Role of Amino Acid Residues for Dioxygen Activation in the Second

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

Role of Amino Acid Residues for Dioxygen Activation in the Second Coordination Sphere of the Dicopper Site of pMMO Mayuko Miyanishi, Tsukasa Abe, Yuta Hori, Yoshihito Shiota, and Kazunari Yoshizawa* Institute for Materials Chemistry and Engineering and IRCCS, Kyushu University, Fukuoka 819-0395, Japan

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S Supporting Information *

ABSTRACT: Formation of an active oxygen species at the dicopper site of pMMO is studied by using density functional theory (DFT) calculations. The role of the amino acid residues of tyrosine (Tyr374) and glutamate (Glu35) located in the second coordination sphere of the dicopper site is discussed in detail. The phenolic proton of the tyrosine residue is transferred to the Cu2O2 core in a twostep manner via the glutamate residue, and an electron is directly transferred to the Cu2O2 core. These proton- and electron-transfer processes induce the O−O bond cleavage of the μ−η2:η2-peroxodicopper(II) species to form the (μ-oxo)(μhydroxo)CuIICuIII species, which is able to play a key role of methane hydroxylation at the dicopper site of pMMO (Inorg. Chem. 2013, 52, 7907). This proton-coupled electron-transfer mechanism is a little different from that in tyrosinase in that the proton of substrate tyrosine is directly transferred to the dicopper site (J. Am. Chem. Soc. 2006, 128, 9873) because there is no proton acceptor in the vicinity of the dicopper site of tyrosinase. The rate-determining step for the formation of the (μ-oxo)(μ-hydroxo)CuIICuIII species is determined to be the O− O bond cleavage. These results shed new light on the interpretation of the role of the tyrosine and glutamate residues located in the second coordination sphere of the dicopper site of pMMO.

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INTRODUCTION Amino acid residues around the active metal sites in oxygenation enzymes finely tune the reductive activation of dioxygen. The second coordination sphere is considered to play an important role in the formation of transition metal active-oxygen species in oxygenation enzymes.1−3 A copperbased oxygenation enzyme particulate methane monooxygenase (pMMO), which is an integral membrane protein occurring in methanotrophs, is able to convert methane to methanol under ambient temperature and pressure using dioxygen as an oxidant.4−7 In 2005, Rosenzweig and coworkers revealed the crystal structure of pMMO by X-ray structural analysis at 2.8 Å resolution.8 According to their report, pMMO has three kinds of residues in the vicinity of the monocopper and dicopper centers, such as histidine (His), that is directly coordinated to the copper centers (primary coordination sphere) and tyrosine (Tyr) and glutamate (Glu) (second coordination sphere).9−11 In 2011, the crystal structure of pMMO was improved in higher resolution (2.68 Å),12 but the structural analysis did not settle the location and composition of the pMMO active site. In a computational study, we previously compared the formation energy between the mononuclear and dinuclear copper−oxo or −oxyl species in pMMO by using QM/MM and DFT methods.13 The formation of the dinuclear copper−oxo species is more stable in energy than that of the mononuclear copper−oxo species although both the species have catalytic ability sufficient for the C−H activation of methane. We therefore consider that the dicopper site is more favorable as the active site of pMMO for © XXXX American Chemical Society

methane hydroxylation from the viewpoint of formation energy for the two active species. On the other hand, Rosenzweig and co-workers proposed recently the mononuclear copper site to play the catalytic role instead of the dinuclear copper site on the basis of quantum refinement, spectroscopic, and crystallographic results of pMMO.14−16 Figure 1 shows the three main

Figure 1. Three main proposals for the active site of pMMO.6,8,14,15

proposals for the active site of pMMO. Although there has been debate about the monocopper or dicopper species from the viewpoint of computational quantum chemistry,13,14 the active species of pMMO has been widely believed so far to be the dinuclear copper−oxygen species for a long time.17−19 According to the X-ray crystallographic and EXAFS analyses of pMMO,12,20−23 several types of dinuclear copper−oxygen species have been proposed as possible candidates for the active species of pMMO, as shown in Figure 2.24−27 For example, μ-η2:η2-peroxodicopper(II) complexes have been Received: June 12, 2019

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DOI: 10.1021/acs.inorgchem.9b01752 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Figure 2. Dinuclear copper−oxygen complexes.

pMMO look similar. We previously proposed from QM/MM and DFT calculations that the H atom transfer from substrate tyrosine to the dicopper active site effectively induces the O− O bond cleavage of the peroxo moiety in tyrosinase.50 The proton of substrate tyrosine is directly transferred to the dicopper site because there is no proton acceptor in the vicinity of the dicopper site in tyrosinase. In this study, we investigate the reaction mechanism how OHO is formed from PO in pMMO, as shown in Scheme 1. We set up a model of PO having three histidine residues (His33, His137, and His139), one glutamate residue (Glu35) and one tyrosine residue (Tyr374), in order to consider effects of the residues located around the dicopper site, as shown later in this article.

developed as a model complex of the oxy forms of the O2binding copper proteins such as hemocyanin,28 tyrosinase,29 and catechol oxidase.30 Then, two electrons are injected into the peroxide moiety from the two copper ions, and the resultant O−O bond homolytic cleavage leads to the formation of bis(μ-oxo)dicopper(III) complexes. Although the bis(μoxo)dicopper(III) species has not been observed in any natural systems, it is widely characterized as a transient reactive intermediate in synthetic model complexes. Furthermore, the bis(μ-oxo)dicopper(III) species with a triplet ground state31 and the mixed-valent bis(μ-oxo)dicopperCu(II)Cu(III) species32−34 have also been suggested as the active species of pMMO. In addition, the (μ-oxo)dicopper(II) species is considered to be one of the reasonable candidates for the active species in Cu-exchanged zeolite catalysts that efficiently convert methane to methanol.35−42 Along with the experiments mentioned above, we carried out an initial theoretical study to clarify the reactivity for a dicopper−oxygen species of pMMO.43 Equilibrium between the μ-η2:η2-peroxodicopper(II) and the bis(μ-oxo)dicopper(III) complexes was calculated in order to discuss the activation energy for the O−O bond cleavage as well as these electronic structures.44,45 Moreover, we found that the mixed-valent bis(μ-oxo)CuIICuIII species is more reactive than the symmetric bis(μ-oxo)CuIIICuIII species because in the mixed-valent bis(μ-oxo)CuIICuIII complex the amplitude of the σ* SOMO localized on the bridging oxo moieties is large enough for the C−H bond homolytic cleavage of methane.13,46 We thus propose that the acceptance of one electron by the Cu2O2 core is essential for the methane activation by pMMO. As continuing research efforts in the reaction mechanism for pMMO, we considered that the hydrogen atom transfer or the proton-coupled electron transfer (PCET; the transfer of electron and proton)47,48 from the tyrosine residue (Tyr374) to the μ-η2:η2-peroxodicopper(II) species (PO) can lead to the formation of the reactive (μ-oxo)(μ-hydroxo)CuIICuIII species (OHO), which is able to work for methane hydroxylation, as shown in Scheme 1.49 The mechanism of the formation of OHO as an important step is still unclear about the role of Tyr374 in the second coordination sphere around the dicopper site. The structures of the dicopper sites of tyrosinase and

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COMPUTATIONAL METHOD

We used the B3LYP functional51 combined with the (16s10p6d) primitive set of Wachters−Hay supplemented with one polarization ffunction (α = 1.44 for Cu)52 for the Cu atoms and the D95** basis set53 for the H, C, N, and O atoms. The program used was the Gaussian 09 program package.54 After geometry optimizations, vibrational analyses were performed for the estimation of the zeropoint vibrational energies. For singlet-state calculations, we checked the instability of restricted SCF solutions using the unrestricted broken-symmetry approach. All singlet energies from the unrestricted calculations were computed by using the raw broken-symmetry SCF energy without modification. In a previous study, we constructed a cluster model of the enzyme active center of 1YEW (2.8 Å resolution) and performed constraint optimizations, which are useful to reproduce the rigidity of the active site involving some residues at the second coordination sphere.49 Referring to this constraint method, we also added His33, His137, His139, Glu35, and Tyr374 to the cluster model in the dicopper model and performed constraint optimizations by fixing the C-α atoms of the Glu35, His137, His139, and Tyr374 residues to their positions in the crystal structure, as shown in Figure 3. We consider a peroxo species with the CuIICuII oxidation state, which has two spin

Scheme 1. Formation of the (μ-Oxo)(μ-hydroxo)CuIICuIII Species (OHO) and Its Reaction with Methane from ref 49

Figure 3. Calculation model for the μ-η2:η2-peroxo CuIICuII species (PO). The C-α atoms indicated by asterisk in the Glu35, His137, His139, and Tyr374 residues are constrained to their positions in the crystal structure. The N-terminus of His33 is not constrained. B


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Inorganic Chemistry

to form the (μ-oxo)(μ-hydroxo)CuIICuIII species together with the tyrosyl radical and “deprotonated” Glu35 (OHO) . Figure 4 shows an energy diagram for the formation of OHO from PO. Let us first look at the initial complexes. From geometry optimizations by DFT calculations, we obtained two kinds of PO, i.e., POG and POT, as starters of this reaction. There is a clear difference in the way of binding to the Cu2 atom between POG and POT, in which Glu35 and Tyr374 coordinate to the Cu2 atom, respectively. POG lies 5.8 kcal/ mol below POT in the open-shell singlet state because of the electrostatic stabilization by coordinating the negative charges of Glu35 to the Cu2 atom in POG. However, according to the relation of the acidity between Glu35 and Tyr374, the proton transfer (PT) process can more easily occur from less stable POT. The PT from Tyr374 to Glu35 provides the peroxo intermediate with protonated Glu35 (Int1a), a calculated energy of which is 3.0 kcal/mol lower than POT. We see that the direct PT from Tyr374 to the Cu2O2 core without involving Glu35 in POT, such as in tyrosinase, is energetically unfavorable with a high activation energy of 29.2 kcal/mol relative to POT, as shown in Figure S1 of the Supporting Information. Thus, the PT from Tyr374 to Glu35 is more favorable in energy rather than the direct PT from Tyr374 to the Cu2O2 core. Then, Int1a is reversibly converted to Int1b, as a structural isomer of Int1a, because the energy difference between Int1a and Int1b is negligibly small. The two structural isomers differ only in the hydrogen bonding orientation of the migrated H atom. Next, the consecutive electron transfer (ET) takes place from Int1b. The first ET occurs from Tyr374 to the Cu2 atom at the Cu2O2 core to give the peroxo intermediate with tyrosyl

states with the triplet and open-shell singlet states, corresponding to the ferromagnetic and the antiferromagnetic coupled binuclear CuII(d9) centers, respectively. The energy difference between the two spin states is small during all the reaction pathways; therefore, the energy profiles based on the two spin states are essentially identical. To avoid duplication of description, we refer to only the energies in the open-shell singlet state.

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RESULTS AND DISCUSSION Mechanism for Dioxygen Activation. We show a reaction pathway from PO to OHO and the relevant electronic configurations in Scheme 2 within the framework of the openScheme 2. Possible Formation Process of OHO from POG

shell singlet state. In fact, the μ-η2:η2-peroxo CuIICuII species is EPR silent.55 The first step is initiated by the transfer of a proton and an electron, leading to the formation of the μ-η1:η2peroxoCuICuII species with tyrosyl radical and “protonated” Glu35 (Int3). Because the phenoxyl radical is considered to be a stable radical,56 this intermediate is rather stable in energy, as seen later in Figure 4. The next step undergoes the O−O bond cleavage of the peroxo species with the two electron transfer from the dicopper centers and the proton transfer from Glu35

Figure 4. Energy profile for the formation of OHO from PO in the open-shell singlet state. The relative energies with respect to POG are in kcal/ mol. Those of the triplet state are shown in the parentheses. C


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Inorganic Chemistry

Figure 5. Optimized Cu2O2 core structures with Glu35 and Tyr374 of intermediates POG, POT, Int1a, Int1b, Int2, Int3, BO, and OHO and transition states TS1, TS2, and TS3 in the open-shell singlet state. The units are in Å. His33, His137, His139, and hydrogen atoms are omitted for clarity.

radical and protonated Glu35 (Int2) with an activation barrier of 13.3 kcal/mol in TS1 in the open-shell singlet state. Then,

Int2 causes the second ET from the Cu2 atom to the Cu1 atom via TS2 with an activation barrier of 2.3 kcal/mol to give D


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Inorganic Chemistry another peroxo intermediate species Int3, which is the μ-η1:η2peroxo CuICuII species together with tyrosyl radical and protonated Glu35. This is a stable intermediate. In their experimental studies, Itoh, Fukuzumi, and co-workers proposed from the observation of C−C coupling dimer of phenol substrates that a similar phenoxyl radical species should be generated in the reaction of a μ-η2:η2-peroxo CuIICuII complex with a phenol substrate.57,58 We previously reported a possible mechanism for the biological conversion of tyrosine to dopaquinone by tyrosinase with a dicopper active site,50 where substrate tyrosine is located near the dicopper active site. The H atom of substrate tyrosine is directly transferred to the dicopper site for the formation of an intermediate similar to OHO, whereas in pMMO the H atom of Tyr374 migrates to the Cu2O2 core in a two-step manner via Glu35. The active site of tyrosinase has no specific amino acid residue (in the second coordination sphere) that can work as a base to abstract the proton from the phenol substrate.29,58−60 Therefore, the mechanism to form OHO is slightly different between pMMO and tyrosinase. Additionally, in the pMMO model, the protein provides only two nitrogen-based-histidine ligands to each copper center in pMMO, which is different from tyrosinase that has three nitrogen-based-histidine ligands. Therefore, one of the copper atoms is reduced from copper(II) to copper(I) to take the Tshape geometry in Int2 and Int3, as shown in Figure 5. In general, the structure of copper(I) complexes takes a threecoordinate T-shape geometry or a four-coordinate tetrahedral geometry.24 In addition, Int3 is energetically more stable than Int2. The Cu1 atom coordinates the separate two nitrogen atoms, whereas the Cu2 atom coordinates the chelated two nitrogen atoms as shown in Figure 3, in which the geometry of the Cu1 atom is more flexible than that of the Cu2 atom. Thus, the Cu1 atom can take an ideal T-shape geometry of copper(I) state, leading to stabilizing the structure of Int3. Next, we consider the transformation of Int3 to OHO, which involves the O−O bond cleavage and the PT from Glu35 to the Cu2O2 core. The O−O bond cleavage takes place from Int3 to give the bis(μ-oxo)CuIICuIII species together with tyrosyl radical and the protonated Glu35 (BO) with an activation barrier of 14.7 kcal/mol (TS3). Because the energy barrier is less than 20 kcal/mol, this process is generally expected to occur under physiological conditions. Because TS3 is higher than TS1 and TS2 in energy, the rate-determining step can be determined to be the O−O bond cleavage of Int3 in the overall reaction for the formation of OHO. Without the formation of Int3, the O−O bond cleavage from POT or Int1a would be energetically unfavorable (17.2 kcal/mol for POT and 25.1 kcal/mol for Int1a, see Supporting Information, Figures S2 and S3). This result is in good agreement with the previous calculation for tyrosinase, which suggests that the O− O bond cleavage of the peroxo intermediate is the ratedetermining step with an activation energy of 14.9 kcal/mol.50 After the O−O bond cleavage, the PT from Glu35 to the O1 atom takes place to provide OHO. This is actually a barrierless process. The tyrosyl radical is kept located in the second coordination sphere during the reaction. Reaction Species for Dioxygen Activation. Let us look next at the geometrical changes of the reaction species involved in dioxygen activation in detail on the basis of the optimized structures shown in Figure 5. The structure of initial complex POG shows that Glu35 is coordinated to Cu2, the Cu2−OTyr

and Cu2−OGlu distances being 3.853 and 2.177 Å, respectively. The acidity of Glu35 as a carboxylic acid is higher than that of Tyr374 as a phenol, and it is more increased by coordinating to the copper ion. On the other hands, POT shows that Tyr374 is coordinated to Cu2, the Cu2−OTyr and Cu2−OGlu distances being 2.269 and 3.711 Å, respectively. Owing to the increased acidity of Tyr374 caused by the binding to the copper ion, the proton can move from Tyr374 to Glu35 without activation barrier. The ligand exchange is expected to promote the PT process from Tyr374 to Glu35, leading to form Int1a. The transformation of POT to Int1a shows that the Cu2−O2 bond distance increases from 2.015 Å in POT to 2.626 Å in Int1a along with the decrease of the Cu2−Otyr bond distance from 2.269 Å in POT to 1.950 Å in Int1a via the PT process while the other bond distances (O1− O2, Cu2−O1, Cu2−O2, and Cu2−O Glu ) are almost unchanged. The Cu2 atom strongly coordinates to Tyr374 together with the cleavage of the Cu2−O2 bond in Int1a. Although the energy difference between Int1a and Int1b is negligibly small, as shown in Figure 4, there is a significant difference in the hydrogen bonding orientation of the migrated proton between Int1a and Int1b. The migrated proton interacts with OTyr in Int1a, while it is anchored by the O1 atom of the Cu2O2 core in Int1b. The Cu2−OGlu bond distance increases from 3.434 to 4.561 Å, whereas the Cu2− OTyr bond distance slightly decreases from 1.950 to 1.900 Å, with the internal conversion of Int1a to Int1b. To cause the ET process without returning the proton to Tyr374, the proton is needed to anchored by the O1 atom to generate a hydrogen bond. As a result, we consider that the ET process can easily occur in Int1b instead of Int1a because the proton can be held at Glu35 in Int1b. In the first ET from Int1b to Int2, the Cu2−OTyr bond length is increased from 1.900 Å in Int1b to 3.003 Å in TS1 and 4.519 Å in Int2, while the O1− O2 bond and the other Cu−O bond distances show almost no changes. Then, the second ET from Int2 to Int3 induces a decrease in the Cu2−O1 bond lengths in going from 2.818 Å in Int2 to 2.394 Å in TS2 and 1.931 Å in Int3 along with an increase in the Cu1−O1 bond lengths from 1.942 Å in Int2 to 1.992 Å in TS3 and 3.088 Å in Int3, so that the Cu2−O1 bond is formed together with the Cu1−O1 bond cleavage. Then, the O−O bond cleavage takes place concomitantly with the formation of the Cu1−O1 bond. The O1−O2 bond length increases from 1.459 Å in Int3 to 1.753 Å in TS3 and 2.426 Å in BO, while the Cu1−O1 distance is shortened from 3.088 Å in Int3 to 1.969 Å in TS3 and 1.952 Å in BO. The O− O bond distance of TS3 is reasonable for a transition state structure; the value is consistent with previous DFT results for the O−O bond cleavage in tyrosinase (the O−O distance 1.74 Å)50 and the dicopper model complex (1.85 Å).45 Consequently, pMMO and tyrosinase are quite similar in both of the energetic and structural aspects for the O−O bond cleavage. Finally, BO is easily converted to OHO with the PT from Glu35 to the Cu2O2 core. The OGlu−H bond distance increases from 1.094 Å in BO to 1.429 Å in OHO along with a decrease in the O1−H bond distance from 1.380 Å in BO to 1.066 Å in OHO. In addition, the Cu−Cu distance of 4.088 Å in Int3 decreases to 2.813 Å in BO and 2.842 Å in OHO, which is in good agreement with the experimental results by X-ray crystallography and EXAFS analysis for bis(μoxo)dicopper complexes (∼2.8 Å).61,62 Thus, we consider that the choice of our calculation model should be reasonable in the E


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Inorganic Chemistry

consistent with the first-step PT from Tyr374 to Glu35 during the complexation from POT to Int1b. Then, in Int1b, TS1, and Int2, the spin density of the Cu1 atom is 0.42 in Int1b, 0.37 in TS1, and 0.38 in Int2, whereas that of the Cu2 atom increases from −0.53 in Int1b to 0.01 in TS1 and 0.05 in Int2, as shown in Table 1 and Figure S4 in the Supporting Information. Furthermore, the spin density of OTyr decreases from −0.14 in Int1b to −0.37 in TS1 and −0.38 in Int2, which means that the OTyr atom has a significant radical character in particular in Int2. The ESP charge of the Cu2O2 core decreases from 1.40e in Int1b to 0.89e in TS1 and 0.81e in Int2 due to the reduction of the Cu2 atom, as listed in Table 2. These results clearly demonstrate that the Cu2−OTyr bond in Int1b is homolytically cleaved together with a change in the formal charge of the Cu2 atom from +2 in Int1b to +1 in Int2 with tyrosyl radical. We see that the ESP charges of Tyr 374 as well as the Cu2O2 core are changed depending on the Cu2−OTyr bond distance, as shown in Supporting Information, Figure S5. Next, in Int2, TS2, and Int3, the spin density of the Cu1 atom decreases from 0.38 in Int2 to 0.29 in TS2 and 0.02 in Int3, whereas that of the Cu2 atom increases from 0.05 in Int2 to 0.15 in TS2 and 0.39 in Int3, as shown in Table 1 and Supporting Information, Figure S4. The spin density of OTyr is −0.38 in Int2, −0.38 in TS2, and −0.36 in Int3. These results suggest that the Cu1 atom is reduced from +2 to +1, while the Cu2 atom is oxidized from +1 to +2 in the transformation from Int2 to Int3. Because the second ET takes place within the Cu2O2 core, the ESP charge of Cu2O2 remains almost unchanged, 0.81e in Int2, 1.00e in TS2, and 0.94e in Int3, as listed in Table 2. In the O−O bond cleavage from Int3 to BO via TS3, the spin density of the Cu1 atom increases from 0.02 in Int3 to 0.31 in TS3 and 0.54 in BO, whereas that of the Cu2 atom decreases from 0.39 in Int3 to 0.39 in TS3 and −0.12 in BO as shown in Table 1. These results clearly tell us that the Cu1 atom is oxidized from +1 to +2 and the Cu2 atom is oxidized from +2 to +3 when the O−O bond in Int3 is cleaved. Interestingly, we found that the ET from the two copper atoms to the peroxo moiety is not simultaneous but sequential. Because the spin density of the Cu1 atom begins to change before TS3, the initial ET takes place from the Cu1 atom. Then, the second ET takes place from the Cu2 atom because the spin density of the Cu2 atom starts to change after TS3. Because the O−O bond cleavage takes place within the Cu2O2 core, the ESP charge of Cu2O2 is almost unchanged, 0.94e in Int3, 0.96e in TS3, and 0.98e in BO, as listed in Table 2. In the final step from BO to OHO, calculated spin densities of all the atoms are listed in Table 1. The ESP charge of the Cu2O2 core increases from 0.98e in BO to 1.57e in OHO, whereas that of Glu35 decreases from −0.05e in BO to −0.64e in OHO. These results indicate that a proton is released from Glu35 to the Cu2O2 core, resulting in the formation of OHO. Because BO and OHO have large spin densities at the O2 atom, these two species are expected to be active for the C−H activation of methane, as discussed in a previous study.49 The values obtained from the Mulliken spin population (Table 1) and ESP charge (Table 2) analyses fully explain the PCET mechanism for dioxygen activation at the dicopper site.

O−O bond cleavage and the Cu−Cu distance. Because Tyr374 located in the second coordination sphere is relatively flexible, the significant geometrical change can be allowed during the reaction. Population Analysis for PCET. To elucidate the PCET process associated with Tyr374 and Glu35 during the O−O bond cleavage of the μ-η2:η2-peroxodicopper(II) species (PO) to form the (μ-oxo)(μ-hydroxo)CuIICuIII species (OHO), we calculated spin density by the Mulliken spin population analysis and electrostatic potential fit (ESP) charge by the Merz−Kollmann (MK) method,63 as summarized in Tables 1 Table 1. Calculated Mulliken Spin Populations in the OpenShell Singlet Statea species

Cu1

Cu2

O1

O2

OTyr

POG POT Int1a Int1b TS1 Int2 TS2 Int3 TS3 BO OHO

0.44 0.44 0.39 0.42 0.37 0.38 0.29 0.02 0.31 0.54 0.57

−0.53 −0.51 −0.55 −0.53 0.01 0.05 0.15 0.39 0.39 −0.12 −0.11

0.05 0.06 0.10 0.23 0.25 0.26 0.27 0.25 0.10 0.20 0.14

0.08 0.06 0.24 0.16 0.24 0.23 0.22 0.24 0.06 0.31 0.33

0.00 0.00 −0.09 −0.14 −0.37 −0.38 −0.38 −0.36 −0.36 −0.36 −0.36

a

See Figure 3 for the atom labeling.

Table 2. Calculated Electrostatic Potential Fit (ESP) Charge in the Open-Shell Singlet State species G

PO POT Int1a Int1b TS1 Int2 TS2 Int3 TS3 BO OHO a

Cu2O2a

Glu35

Tyr374

1.70e 1.71e 1.64e 1.40e 0.89e 0.81e 1.00e 0.94e 0.96e 0.98e 1.57e

−0.70e −0.67e 0.10e 0.12e 0.14e 0.15e 0.01e 0.00e −0.01e −0.05e −0.64e

0.00e −0.04e −0.74e −0.52e −0.03e 0.04e 0.00e 0.06e 0.05e 0.06e 0.07e

The Cu2O2 core including His33, His137, and His139. protonated Cu2O2 core.

b

The

and 2, respectively. It is confirmed that these electrostatic figures reasonably change along the PT and ET processes. In POT, Int1a, and Int1b, calculated spin densities of the Cu1 and Cu2 atoms are 0.44 and −0.51 in POT, 0.39 and −0.55 in Int1a, and 0.42 and −0.53 in Int1b, which are identical among those species, as listed in Table 1. The two Cu atoms have significant spin densities, which are antiferromagnetically coupled in the open-shell singlet state. These results can tell us that the formal charges of the Cu1 and Cu2 atoms remain to be +2, i.e., d9 in these initial complexes. Then, the ESP charge of Tyr374 decreases from −0.04e in POT to −0.74e in Int1a and −0.52e in Int1b, while that of Glu35 increases from −0.67e in POT to 0.10e in Int1a and 0.12e in Int1b, as shown in Table 2. These results are fully

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CONCLUSIONS We have studied that the tyrosine and glutamate residues in the second coordination sphere of the dicopper site of pMMO play an important role in the formation of an active species for F


Article

Inorganic Chemistry

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methane activation. The phenolic proton of the Tyr374 residue is transferred to the Cu2O2 core in a two-step manner via the Glu35 residue, while an electron is directly moved to the Cu2O2 core. These PT and ET processes effectively induce the O−O bond cleavage of the μ-η2:η2-peroxodicopper(II) species to form the (μ-oxo)(μ-hydroxo)CuIICuIII species. In contrast, in the case of tyrosinase the proton of substrate tyrosine is directly transferred to the dicopper site because there is no proton acceptor in the vicinity of the dicopper site in tyrosinase. The glutamate residue in the second coordination sphere of pMMO promotes the two-step proton transfer, and the resultant (μ-oxo)(μ-hydroxo)CuIICuIII species is able to oxidizes methane in place of tyrosine. We see that in this mechanism, phenoxyl radical, which is known as a stable radical, is formed in the vicinity of the dicopper site of pMMO. The suitable position of the tyrosine and glutamate residues should lead to form the active species efficiently under physiological conditions. Recent studies suggest that the activation of O2 and oxidation of methane should take place at the monocopper center, as mentioned earlier in this article.14−16 However, we cannot rule out the possibility of O2 activation at the dicopper site of pMMO, as actually observed at the dicopper sites of hemocyanin, tyrosinase, and catechol oxidase.28−30

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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.9b01752. Additional figures (energy diagrams and plots of spin densities and ESP charges) and Cartesian coordinate (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Tsukasa Abe: 0000-0002-2400-3797 Yuta Hori: 0000-0002-3212-1996 Yoshihito Shiota: 0000-0003-2054-9845 Kazunari Yoshizawa: 0000-0002-6279-9722 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.Y. acknowledges KAKENHI grant nos. JP15K13710 and JP17H03117 from the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), the MEXT Projects of “Integrated Research Consortium on Chemical Sciences”, “Cooperative Research Program of Network Joint Research Center for Materials and Devices”, “Elements Strategy Initiative to Form Core Research Center”, and JSTCREST “Innovative Catalysts JPMJCR15P5”.

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REFERENCES

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