Base Mechanism to the Hydrolysis of Phosphate Triester Promoted

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Base Mechanism to the Hydrolysis of Phosphate Triester Promoted by the Cd2+/Cd2+ Active site of Phosphotriesterase: A Computational Study Marcelo A. Chagas,† Eufrásia S. Pereira,† Marina P. B. Godinho,† Júlio Cosme S. Da Silva,*,†,‡ and Willian R. Rocha*,† †

LQC-MM: Laboratório de Química Computacional e Modelagem Molecular Departamento de Química, ICEx, Universidade Federal de Minas Gerais 31270-901 Pampulha, Belo Horizonte, Minas Gerais, Brazil ‡ GQC: Grupo de Química Computacional Instituto de Química e Biotecnologia, IQB, Universidade Federal de Alagoas Campus A. C. Simões, 57072-900 Maceió, Alagoas, Brazil S Supporting Information *

ABSTRACT: In the present work, density functional theory (DFT) calculations at the B3LYP/6-31+G(d) and including dispersion effects were used to investigate the hydrolysis of paraoxon, using a cluster model of the active site of Cd2+/Cd2+phosphotriesterase (PTE) from Pseudomonas diminuta. The mechanism proposed here consist of (i) Exchange of the coordinated water molecule and coordination of the substrate to the more solvent exposed Cdβ center in monodentate fashion, (ii) protonation of the μ-hydroxo bridge by the uncoordinated water molecule and in situ formation of the nucleophile, (iii) formation of a pentacoordinate intermediate with significant bond breaking to the leaving group and bond formation to the nucleophile, and (iv) protonation of the Asp301 residue and restoration of the active site through the coordination of another water molecule of the medium. The water molecules initially coordinated to the active site play a crucial role in stabilizing the transition states and the pentacoordinate intermediate. The reaction takes place in a two-step (AN + DN) mechanism, with energy barriers of 12.9 and 1.9 kcal/mol for the first and second steps, respectively, computed at the B3LYP-D3/6-311++G(2d,2p) level of theory, in excellent agreement with the experimental findings. Dispersion effects alone contribute to diminish the energy barriers as much as 26%. The base mechanism for the Cd2+/Cd2+-PTE proposed here, in conjunction with the agreement found with the experimental energetic value for the energy barrier, makes it a consistent and kinetically viable mechanistic proposal for the hydrolysis of phosphate triesters promoted by the Cd2+ substituted PTE enzyme.

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with either Cd2+, Co2+, Ni2+, or Mn2+ with the restoration of the full catalytic activity.9 The remarkable enhancement of the hydrolysis of organophosphates catalyzed by the wild-type PTE can be exemplified with paraoxon (diethyl 4-nitrophenyl phosphate), which is a potent acetylcholinesterase inhibitor, derived from the insecticide parathion10 and as potent as the nerve agent sarin.11 Aubert, Li, and Raushel9 measured the kinetic rate constant for the hydrolysis of paraoxon by wild-type PTE as kcat = 2300 ± 60 s−1 at 298 K, which can be translated into a free-energy barrier of 12.9 kcal/mol. The alkaline hydrolysis of paraoxon in aqueous solution takes place with a second-order rate constant of 7.5 × 10−2 M−1 s−1 at 298 K with a free energy barrier of 18.9 kcal/mol. Therefore, the catalytic efficiency of PTE in the hydrolysis of paraoxon approaches the

INTRODUCTION Phosphate triesters have been employed as agricultural pesticides, insecticides, and chemical warfare nerve agents.1−4 These organophosphate pesticides act on the nervous system, inhibiting acetylcholinesterase, and human exposure to these compounds can lead to brain damage. Therefore, their use as pesticides is under intense debate.5 In fact, their use in agriculture is banned in some countries. Due to the high stability of these compounds in the environment and their malign effects, there is a great interest in developing bioremediation technologies to facilitate the degradation of organophosphate contaminants including the disposal of chemical weapons. Phosphotriesterease (PTE) enzyme from Pseudomonas diminuta is particularly attractive as a biocatalyst because it can hydrolyze a large number of phosphate triesters.6−8 This enzyme, in its native form, has an active site containing divalent zinc metal cations. The two native Zn2+ ions can be substituted © XXXX American Chemical Society

Received: February 7, 2018

A

DOI: 10.1021/acs.inorgchem.8b00361 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry limits of diffusion,12 and the high specificity of PTE makes paraoxon the best substrate for mechanistic studies. The mechanistic aspects involved in the enzymatic hydrolysis of organophosphates is of great interest, due to the potential application in bioremediation, and has been the subject of intense research.13,14 The catalytic mechanism proposed for the phosphotriesterase enzyme, from P. diminuta, using different experimental techniques suggests the following: (i) The nucleophilic attack is directed at the phosphorus center rather than at the leaving group.6 (ii) An associative mechanism has significant changes in bond order to both phosphoryl and phenolic oxygen of the leaving group at the transition state.12 (iii) The participation of the μ-hydroxo bridge is as the nucleophile for the enzymatic hydrolysis of paraoxon.15,16 However, organophosphate-degrading enzymes from Agrobacterium radiobacter (OpdA) exhibit more flexibility in their mechanism of action.17 PTEs from P. diminuta and OpdA share more than 90% of sequence identity.18 They have the same metal-binding amino acids, and the only significant variation for catalysis is found in three residues in the substrate binding pocket at positions 254 (Arg/His), 257 (Tyr/His), and 272 (Phe/Leu) of OpdA/PTE from P. diminuta, respectively. Interestingly, despite the high similarity, these two enzymes exhibit significant variations in reaction mechanism and substrate specificity. The mechanism proposed for PTE from P. diminuta suggests the coordination of the substrate to the β metal ion more exposed to the solvent and subsequent hydrolysis initiated through the attack of the hydroxide molecule bridging the two metallic centers.9 On the basis of their crystallographic analyses of bacterial Fe2+/Zn2+ OpdA, Jackson et al.20 have shown that the μ-hydroxo bridge is not a nucleophile but acts by assisting the deprotonation of the nucleophilic water molecule terminally coordinated to the αmetal which initiates the hydrolysis reaction. Studies with biomimetic compounds also shows the α metal coordinated OH−/water as the nucleophile for the reaction.21 Schenk and co-workers17 found that for the Zn2+ and Cd2+ derivatives of OpdA only one relevant protonation equilibrium (pKa ∼ 4−5) was found consistent with μ-hydroxo acting as nucleophile. However, the authors also found for the Co2+ derivative of OpdA that two protonation equilibria (pKa1 ∼ 5 and pKa2 ∼ 10) were identified as relevant for catalysis suggesting that a terminal hydroxide acts as the nucleophile. Schenk and coworkers22 have also demonstrated that in OpdA there is an extensive hydrogen bond network that connects the substrate binding pocket to the metal center, involving three amino acids (Arg254, Tyr257, and Phe272) which is responsible for the substrate specificity and mechanistic flexibility. In the homologous enzyme Cd2+/Cd2+-PTE this hydrogen bond network is not present once the His254 and His255 residues are present instead of Arg254 and Tyr257. In principle, this lack of the hydrogen bond network in Cd2+/Cd2+-PTE makes the mechanism less influenced by the residues of the second coordination sphere and more dependent on the local chemical environment around the active site, especially in the beta site. Thus, although PTE and OpdA share ∼90% of sequence identity, the difference with respect to these key residues around the active site can yield considerable variation regarding to their catalytic efficiency and selectivity. Therefore, these experimental findings show that the mechanism involved in the organophosphate degradation by PTEs is pH and metal composition dependent.

Computational studies have confirmed some mechanistic proposals for PTEs. However, most of the theoretical studies on the mechanism of triesters hydrolysis by PTEs have been focused on the native Zn2+/Zn2+-phosphotriesterase, although it is worth mentioning that other classes of triesterases containing different transition metal ions have also recently been the subject of computational studies.23−25 This can in part be attributed to the lack of force field parameters to describe the active site of PTEs, substituted by Cd2+, Co2+, Ni2+, or Mn2+, in QM/MM calculations and that the closed-shell electron configuration of the Zn2+/Zn2+-PTE makes this system more attractive for full quantum mechanical or cluster model calculations. Using a cluster model of the active site of Zn2+/ Zn2+-PTE enzyme, composed of 82 atoms, Chen, Fang, and Himo26 investigated the hydrolysis reaction of methylparaoxon and obtained a two-step mechanism with formation of a pentacoordinate intermediate. The rate-determining step was found to have an energy barrier of 10.8 kcal/mol, at the B3LYP/6-311++G(2d,2p) level of theory, for the direct attack of the bridging hydroxide to the substrate, comparing favorably with the experimental value of 12.9 kcal/mol. The ability of the bridging OH− to act as base was investigated by Kim et al.27 adding a water molecule to the Znα center and using the same cluster approach. The authors found that the water molecule does not stay coordinated to the Znα, which prefers to adopt pentacoodinated geometry. The water molecule instead prefers to interact with the bridging OH− ligand, which acts as base, deprotonating the water molecule and in turn attacking the substrate. In contrast to the previous work, the authors obtained a single-step mechanism with energy barrier computed as 17.3 kcal/mol at the same level of theory. Therefore, the difference in energy barriers shows that the bridging OH− acting as nucleophile may be the preferential pathway. Wong and Gao28 investigated the hydrolysis of paraoxon by Zn2+/Zn2+-PTE treating the whole enzyme by means of hybrid QM/MM calculations and including the protein dynamic effects treated by molecular dynamics simulations (QM/MM/MD). The authors showed the importance of the second amino acid residue shell, not included in the previous cluster model studies. In their mechanism, the His254 residue is protonated and loses the proton to the solvent as the leaving group departs from the active site. His254 also acts as base, activating and deprotonating the water nucleophile. The product was found to remain protonated and coordinated to only the Znβ center. The authors obtained a mechanism with a single transition state. However, a transition state plateau in the potential of mean force coordinate was found, similar in nature to the intermediate state obtained with the cluster model.26 The computed energy barrier of 18.3 kcal/mol, at the B3LYP// AM1:MM level, was ∼6 kcal/mol higher than the value derived from the experimental rate constant for the wild-type PTE.8 It is important to mention here that Pedroso et al.19 have shown that the second sphere amino acid in position 254 (Arg in OpdA and His in PTE from P. diminuta) plays a crucial role in modulating the substrate preference and binding of these enzymes. In the case of OpdA, position 254 is occupied by an arginine amino acid, involved in an extensive hydrogen bond connecting the substrate binding pocket to the active site which is associated with the substrate specificity. Therefore, these amino acids have a different role from that found by Wong and Gao for the Zn2+/Zn2+-PTE. Second-sphere effects have also been demonstrated to play an important role in the catalytic B


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Inorganic Chemistry

Figure 1. (A) Crystallographic structure of the Cd2+/Cd2+ phosphotriesterase from P. diminuta34 with zoomed view of the active site showing the residues and water molecules coordinated to the metallic centers. (B) Structural model of the active site of Cd2+/Cd2+ phosphotriesterase used in this work. Carbon atoms marked with asterisk were kept frozen during the geometry optimizations to take into account the steric effects of the enzyme environment.

nature of the ions on the active site of PTEs may have a significant impact on the reactivity toward hydrolysis of triesters.9,32 For instance, Aubert, Li, and Raushel,9 have shown that the Cd2+/Cd2+-PTE has a catalytic efficiency around 5 times greater than the efficiency of the wild type enzyme. This catalytic enhancement promoted by Cd2+ substitution is also observed in OpdA19 and in the glycerophosphodiesterase enzyme from Enterobacter aerogenes (GpdQ).33 Therefore, a mechanistic study, at the atomistic level, is of great interest and can provide important information on the effects of the different metallic ions on the mechanism and catalytic performance of the PTEs. In this article, we performed a systematic study of the hydrolysis of the organophosphate paraoxon (diethyl 4nitrophenyl phosphate), promoted by Cd2+/Cd2+-phosphotriesterase. Mechanistic details were obtained by full quantum mechanical calculations, at the density functional theory (DFT) level,35 using a structural model of the active site of the Cd2+/ Cd2+-PTE enzyme. Structural aspects of the active site and comparison with wild-type Zn2+/Zn2+-PTE are performed and their interactions with the substrate paraoxon analyzed. As we shall see, the interaction of the substrate with the active site is

performance of biomimetic compounds as was demonstrated recently by Camargo et al.29 with biomimetic compounds of purple acid phosphatase. Mo and co-workers30 employed QM(PM3)/MM/MD simulations to investigate the hydrolysis of paraoxon by Zn2+/Zn2+-PTE, using reoptimized PM3 parameters for the phosphorus atom. According to the authors, the substrate binds to the more solvent exposed Znβ center, and the μ-hydroxo ligand acts as the nucleophile. Interestingly, the authors found that the rate-limiting step is associated with the distortion of paraoxon to access the bridging OH− nucleophile and the subsequent SN2 reaction proceeds quickly. The energy barrier was computed as 18.7 kcal/mol which was also high compared with the experimental data available. Later, Tuñoń and coworkers31 studied the hydrolysis of paraoxon by Zn2+/Zn2+PTE and in aqueous solution, using hybrid QM(AM1-d)/MM/ MD simulations. The authors found that in both aqueous solution and in the active site of PTE the hydrolysis of paraoxon follows an ANDN or associative mechanism with the bridging hydroxide ligand acting as nucleophile. The barrier was computed as 10.1 kcal/mol, which is only 2.8 kcal/mol below the experimental value. In addition, studies have shown that the C


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Inorganic Chemistry

Figure 2. Crystallographic structures of the active site of Cd2+/Cd2+ phosphotriesterase (A) and Zn2+/Zn2+ phosphotriesterase (B), highlighting the main structural features of the active sites. Bond distances are given in Å. Therefore, the cluster model of the active site presents a total of 74 atoms, 370 electrons, +1 charge, and a singlet spin state. Geometry optimizations were performed at the DFT level35 using the hybrid GGA B3LYP functional36,37 including the D3 dispersion correction proposed by Grimme and co-workers38 employing the full electron 6-31+G(d) basis set39−41 for C, H, O, P, and N atoms. The core electrons of Cd and Zn were treated with LANL2DZ pseudopotential42 and the valence electrons with the LANL2DZ associated double-ξ basis set. Restraints were applied to the atomic coordinates of the terminal carbon atoms of the model, as shown in Figure 1, in order to take into account the steric effects of the protein environment. The electrostatic effects of the broader protein environment were modeled using the SMD continuum solvation model43 with a dielectric constant equal to 4. Frequency calculations were carried out at the optimized structures to characterize the stationary points and to obtain the thermodynamic properties within the harmonic oscillator and rigid rotor approaches. In order to verify the applicability of our cluster model to describe the structural features of the active site, we compared the full DFT calculations on the cluster model with a hybrid QM/MM calculation of the ONIOM44,45 type, performed on the entire Cd2+/Cd2+-PTE enzyme. The QM part of the ONIOM calculation were described by the same B3LYP/6-31+G(d)/LANL2DZ level of calculation em-

quite different for these two PTEs. In contrast with the accepted mechanism for the Zn2+/Zn2+-PTE, which involves the μ-hydroxo species as nucleophile, we propose a new mechanism for the hydrolysis of paraoxon promoted by the Cd2+/Cd2+-PTE enzyme in which the nucleophile species is formed in situ, through the activation of a water molecule present on the active site of the enzyme. To the best of our knowledge, this is the first computational study on the mechanism of Cd2+ substituted phosphotriesterase.

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COMPUTATIONAL DETAILS

The active site model of the Cd2+/Cd2+-PTE enzyme was built based on the high-resolution crystal structure reported by Benning et al.34 (Protein Databank code: 1JGM). As can be seen in Figure 1, the five amino acids of the first shell (His55, His57, His201, His230, and Asp301) directly coordinated to the Cd2+ ion are included in the model. The metals are bridged by the carboxylate group of lysine (Lys169) and a μ-hydroxo ligand. The two metal ions are designated as α and β. The β metal is more solvent-exposed and coordinated by two water molecules. The histidine residues were modeled as methyl imidazole, Lys169 as carboxylated methylamine and Asp301 as acetate. D


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Inorganic Chemistry ployed in the model system and, the MM part was described with the UFF force field.46 The QM part included the same residues and water molecules of the cluster model. Hydrogen atoms were used as link atoms connecting the two parts. Also, in order to verify the flexibility of the cluster model to describe the structural features of the active site, geometry optimization with a larger model was also carried out. In this model, the entire coordinating amino acids were included, generating a model with 142 atoms, 550 electrons, and +1 charge, with the same restraints to the terminal carbon atoms. Given that the positions of some atoms were maintained frozen during the optimization procedure, the full Hessian matrix was obtained from a standard frequency calculation, but then only the subblock corresponding to the N atoms whose positions were optimized was mass-weighted and diagonalized using the block-Hessian procedure.47 This analysis was carried out using an in house software developed in the group of Prof. Jeremy Harvey.48 Mechanistic insights were obtained through intrinsic reaction coordinate (IRC) calculations,49 performed with the Gonzalez− Schlegel second-order path50 starting from the optimized transition states structures, with a step length of 0.10 (a.m.u.)1/2·Bohr. The final structures of the IRC calculations were then fully optimized and characterized with frequency calculations at the same level of theory. Aiming at obtaining better energetic results, single-point energy calculations at the B3LYP-D3/6-31+G(d)/LANL2DZ optimized structures were carried out with the extended basis set 6-311+ +G(2d,2p) and including the solvent effects with the SMD continuum model. All geometry optimization and frequency calculations were performed using the C.01 version of the Gaussian 09 program.51

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RESULTS AND DISCUSSION Structure of the Active Site. Figure 2 shows the initial structure of the Cd2+/Cd2+ and Zn2+/Zn2+ active sites of PTE, obtained from the X-ray structures.34 The main difference among them is the presence of two coordinated water molecules in the Cd atom more exposed to the solvent, Cdβ, with bond distances of 2.339 and 2.463 Å. This makes Cdβ adopt a distorted octahedral geometry composed by the residues His230, His201, Lys169, two water molecules, and the μ-OH ligand. In contrast, the Znβ adopts a trigonal bipyramidal geometry composed by the residues His230, His201, Lys169, one water molecule distant 2.097 Å from the metal, and the bridging hydroxide ligand. For both enzymes, the metallic site less exposed to the solvent (Znα and Cdα) adopts distorted trigonal bipyramidal geometry composed by His55, His57, Asp301, Lys169, and the hydroxide bridge. As we shall see later, the presence of this second water molecule coordinated to the Cdβ will have a great impact on the reactivity of the enzyme. Optimization of the active sites at the B3LYP-D3/631+G(d)/LANL2DZ level leads to the release of one water molecule. As can also be seen in Figure 3, the water molecule coordinated to the Znβ leaves the coordination sphere of the metal and makes hydrogen bond with the μ-hydroxo ligand, with a HO---H distance of 1.836 Å. In the optimization of the Cd2+/Cd2+-PTE active site, a water molecule coordinated to the Cdβ site leaves the coordination sphere and makes a hydrogen bond network involving the μ-hydroxo ligand (with HO---H distance of 1.504 Å); the other water molecule remained coordinated to the metal (with H2O---H2O distance of 1.543 Å) and the Asp301 residue. The geometry around the β center changes drastically, assuming a tetrahedral-like geometry for the Zn2+/Zn2+-PTE and distorted trigonal bipyramidal geometry, with high degree of piramidality, for the Cd2+/Cd2+-PTE. Krauss et al.52 also obtained a hydrogen bond network involving the water molecule coordinated to the Cdβ site, the μ-hydroxo ligand and the Asp301 residue in their investigation

Figure 3. B3LYP-D3/6-31+G(d)/LANL2DZ optimized structure of the structural model of the active sites of Cd2+/Cd2+-PTE (A) and Zn2+/Zn2+-PTE. The main structural features are highlighted. Bond distances are given in Å.

of the coordination geometries of Zn2+ and Cd2+ in PTE from P. diminuta, using the effective fragment potential approach to represent the protein environment. The authors also obtained distorted trigonal bipyramidal geometry around the α metallic site and a square pyramidal geometry around the β site. It is worth noting that the more solvent exposed β center is where the substrate coordinates to the enzyme. Changing the geometry from octahedral to square pyramidal at this position opens one coordination site for the substrate binding and therefore, as we shall see later, can affect the reaction energetics and mechanism. The Zn2+/Zn2+-PTE enzyme, however, may have an additional energy penalty to change the tetrahedral-like geometry at the Znβ site to accommodate the substrate. One important discussion when dealing with theoretical modeling of enzymatic reactions is whether the cluster model can indeed reproduce the structural characteristics of the active site of the whole enzyme since the enzyme environment is not properly taken into account. One way to introduce such effects is to use the QM/MM approaches in which the nonreactive part of the enzyme is described by appropriate empirical force fields.53,54 As pointed out by Siegbahn and Himo,55 geometry optimization is a fundamental ingredient when cluster model of the active site is used to describe enzymatic reactions. Therefore, in order to verify the adequacy of our cluster model to describe the structure of the active site of the enzyme, we performed an ONIOM geometry optimization on the entire E


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Inorganic Chemistry

In contrast, for the Zn2+/Zn2+-PTE, an additional energy penalty may be involved in the coordination of the substrate at the Znβ site. The water molecule that leaves the β site makes strong hydrogen bonds with the μ-hydroxo ligand and as we shall see may be involved in proton transfer reactions, forming nucleophilic species in situ and therefore altering the reactivity and changing the mechanism. The next step was to analyze the interaction of the substrate with the active site and to explore the role of these water molecules on the substrate binding process. Interaction of the Substrate Paraoxon with the Active Site of Cd2+/Cd2+-PTE. In order to analyze the structural changes occurring along the pathway for the interaction of paraoxon with the active site of Cd2+/Cd2+-PTE, a potential energy surface (PES) was constructed varying the distance between the phosphoryl oxygen atom of paraoxon to the β metallic center of the active site from 4.0 to 2.2 Å in intervals of 0.2 and 0.5 Å. This distance range was based on crystallographic data56 which shows that the nearest distance between the phosphoryl oxygen atom of analogue substrate diethyl 4methylbenzylphosphonate and the zinc atom of the native PTE is ∼3.4 Å. The substrate/active site interaction distances were kept frozen, and all other geometric parameters were fully optimized, keeping the terminal carbon atoms of the active site fixed, as cited before. Figure 4 shows the PES for the interaction of paraoxon with the Cdβ of the active site of Cd2+/Cd2+-PTE. As can be seen, the interaction is downhill as the substrate approaches the active site and the geometry around the β cadmium center changes from square pyramidal to distorted octahedral without energy barrier. For all structures obtained along the PES, it is observed that one water molecule remains coordinated to the metallic center with an average distance of 2.360 Å. The other water molecule also remains involved in the hydrogen bond network involving the μ-hydroxo ligand, with an average distance of 1.640 Å, and with the coordinated water, with an average distance of 1.690 Å. These hydrogen bonds are important to stabilize the water molecules at the active site. Our results, therefore, show that even after the interaction with the substrate the water molecule remains coordinated to the Cdβ center and the other water molecule still remains making strong hydrogen bonds with the bridging hydroxide ligand. As can be seen in Figure 4c, the final structure of the PES, with Cdβ---OP distance of 2.200 Å, shows the oxygen of the water molecule and the μ-hydroxo ligand at almost the same distance (∼ 3.8 Å) from the phosphorus atom of the substrate. Additionally, these groups do not have favorable orientation for attacking the substrate in line with the leaving group, passing through a trigonal bipyramidal-like transition state geometric arrangement. Therefore, the ∠(Cd−O−P−O) dihedral angle was rotated by 180°, and the system reoptimized without any constraint in the Cdβ---O distance. The final optimized structure, shown in Figure 5, is ∼3.5 kcal/mol more stable than the previous structure without rotation of the dihedral angle. The distance between the oxygen atom of the phosphoryl group and the β cadmium is 2.458 Å. In this new structure the water molecule and the μ-hydroxo ligand are 3.507 and 3.800 Å distant from the phosphorus atom of the substrate, respectively. The new orientation of the ∠(Cd−O− P−O) angle allows for a more favorable configuration for the in line attack of the nucleophile with the leaving group and favoring the trigonal bipyramidal transition state arrangement. Also important are the facts that the water molecule remains coordinated to the cadmium atom and that the other water

enzyme, with the QM part described at the B3LYP-D3/631+G(d)/LANL2DZ level and the MM part described with the UFF force field. Also, in order to check the flexibility of the model used, geometry optimization with a larger model with 142 atoms was also carried out. We also compared the structure obtained with geometry optimization of the cluster without any geometric constraint. Table 1 shows the RMSD obtained when Table 1. Root Mean Square Deviation (RMSD) between the Optimized Cluster Model of the Cd2+/Cd2+-pdPTE Active Site, The Experimental X-ray Diffraction Structure and the ONIOM Optimized Structure experimentala

model (frozen)b model (free)d ONIOMe

ONIOM

bonds

angles

bonds

angles

0.105 (0.144)c 0.095 0.181

6.3 (15.4) 16.6 7.6

0.056 (0.085) 0.080

5.6 (12.0) 13.1

a

Experimental X-ray structure taken from reference 34. bCluster model optimized at the B3LYP/6-31+G(d) level of theory with fixed coordinates of the peripheral carbon atoms as described in the text. c Values in parentheses were obtained for the larger cluster model with 142 atoms. dCluster model fully optimized at the B3LYP/6-31+G(d) level of theory without fixing coordinates. eGeometry optimization of the full enzyme with the hybrid ONIOM (DFT/6-31+G(d):UFF) method, as described in the text.

comparing the cluster optimization of the active site of Cd2+/ Cd2+-PTE, with and without frozen coordinates, with the experimental and ONIOM optimized structures. The data in Table 1 clearly shows that using the cluster approach with, frozen coordinates of the peripheral carbon atoms, produces essentially the same geometry of the active site as the ONIOM calculation, with RMSD over the bonds and angles of 0.056 Å and 5.6°, respectively, when compared with each other. Both methods produce essentially the same deviation in the bond angles from the experimental structure, with RMSD around 7° and the deviation over the bonds being slightly better using the cluster model, with RMSD 0.105 against 0.181 for the ONIOM. Clearly, the use of a cluster model without proper restraint to reproduce the enzyme environment produces larger bonds and angles deviations, as can be seen by the larger RMSD obtained when comparing with the experimental and ONIOM geometries. Therefore, the use of this approach may lead to incorrect representation of the active site and may affect the energetics and mechanism of the reaction. As can be seen in Table 1, the increase of cluster size does not promote a substantial improvement in the description of structural parameters of the active site of Cd2+/Cd2+-PTE in comparison to results achieved by using the small model. In fact, the RMSD computed over the angles and the bonds is worse. The data reported in Table 1 for the RMSD calculated in relation to Xray indicate that our small model describe well the main structural parameters of active site of PTE. To summarize, our results shows that the water molecules coordinated to β site, which are more exposed to the solvent, may have an important role on the reactivity of PTE. Upon optimization one water molecule leaves the coordination sphere changing the geometry at the β metallic site, opening a coordination position for substrate binding. For Cd2+/Cd2+PTE, the geometry change from octahedral to square pyramidal leaves a coordination site ready for the substrate coordination. F


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Inorganic Chemistry

metal site upon coordination of paraoxon. Coordination of the substrate to the β site was also observed in the Mn(II) derivative of glycerophosphodiesterase enzyme from E. aerogenes (GpdQ).33 This enzyme has a more complex mechanism for catalysis in which the coordination of the substrate promotes the binding of a second metal ion to the β site. For this enzyme, the likely nucleophile is a water molecules terminally bound to the α site.67 Therefore, comparing with our results, it seems that the β site of PTE, OpdA, and GpdQ behaves differently upon substrate coordination and may be responsible for the functional differences of these enzymes. It is also worth mentioning that other bimetallic hydrolases such as purple acid phosphatase (PAP) also exhibit catalytic activity only when the substrate is coordinated to the divalent metal ion in a monodentate coordination mode as obtained for Cd2+/ Cd2+-PTE, as was demonstrated by Mitić et al.68 The positioning of the substrate in the active site is an important factor related to the possible interactions involving the spectator groups of the substrate and also the leaving group. Ornstein and co-workers57 in their MD simulations and QM studies on the zinc-substituted PTE-substrate complexes showed that the enzyme exhibit two hydrophobic pockets related to the interactions of the alkyl spectator groups and a third pocket for the correct positioning of the leaving group. The pocket in which the substrate is localized exhibit flexibility and interacts with the substrate with conformational adjustments. Molecular dynamics results65 also have shown that the orientation of the leaving group is a key factor determining the selectivity of quiral substrates derived from paraoxon. Mo and co-workers30 have shown that the rate-limiting step of the reaction is indeed associated with distortions of the paraoxon for the attack of the nucleophile. Therefore, in order to verify the orientation of the substrate in the active site, a conformational search around the ∠(Cd−O−P−O) angle was performed and confirmed that the optimized structure shown in Figure 5b is indeed the lowest energy geometry. This structure was then used to analyze the hydrolysis mechanism. Proposed Reaction Mechanism for the Hydrolysis of Paraoxon. Figures 6 and 7 show the structures of the stationary points obtained along the reaction coordinate for the hydrolysis of paraoxon catalyzed by the Cd2+/Cd2+-PTE active site. The reaction starts with formation of the enzyme− substrate complex, through the coordination of paraoxon to the β cadmium center, forming the reactant species, Reac, shown in Figure 6. Paraoxon coordinates to the metallic β center in monodentate fashion though the phosphoryl oxygen with a distance of 2.393 Å. The local geometry of the β cadmium center changes from square pyramidal to octahedral and the α cadmium center remains with a distorted trigonal bipyramidal arrangement. In this Reac structure, the water molecule approaches in line to the leaving group with a distance of 3.935 Å to the phosphorus atom and attack angle ∠(H2O−P− OPh) of 167°. The distance of the leaving group to the phosphorus atom, P−OAr, is 1.618 Å. The attacking water molecule makes strong hydrogen bond with the μ-OH ligand, with a distance of 1.669 Å and with the coordinated water molecule, with a distance of 1.841 Å. The water molecule also makes hydrogen bond with the carboxylate group of the bridging Lys169 with a distance of 1.924 Å. In the Reac structure, the cadmium atoms are 3.687 Å distant from each other. It is worth noting that the substrate coordinates only in the Cdβ center, in contrast with the native Zn2+/Zn2+-PTE for which a symmetrical bidentate coordination mode of paraoxon

Figure 4. Potential energy surface for the interaction of paraoxon with the structural model of Cd2+/Cd2+ phosphotriesterase (A). Structures obtained with r(Cdβ---OP) = 4.0 Å (B) and 2.2 Å (C) are shown.

molecule also remains involved in the hydrogen bond network. Ely et al.22 have applied magnetic circular dicroism (MCD) to investigate the electronic and geometric structures of the Co2+/ Co2+-OpdA. The authors observed a reduction in the number of the ligands coordinated to the β metal site and a reduction of the exchange coupling constant upon substrate binding. These data were interpreted as a shift of the bridging hydroxide ligand into a pseudoterminal position which in turn attacks the substrate. Our results for the Cd2+/Cd2+-PTE show that the bridging hydroxide remains coordinated to the β metal site, with only a small increase of 0.033 Å in the distance to the β G


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Inorganic Chemistry

Figure 5. Optimized structure of the substrate-enzyme complex without rotation of the ∠(Cd−O−P−O) dihedral angle (A) and after rotation (B).

structure exhibits an imaginary frequency of 230.2i cm−1 associated with the synchronized motion involving a proton transfer from the water molecule to the bridging OH− ligand and attack to the phosphorus atom. Therefore, this proton transfer will generate an OH− nucleophile in situ, which will attack the phosphorus atom of the substrate, in line with the leaving group, with an angle ∠(HO−P−OAr) of 180°. The OH bond of the water molecule is stretched to 1.383 Å, and the distance to the phosphorus atom decreases to 2.173 Å. At the same time, the distance of the leaving group to the phosphorus center increases to 1.710 Å. It is important to emphasize the important role of the coordinated water molecule in stabilizing the structure of the transition state and to orient the nucleophile for the attack, maintaining hydrogen bonds with the carboxylate of Lys169 and the OH− nucleophile in formation, with distances of 1.913 and 1.720 Å, respectively. At the transition state TS1, the distance between the cadmium atoms increases to 3.799 Å, and all bond distances around the phosphorus atoms increases as well. Transition state structure TS1 leads to the formation of a pentacoordinate intermediate, Int, shown in Figure 6. In the intermediate, the proton of the water molecule is completely transferred to the bridging ligand and the formed OH− nucleophile binds to the phosphorus, occupying the axial

to the two zinc centers has been proposed9 that facilitates the nucleophilic attack of the bridging OH− ligand. For the cadmium enzyme, this bidentate coordination mode is sterically forbidden. The main structural change that prevents this coordination mode is the presence of two water molecules coordinated to the β metal site34 of Cd2+/Cd2+-PTE in an octahedral coordination environment, compared with only one terminally coordinated in Zn2+/Zn2+-PTE forming a trigonal bipyramidal geometry. Upon coordination of paraoxon to the β metal site, the more labile water molecule is displaced and makes strong hydrogen bond with the bridging hydroxide ligand as shown in Figure 6. The presence of this water molecule highly stabilized through hydrogen bond with the bridge, prevents the substrate to adopt a second coordination to the α metal site. Therefore, one of the water molecules coordinated to the β metal site has a very important catalytic role in generating the nucleophilic species and somehow orienting the substrate. The only way for this substrate to adopt a bidentate coordination would be to remove the water molecules near the active site and thus lose their catalytic effects. Further interaction of the water molecule with the phosphorus atom leads to the formation of the first transition state (TS1) of the reaction, as can be seen in Figure 6. This H


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Inorganic Chemistry

Figure 6. B3LYP-D3/6-31+G(d)/LANL2DZ Optimized structure obtained along the first step of the reaction coordinate for the hydrolysis of paraoxon by the active site of Cd2+/Cd2+ phosphotriesterase. Reactant species, Reac, first transition state, TS1, and the pentacoordinate intermediate, Int. Bond distances are given in Å.

net charge of −0.983e in the reactant state, which facilitates the deprotonation of the hydrogen bonded water molecule. The proton transferred to the bridge also has a high positive charge of +0.653e. The pentacoordinate phosphorane intermediate, Int, is stabilized by strong hydrogen bonds with the bridging hydroxide ligand, with a distance of 1.648 Å, and the with

position of a trigonal bipyramidal geometry with a distance of 1.834 Å. The other axial position is occupied by the leaving group, with a P−OAr distance of 1.787 Å, with the spectator ligands and the phosphoryl oxygen forming the equatorial plane. It is worth mentioning that despite being coordinated to two Cd2+ ions the oxygen atom of the μ-hydroxide bridge has a I


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Inorganic Chemistry

Figure 7. B3LYP-D3/6-31+G(d)/LANL2DZ Optimized structure obtained along the second step of the reaction coordinate for the hydrolysis of paraoxon by the active site of Cd2+/Cd2+ phosphotriesterase. Second transition state, TS2, and the final product, Prod. Bond distances are given in Å.

A further approach of the nucleophile leads to the second transition state (TS2) of the reaction, shown in Figure 7, having an imaginary frequency of 63.8i cm−1, related to the stretching of the P−OAr bond. The P−OAr bond of 2.431 Å represents ∼50% of bond breaking when compared with the same bond in the reactant species. In contrast, the P−OH bond of 1.704 Å shows ∼96% of bond formation at TS2, as compared with the same bond distance at the product. The hydrogen bonds involving the nucleophile, the bridging hydroxide ligand, and the coordinated water molecule weakens, showing distances of 1.920 and 2.058 Å, respectively, and the geometry around the phosphorus atom changes to a distorted tetrahedral. The final product of the reaction, Prod, shown in Figure 7, is characterized by the complete breaking of the bond involving the nucleophile and the leaving group, with P−OAr bond distance of 3.600 Å and complete formation of the bond involving the nucleophile, with P−OH bond distance of 1.640 Å. The P−O bond of the phosphoryl group restores its double bond character, with PO bond distance of 1.487 Å and the hydrogen bonds involving the diphosphate species formed being weakened. It is important to emphasize that during the

coordinated water molecule has a distance of 1.812 Å. The Cd−Cd distance increases to 3.912 Å at Int structure. The coordinated water molecule plays an important role in stabilizing the pentacoordinate intermediate, maintaining hydrogen bonds with the carboxylate and with the nucleophile. It is important to mention that the existence of this pentacoordinate intermediate is a matter of debate.58,59 In fact, the phosphorane intermediate appears in some theoretical studies of biomimetic compounds of PAP,60,61 cluster model of PTE,26 and on QM/MM investigations of the Zn2+/Zn2+-PTE enzyme, where a transition state plateau in the reaction coordinate was found, similar in nature to the intermediate state.28 However, to the best of our knowledge this intermediate has never been experimentally detected in the enzymatic reaction of organophosphate degrading enzymes.34,62,63 One of the reasons may be the short lifetime of this intermediate. As we demonstrated by ab initio molecular dynamics studies on the hydrolysis of phosphate mono and diester in aqueous solution,64 this phosphorane intermediate has a short lifetime of ∼1 ps. J


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Inorganic Chemistry

distances along the reaction coordinate. As can be seen, the bond of the leaving group to the phosphorus atom remains almost unaltered until the pentacoordinate intermediate is reached. From Reac to Int, the P−OAr bond dissociates only 10% compared to Reac, and at the second transition state, TS2, the bond is 50% dissociated. That is, the P−OAr bond dissociates significantly only in the second step of the reaction. In contrast, in the first part of the reaction the bond of the nucleophile to the phosphorus atom is almost complete. The P−OH2 bond is 67% formed already at TS1 and is 88% at intermediate Int. DFT calculations on the hydrolysis of the analogous compound methylparaoxon, promoted by the native Zn2+/Zn2+-PTE enzyme, predicts the reaction to takes place in two steps (AN + DN mechanism) with formation of the pentacoordinate intermediate.26 However, QM/MM/MD calculations on hydrolysis of paraoxon by the same Zn2+/ Zn2+-PTE enzyme shows a single-step associative ANDN mechanism31 or SN2 mechanism with a transition plateau.28 The structural analysis performed here for the active site model of Cd2+/Cd2+-PTE enzyme clearly shows that the hydrolysis of paraoxon promoted by model Cd2+/Cd2+-PTE active site follows a two-step associative mechanism of the (AN + DN) type, passing through a pentacoordinate intermediate with significant bond breaking to leaving group and bond formation to the nucleophile. The difference here is that in our study with the model active site we have shown that the nucleophilic species is formed in situ trough proton transfer from a water molecule to the bridging ligand. The water molecules initially present as coordinated to the β center of the active site of Cd2+/Cd2+-PTE thus play an important role in the mechanism proposed in this work. The energetics involved in the proposed mechanism for the hydrolysis of paraoxon by Cd2+/Cd2+-PTE are quoted in Table 3. The energy values were obtained at the B3LYP-D3/6-311+ +G(2d,2p) through single-point energy calculations at the B3LYP/6-31+G(d)/LANL2DZ optimized structures. The inclusion of different effects on the final energy results is compared. It is clear from inspection of Table 3 that the first step of the reaction is the rate-determining step of the reaction with activation energy of 19.3 kcal/mol in gas phase. Dispersion effects alone contribute to reduce drastically this energy barrier by ∼26%, and inclusion of the solvent effects leads to a final value of 12.9 kcal/mol. Dispersion effects also affect drastically the stability of the pentacoordinate intermediate, Int, from 16.4 to 12.7 kcal/mol, representing ∼23% increase in the stability of Int, and reduce the second energy barrier from Int1 to TS2 by 0.9 kcal/mol (∼12.6%). Dispersion effects are less pronounced in the stability of the product, Prod, representing an increase in stability of 1.2 kcal/mol (∼8.0%). From these results, the important role played by dispersion effects on the entire energy

entire reaction coordinate the substrate remains coordinated only to the β cadmium center in a monodentate fashion. A possible mechanism to restore the active site in the resting state can be inferred by analyzing the distances involving the bridging OH− ligand, the phosphoryl oxygen atom to the Cdβ center, and the proton transfer to the Asp301 residue. As can be seen in Figures 6 and 7, as the μ-hydroxo bridge deprotonates the nucleophilic water molecule, its bond distance to the Cdβ center increases, varying 0.254 Å from the reactant to the product. This can be explained by the change of the anionic character of the OH− bridge in Reac to the neutral character of water in the product. As the reaction proceed the hydrogen bond involving the μ-hydroxo bridge and the Asp301 residue becomes stronger, and the hydrogen bond distance varies 0.405 Å from the reactant to the product. The PO---Cdβ bond, however, increases 0.080 Å from the second transition state to the product. Therefore, a possible mechanism for the restoration of the active site may be associated with the protonation of the Asp301 residue by the bridged water molecule, restoring the μ-hydroxo bridge and contributing to the decrease in the PO---Cdβ polarization, which releases the product. Coordination of a water molecule from the medium would restore the active site resting state. The Asp301 may be involved in a proton relay mechanism to the medium, involving the Asp301 and the His254 residue, which is oriented toward the Asp301, in the enzyme. The role played by the Asp301 in active site of PTE was demonstrated by Aubert, Li, and Raushel9 by the drop of catalytic activity of 2−3 orders of magnitude when this residue is mutated to alanine or asparagine. Therefore, our mechanistic proposal is in line with the experimental findings. The classification of the type of mechanism involved in the enzymatic hydrolysis of paraoxon by Cd2+/Cd2+-PTE enzyme can be obtained through the analysis of the extent of P−OAr bond breaking and P−nucleophile bond formation along the reaction coordinate, providing a structural characterization of the mechanism. Table 2 shows the P−OAr and P−OH2 bond Table 2. Variation of the Phosphorus−Nucleophile (P− OH2O) and Phosphorus Leaving Group (P−OAr) Bond Distances and Percentage of P−OAr Bond Breaking (%B.B) and Percentage of P−OH2O Bond Forming (%B.F) along the Reaction Coordinatea P−OAr %B.B P−OH2O %B.F a

Reac

TS1

Int

TS2

Prod

1.62 0 3.93 0

1.71 6 2.17 67

1.79 10 1.83 88

2.43 50 1.70 96

3.60 100 1.64 100

Bond distances in Å.

Table 3. Relative Energies (in kcal/mol) Obtained for the Hydrolysis Reaction of Paraoxon Promoted by Cd2+/Cd2+-pdPTE Active Site, With Different Levels of Calculationa species method

Reac

TS1

Int

TS2

Prod

B3LYP/6-311++G(2d,2p) B3LYP/6-311++G(2d,2p) + D3 B3LYP/6-311++G(2d,2p) + D3 + SMD

0.0 0.0 0.0

19.3 14.2 12.9

16.4 12.7 9.9

24.2 19.2 11.8

14.4 13.3 4.8

a

D3 is including the D3 dispersion correction of Grimme.38 SMD includes solvation free energies of the species computed with the SMD continuum model of Thrular and Cramer,43 with dielectric constant 4.0. Results with the 6-311++G(2d,2p) basis set were obtained through single-point energy calculations at the B3LYP/6-31+G(d) optimized geometries. K


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et al.69 Fluoride is a potent inhibitor of OpdA, displacing the hydrolysis initiating μ-OH ligand.19 However, OPH enzyme, which shares more than 90% of similarity, does not exhibit inhibition upon complexation with fluoride. Two hypotheses were raised to explain the lack of inhibition: The binding of the substrate may somehow prevent the fluoride from binding or the fluoride is not displacing the relevant nucleophile that initiates catalysis, which is supposedly the bridging hydroxide ligand. Our mechanistic proposal is in line with this last hypothesis since the hydrolysis-initiating species is, in fact, a water molecule in the second coordination sphere, previously coordinated to the Cdβ site. In this case, displacement of the μhydroxide ligand would not lead to the enzyme inhibition. A water molecule in the second coordination sphere was also found to be the hydrolysis-initiating nucleophile for the PAP, containing the Fe3+−M2+ (M = Fe2+, Mn2+) active site.68 In this case, the hydrolysis of the substrate is initiated by a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe3+. Therefore, we may say that our mechanistic proposal is consistent and can complement some available experimental studies on the mechanism of phosphotriesterase enzymes. The computed energy barrier of 12.9 kcal/mol for the mechanism proposed here is in very good agreement with the experimental value and, it is interesting to note that if we compare this energy barrier with the barrier of 17.3 kcal/mol computed by Kim et al.27 for a similar base mechanism in the Zn2+/Zn2+-PTE our computed value is 4.4 kcal/mol smaller. Also, our computed barrier is of the same magnitude than those computed for the nucleophilic attack of the bridging hydroxide in Zn2+/Zn2+-PTE. As was mentioned previously, we obtained strong steric hindrance for the coordination of the substrate in a way to allow the direct attack of the bridging hydroxide. Therefore, the mechanism proposed in this study is also kinetically viable. The Cd2+/Cd2+-PTE has optimal catalytic activity at high pH. In the mechanism proposed here, at low pH the oxygen atom of the bridge can be protonated, due to the high negative charge over the oxygen, avoiding the in situ formation of the proton by the hydrogen bonded water molecule, which may explain the low reactivity at low pH values. At high pH, in situ formation of the OH− nucleophile is more favorable. Therefore, the base mechanism for the Cd2+/Cd2+-PTE proposed here, in conjunction with the agreement found with the experimental energetic value for the energy barrier, makes it a consistent and kinetically viable mechanistic proposal for the hydrolysis of phosphate triesters promoted by the Cd2+ substituted PTE enzyme.

profile of the reaction is apparent and, as was also pointed out by other authors,54 makes the inclusion of these effects essential in the study of enzymatic reactions, no matter which approach is used (QM, Cluster, QM/MM, etc.). Our final energetic results at the B3LYP-D3/6-311++G(2d,2p) level and including dispersion and solvent effects show that the hydrolysis of paraoxon by Cd2+/Cd2+-PTE follows a two-step (AN + DN) mechanism, with energy barriers of 12.9 and 1.9 kcal/mol for the first and second steps, respectively. For the sake of comparison, calculations on the cluster model of the Zn-PTE active site carried out by Kim et al.27 revealed an energy barrier of 17.3 kcal/mol for the mechanism in which the bridging hydroxide acts as base, deprotonating the water molecule. For the direct nucleophilic attack of the bridging hydroxide ligand, calculations carried out by Chen et al.26 produced an energy barrier of 10.8 kcal/mol. It is worth noting that our mechanistic proposal shows an energy barrier that is almost half the energy barrier of 23.9 kcal/mol computed for the Zn2+/Zn2+ biomimetic models of phosphodiesterase, carried by Brown et al.,61 in which a terminally coordinated hydroxide acts as nucleophile. It is also interesting to note that our computed energy barrier is only 0.7 kcal/mol higher than the value of 12.3 kcal/mol that we computed for a biomimetic model of PAP for a terminally coordinated hydroxide ligand acting as nucleophile.60 Experimentally, the hydrolysis of paraoxon by Cd2+/Cd2+-PTE has a catalytic constant of kcat = 2500 s−1, measured at pH 9.0 and 20% methanol.9 This experimental rate constant translate in an activation energy of 12.8 kcal/mol. Rochu and co-workers32 also determined the catalytic activity of PTEs and found that the hydrolysis of paraoxon by Cd2+ substituted PTE has kcat = (3900 ± 300) s−1 at pH 9.0 and 25 °C, which gives an activation energy of 12.5 kcal/mol. In our mechanistic proposal, the bridging hydroxide ligand is not the hydrolysis-initiating species, attacking directly the coordinated substrate. Instead, it acts as base deprotonating a water molecule, previously coordinated to the Cdβ center, which in turn attacks the phosphorus atom of the ligand. The role of the bridging hydroxide to act as a nucleophile, attacking the substrate directly, or to act as a base, generating an OH− nucleophile which attacks the substrate, is in fact one important mechanistic aspect of the phosphotriesterases. Gouré et al.,66 investigating the hydrolysis promoted by a FeIIIFeII complex, have shown by means of 1H NMR and Mössbauer spectroscopies, that the incoming substrate turns the μ-hydroxo nucleophile into a terminal one. This shifting of μ-hydroxide ligand into a terminal one was also observed by Ely et al.22 for Co2+/Co2+-OpdA. The presence of two coordinated water molecules at the more exposed Cdβ center of the active site, which are not present in the native Zn2+/ Zn2+ enzyme, are responsible for the main mechanistic differences found for the Cd2+/Cd2+-PTE enzyme. These two water molecules play a very important catalytic role in the entire mechanism. One of the water molecules leaves the coordination sphere of the Cdβ center, in order to allow the substrate binding, and makes strong hydrogen bonding interaction with the bridging hydroxide ligand, facilitating the deprotonation and formation of the OH− nucleophile. This water molecule, now on the second coordination sphere thus acts as the hydrolysis-initiating species. Strong evidence in favor of this mechanistic proposal comes from the high-resolution crystal structure of fluorideinhibited organophosphate OpdA enzyme, obtained by Selleck

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FINAL REMARKS In the present work, DFT calculations at the B3LYP/631+G(d) and including dispersion effects were performed, for the first time, on a cluster model of the active site of Cd2+/Cd2+ phosphotriesterase from P. diminuta to investigate the hydrolysis mechanism of the phosphotriester paraoxon. The mechanism proposed here consist of (i) Exchange of the coordinated water molecule and coordination of the substrate to the more solvent exposed Cdβ center in monodentate fashion, (ii) protonation of the μ-hydroxo bridge by the uncoordinated water molecule and in situ formation of the nucleophile, (iii) formation of a pentacoordinated intermediate with significant bond breaking to leaving group and bond formation to the nucleophile, and (iv) protonation of the L


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(4) Kirby, A. J.; Nome, F. Fundamentals of Phosphate Transfer. Acc. Chem. Res. 2015, 48, 1806−1814. (5) Erickson, B. E. U.S. EPA’s chlorpyrifos decision spurs pushback. C&EN 2017, 95, 27−30. (6) Lewis, V. E.; Donarski, W. J.; Wild, J. R.; Raushel, F. M. Mechanism and Stereochemical Course at the Phosphorus of the Reaction Catalyzed by a Bacterial Phosphoreiesterase. Biochemistry 1988, 27, 1591−1597. (7) Donarski, W. J.; Dumas, D. P.; Heitmeyer, D. P.; Lewis, V. E.; Raushel, F. M. Structure-Activity Relationships in the Hydrolysis of Substrates by the Phosphotriesterase from Pseudomonas diminuta. Biochemistry 1989, 28, 4650−4655. (8) Dumas, D. P.; Caldwell, S. R.; Raushel, F. M. Purification and Properties of the Phosphotriesterase from Pseudomonas Diminuta. J. Biol. Chem. 1989, 264, 19659−19665. (9) Aubert, S. D.; Li, Y.; Raushel, F. M. Mechanism for Hydrolysis of Organophosphates by Bacterial Phosphotriesterase. Biochemistry 2004, 43, 5707−5715. (10) Kennedy, D. J.; Mayer, B. P.; Baker, S. E.; Valdez, C. A. Kinetics and Speciation of Paraoxon Hydrolysis by Zinc(II)-Azamacrocyclic Catalysts. Inorg. Chim. Acta 2015, 436, 123−131. (11) Benschop, H. P.; De Jong, L. P. A. Nerve Agent Stereoisomers: Analysis, Isolation, and Toxicology. Acc. Chem. Res. 1988, 21, 368− 374. (12) Caldwell, S. R.; Newcomb, J. R.; Schlecht, K. A.; Raushel, F. M. Limits of Diffusion in the hydrolysis of Substrates by the Phosphotriesterase from Pseudomonas diminuta. Biochemistry 1991, 30, 7438−7444. (13) Bigley, A. N.; Raushel, F. M. Catalytic Mechanisms for Phosphotriesterases. Biochim. Biophys. Acta, Proteins Proteomics 2013, 1834, 443−453. (14) Schenk, G.; Mateen, I.; Ng, T.-K.; Pedroso, M. M.; Mitić, N.; Jafelicci, M., Jr.; Marques, R. F. C.; Gahan, L. R.; Ollis, D. L. Organophosphate-degrading Metallohydrolases: Structure and Function of Potent Catalysts for Applications in Bioremediation. Coord. Chem. Rev. 2016, 317, 122−131. (15) Omburo, G. A.; Kuo, J. M.; Mullins, L. S.; Raushel, F. M. Characterization of the Zinc Binding Site of Bacterial Phosphotriesterase. J. Biol. Chem. 1992, 267, 13278−13283. (16) Samples, C. R.; Howard, T.; Raushel, F. M.; DeRose, V. J. Protonation of Binuclear Metal Center Within the Active Site of Phosphotriesterase. Biochemistry 2005, 44, 11005−11013. (17) Ely, F.; Hadler, K. S.; Gahan, L. R.; Guddat, L. W.; Ollis, D. L.; Schenk, G. The Organophosphate-Degrading Enzyme from Agrobacterium Radiobacter Displays Mechanistics Flexibility for Catalysis. Biochem. J. 2010, 432, 565−573. (18) Yang, H.; Carr, P. D.; McLoughlin, S. Y.; Liu, J. W.; Horne, I.; Qiu, X.; Jeffries, C. M. J.; Russell, R. J.; Oakeshott, J. G.; Ollis, D. L. Evolution of an Organophosphate-degrading Enzyme: A Comparison of Natural and Direct Evolution. Protein Eng., Des. Sel. 2003, 16, 135− 145. (19) Pedroso, M. M.; Ely, F.; Mitić, N.; Carpenter, M. C.; Gahan, L. R.; Wilcox, D. E.; Larrabee, J. L.; Ollis, D. L.; Schenk, G. Comparative Investigation of the Reaction Mechanisms of Organophosphatedegrading Phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH). JBIC, J. Biol. Inorg. Chem. 2014, 19, 1263−1275. (20) Jackson, C. J.; Foo, J. L.; Kim, H. K.; Carr, P. D.; Liu, J. W.; Salem, G.; Ollis, D. L. In Crystallo Capture of a Michaelis Complex and Product-binding Modes of a Bacterial Phosphotriesterase. J. Mol. Biol. 2008, 375, 1189−1196. (21) Daumann, L.; Schenk, G.; Ollis, D. L.; Gahan, L. R. Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes. Dalton Trans. 2014, 43, 910−928. (22) Ely, F.; Hadler, K. S.; Mitić, N.; Gahan, L. R.; Ollis, D. L.; Plugis, N. M.; Russo, M. T.; Larrabee, J. A.; Schenk, G. Electronic and geometric structures of the organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA). JBIC, J. Biol. Inorg. Chem. 2011, 16, 777−787.

Asp301 residue and restoration of the active site by the coordination of another water molecule of the medium. The water molecules initially coordinated to the active site plays a crucial role in stabilizing the transition states and the pentacoordinate intermediate. The reaction takes place in a two-step (AN + DN) mechanism, with energy barriers of 12.9 and 1.9 kcal/mol for the first and second steps, respectively, computed at the B3LYP-D3/6-311++G(2d,2p) level of theory, in excellent agreement with the experimental findings. We found that dispersion effects alone contributes to diminish the energy barriers as much as 26% attesting once more to the necessity to include dispersion effects on this kind of enzymatic reaction. Also important is the sampling of the conformational space of the coordinated substrate. In the case of paraoxon, the orientation of the leaving group resulted in an additional stabilization of the substrate-enzyme complex of more than 4 kcal/mol. Finally, it is important to mention that the base mechanism for the Cd2+/Cd2+-PTE proposed here, in conjunction with the agreement found with the experimental energetic value for the energy barrier, makes it a consistent and kinetically viable mechanistic proposal for the hydrolysis of phosphate triesters promoted by the Cd2+ substituted PTE enzyme.

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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.8b00361. Optimized Cartesian coordinates of all species obtained along the reaction coordinate and total energies computed with different levels of calculations (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Fax: +55 31 34095700. ORCID

Willian R. Rocha: 0000-0002-0025-2158 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the CNPq (Conselho Nacional de Desenvolvimento ́ Cientifico e Tecnológico, INCT-Catálise) and FAPEMIG (Fundaçaõ de Amparo à Pesquisa do Estado de Minas Gerais) for the financial support and research grants. We thank the ́ LQTC (Laboratório de Quimica Teórica e Computacional Universidade Federal de Pernambuco) for the computational resources used in some calculations. We also to thank Prof. Jeremy N. Harvey for providing his code for block-Hessian diagonalization.

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REFERENCES

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