MM Calculation of the Enzyme Catalytic Cycle Mechanism for

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QM/MM Calculation of Enzyme Catalytic Cycle Mechanism for Copper- and Zinc-Containing Superoxide Dismutase Masami Lintuluoto, Chiaki Yamada, and Juha Mikael Lintuluoto J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b03589 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 15, 2017

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The Journal of Physical Chemistry

QM/MM Calculation of the Enzyme Catalytic Cycle Mechanism for Copper- and Zinc-containing Superoxide Dismutase

Masami Lintuluoto*,1, Chiaki Yamada1, and Juha M. Lintuluoto2

1

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamohanki-cho, Sakyo, Kyoto 606-8522, Japan

2

Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8530, Japan

1

ACS Paragon Plus Environment


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ABSTRACT The entire enzyme catalytic mechanism including the electron and the proton transfers of the copper- and zinc-containing extracellular superoxide dismutase (SOD3) was investigated by using QM/MM method. In the first step, the electron transfer from

O2•− to SOD3 occurred without the bond formation between the donor and the acceptor and formed the triplet oxygen molecule and reduced SOD3. In the reduced SOD3, the distorted tetrahedral structure of Cu(I) atom was maintained. The reduction of Cu(II) atom induced the protonation of His113, which bridges between the Cu(II) and Zn(II) atoms in the resting state. Since the protonation of His113 broke the bond between Cu(I) and His113, three-coordinated Cu(I) was formed. Further, we suggest the binding of O2•− formed hydrogen peroxide and the resting state after both the Cu reduction and the protonation of His113. The protonation of His113 caused the conformational change of Arg186 located at the entrance of the reactive site. The electrostatic potential surface around the reactive site showed that Arg186 plays an important role as electrostatic guidance for the negatively charged substrates only after the protonation of His113. The rotation of Arg186 switched the proton supply routes via Glu108 or Glu179 for transferring two protons from the bulk solvent.

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INTRODUCTION The

superoxide

dismutases

(SODs)

protect

cells

by

catalyzing

the

disproportionation conversion of superoxide anion radical (O2•−) into molecular oxygen and hydrogen peroxide, H2O2. Reactive oxygen species (ROS) including O2•− are generated in a wide range of life processes and have toxicity due to their reactivity. There are three classes of human SODs, with differing structural features and the containing metal atoms. Both SOD1 and SOD3 include Cu and Zn atoms, whereas Mn atom is found in SOD2. Cytosolic SOD1 is a homo dimer and is expressed in almost all cells. Extracellular SOD3, a tetramer containing two identical homo dimers (Figure 1), has important roles in the protection of human brain and lungs from hyperoxia and in the adjustment of the concentration of nitric oxide.1-2 The reactive sites of both SOD1 and SOD3 are conservative, and their reactive site structures are similar (Figure 1). Both of the metal atoms are coordinated to four ligands, and one His ligand bridges between the Cu and Zn atoms. A single site mutation of SOD1 is associated with the neurodegenerative disorder known as familial amyotrophic lateral sclerosis (fALS),2-5 and SOD3 is thought to be associated with the risk of myocardial infarction, cardiovascular death and all-cause mortality in people with diabetes.6-7 SOD3 is the only glycosylated SOD isoenzyme,1 and it has affinity for heparin8

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and sulfated glycosaminoglycans.9 The C- and N-terminal end regions of SOD3, which are extended compared to the equivalent loops in SOD1, are required for tetramerization and heparin binding.1 SOD3 is highly expressed in the blood vessel walls and in the lungs, and it has been suggested to play important roles in various oxidative stress-dependent

pathophysiologies,

including

hypertension,

heart

failure,

ischemia-reperfusion injury, and lung injury.10-12 The disproportionation reaction of O2•− by SODs is very rapid and reaches the diffusion limit. Kinetic studies of SOD1 have indicated that the disproportionation reaction of O2•− includes two irreversible reactions as follows:13-15 Protein-Cu2+ + O2•− → Protein-Cu+ + O2

(1)

Protein-Cu+ + O2•− + 2H+ → Protein-Cu2+ + H2O2

(2)

The high-resolution structures of SOD1 from the eukaryotic thermophile Alvinella pompejana and its complex with H2O2 support an inner sphere mechanism rather than an outer sphere mechanism.16 At the first step, the O2•− binds to Cu(II), and Cu(II) is reduced and molecular O2 is formed. The bond between Cu and the His ligand bridging the two metal sites is broken, and the His ligand is protonated. In the second step, a proton from the His ligand and an electron from Cu(I) transfer to O2•− binding at Cu(I) to form H2O2 or HO2−. Cu(I) and the bridged His ligand are reformed.

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The highly conservative Arg residue located at the entrance of the narrow reactive site in SOD1 has been suggested to have an important role as electrostatic guidance for the negatively charged substrates.17-20 Arg141, Arg143 and Arg186 are located at the entrance of the reactive site in bovine SOD1, human SOD1 and human SOD3, respectively. The Arg residue of SOD1 has appeared to interact with the O2•− binding on Cu in the supposed catalytic mechanisms.17,21 Although there have been numerous structural and mechanistic studies of SOD1, the details of the reaction mechanism including the order of electron and proton transfers have not been established. In addition, the mechanism of SOD3 has not been elucidated. Quantum chemical studies for bovine SOD1 have shown the existence of the O2•− binding state stabilized by Arg141 and the inner-sphere electron transfer by using active site models containing Cu, its four ligands and Arg141.22-24 The inner-sphere electron transfer includes the bond formation between the electron donor and acceptor.21,25 Estimations of pKa and the reduction potential of SOD have been performed by using the density functional theory (DFT) method and electrostatic modeling with a relatively large model composed of both metals and their ligand residues.26 In another DFT study, a unique assumption was introduced to investigate the catalytic cycle of SOD1 and the role of Asp121 interacting with two His ligands

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coordinated to Cu and Zn, respectively.27 In the proposed mechanism, O2•− was assumed to be protonated during the approach to the reactive site before the binding to Cu(II), and OOH radical was bound on Cu(II).27 Cu(II)OOH was formed while keeping the four-coordination of Cu(II), and then the protonation of bridging His residues occurred.27 Another DFT study that used a model including the Cu atom and its four His ligands was also reported.28 The reaction proceeded by two steps, and the catalytic process started with the binding of O2•− to Cu(II). The rate-determining step was the second step, and it included the H2O2 formation. A molecular dynamic simulation of SOD1 has also been performed to clarify the structure of the active site.29 That study's solvent shell analysis revealed that there was not any tightly bound water molecule near the Cu ion, but there were loosely bound water molecules located approx. 4–7 Å from Cu atom.29 In the present study, we investigated the enzyme catalytic cycle of human SOD3 and

the

roles

of

second-sphere

amino

residues

by

using

the

quantum

mechanics/molecular mechanics (QM/MM) method. Two positions for a Cu atom in the crystal structure were suggested to be due to not only the reduction of Cu but also the protonation of bridging His113. In the first step of the catalytic cycle, the electron

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transfer from O2•− to Cu(II) occurred without bond formation between them. The Cu reduction by the electron transfer from O2•− induced the protonation of bridging His113. The binding of O2•− on Cu occurred only after the reduction of Cu and the protonation of His113, and then the protonation of O2•− on Cu followed to form H2O2 and the resting state of SOD3. We found that the Arg186 located at the entrance of the narrow reactive site can play a role as electrostatic guidance due to the conformational change induced by the protonation of bridging His113. The conformational change of Arg186 was also found to be related to the switching of two proton supply routes starting from Glu108 and Glu179, respectively.

METHOD The initial coordinates of human SOD3 were derived from the X-ray structures (Protein Data Bank [PDB] 2JLP).1 Hydrogen atoms were added to each residue in the standard manner by assuming a standard protonation state under physiological pH conditions. The Cu and Zn atoms and their seven ligands His96, His98, His113, His121, His124, His163 and Asp127 are included in the QM region, as shown in Figure 2. His96, His98 and His 163 coordinate to the Cu, and His121, His124 and Asp127 coordinate to

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the Zn atom. His113 coordinates to both metal centers and is assumed to play an important role in the catalytic cycle of the Cu and Zn-containing SODs. Three water molecules, named WAT1 to WAT3, located above the reactive site are also included in the QM region. The amino residues and water molecules within a 10 Å radius from Cu and Zn are included in the MM region. Two water molecules, WAT4 and WAT5, and so-called second-sphere amino residues, i.e., Asp167, Asn180, Gly184 and Arg186 in the MM region are relaxed during geometry optimizations. Two water molecules, WAT4 and WAT5, interact with three other water molecules of the QM region, and build the hydrogen bond network with the second-sphere residues, i.e., Asn180 and Arg186. The residue Asp167 interacts with the Nε atoms of His96 and His121, and residue Asp122 located at the same corresponding position of bovine SOD1 interacts with His44 and His69; Asp122 has been supposed to stabilize the conformation of reaction site.27 Arg143 and Thr137 in human SOD1, which are equivalent residues to Arg186 and Asn180 in SOD3,1 play the role of electrostatic guidance for the negatively charged substrates to the Cu located at the bottom of the narrow reactive site.17-19 We performed the QM/MM calculation by using the two-layer ONIOM scheme.30 We used the B3LYP functional for geometry optimization and single-point calculations of the QM region. The basis sets 6-31G(d,p) for N, O, S and H atoms and the

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Stuttgart/Dresden ECP valence basis sets (SDD)31-32 for Cu and Zn atoms were used. We used the universal force field (UFF)33-34 for the MM region, and the charge equilibration (QEq)35 approach was used for the atomic charges of the MM region. The UFF was developed for the investigation36-37 of inorganic and organic materials and compounds of elements throughout the periodic table,33 and the extent development of the UFF has been performed for organic compounds including the transition metals.34 The UFF has also been successfully used with the ONIOM method for the investigations of proteins38-40 and metalloproteins.41-44 We estimated the Gibbs free energy changes along the reaction steps, i.e., the electron transfer from O2•− to Cu(II) to form the molecular oxygen and Cu(I), the binding of O2•− on Cu, and the formation of H2O2. We calculated the values for the Gibbs free energy by using the optimized structures of the QM region without the MM region. The protonation of NεHis113, which is bridging between the Cu and Zn atoms, occurred at the early step of reaction, and the protonation of O2•− binding on Cu (Cu-O-O) is involved in the formation of H2O2. We therefore estimated the pKa values from the Gibbs free energy differences (∆G) for the HNεHis113 and Cu-O-O-H by using the Born-Haber thermodynamic cycle. The values of the solvation energy were calculated by using a polarizable

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continuum model (PCM) with an ε = 4.0, 20.0, and 78.4 as an implicit solvent model.45 The solvation energy for the proton estimated by the experimental and computational methods are in the range between −254 and −270 kcal·mol−1.46-54 We used the value −270 kcal·mol−1. The value of −6.32 kcal·mol−1 was used for the gas-phase free energy of the proton at 298 K and the standard pressure.55 The error of 1 kcal·mol−1 in the Gibbs free energy difference translates to an error of approx. 0.73 in the estimated pKa. The error in the estimation of Gibbs free energy by the DFT method has been reported as 2–3 kcal·mol−1.56 The calculated values of pKa based on the Born-Haber cycle have been comparable to the experimental results and have been used for qualitative discussions in several studies.46-48,52-54 We have used the same methods to estimate the values of pKa for the enzyme catalytic reaction of Cu-containing nitrite reductase, and we used the values in discussions of the complicated reaction mechanism containing the electron and proton transfers.57-58 We thus contend that the comparison of pKa values among our calculation models is qualitatively correct. All calculations were carried out by using the Gaussian 09 program.59 Facio and CueMol were used for the visualization of results.60-61

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RESULTS AND DISCUSSION Conformation and electronic structure of the resting state Four His ligands are tetrahedrally coordinated to Cu(II), and three His and an Asp ligands are tetrahedrally coordinated to Zn(II) as shown in Figure 3(a),(b). The His113 is coordinated to both Cu and Zn atoms. The distances between the metal atoms and their ligands were approx. 2 Å, as shown in Figure 3(b). There were five water molecules (WAT1–5) above the reactive site. These water molecules were far from the Cu atom located at the bottom of the reactive site, and the distance between the O atom of the nearest water (WAT1) and Cu(II) was 4.91 Å, which is in good agreement with the results from the solvent sphere analysis of a molecular dynamics (MD) simulation.29 The five water molecules built the hydrogen bond network with Asn180, Arg186 and Glu108 as shown in Figure 3(a),(c). Four water molecules, WAT1–3 and WAT5 (WAT4 is the exception), are in line starting from the Glu108 located at the entrance of the reactive site. The net atomic charges and spin density on both metal atoms in the resting state are listed in Table 1. The spin density localized at the Cu atom shows that Cu is in the oxidized state.

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Reduction and protonation of the resting state We next investigated the addition of an electron to the resting state, [Cu(II)-His113-Zn(II)]. The geometrical structure was not affected by the electron addition, so the tetrahedral conformation around both Cu and Zn was kept. The net atomic charge and spin density of the Zn atom did not change by adding an electron, whereas the net atomic charge of Cu decreased from 0.443 to 0.115, and the spin density located at the Cu atom disappeared (as shown in Table 1). Therefore, the addition of an electron to the resting state, [Cu(II)-His113-Zn(II)], forms the reduced state of Cu, [Cu(I)-His113-Zn(II)]. The standard reduction potentials in the aqueous solution for Cu2+/Cu, Cu2+/Cu+ and Zn2+/Zn are 0.34, 0.16 and −0.76 V, respectively.62 Since the electron affinity of Cu2+ is higher than that of Zn2+, the Cu(II) rather than Zn(II) was reduced in human SOD3. Crystallographic and kinetic studies have shown that the zinc atom plays two important roles in human SOD1, maintaining the structural integrity and the catalytic ability over a wide range of pH values (5.0–9.5).63-66 The protonation of the bridging Nε-His113 of the resting state yielded [Cu(II) HNε-His133-Zn(II)]. The conformational changes by the protonation are shown in Figure 4(a). The coordination number of Cu decreased from four to three due to the

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protonation of Nε-His113, and the Cu moved away from the His113 by 1.2 Å. This result is in good agreement with the X-ray structure, in which the Cu atom was located at two positions, and the distance between the two positions was 1.2 Å.1 The conformations of Arg186 and three His ligands of Cu were changed by the protonation of Nε-His113, as shown in Figure 4(a),(b). The dihedral angle of Cu-Nδ-Cε-Nε of His96 is changed from 170.0° to 163.2° by the protonation of Nε-His113. The dihedral angles of the Cu-Nε-Cε-Nδ of His98 and His163 are changed from −161.9° and −167.4° to −169.2° and −145.4°, respectively. The dihedral angle of the Cβ-Cγ-Cδ-Nε of Arg186 is changed from −96.8° to −174.0°. In the X-ray structure,1 the dihedral angle of the Cβ-Cγ-Cδ-Nε of Arg186 in subunits A and D without SCN− ligand are −174.4° and −168.7°, respectively, and those in subunits B and C with SCN− ligand are −170.2° and −172.1°, respectively. The conformation of Arg186 after the protonation of His113 in our calculation corresponds to the X-ray crystal structure of SOD3. The protonation of Nε-His113 changed the hydrogen bond network of WAT1–5. WAT5 was separated from the other water molecules. WAT1 interacted with the HNε-His133 and shortened the distance to Cu to 3.55 Å. WAT2 moved away from Arg186 and interacted with His163 and the backbone of Asn180. WAT3 was located

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above the His98 before the protonation of Nε-His113 and moved to near His113 by the protonation of Nε-His113. Three water molecules (WAT1–WAT3) formed a line along the narrow channel composed by the Asn180 and Arg186 as shown in Figure 4(a),(c). The interactions between the three His ligands and the second-sphere residues were also changed by the protonation of Nε-His113 as shown in Figure 4(b),(c). Before the protonation, His98 interacted with the WAT3 and backbone of Gly111. The protonation of Nε-His113 induced the rotation of Arg186 and the movement of WAT3, and His98 consequently changed the interaction with the surroundings. His98 interacted with Arg186 and the backbone of Thr110 and Gly111 after the protonation of Nε-His133. The conformational changes induced by the protonation of His113 in [Cu(I)-His113-Zn(II)] were the same as those in [Cu(II)-His113-Zn(II)], so the geometries of all atoms in [Cu(I) HNεHis113 Zn(II)] were the same as those in [Cu(II) HNεHis113 Zn(II)]. The net atomic charge of Cu atom was not changed by the protonation of His113 in either of the Cu oxidation states, but the spin density of Cu atom was decreased from 0.635 to 0.302 by the protonation of His113 in the Cu(II) state, as shown in Table 1. We calculated the pKa of HNε-His113; the results are shown in Table 2. The value

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of pKa for the reduced bovine SOD1 has been reported as 10.7.67 The calculated values of pKa for [Cu(I) HNεHis113-Zn(II)] depended on the value of ε and varied from 15.9 to 28.5 (Table 2). The copper atom is positioned at the bottom of the narrow reaction site, and there was no water molecule around the reaction site. The nearest water molecule was located 4.91 Å away from the Cu(II) reaction site. The MD simulation of SOD1 also showed that there are secondary spheres of water molecules, which are loosely bound and 4–7 Å away from the Cu atom.29 We therefore consider the assumption of the low solvent dielectric constant around the reactive site to be correct. The value of pKa for the [Cu(I) HNεHis113-Zn(II)] calculated with ε = 4.0 is reasonable and comparable to the experimental result,67 and from this point forward we will discuss the values calculated with ε = 4.0. The protonation of His113 in the resting state [Cu(II)-His113-Zn(II)] seems to be unlikely to occur due to the very low pKa, whereas in the reduced state of Cu [Cu(I)-His113-Zn(II)] the protonation of His113 can occur easily. The β-LUMO of [Cu(II)-His113-Zn(II)] is a σ*(Cu-NεHis113) orbital composed of the NεHis113 p-orbital and Cu d-orbital, and the orbital spread over the Cu atom as shown in Figure 5. The addition of an electron to [Cu(II)-His113-Zn(II)] weakens the Cu-NεHis113 bond and makes the protonation of NεHis113 occur easily, and forms [Cu(I) HNεHis113-Zn(II)].

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The X-ray structure analyses and the extended X-ray absorption fine structure (EXAFS) for SOD1 have shown that Cu occupied two positions.68-69 These two positions for the Cu atom in SOD1 and SOD3 are assigned the possibility of both the Cu oxidized state and the ligand binding state.1,68-69 For human SOD1, the presence of the reduced Cu was not found to be related to the X-ray-induced photoreduction in the EXAFS and crystallographic studies.68-69 The conformation of Arg186 and the position of Cu were changed by the protonation of His113 in our present study. In addition, the value of pKa in [Cu(I) HNεHis113-Zn(II)] is large enough to protonate and break the His113 bridging between the Cu and Zn atoms. We thus think that the two positions correspond to [Cu(II)-His113-Zn(II)] and [Cu(I) HNεHis113-Zn(II)].

Approach of O2•− to Cu We next investigated the binding of O2•− to the resting state. Although we tried two O2•− binding orientations, i.e., the end-on and the side-on at the Cu(II) atom, neither binding form was stable. The formation of triplet O2 and the reduction of Cu occurred at the same time, and the triplet O2 was released. We next tried to set the approach for the O2•− to the Cu through the narrow channel composed by the Asn180 and Arg186. The electron transfer from O2•− to Cu(II) occurred even at the range from 6 to 8 Å, and the

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triplet O2 molecule was formed, regardless of whether or not the His113 was protonated. The tetrahedral structure centered by the Cu atom was distorted, especially around the Cu-NεHis113. The Nδ or Nε lone pair of His ligands interacted with the Cu atom, and the Cu and imidazole rings of His residues were in-plane except for that of His113. The lone-pair of Nε-His113 was spread over the Cu(II) atom due to the structural distortion, as shown in Figure 5. The negatively charged substrates may not approach due to the repulsion with the lone pair of Nε-His113. The reduction potential of O2•−/O2 is −0.160 V vs. NHE, whereas that of bovine SOD1 has been reported as 403 mv vs. NHE.70 The electron can travel over 10 Å between the electron donor and acceptor in protein.71-74 The high-resolution crystal structure of the electron donor-acceptor complex between the Cu-containing nitrite reductase and cytochrome c has been reported, and the distance between the redox centers was 10.5 Å.75 We think that the electron transfer can easily occur without the bond formation due to the great difference of reduction potentials. Since the O2•− is solvated by water molecules in vivo, our models may not be sufficient to describe the electron transfer in vivo. However, we note that our results are available for qualitative discussion. We investigated the effect of the structural distortion of bridged His113 on the

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reduction potential of Cu(II) by using a smaller model extracted from the optimized structure of the ONIOM models. The smaller models contained a Cu atom and four imidazole rings instead of four His ligands, as shown in Figure S1. The imidazole rings of three His ligands, with the exception of His113 and the Cu atom in the resting state, are in-plane. The dihedral angles of N-C-N-Cu in His96, His98 and His163 are in the range between 162.0 and 170.0°, and that in His113 is 153.5°. After the optimization of the smaller model, four imidazole rings and Cu atom became in-plane, and the dihedral angles of N-C-N-Cu for the four imidazole rings were in the range between 178.2 and 178.6°. The Gibbs free energy differences by the geometry optimization were 26.0 and 24.2 kcal•mol−1 at ε = 0.0 and 4.0, respectively. We calculated the redox potentials by using the Born-Haber thermodynamic cycle. The value of the free energy change associated with the reference normal hydrogen electrode (NHE) was set to −4.44 V.76 The reduction potentials with ε = 4.0 were 0.264 and −0.396 V before and after the geometry optimization, respectively. The structural distortion of His113 changed the reduction potential, and raising the reduction potential accelerated the electron transfer from O2•− to Cu(II). The electron transfer processes from O2•− to Cu(II) are summarized in the following equations:

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CuII˗His113˗ZnII + O∙ ⇌ CuI˗His113˗ZnII + O

(3)

CuII HN His113˗ZnII + O∙ ⇌ CuI HN His113˗ZnII + O

(4)

Experimental and theoretical studies24,69 of SOD1 suggested that Cu is coordinated by three His residues and that the His133 bridging bond is broken in the intermediate as a reduced state of SOD1. On the other hand, experimental results with the competitive oxygen (18O) kinetic isotope effects (KIEs) have shown evidence of the reduced Cu coordinated by four His ligands, including the bridging His residue.25 In addition, the electron transfer (ET) from O2•− to SOD was not found as outer-sphere ET but rather as inner-sphere ET, which includes the bond formation between the electron donor and acceptor.25 Our results showed the existence of four coordinated reduced SOD, but they do not support the inner-sphere ET including the bond formation between the electron donor and acceptor. It is difficult for the negatively charged substrates to approach the Cu atom due to the repulsive interaction with the lone pair of NεHis113, as mentioned earlier. We suspect that the electron transfer occurs from O2•− to SOD3 without the bond formation, and that it forms the tetrahedrally coordinated reduced SOD3. The binding of O2•− did not occur on [Cu(I)-His113-Zn(II)], whereas the O2•− was bound on Cu(I) in [Cu(I) HNεHis113-Zn(II)]:

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CuI HN His113˗ZnII + O∙ ⇌ CuI˗O˗O HN His113˗ZnII

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(5)

The Gibbs free energy differences for these reactions, i.e., Eqs. (3)–(5) are shown in Table 3. All of the reactions are exothermic with ε = 4.0. The structure of O2•− binding to Cu(I) in [Cu(I) HNεHis113-Zn(II)] is illustrated in Figure 6. The O2•− binding on Cu(I) was in the end-on binding form, in which one O atom (Oa) was coordinated to Cu(I) and the other O atom (Ob) interacted with the HNεHis113 (Figure 6). The O-O bond length (1.40 Å) was larger than the 1.24 Å of free O2•− and the 1.34 Å of free O2 in the gas phase. The short distance between Ob and HNεHis113 and the long bond length of H-Nε may indicate that the protonation of O2•− already occurs in this state. The distance between Cu and Oa was 1.95 Å and the angle of Oa-Cu-NεHis163 was 91.2°. The distances between the Cu and N atoms of three ligands, i.e., His96, His98 and His163 were 1.98, 1.93 and 2.17 Å, respectively, and these values are almost the same as those in [Cu(I) HNεHis113-Zn(II)]. The Oa and three His ligands formed the Cu-centered pyramidal structure. The two water molecules, WAT1 and WAT3, interacted with Ob and Oa, respectively, in which the distances Ob-WAT1 and Oa-WAT3 were 1.81 and 1.74 Å, respectively. The Cu had the spin density 0.447 (Table 1), and thus it seems that Cu is in the oxidized state.

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Formation of H2O2 We investigated the protonation of Cu-O-O− to form H2O2 and the resting state. As already mentioned, the proton transfer from HNεHis113 seemed to have already occurred in [Cu(I)-O-O− HNεHis113-Zn(II)]. We assumed that the second proton is carried from the bulk solvent via WAT1 or WAT3 interacting with the binding O2•− in [Cu(I)-O-O− HNεHis113-Zn(II)], as shown in Figure 7. We further think that the proton carried by WAT1 or WAT3 was transferred to Ob or Oa, respectively. However, both reaction routes yielded [Cu(I)-O-O-H HNεHis113-Zn(II)], and HNεHis113 interacted with the Oa atom. The pKa for the Cu(I)-O-O-H was relatively low compared to the HNεHis113 in [Cu(I) HNεHis113-Zn(II)], as shown in Table 2 (∆pKa = 8.9). The optimized structure of [Cu(I)-O-O-H HNεHis113-Zn(II)] is illustrated in Figure 8. The O-O bond length was elongated by the protonation to 1.42 Å, which is close to 1.44 Å of the free H2O2 in the gas phase. WAT1 and WAT3 have close contacts with HOb and Oa atoms, 1.62 and 1.86 Å, respectively, and the 1.60 Å proximity of HNεHis113 with the Oa atom suggests considerable interaction. The net atomic charge and spin density of Cu became larger and closer to the values in [Cu(II)-His113-Zn(II)], as shown in Table 1. In the last step, the proton transferred from the HNεHis113 to the Oa atom without

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an energy barrier to form H2O2 and the resting state as shown in the following equation: CuI˗O˗OH HN His113˗ZnII ⇌ CuII˗His113˗ZnII + H2 O2 ↑

(6)

The hydrogen peroxide was released from the Cu atom simultaneously with the formation of the resting state. The Gibbs free energy differences from Eq. (6) with three different ε values all show exothermic behavior as shown in Table 3.

Enzyme catalytic cycle We summarized the suggested catalytic cycle of SOD3 as shown in Figure 9. First, O2•− approaches the resting state [Cu(II)-His113-Zn(II)] and the electron transfer from O2•− to SOD3 occurs without the bond formation between the electron donor and acceptor. The electron addition to the σ*(Cu-NεHis113) increases the proton affinity of NεHis113. The binding of O2•− does not occur in either of the Cu oxidized states before the protonation of His113, but it binds to Cu in [Cu(I) HNεHis113-Zn(II)] to form Cu(I)-O-O− with the stabilization energy of 19.6 kcal·mol−1. Under the physiological conditions, the protonation of Cu(I)-O-O− occurs to form Cu(I)-O-O-H by the proton transfer from bulk solvent due to its pKa of 7.0. At the last step, the proton transfer from HNεHis113 yields H2O2 and the resting state with the stabilization energy of −1.8 kcal·mol−1.

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The role of Arg186 The distance between Arg186 and Asn180 was changed by the protonation of His113 from 7.66 Å to 5.43 Å. These amino residues are identical to Arg143 and Thr137 in human SOD1, and they have been suggested to play a role as electrostatic guidance for the negatively charged substrate and to exclude the large negatively charged non-substrate compounds.18-20 In some catalytic mechanisms proposed based on experimental results, Arg141 of bovine SOD1 and Arg143 of human SOD1 interact and stabilize the O2•− binding on Cu.17,21 However, Arg186 of human SOD3 could not interact with the O2•− binding on Cu in our suggested mechanism due to its long distance from the active site and the existence of water molecules (WAT1 and WAT3) in the region between Arg186 and binding O2•− in [Cu(I)-O-O− HεNHis113-Zn(II)]. The shortest distance between Arg186 and Oa of binding O2•− was 5.30 Å. The side-chain of Arg143 in human SOD1 has been suggested to be constrained due to being sandwiched between His48 (the Cu ligand) and Thr58 (next to the disulfide bond site).18 There are His98 and Glu108 residues in human SOD3, which are identical to these two residues in human SOD1. As discussed earlier and shown in Figures 3 and 4, the conformation of Arg186 was changed by the protonation of His113 in human SOD3. The electrostatic potential on the solvent-accessible surface is shown in Figure

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10. The adaptive Poisson-Boltzmann solver (APBS) and PDB2PQR tool77-79 were used for the solving the PB equation. The atomic radii and charges of AMBER9980 for the estimation of electrostatic potential was used. The electrostatic potential around the narrow reaction site looks neutral before the protonation of His113 in the Cu oxidized state. After the protonation of His113 in Cu reduced state, the electrostatic potential around the reaction site becomes positive. The electrostatic potential on the molecular surface illustrated in Figure 11 shows that negatively charged Glu108 is located just behind Arg186 before the protonation of His113, and their charges are cancelled. The rotation of the side-chain of Arg186 by the protonation of His113 results in the separation of Glu108 from Arg186, and thus the positive charge of Arg186 starts to work as electrostatic guidance (Figure 11). In the resting state, Arg186 does not work as electrostatic guidance and does not exclude large negatively charged substrates. Since the electron transfer from the first O2•− to Cu(II) occurs without the bond formation between them, the electrostatic guidance for taking O2•− to the narrow reactive site is not required. Any negatively charged substrates do not bind to Cu(II) because of the repulsive interaction, as mentioned earlier. The distance between Asn180 and Arg186 is relatively large (7.66 Å) for the exclusion of the large substrates. On the other hand, the reduction

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of O2•− to form H2O2 requires the bond formation to the Cu atom, and Arg186 and Asn180 become important as electrostatic guidance and to exclude the larger negatively charged substrates. The rotation of Arg186 shortens the distance from Asn180 to 5.43 Å, and the positive charge of Arg186 becomes effective as electrostatic guidance due to the separation from Glu108. We confirmed the conformational change of Arg186 induced by the protonation of His113 by using a new ONIOM model in which the QM region was extended by including Arg186 (QM2). The dihedral angle of Cβ-Cγ-Cδ-Nε of Arg186 in the ONIOM calculation with the QM2 model was changed from −107.5º to −157.3º by the protonation of His113. The distance between Glu108 and Arg186 changed from 2.63 to 4.68 Å in the ONIOM calculation with the QM model not including Arg186, whereas it changed from 2.91 to 3.95 Å in the ONIOM calculation with the QM2 model. There were no remarkable effects of including Arg186 to the QM region in the ONIOM calculation.

Two proton supply routes The rotation of Arg186 induced by the protonation of His113 affects the hydrogen bond network composed of the water molecules, Glu108, Asn180, and Arg186 above

25



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the reaction site as discussed and shown in Figures 1 and 2. The water molecules build the hydrogen bond network with Glu108 and Arg186 before the protonation of His113, while the water molecules are in line starting from Glu179 after the protonation of His113 as shown in Figure 12. Before the protonation of His113, the hydrogen bond network above the reactive site was divided into two parts. The first network was composed of Glu108, Arg186, Asn180 and water molecules, and another network existed around the Glu179. There was no water around the Cu atom. This distribution of water molecules around the reactive site is similar to the results of an MD simulation for the resting state of SOD1.29 After the protonation of His113, the hydrogen bond network was formed along the shallow cleavage starting from Glu179, and WAT5 and Glu108 were separated from the network due to the rotation of Arg186. We propose that there are two proton supply routes starting from Glu108 or Glu179, and both are regulated by Arg186. Two protons must be supplied from the bulk solvent for the protonation of His113 and the binding O2•− on Cu(I). The proton supply routes via Glu108 and Glu179 work for the protonation of His113 and the binding of O2•− on Cu(I), respectively. Thr58 and Lys136 in human SOD1 correspond to Glu108 and Glu179, respectively. The routes and mechanism of proton supply to the reaction site may

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therefore be different between human SOD1 and SOD3.

Conclusion Our QM/MM calculation results clarify the human SOD3 enzyme turnover mechanism including the electron and proton transfer processes. At the first step, the electron transfer from O2•− to SOD3 occurs without the bond formation between the electron donor and acceptor. Our QM/MM results are in good agreement with the MD study of the active site of human SOD1.29 The distorted tetrahedral structure of Cu(II) in the resting state of SOD3 also caused the repulsive interaction for the negatively charged substrates due to the lone pair of NεHis113. The electron transfer from O2•− to SOD3 forms triplet O2 and reduced SOD3 while maintaining the four-coordination of Cu. The existence of the intermediate with the four-coordinated Cu(I) confirms the results from the 18O KIEs.25 The reduction of the Cu atom induces the protonation of NεHis113 and the breaking of the bridge between the Cu and Zn atoms. The conformation formed by the reduction of Cu and the protonation of NεHis113 is in agreement with the two positions of the Cu atom and the short distance between Arg186 and Asn180 in the X-ray structure.1 The distance between Arg186 and Asn180 is changed from 7.66 Å to 5.43 Å

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by the protonation of His113. Before the protonation of His113, Arg186 is positioned near the negatively charged Glu108, and therefore the negative and positive charges of two amino residues are cancelled, while the rotation of Arg186 induced by the protonation of His113 increases the distance between Arg186 and Glu108, and the positive charge of Arg186 begins to work as electrostatic guidance. We suggest that the different proton supply routes work to supply the two protons for producing H2O2. The proton supply route via Glu108 works for the protonation of His113, and the route via Glu179 functions to transfer the proton from the bulk solvent to the binding of O2•−. There is no energy barrier in our suggested catalytic cycle, and it is in good agreement with the experimental results in which the reaction rate reaches the diffusion limit.

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ASSOCIATED CONTENT

Supporting Information

The optimized geometric parameters of the resting state, [Cu(II)-His113-Zn(II)] and the reduced and protonated SOD3, [Cu(I) HNεHis113-Zn(II)], the QM2 model used for the estimation of the distortion effect of His113 on the reduction potential and its optimized structure.

AUTHOR INFORMATION

Corresponding Author

*Prof. Masami Lintuluoto, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamohanki-cho, Sakyo, Kyoto 606-8522, Japan. Tel: +81-75-703-5445. E-mail: [email protected]

ACKNOWLEDGMENT

The computations in this work were performed at the Research Center for Computational Science, Okazaki, Japan. 29



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Page 44 of 75

Table 1. The net atomic charges and spin densities on Cu and Zn atoms in the human SOD3 Cu Net atomic

Zn Spin density

charge

Net atomic

Spin density

charge

[Cu(II)-His113-Zn(II)]

0.443

0.635

0.641

0.0

[Cu(I)-His113-Zn(II)]

0.115

0.0

0.627

0.0

[Cu(II) HNεHis113-Zn(II)]

0.344

0.302

0.657

0.0

[Cu(I) HNεHis113-Zn(II)]

0.177

0.0

0.646

0.0

[Cu(I)-O-O− HNεHis113-Zn(II)]

0.362

0.447

0.615

0.0

[Cu(I)-O-OH HNεHis113-Zn(II)]

0.418

0.537

0.637

0.0

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Table 2. The values of pKa calculated with ε = 78.4, 20.0 and 4.0 for HNεHis113 and Cu(I)-O-O-H [Cu(II) HN His113-Zn(II)]

[Cu(I) HN His113-Zn(II)]

[Cu(I)-O-O-H HNεHis113-Zn(II)]

78.4

6.71

28.5

22.7

20.0

4.35

26.3

20.3

4.0

−15.7

15.9

7.0

ε

ε

ε

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Table 3. The Gibbs free energy differences for Eqs. (3)–(6) in kcal·mol−1 ε

Eq. (3)

Eq. (4)

Eq. (5)

Eq. (6)

78.4

40.0

10.1

22.9

−4.3

20.0

−35.7

−65.8

14.6

−0.9

4.0

−62.0

−105.4

−19.6

−1.8

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Figure legends

Figure 1. Structure of the extracellular human SOD3 (PDB 2JLP) and its reactive site including the Cu and Zn atoms.

Figure 2. Model used in the QM/MM calculation. The thin gray wireframe denotes a monomer of SOD3 tetramer. The pink tubes and balls denote the metal atoms, their ligand residues and water molecules included in the QM region. The amino acid residues and water molecules represented by the blue tubes and balls were included in the MM region, and were relaxed during the geometry optimization of the QM region. The deep gray wireframe denotes the MM regions which were fixed during the optimization.

Figure 3. The optimized structure of the resting state, [Cu(II)-His113-Zn(II)]. (a) The conformation around the reaction site. (b) The optimized geometric parameters around both metal atoms. (c) The hydrogen bond network composed of five water molecules, Arg186, Asn180 and Glu108. The red broken lines denote the hydrogen bonds. The values indicate the distances in Å.

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Figure 4. The optimized structure of [Cu(II) HNεHis113-Zn(II)]. (a) The conformation around the reaction site. (b) The optimized geometric parameters around both of the metal atoms. (c) The hydrogen bond network composed of five water molecules, Arg186, Asn180 and Glu108. The red broken lines denote the hydrogen bonds. The values indicate the interaction distances in Å.

Figure 5. The occupied molecular orbital and β-LUMO (the p-orbital of NεHis113 is included) of [Cu(II)-His113-Zn(II)]. The left figure shows the occupied orbital composed of the p-orbital of NεHis113 and Cu d-orbital, and the right figure shows the β-LUMO.

Figure 6. The optimized structure of [Cu(I)-O-O− HNεHis113 Zn(II)]. The red dashed lines denote the hydrogen bonds. The values denote the bond lengths and the interaction distances.

Figure 7. The suggested routes for the protonation of [Cu(I)-O-O− HNεHis113-Zn(II)].

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Figure 8. The optimized structure of [Cu(I)-O-O-H HNεHis113-Zn(II)]. The red dashed lines denote the hydrogen bonds. The values denote the bond lengths and the interaction distances.

Figure 9. The proposed enzyme catalytic enzyme turnover mechanism. The values of Gibbs free energy are in kcal·mol−1.

Figure 10. The electrostatic potential on the solvent-accessible surfaces for [Cu(II)-His113-Zn(II)] and [Cu(I) HNεHis113-Zn(II)]. Blue and red denote the positive and negative values in the range of +10.00 to −10.00 kT·e−1. The red dashed circle denotes the reactive site. CueMol was used for the plot of the electrostatic potential surface.61

Figure

11.

The

electrostatic

potential

on

the

molecular

surface

for

[Cu(II)-His113-Zn(II)] and [Cu(I) HNεHis113-Zn(II)]. Blue and red denote the positive and negative values in the range of +10.00 to −10.00 kT·e−1. CueMol was used for the plot of the electrostatic potential surface.61

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Page 50 of 75

Figure 12. The change of hydrogen bond network composed of the water molecules, Glu108,

Glu179,

Asn180

and

Arg186

by

the

protonation

of

His113

in

[Cu(II)-His113-Zn(II)]. Red and pink ball and stick models denote the water molecules and Glu residues, respectively, and the blue model denotes the other amino acid residues.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Figure 7.

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Figure 8.

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Figure 9.

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Figure 10.

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Figure 11.

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Figure 12.

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Figure 5. The occupied molecular orbital and β-LUMO (the p-orbital of NεHis113 is included) of [Cu(II)His113-Zn(II)]. The left figure shows the occupied orbital composed of the p-orbital of εHis113 and Cu dorbital, and the right figure shows the β-LUMO. 402x204mm (300 x 300 DPI)


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Figure 6. The optimized structure of [Cu(I)-O-O− HNεHis113 Zn(II)]. The red dashed lines denote the hydrogen bonds. The values denote the bond lengths and the interaction distances. 297x210mm (300 x 300 DPI)



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Figure 8. The optimized structure of [Cu(I)-O-O-H HNεHis113-Zn(II)]. The red dashed lines denote the hydrogen bonds. The values denote the bond lengths and the interaction distances. 297x210mm (300 x 300 DPI)



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Figure 10. The electrostatic potential on the solvent-accessible surfaces for [Cu(II)-His113-Zn(II)] and [Cu(I) HNεHis113-Zn(II)]. Blue and red denote the positive and negative values in the range of +10.00 to −10.00 kT∙e−1. The red dashed circle denotes the reactive site. CueMol was used for the plot of the electrostatic potential surface.61 297x210mm (300 x 300 DPI)



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Figure 11. The electrostatic potential on the molecular surface for [Cu(II)-His113-Zn(II)] and [Cu(I) HNεHis113-Zn(II)]. Blue and red denote the positive and negative values in the range of +10.00 to −10.00 kT∙e−1. CueMol was used for the plot of the electrostatic potential surface.61 283x135mm (300 x 300 DPI)


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Figure 12. The change of hydrogen bond network composed of the water molecules, Glu108, Glu179, Asn180 and Arg186 by the protonation of His113 in [Cu(II)-His113-Zn(II)]. Red and pink ball and stick models denote the water molecules and Glu residues, respectively, and the blue model denotes the other amino acid residues. 297x210mm (300 x 300 DPI)


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