussian09 program64 with the B3LYP-D3 functional, which includes Grimme’s empirical D3 dispersion correction (with the default D3 damping function).65,66 Geometry optimizations were performed using the SDD67 pseudopotential for Fe, the 6-311+G(d) basis sets for S, and the 6-31G(d,p) basis sets for C, N, O, and H. To obtain more accurate energies, single-point calculations were performed on the optimized structures using larger basis sets 6-311+G(2d,2p) for all elements except Fe, for which the SDD pseudopotential was used. The

Figure 3. Optimized structures during the first step. Energies are given in kcal/mol relative to Reactseptet. Distances are given in Angstroms, and Mulliken spin densities are shown in red italic. For clarity, only the core of the model is shown. For full model, see Figure 2. 3591


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and singlet barriers were found to be very high. At TS1, the nascent S−O1 bond is 2.40 Å, which is significantly shorter from 3.30 Å in React. The spin densities on S, O1, and Fe are 0.60, −0.49, and 3.84, respectively. The opposite spin densities on S and O1 substantially facilitate the development of a σ bond between S and O1. TS1 has an imaginary frequency of 186.8i cm−1, which mainly corresponds to the S−O1 bond formation. Int1 is a quintet and lies at −13.7 kcal/mol relative to React. At Int1, the S−O1 and Fe−S distances are 1.70 and 2.72 Å, respectively. The following O−O bond cleavage takes place via TS2 (Figure 5) with a very facile barrier, being only 2.7 kcal/mol, and this step was calculated to be exothermic by as much as 21.6 kcal/mol. TS2 has an imaginary frequency of 491.2i cm−1, with the vibrational mode corresponding to mainly O1−O2 bond cleavage. At TS2, the O1−O2 distance is 1.79 Å. The spin densities on O1 and O2 are −0.23 and 0.26, respectively, indicating a homolytic O−O bond cleavage. These spins are further reinforced at the resulting intermediate Int2, in which the spin density on the S−O1 moiety becomes −0.78 and 0.64 on O2. The electronic structure of Int2 can be interpreted as a hybrid state of RSO−−FeIVO/RSO•−FeIIIO, with the latter one formed by one-electron transfer from the RSO− moiety to the ferryl center. The S−O1 bond length decreases from 1.70 Å at Int1 to 1.62 Å at TS2 and further to 1.53 Å at Int2, indicating the formation of an SO double bond during the O−O bond cleavages. From Int2, the iron−oxo moiety can abstract a proton from the adjacent residue Tyr377, leading to the formation of Int3. In the quintet and triplet states, this step was found to be barrierless, and the barrier in the singlet state was calculated to be only 1.0 kcal/mol relative to Int2. In Int3, a tyrosine anion is formed, as evidenced by little spin density on the phenolate moiety. This pathway is different from cysteine dioxygenase, in which the FeIVO moiety further oxidizes the sulfoxide to generate the cysteine sulfinic acid product.23,74 The difference stems from the presence of a tyrosine residue, which reacts with the FeIVO moiety with a very low barrier. There are two possible pathways starting from Int3. First, the sulfur can attack the imidazole Cε (C1) directly via TS4′ (see Supporting Information Figure S2), which is coupled with proton transfer from C1 to the tyrosine anion. This step has a barrier of 25.7 kcal/mol, which is much less favorable compared with the alternative pathway. In this pathway, the imidazole NH group gets deprotonated coupled with partial electron transfer to the iron center, which previously has been proposed by Gauld and co-workers on the basis of thermodynamics calculations.79 Here we show that this process is both thermodynamically and kinetically very feasible. Tyr377 functions as a general base to abstract a proton from the imidazole, which is facilitated by two water molecules as found in the crystal structure. The structures for the proton transfer transition state TS4 and the resulting intermediate Int4 are shown in Figure 5. The barrier for this step in the quintet state was calculated to be 5.4 kcal/mol relative to Int3triplet. TS4 has an imaginary frequency of 309.7i cm−1, which mainly corresponds to proton transfer from N1 to O3. At TS4, the distances of N1−H4 and O5−H4 are 1.39 and 1.13 Å, respectively. Besides, the bond lengths of O4−H3 and O3−H2 are 1.52 and 1.71 Å, respectively. At Int4, the total spin density on the imidazole ring is only 0.12, suggesting that its electronic structure is better described as an imidazole anion with quite small radical character.

Table 1. Relative Energies (in kcal/mol) and Spin Densities on Fe and O for Different Spin States of React spin

singlet(1α1β) triplet(1α1α) triplet(3α1β) quintet(3α1α) quintet(5α1β) septet

relative energy (kcal/mol)

Fe

O1

O2

S

7.9 9.5 10.1 7.9 1.4 0

1.15 1.04 3.38 3.08 4.1 4.03

−0.62 0.57 −0.68 0.61 −0.58 0.7

−0.43 0.39 −0.52 0.47 −0.28 0.61

0 0 0 −0.22 0.43 0.32

density of −0.86). The ferromagnetically coupled quintet state (3α on Fe and 1α on superoxide) lies at +7.9 kcal/mol above the septet. In addition, the ferromagnetically coupled and antiferromagnetically coupled triplet states were calculated to be 9.5 and 10.1 kcal/mol, respectively, higher than the septet. Furthermore, the broken-symmetry open-shell singlet has an energy of 7.9 kcal/mol relative to the septet, while the energy of the closed-shell singlet is 23.7 kcal/mol higher. These results are quite similar to those for many other iron-dependent dioxygenases,23,34−37,39,41−44,47,76−78 except for cysteine dioxygenase.23,74 QM and QM/MM calculations by de Visser and co-workers showed that for cysteine dioxygenase the open-shell singlet is the ground state, and the triplet and quintet states are higher in energy.23,74 The reason for this major difference could come from the different coordination environment of the iron. In cysteine dioxygenase, the amino group and the thiolate group of the cysteine substrate are connected by two carbon atoms, while the imidazole and the thiolate moieties of the two substrates are separated in EgtB. From React, the thiolate can attack the distal oxygen of the superoxide via TS1, which is coupled with one-lectron transfer to the ferric center, generating a ferrous peroxysulfur (FeIIIOOS) intermediate Int1. This mechanism is similar to the one for cysteine dioxygenase23,74 and has also been suggested for EgtB and OvoA by Gauld and co-workers.79 In their work, a 2His/1Asp ligand framework was used as the crystal structure of EgtB and was not available at that time. Consequently, only thermodynamics for certain steps was considered, while transition states and barriers were not calculated.79 The optimized transition state TS1 is displayed in Figure 3. TS1 is a quintet, and the barrier was calculated to be only 7.8 kcal/mol (Figure 4) relative to React. The triplet

Figure 4. Calculated energy profile (in kcal/mol) for the suggested mechanism (from React to Int4). 3592


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Figure 5. Optimized structures for the second, third, and fourth steps. Energies are given in kcal/mol relative to React. Distances are given in Angstroms, and Mulliken spin densities are shown in red italic.

Next, the C−S bond formation between γGC and TMH proceeds via TS5 in the quintet state (Figure 6), which is coupled with one-electron transfer to the ferric ion, generating a ferrous complex Int5. The barrier was calculated to be 10.9 kcal/mol relative to Int4, and this step is endothermic by 6.0 kcal/mol (Figure 7). The singlet and triplet states have much higher barriers. During C−S bond formation, the spin densities on S and O1 vanish, and its value on Fe also decreases from 4.07 to 3.75. At TS5, the critical S−C1 distance is 2.48 Å, which is further decreased to 2.00 Å at Int5. After S−O bond formation, the imidazole ring dissociates slightly from the iron center, with a Fe−N distance of 2.36 Å at Int5. From Int5, a proton transfer from C1 to N1 on the imidazole ring leads to the formation of the final product. This reaction takes place in a stepwise manner with the help of Tyr377 and two water molecules. The proton is first transferred to one water molecule (O4) via TS6 to generate a hydronium ion intermediate Int6. The calculated barrier for TS6 is 11.9 kcal/ mol relative to Int5 and 17.9 kcal/mol relative to Int4. TS6 was characterized by an imaginary frequency of 1353.7i cm−1, corresponding to proton H5 transferring from C1 to O3 and H2 from O3 to O4. At TS6, the critical C1−H5 and O3−H5 distances are 1.40 and 1.24 Å, respectively. The final proton transfer from the hydronium ion to the imidazole nitrogen is barrierless, and the whole reaction is exothermic by 49.6 kcal/ mol. The reaction energy for the nonenzymatic reaction using ethanethiol and 4-methyl-1H-imidazole as the model substrates

(Scheme 3) has been considered. The reaction in water solution (ε = 80) was calculated to be exothermic by 71.1 kcal/ mol (68.3 kcal/mol using ε = 4). This suggests that the process for the release of the two products and the binding of two new substrates and oxygen molecule is exothermic by 21.5 kcal/mol (from −49.6 kcal/mol in Figure 7 to −71.1 kcal/mol). Consequently, the product release and substrate binding are not likely contribute to the total barrier. To summarize, the reaction mechanism suggested from the present calculations is shown in Scheme 4, and the potential energy profile obtained for the entire reaction is shown in Figures 4 and 7. It can be seen that TS6 is the rate-limiting step for the whole reaction, with a total barrier of 17.9 kcal/mol. The experimental kinetic studies on EgtB and OvoA gave rate constants in the range of 0.03−9.87 s−1,14,25−28 which can be converted into barriers in the range of 16.1−19.5 kcal/mol using classical transition state theory. Our calculations are thus in good agreement with experimental kinetic data. Since a proton transfer is involved in the rate-limiting transition state TS6, we also calculated the deuterium kinetic isotope effect (KIE) for this reaction mechanism. This KIE value was simply derived by recalculating the zero-point energies using the mass of deuterium in place of proton. When the TMH substrate imidazole CεH was replaced by CεD, the KIE was calculated to be 5.7. When only protons of the seven crystal water molecules were replaced by deuteriums, the KIE was estimated to be 3.5. The tunneling effect was not 3593


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Figure 6. Optimized structures for the fifth, sixth, and final step in the quintet state. Energies are given in kcal/mol relative to React. Distances are given in Angstroms, and Mulliken spin densities are shown in red italic.

this enzyme. It should be mentioned that the deuterium KIE on the imidazole has been measured to be around 1.0 for OvoA.25 This suggests that OvoA should have a different rate-limiting step without involving hydrogen transfer. The exact mechanism and the reason for the different regioselectivity for OvoA cannot be elucidated at the present stage as the crystal structure of OvoA is not available. 3.2. Mechanism B: Thiyl Radical Attack Followed by Sulfoxidation. In the alternative thiyl radical attack mechanism, two different pathways (pathway B and B′) have been located (Scheme 5), with the energy profiles shown in Figure 8. In this mechanism, the quintet state is always preferred and thus presented here (for the singlet and triplet energy profiles, see Supporting Information Figures S3 and S4). Different from mechanism A, the reaction starts with a hydrogen atom transfer from Tyr377 to the ferric superoxide, and the optimized structures of the transition state B-TS1 and the resulting intermediate B-Int1 are shown in Figure 9. The barrier was calculated to be 9.1 kcal/mol in the quintet state, which is slightly higher than that for S−O bond formation in mechanism A (7.8 kcal/mol for TS1). Importantly, this step is endothermic by 2.7 kcal/mol, while it is exothermic by 13.7 kcal/mol for the S−O bond formation. B-TS1 has an imaginary

Figure 7. Calculated energy profile (in kcal/mol) for the suggested mechanism (from Int4 to Prod).

included here, and it should further increase the KIE value. In addition, the inclusion of the tunneling effect should decrease the total barrier slightly. Further experimental studies are needed to verify our calculated KIE data and also the proposed mechanism for EgtB, as we are not aware of any KIE study on 3594


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Inorganic Chemistry Scheme 3. Nonenzymatic Reaction Using Model Substrates

Scheme 4. Suggested Mechanism of EgtB on the Basis of Calculations

Scheme 5. Alternative Mechanism via Thiyl Radical Attack

frequency of 1238.1i cm−1, corresponding to H1 transfer from O3 to O1. At B-TS1, the key distances of O1−H1 and O3−H1 are 1.10 and 1.35 Å, respectively. After the hydrogen atom transfer, a tyrosyl radical (total spin density of −1.0) is formed at B-Int1, and this radical is antiferromagnetically coupled with the high-spin ferric center to form a quintet species. From B-Int1, there are two possible pathways. First, the thiolate can perform a radical attack on the imidazole ring upon one-electron transfer to the ferric center, generating a tetrahedral intermediate. As shown in Figure 8, the barrier for this step (B-TS2, Figure 9) is very high, being 32.4 kcal/mol relative to React. In addition, formation of the S−C bond via

this mechanism is endothermic by as much as 27.3 kcal/mol. These results can safely rule out pathway B as a viable option. In the alternative pathway B′, proton-coupled electron transfer takes place via B′-TS2, in which Tyr377 abstracts a proton from the imidazole NH group, coupled with oneelectron transfer from the peroxide anion to Tyr377. This leads to the formation of a ferrous hydroperoxyl radical intermediate B′-Int2, which lies at +15.8 kcal/mol relative to React. In B′Int2, the spin density on the hydroperoxyl radical is −0.84, while it is 0.15 on the imidazole ring. This partial radical character on the imidazole moiety could facilitate the subsequent S−C bond formation. It should be pointed out that in mechanism A the proton transfer from the imidazole to 3595


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choice of functional. It is found that M06-D3 also favors pathway A (Figures S5 and S6), thus giving the same mechanistic conclusion. The energy difference between TS1 and B-TS3′, which determines the reaction pathway, was calculated to be 12.5 kcal/mol. However, M06-D3 somewhat overestimates the barriers for pathway A as compared with the experimental kinetic data, as the total barrier was calculated to be 23.6 kcal/mol.

4. CONCLUSION In the present paper, the reaction mechanism of EgtB has been investigated using density functional calculations. Two experimentally suggested mechanisms are given depending on whether S−O bond formation or S−C bond formation takes place first. On the basis of our calculations, we proposed a reaction mechanism with S−O bond formation prior to S−C bond formation as presented in Scheme 4, which is energetically more feasible. The reaction starts with the binding of an oxygen molecule to the ferrous center in an end-on fashion to generate a ferric superoxide complex. Next, S−O bond formation takes place in the quintet state to form ferrous thiyl peroxide intermediate, with a barrier of 7.8 kcal/mol. This is followed by the homolytic O−O bond cleavage in the quintet state to form an RSO−− FeIVO intermediate, associated with a barrier of only 2.7 kcal/mol. These two steps are reminiscent of cysteine dioxygenase, which uses a similar mechanism for the first oxygenation of cysteine. Subsequently, proton relays from Tyr377 to the FeIVO moiety, followed by further proton transfer from the TMH imidazole to Tyr377, facilitated by two crystal water molecules. This proton transfer results in partial radical character on the anionic imidazole, which helps the following critical S−C bond formation via radical coupling. This S−C bond formation step has a barrier of 10.9 kcal/mol in the quintet state, generating a tetrahedral intermediate. Finally, a tautermerization of this intermediate leads to the formation of

Figure 8. Energy profile (in kcal/mol) for the thiyl radical attack mechanism in the quintet state.

the FeIVO moiety (from Int2 to Int4) is exothermic by 3.8 kcal/mol (Figure 4), while it is endothermic by 13.1 kcal/mol from B-Int1 to B′-Int2 (Figure 8). This large difference stems from the different oxidation power as well as proton affinity of FeIVO (mechanism A) and FeIII−superoxide (mechanism B). Importantly, this extra 15.8 kcal/mol has to be overcome for the following S−C bond formation in pathway B′. The associated barrier for the thiyl attack (B′-TS3) was calculated to be 25.8 kcal/mol (15.8 + 10.0 kcal/mol), and the formation of the tetrahedral intermediate B′-Int3 is endothermic by 8.1 kcal/mol. Compared with the sulfur oxidation step (TS1) in mechanism A, the barrier for pathway B′ is 17.0 kcal/mol higher, and thus, this pathway can be discarded as well. In this case, it is very important that the relative barriers for TS1 and B-TS3′ should be used to analyze whether pathway A or pathway B′ is favored, rather than comparing TS6 and B-TS3′. Single-point calculations using the M06-D380 functional have also been performed to check the sensitivity of the results to the

Figure 9. Optimized structures for pathway B in the quintet state. Energies are given in kcal/mol relative to Reactseptet. Distances are given in Angstroms, and Mulliken spin densities are shown in red italic. 3596


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Figure 10. Optimized structures during the second and third steps of pathway B′ in the quintet state. Energies are given in kcal/mol relative to React. Distances are given in Angstroms, and Mulliken spin densities are shown in red italic.

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the final product via a stepwise proton transfer from the imidazole Cε to its neighboring nitrogen, facilitated by Tyr377 and two water molecules. Proton transfer from the imidazole carbon to Tyr377 turns out to be the rate-limiting step for the whole reaction, with a total barrier of 17.9 kcal/mol in the quintet state, in good agreement with experimental kinetic data. The triplet and singlet states have much higher barriers. Very importantly, a proton transfer is involved in the rate-limiting step, and the deuterium KIE for the TMH imidazole CεH/CεD was calculated to be 5.7, which requires further experimental studies to verify our predication. For the alternative thiyl radical attack mechanism, S−C bond formation between a thiolate and a neutral imidazole has a barrier of 32.4 kcal/mol, while the barrier decreases to 25.8 kcal/mol when the imidazole NH group gets deprotonated by Tyr377. Compared with mechanism A, this mechanism can be ruled out as its barrier is 7.9 kcal/mol higher and thus kinetically less favorable.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21503083) and startup funding from Huazhong University of Science and Technology.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b03177.

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REFERENCES

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Structures, energy profiles, and coordinates (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Rong-Zhen Liao: 0000-0002-8989-6928 Notes

The authors declare no competing financial interest. 3597


Article

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Theoretical Study of the Mechanism of the Nonheme Iron Enzyme

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