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Mar 23, 2016 - Japan. ∥. Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata Gakuen-machi, Akita, Akita 010-8502, Japa...
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Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study Megumi Kayanuma,*,† Mitsuo Shoji,†,‡ Masafumi Yohda,§ Masafumi Odaka,∥ and Yasuteru Shigeta†,‡ †

Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan § Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan ∥ Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata Gakuen-machi, Akita, Akita 010-8502, Japan ‡

S Supporting Information *

ABSTRACT: The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from βTyr72 to the substrate via αSer113, and cleavage of the S−O bond of αCys114−SO− and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of βArg56K, which is one of the essential amino residues in the enzyme, we analyzed a βR56K mutant in which βArg56 was replaced by Lys. The results suggest that βArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S−O bond via a hydrogen bond with the oxygen atom of αCys114−SO−.

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INTRODUCTION Nitrile hydratases (NHases),1 which catalyze the hydration of nitriles to the corresponding amides, are one of the most widely used biocatalysts in the chemical industry.2−6 For example, nearly 25% of the world’s total production of acrylamide (95,000 tons per year) is provided via the biotechnological process. The efficient enzymatic hydration of nitriles is the preferred method compared with chemical synthesis because the reaction occurs under mild conditions (neutral to slightly basic pH at mild or low temperature) and achieves a high yield without forming any byproducts. NHases also have a potential application in environmental biotechnology as bioremediation of toxic nitriles from contaminated air, soil, and water systems.3−6 NHases are microbial enzymes that are involved in nitrile metabolism together with amidases, which catalyze the hydrolysis of amides to carboxylic acids and ammonia. They are composed of two subunits (α and β subunits) and have a unique active site structure that includes either a low-spin nonheme iron(III)7−9 or a non-corrinoid cobalt(III)10 ion coordinated by two deprotonated main-chain amide nitrogens, a side-chain sulfur of a cysteine (Cys−S−), and two posttranslationally modified cysteine sulfurs, i.e., a cysteine−sulfenic (Cys−SO−) and a cysteine−sulfinic (Cys−SO2−) group, which were shown to be deprotonated by Fourier transform infrared spectroscopy11 and a combination of electron paramagnetic © XXXX American Chemical Society

resonance, magnetic circular dichroism, low-temperature absorption spectroscopy, and density functional theory (DFT) calculations.12 It is known that the oxidation of the active site cysteine residues is essential for the catalytic activity of NHases.13 The possible role of the sulfenic moiety as a nucleophile was first proposed by Heinrich et al. based on the study of mimetic complexes of NHases.14 Several plausible catalytic mechanisms of NHases have been proposed.4,9,15−19 Recent experimental20,21 and theoretical 12,22,23 studies indicated the formation of a cyclic intermediate via the nucleophilic attack of the oxygen atom of Cys−SO− on the metal-coordinated substrate (Scheme 1), which had been first proposed by Hopmann in 2008 based on the quantum chemical studies of active site models of Scheme 1

Received: November 20, 2015 Revised: March 10, 2016

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The Journal of Physical Chemistry B NHases.24 However, the subsequent reactions involved in the production of the amide product and reproduction of the active site structure remain unclear. Hopmann put forward a reaction mechanism involving disulfide bond formation between αCys109−S− and αCys114−SO− concomitant with S−O bond dissociation and proton transfers from βTyr72 to the substrate via αSer113 followed by proton transfer from βArg56 to the substrate nitrogen, based on DFT calculations with active site models.22 Light et al. proposed a mechanism in which a water molecule directly attacked the S atom of the cyclic structure based on DFT calculations with active site models, in which both βTyr72 and βTyr37 were not included.12 At the transition state, the distance between the S atoms of αCys109− S− and αCys114−SO− was shortened, which was similar to the disulfide intermediate in Hopmann’s mechanism.22 Conversely, Yamanaka et al. mentioned that disulfide bond formation was unlikely because the distance between the S atoms of αCys109−S− and αCys114−SO− did not change in the timeresolved X-ray crystallography analysis of a mutant NHase containing Lys in place of βArg56 (βR56K), although βArg56 does not seem to be essential for the disulfide bond formation.21 In a previous theoretical study that used a hybrid quantum mechanics/molecular mechanics (QM/MM) method, the initial step of the catalytic mechanism of NHase and the effects of the protein environment on the reactions were analyzed.23 The most noteworthy difference between the theoretical results of the active site models18,25 and the QM/ MM model23 was that the proton transfer from βArg56 to the oxygen atom of αCys114−SO− that had been proposed in the former18,25 was not observed in the latter. This can be attributed to the weaker proton donor ability of βArg56 in the protein environment compared with the active site models. As βArg56 is one of the amino acid residues that is essential for the catalytic activity of NHases,26 there is no doubt that explicit treatment of the protein environment is desirable when analyzing the reaction mechanism of NHases theoretically. Furthermore, in the previous theoretical study based on the active site models mentioned above, the transition state for proton transfer from βArg56 to the substrate nitrogen, resulting in an intermediate with deprotonated βArg56, was almost identical in energy to that for the formation of the cyclic intermediate, which was the highest among the total reaction path.22 In the present study, we reexamined the catalytic mechanism of an Fe-containing NHase after the formation of the cyclic intermediate using the QM/MM method, to identify the most plausible reaction path for amide formation catalyzed by NHases and discuss the effect of the protein environment on the reaction. In addition, the role of βArg56, which is one of the essential amino residues in this protein, was examined by the analyses of a mutant NHase, βR56K.

Figure 1. Atoms included in the QM region are indicated as follows: hydrogen (white), carbon (gray), nitrogen (blue), oxygen (red), sulfur (yellow), and iron (purple).

βTyr37). The two arginine residues formed hydrogen bonds with the oxygen atoms of the post-translationally modified cysteine residues (αCys112−SO2− and αCys114−SO−), and two tyrosine residues formed a hydrogen-bond network with αSer113. Note that the water molecule formed a hydrogen bond with the backbone oxygen of αAla164 and was suggested to be involved in the catalytic reaction by Yamanaka et al.21 For QM calculations, DFT with B3LYP functional27,28 was used. LanL2DZ (Los Alamos ECP plus DZ) basis sets29 were employed for the iron atom, and 6-31G(d) basis sets30−32 were used for the other atoms during geometry optimizations, while 6-311++G(d,p) basis sets33,34 were used for nonmetal atoms in energy calculations at the optimized structures. The MM atoms were subjected to an AMBER-99 force field.35 Hydrogen link atoms were used for the QM/MM boundary. Nonbonded QM−MM interactions were calculated with a cutoff of 9 Å during geometry optimizations, while interactions of QM atoms with all MM charges were included in energy calculations. All calculations were performed using the NWChem program package (version 6.3).36 The initial coordinates were taken from the X-ray crystal structure of NHase from Rhodococcus erythropolis N771, which includes a water molecule at the sixth ligand site of the iron ion and a product molecule, trimethylacetamide, in the active site pocket, as determined at a resolution of 1.47 Å (PDB ID: 3A8O).37 After protonation and addition of 24 counterions of Na, the system was solvated in a cubic box of water with a 100 Å edge, and initial geometry optimization was performed at the QM/MM level. Subsequently, the trimethylacetamide and the water molecules were removed and an acetonitrile molecule, which is the smallest aliphatic nitrile, was placed in the active site as a substrate. The transition states were searched using a nudged elastic band (NEB) algorithm.38 Normal mode analyses of the QM region were performed for all the optimized structures, to confirm whether they were energy minima or transition states. In the present study, the electronic energy profile along possible reaction pathways was discussed. The relative energy was calculated with reference to that of the five-coordinated structure with an acetonitrile molecule in the active site (Figure 1). In the resting state, the sixth ligand is expected to be a water molecule as seen in X-ray crystal structures,21 though, in Fecontaining NHases in the dark, the water molecule is replaced

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THEORETICAL METHODS The atoms included in the QM region are shown in Figure 1. The QM region consists of an Fe(III) ion, an acetonitrile molecule (as the substrate), a water molecule located near the sulfenic sulfur, four metal-coordinated residues (αCys109−S−, αCys112−SO2− with the exception of the main-chain NH, αSer113, and αCys114−SO−) with adjacent main-chain atoms (CO of αVal108, NH of αSer110, and NH of αThr115), part of the side chains of two arginine residues (βArg56 and βArg141), and the side chains of two tyrosine residues (βTyr72 and B

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Figure 2. Preferred reaction mechanism analyzed in this study.

by a nitric oxide which is inactive and can be photoactivated.39 Green et al. analyzed the thermodynamic cycle of ligand exchange of the water for acetonitrile by DFT calculations with active site models and showed that the cost was about +30 kJ/ mol, in which the dissociated ligand existed in an aqueous solution.40 In the previous QM/MM calculation, the difference in electronic energy between the water- and acetonitrilecoordinated structures was rather small (3.92 kJ/mol), in which the released ligand was considered to stay in the active site of NHase. Therefore, the dissociated water molecule was stabilized by a hydrogen bond with αCys114−SO−.23 In both cases, the water-coordinate structure is expected to be more stable than the acetonitrile-coordinated one. Thus, including the effect of ligand exchange would shift the relative energies higher compared with the values referring the five-coordinated structure. On the other hand, if the water molecule, which was released from the iron ion, stays in the active site, it might be able to be involved in the catalytic reaction in place of the water molecule we used in the present study. As shown below, the cost of the relocation of the water molecule was not negligible, including a water molecule which was first coordinated to the iron ion would also lower the relative energies of the intermediates after water relocation.

Figure 3. Calculated electronic energy profile of NHase from the cyclic intermediate to the amide product. The energies are relative values to that of the initial 5-coordinated structure with an acetonitrile molecule in the active-site pocket shown in Figure 1.

hydrogen-bond network, in which the Nsubstrate−HαSer113, OαSer113−HβTyr72, and OβTyr72−HβTyr37 distances were 1.64, 1.56, and 1.68 Å, respectively. The activation barrier (TS-1, Figure 4a) for proton transfers from βTyr72 to αSer113 and from αSer113 to the substrate resulting in the protonated cyclic intermediate (I-2, Figure 5a) was 16.2 kJ/mol (Figure 3). βTyr72 was easily deprotonated, as it was stabilized by hydrogen bonds with βTyr37 and αSer113. Proton transfers from the tyrosine residue to the substrate via the serine residue was proposed by Holz and co-workers based on the pH and temperature dependences of the kinetic parameters of Cocontaining15 and Fe-containing16 NHases, though they

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RESULTS AND DISCUSSION Most Preferred Reaction Mechanism. As proposed in Hopmann’s theoretical study using active site models of NHase,22 disulfide bond formation between αCys109 and αCys114 occurred easily from the cyclic intermediate (I-1 → TS-1 → I-2 → I-3; Figures 2 and 3). In the cyclic intermediate (I-1, Figure S1a), the nitrogen atom of the substrate and the hydroxyl groups of αSer113, βTyr72, and βTyr37 formed a C

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Figure 4. Optimized geometries of (a) the transition state for proton transfers from βTyr72 to αSer113 and from αSer113 to the cyclic structure (TS-1), (b) the transition state for the formation of the imidic acid intermediate from the disulfide intermediate by direct water attack (TS-4), (c) the transition state for isomerization from the imidic acid intermediate to the amide product (TS-5), and (d) the amide product (P). For clarity, atoms mainly involved in the reaction or coordinated to the iron ion (i.e., the substrate, the water molecule, the hydroxyl groups of βTyr72 and αSer113, the sulfur atom of αCys109, the sulfinic group of αCys112, the backbone nitrogen atoms of αSer113 and αCys114, the sulfenic group of αCys114, and the iron ion) are shown with thick lines, and the other atoms are shown in thin lines.

Figure 5. Optimized geometries of (a) the protonated cyclic intermediate (I-2) and (b) the disulfide intermediate (I-3) of wildtype NHase and (c) the protonated cyclic intermediate (I-2_βR56K) and (d) the disulfide intermediate (I-3_βR56K) of the βR56K mutant. The bond length (in red in panel d) was fixed during geometry optimization.

distance was 3.38 Å) and the backbone carbonyl oxygen (S−O distance was 3.49 Å) of αThr162. In disulfide intermediate (I3), the latter came closer to the sulfur atoms of αCys109 and αCys114 (S−O distances were 3.28 and 3.26 Å, respectively). Such short S−O distances with the neighboring backbone carbonyl oxygen were also seen in an apo form of a Cocontaining NHase, which lacked the metal ion and formed a disulfide bond between the cysteine residues, and the corresponding S−O distances were 3.27 and 2.78 Å, respectively.42 Therefore, it seems that there was no surrounding component which might preclude the formation of disulfide intermediate. As subsequent reactions, we demonstrated a new mechanism in which a water molecule located near the sulfur atom of αCys114 came closer to the sulfur atom (I-4, Figure S1b) and attacked this atom, resulting in the formation of an imidic acid intermediate (I-5, Figure S1c). In this mechanism, the formation of the renewed S−O bond of αCys114 and the dissociation of the S−S bond between αCys109 and αCys114 occurred simultaneously. When the water molecule moved to a position proper to attack the sulfur atom of αCys114 (I-4, Figure S1b), the energy became higher than that of I-3 by 59.4 kJ/mol (Figure 3). The activation barrier (TS-4, Figure 4b) for the formation of an imidic acid intermediate was 14.2 kJ/mol (Figure 3), which is low enough to be overcome at room temperature. This mechanism is similar to that proposed by Light et al., except for the proton transfers from βTyr72 to the substrate via αSer113 before the water attack.12 On the other hand, Hopmann considered that the water attack on the sulfur atom of αCys114 occurred after the release of the amide product and the coordination of another water molecule to the iron center.22 In the mechanism, a proton transfer from βArg56 was involved in the formation of the amide. Both Hopmann’s

proposed mechanisms in which the tyrosine residue was deprotonated before the coordination of nitrile to the metal center. Hopmann et al. theoretically analyzed these mechanisms and suggested that the tyrosine residue could act as a general acid.41 The protonated cyclic intermediate immediately formed the disulfide bond between αCys109 and αCys114 (Figure 5b). A low-barrier transition state between the protonated cyclic intermediate and the disulfide intermediate obtained in the geometry optimization became lower in energy than that of the cyclic intermediate in the energy calculation, which used larger basis sets and included all interactions between QM atoms and MM charges. However, this transition state was not important, as it was much lower in energy than that of TS-1 (by about 45 kJ/mol in both calculations). When the nitrogen atom of the substrate was protonated, the S−O bond was elongated from 1.72 to 1.78 Å and the C−O bond was shortened from 1.44 to 1.37 Å, which facilitated the dissociation of the S−O bond and the formation of the S−S bond. In addition, charge neutralization of the cyclic intermediate may also contribute to the facilitation of disulfide bond formation, in which an electron was transferred from the S−O bond to the amide group of the intermediate (I-2 in Figure 2). We also checked the atoms around the sulfur of αCys109 to confirm that the protein environment would not be unfavorable to the formation of disulfide bond. In the crystal structure (PDB ID: 3A8O),37 the nearest atoms were a water molecule (S−O D

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latter by 15.6 kJ/mol in the wild type. This difference can be explained by the formation of a hydrogen bond between βArg56 and the oxygen atom of the coordinated CH3CONH intermediate in the disulfide structure (1.98 Å, Figure 5b), which might contribute to the stabilization of the dissociation of the S−O bond, accompanied by electron transfer from the S−O bond to the C−O bond (I-2 in Figure 2). In addition, as βArg56 pulled up the oxygen atom via the hydrogen bond, i.e., the S−O distance of αCys114 was longer in the wild type (2.53 Å, Figure 5b) than it was in the βR56K mutant (2.30 Å, Figure 5d) by 0.23 Å, and the N−Fe−Sαcys114 angle in the former was larger (97.2°, Figure 5b) than in the latter (92.6°, Figure 5d), the NH group of the CH3CONH intermediate became closer to the hydroxyl oxygen of αSer113 (i.e., the NH−O distance was 1.97 and 2.28 Å in the wild type and βR56K mutant, respectively). This might also contribute to the enhancement of the hydrogen bond between them in the wild type compared with the βR56K mutant. These synergetic effects of hydrogen bonds might make the formation of the disulfide bond easy during the catalytic reaction of NHases. When the water molecule moved to form hydrogen bonds with βLys56 and αCys114−SO−, the disulfide intermediate was obtained without any structural constraint (Figure S1d). The structure was similar to the disulfide intermediate after water relocation of wild-type NHase (Figure S1b), though the Sαcys114−Owater distance in panel d was longer (2.66 Å) than that in panel b. The disulfide intermediate was higher in energy than the protonated cyclic intermediate (Figure 5c) by 48.9 kJ/ mol. These results showed that the theoretical studies that showed the involvement of disulfide intermediate (previous22 and present studies) do not conflict with the results obtained by time-resolved crystallography of the βR56K mutant,21 in which the water molecule stayed at the initial position. It might also be possible that a new water molecule would come into the active site and assist the formation of the disulfide bond in βR56K. Even though, the disulfide structure of the βR56K mutant might be a short-lived intermediate, because the transition state for the water attack (TS-4) was low (i.e., only 14.2 kJ/mol higher in energy than the disulfide intermediate after water relocation (I-4)) in the wild-type NHase. Therefore, it is not contradicting that the formation of the disulfide bond was not seen in the experimental study of βR56K while theoretical studies of wild-type NHase suggested it. The analyses of the βR56K mutant resolved the apparently contradictory results between the theoretical studies (present and previous22), which suggested the involvement of the disulfide intermediate in the catalytic mechanism of NHases, and the time-resolved X-ray crystallography analysis of the βR56K mutant,21 in which no significant change in the distance between αCys109 and αCys114 was observed. As βArg56 was suggested to contribute to the disulfide bond formation, as shown above, the absence of disulfide intermediate in the experiment of the βR56K mutant is not a disproof of the formation of disulfide intermediate in the catalytic mechanism of NHase. Other Reaction Mechanisms: Proton Transfer from βArg56. As the subsequent reaction mechanism of the disulfide intermediate, Hopmann considered a proton transfer from βArg56 to the substrate nitrogen, resulting in the formation of the amide product.22 However, our QM/MM study showed that such an intermediate with a deprotonated βArg56 (Figure S3) was unstable, with a relative energy of

and Light’s mechanisms and the effects of proton transfers are discussed later in detail (see the sections Other Reaction Mechanisms: Proton Transfer from βArg56 and Other Reaction Mechanisms: Absence of Disulfide Bond Formation). After the formation of the imidic acid intermediate, the isomerization from the imidic acid to the amide product was catalyzed by the sulfenic acid moiety of αCys114−SOH, involving proton transfers from the imidic acid oxygen to αCys114−SOH and from αCys114−SOH to the imidic acid nitrogen (TS-5, Figure 4c), the activation barrier of which was 47.9 kJ/mol (Figure 3). It would be also possible that the imidic acid might be released from the enzyme and then isomerize to the amide, because the barrier for the isomerization reaction between the imidic acid and the amide supported by a water molecule was reported to be low (20 kJ/mol) in the previous theoretical study.18 If the water attack on the sulfur atom of αCys114 directly generated the amide product (I-4 → P), the activation energy was rather high (93.1 kJ/mol, i.e., the relative energy of the transition state was 76.7 kJ/mol). In the transition state (Figure S2), a proton was abstracted from the water molecule by the nitrogen atom of the CH3CONH intermediate, and the N−H− Owater distances were 1.43 and 1.14 Å, respectively. Conversely, in the transition state of the formation of the imidic acid (Figure 4b), a proton was abstracted from the water molecule by the oxygen atom of the CH3CONH intermediate, and the O−H−Owater distances were 1.28 and 1.13 Å, respectively. Comparing the transition state structures for the formation of the amide and for the imidic acid, it is revealed that the shorter distance for proton abstraction might explain the lower activation barrier in the latter case. The amide product (P, Figure 4d) remained higher in energy compared with the disulfide intermediate (I-3), by 26.1 kJ/mol (Figure 3), and the initial active site structure (Figure 1) was not completely regenerated, i.e., βTyr72 was deprotonated while αCys114−SO− was protonated. When a new water molecule would come in the active site, the dissociation of the generated amide from the iron ion and the coordination of the water molecule to the iron ion might lead to three-step sequential proton transfers from αCys114−SOH to the coordinated water molecule, from the water to αSer113, and from αSer113 to βTyr72, thus resulting in the regeneration of the initial active site structure of NHase. βArg56 Mutant. The disulfide intermediate was expected to occur easily, as mentioned above. However, the disulfide intermediate was not observed in the time-resolved crystallography of the βR56K mutant,21 although βArg56 did not seem to be involved in the disulfide bond formation. To clarify whether βArg56 plays a significant role in the disulfide intermediate, we examined the reactions in the βR56K mutant. When βArg56 was replaced by Lys, which has a shorter side chain than Arg and did not form a hydrogen bond directly with the oxygen atom of αCys114−SO−, the disulfide structure became unstable and changed to the protonated cyclic intermediate (Figure 5c) during geometry optimization. To analyze the cause of the instability of the disulfide intermediate in the βR56K mutant, we optimized the structure by imposing a constraint on the distance between the sulfur atoms of αCys109 and αCys114 (fixed at 2.28 Å), which was the same as that in the disulfide intermediate of wild-type NHase (Figure 5b). In the βR56K mutant, the disulfide structure (Figure 5d) was higher in energy than was the protonated cyclic intermediate, by 19.2 kJ/mol, whereas the former was more stable than the E

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the direct water attack on the disulfide intermediate, in which the water molecule can come closer to the nitrogen atom because there was space between the sulfur atom and the oxygen atom of the cyclic structure (Figure S1b), proton abstraction by the nitrogen atom (Figure S2) was much less favorable than that by the oxygen atom (Figure 4b) because of the shorter distance of proton transfer in the latter reaction, as mentioned above. Thus, in the case of the direct water attack on the cyclic intermediate, proton abstraction by the oxygen atom of the cyclic structure (Figure S5b) seems more likely than that by the nitrogen atom. This mechanism corresponded to that reported by Light et al.12 The relative energy of the generated intermediate (Figure S5c) was +47.2 kJ/mol, which was much higher than that of the transition state for proton transfer to the cyclic intermediate from βTyr72 via αSer113 (TS-1, Figure 4a), i.e., −22.1 kJ/mol. Therefore, the direct water attack on the cyclic intermediate is improbable. In the DFT calculations by Light et al. using active site models, the intermediate was less unstable, i.e., the intermediate was higher in energy than the cyclic intermediate by 41 kJ/mol.12 The difference might be explained by a hydrogen bond network around the attacking water molecule (with side chain of βArg56, main chain oxygen of αAla164, and a water molecule) when it moved to react with the sulfur atom of αCys114 as shown in the present study. In addition, the reaction barrier for this mechanism was reported to be about 60 kJ/mol,12 which was higher compared with that for proton transfer from βTyr72 followed by the formation of the disulfide intermdiate (TS-1).

115.5 kJ/mol, which is much higher than that of the disulfide intermediate (by 191.3 kJ/mol). Thus, this mechanism is not plausible in the protein environment. Here, we explain the difference between the results obtained using active site models and our QM/MM analyses. The main reason for the difference shown between the results of the present and previous22 theoretical studies may be the weaker proton donor ability of βArg56 in the protein environment compared with that in the active site model, which treated the protein environment approximately using the polarizable continuum model (PCM). As discussed in the previous QM/MM study of the initial step of the catalytic mechanism of NHase,23 the explicit protein environment, including the negatively charged amino acid residues located around βArg56, may weaken the proton donor ability of βArg56 compared with that observed in the active site model. For example, in the crystal structure of NHase (PDB ID: 3A8O),37 the shortest distances between the nitrogen atoms of the guanidine group of βArg56 and the oxygen atoms of the carbonyl groups of βAsp53 and αGlu165 are 4.39 and 4.70 Å, respectively. There is also a positively charged amino residue near βArg56, i.e., αArg167, and the shortest NαArg167−NβArg56 distance is 3.42 Å, although αArg167 also forms a hydrogen bond with αGlu165 with the shortest NαArg167−NαGlu165 distance of 3.36 Å. Therefore, in total, the protein environment around βArg56 contributes to the stabilization of the positive charge on βArg56. In addition, the molecular surface of this structure obtained from the eF-site (electrostatic surface of functional site) database43 indicates that polarization of the whole protein might also stabilize the positive charge on βArg56. Figures S4c and S4d shows the electrostatic potentials and hydrophobic properties of the βArg56 side (right side) and the αCys114 side (left side) of NHase (Figure S4a), respectively. The more negative protein environment of the βArg56 side compared with the αCys114 side (i.e., coordination site) might also hamper the proton transfer from βArg56 to the coordinated substrate. Another reason for the difference in the reaction mechanism between active site models and the QM/MM system might be that the side chain of βArg56 was not able to change its orientation freely to form hydrogen bonds with the coordinated intermediate due to interactions with surrounding amino residues and water molecules that had not been described by the active site models. In the intermediate structure containing the deprotonated βArg56 (Figure S3), the position of βArg56 shifted toward the amino group of the amide product that was coordinated to the metal center. For example, the distances between the deprotonated ω-nitrogen atom of βArg56 and the iron ion in the disulfide intermediate (Figure 5b) and that in the intermediate with the deprotonated βArg56 (Figure S3) were 5.96 and 4.08 Å, respectively. Such a shift disturbed the hydrogen bond network around βArg56 and caused destabilization. Other Reaction Mechanisms: Absence of Disulfide Bond Formation. Yamanaka et al. proposed a mechanism without the formation of a disulfide intermediate.21 In this mechanism, the nitrogen atom of the cyclic intermediate abstracts a proton from the water molecule, which then attacks the sulfur atom of αCys114 (Figure S5a). However, proton abstraction by the nitrogen atom seems unlikely, because the water molecule attacks the sulfur atom from the opposite side of the nitrogen atom. Therefore, the proton has to be transferred over a long distance. In fact, even in the case of

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CONCLUSION We proposed a reaction path of an Fe-containing NHase subsequent to the formation of the cyclic intermediate (Scheme 1) using the QM/MM method, as shown in Figure 2. As suggested by Hopmann,22 the disulfide intermediate (I-3, Figure 5b) was formed easily from the cyclic intermediate (I-1, Figure S1a), and a reaction mechanism without the formation of the disulfide intermediate (Figure S5) was shown to be unlikely. However, the subsequent mechanism differed from that proposed by Hopmann. The formation of the imidic acid intermediate via a direct attack of the water molecule on the sulfur atom of αCys114 (I-5, Figure S1c) was preferred compared with the amide production via proton transfer from βArg56 (Figure S3). This might be because βArg56 was a weaker proton donor in the protein environment compared with the active site models, as discussed in the previous QM/ MM study;23 therefore, the intermediate with the deprotonated βArg56 was unstable. The formation of the amide via a direct water attack on the sulfur atom of αCys114 (Figure S2) was also shown to be less favorable than the formation of the imidic acid intermediate. The imidic acid was isomerized to the amide, i.e., the final product (P, Figure 4d), which was catalyzed by the sulfenic acid group of αCys114, or would occur outside the enzyme. In the final structure of our analyses, βTyr72 was deprotonated, whereas αCys114−SO− was protonated, which would be recovered to the initial active site structure when a water molecule would come into the active site and be coordinated to the iron ion, followed by proton transfers from αCys114−SOH to the water molecule, from the water molecule to αSer113, and from αSer113 to βTyr72. By comparing the reactions of wild-type NHase and βR56K mutant (Figure 5), we have shed light on the function of βArg56, which is one of the amino acid residues that are F

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The Journal of Physical Chemistry B essential for the catalytic activity of NHases.26 βArg56 played an important role in the formation of the disulfide intermediate (I-3, Figure 5b). Lys has a shorter side chain compared with Arg and was not able to form a hydrogen bond directly with the oxygen atom of the cyclic structure. The results showed that it is not discrepant that the formation of disulfide intermediate was not observed in the experimental study of βR56K, while theoretical studies of wild-type NHase suggested it.

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(12) Light, K. M.; Yamanaka, Y.; Odaka, M.; Solomon, E. I. Spectroscopic and Computational Studies of Nitrile Hydratase: Insights Into Geometric and Electronic Structure and the Mechanism of Amide Synthesis. Chem. Sci. 2015, 6, 6280−6294. (13) Murakami, T.; Nojiri, M.; Nakayama, H.; Odaka, M.; Yohda, M.; Dohmae, N.; Takio, K.; Nagamune, T.; Endo, I. Post-Translational Modification is Essential for Catalytic Activity of Nitrile Hydratase. Protein Sci. 2000, 9, 1024−1030. (14) Heinrich, L.; Mary-Verla, A.; Li, Y.; Vaissermann, J.; Chottard, J.-C. Cobalt(III) Complexes with Carboxamido-N and Sulfenato-S or Sulfinato-S Ligands Suggest that a Coordinated Sulfenate-S is Essential for the Catalytic Activity of Nitrile Hydratases. Eur. J. Inorg. Chem. 2001, 2001, 2203−2206. (15) Mitra, S.; Holz, R. C. Unraveling the Catalytic Mechanism of Nitrile Hydratase. J. Biol. Chem. 2007, 282, 7397−7404. (16) Rao, S.; Holz, R. C. Analyzing the Catalytic Mechanism of the Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1. Biochemistry 2008, 47, 12057−12064. (17) Hashimoto, K.; Suzuki, H.; Taniguchi, K.; Noguchi, T.; Yohda, M.; Odaka, M. Catalytic Mechanism of Nitrile Hydratase Proposed by Time-Resolved X-ray Crystallography Using a Novel Substrate, tertButylisonitrile. J. Biol. Chem. 2008, 283, 36617−36623. (18) Hopmann, K. H.; Guo, J. D.; Himo, F. Theoretical Investigation of the First-Shell Mechanism of Nitrile Hydratase. Inorg. Chem. 2007, 46, 4850−2856. (19) Hopmann, K. H.; Himo, F. Theoretical Investigation of the Second-Shell Mechanism of Nitrile Hydratase. Eur. J. Inorg. Chem. 2008, 2008, 1406−1412. (20) Martinez, S.; Wu, R.; Sanishvili, R.; Liu, D.; Holz, R. The Active Site Sulfenic Acid Ligand in Nitrile Hydratases Can Function As a Nucleophile. J. Am. Chem. Soc. 2014, 136, 1186−1189. (21) Yamanaka, Y.; Kato, Y.; Hashimoto, K.; Iida, K.; Nagasawa, K.; Nakayama, H.; Dohmae, N.; Noguchi, K.; Noguchi, T.; Yohda, M.; et al. Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile. Angew. Chem., Int. Ed. 2015, 54, 10763−10767. (22) Hopmann, K. H. Full Reaction Mechanism of Nitrile Hydratase: A Cyclic Intermediate and an Unexpected Disulfide Switch. Inorg. Chem. 2014, 53, 2760−2762. (23) Kayanuma, M.; Hanaoka, K.; Shoji, M.; Shigeta, Y. A QM/MM Study of the Initial Steps of Catalytic Mechanism of Nitrile Hydratase. Chem. Phys. Lett. 2015, 623, 8−13. (24) Hopmann, K. H. Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanisms and Selectivities. Ph.D. Thesis, Royal Institute of Technology, April 2008. (25) Dey, A.; Chow, M.; Taniguchi, K.; Lugo-Mas, P.; Davin, S.; Maeda, M.; Kovacs, J. A.; Odaka, M.; Hodgson, K. O.; Hedman, B.; et al. Sulfer K-edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-Heme Iron Active Site. J. Am. Chem. Soc. 2006, 128, 533−541. (26) Piersma, S. R.; Nojiri, M.; Tsujimura, M.; Noguchi, T.; Odaka, M.; Yohda, M.; Inoue, Y.; Endo, I. Arginine 56 Mutation in the β Subunit of Nitrile Hydratase: Importance of Hydrogen Bonding to the Non-Heme iron center. J. Inorg. Biochem. 2000, 80, 283−288. (27) Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. (28) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Function of the Electron Density. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785−789. (29) Hay, P. J.; Wadt, W. R. Ab Initio Effective Core Potentials for Molecular Calculations. Potentials for K to Au Including the Outermost Core Orbitals. J. Chem. Phys. 1985, 82, 299−310. (30) Hehre, W. J.; Ditchfield, R.; Pople, J. A. Self-Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian-Type Basis Set for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 1972, 56, 2257−2261.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b11363. Optimized structures, reaction mechanisms, molecular surface of NHase, and complete refs 21, 25, and 26 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-29-853-6284. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work has been supported by “Interdisciplinary Computational Science Program” at the Center for Computational Sciences, University of Tsukuba, Japan.

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REFERENCES

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DOI: 10.1021/acs.jpcb.5b11363 J. Phys. Chem. B XXXX, XXX, XXX−XXX