DFT Study on Enzyme Turnover Including Proton and Electron

Article pubs.acs.org/biochemistry

DFT Study on Enzyme Turnover Including Proton and Electron Transfers of Copper-Containing Nitrite Reductase Masami Lintuluoto*,† and Juha M. Lintuluoto‡ †

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamohanki-cho, Sakyo, Kyoto 606-8522, Japan ‡ Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8530, Japan ABSTRACT: The reaction mechanism of copper-containing nitrite reductase (CuNiR) has been proposed to include two important events, an intramolecular electron transfer and a proton transfer. The two events have been suggested to be coupled, but the order of these events is currently under debate. We investigated the entire enzyme reaction mechanism of nitrite reduction at the T2 Cu site in thermophilic Geobacillus CuNiR from Geobacillus thermodenitrificans NG80-2 (GtNiR) using density functional theory calculations. We found significant conformational changes of His ligands coordinated to the T2 Cu site upon nitrite binding during the catalytic reaction. The reduction potentials and pKa values calculated for the relevant protonation and reduction states show two possible routes, A and B. Reduction of the T2 Cu site in the resting state is followed by endothermic nitrite binding in route A, while exothermic nitrite binding occurs prior to reduction of the T2 Cu site in route B. We concluded that our results support the random-sequential mechanism rather than the ordered mechanism.

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ransformation of nitrite or nitrate to gaseous dinitrogen is an important step in the geobiochemical nitrogen cycle.1−4 Denitrification, including dissimilatory reduction of nitrate or nitrite, regulates the amount of nitrogen in soil, waters, and atmosphere.2,4 Nitrate is reduced to nitrite by the nitrate reductases, followed by the conversion to nitric oxide by the dissimilatory nitrite reductases (NiRs).1 NiRs contain metal cofactors such as copper or iron at the catalytic sites. Coppercontaining NiRs (CuNiRs) are encoded by the NirK gene, while iron-containing NiRs are encoded by the NirS gene.5,6 CuNiRs are homotrimeric enzymes, and each monomer contains two different Cu sites, type 1 (T1) and type 2 (T2). The T1 Cu site is connected to the T2 Cu site via the Cys-His peptide, where the distances between two Cu sites are over 12.5 Å, as shown in Figure 1. The T1 Cu site is the initial site of reduction by an electron donor, and the electron is transferred to the T2 Cu site via the Cys-His peptide. In the resting state, the T2 Cu site is bound to three His residues and a water molecule, and substitution of the water molecule by nitrite occurs in the initial stage of the catalytic reaction.7−11 The mechanisms of nitrite reduction by CuNiR proposed on the basis of the experimental results7−13 contain an intramolecular electron transfer and protonation of the nitrite,7−9,12,14−16 but the details of the mechanism, such as the order of these two events, the electron and proton transfers, have not been clarified. The properties of CuNiRs, such as gene expression,1,17 the affinity for nitrite, the activity of the catalytic reaction,18 and the redox potential,19,20 are strongly dependent on pH and the nitrite concentration. The activity of CuNiR increases with decreasing pH, and the optimum range of pH for activity is 5 to 6.13,21 The nitrite concentration dependence of inhibition of the intramolecular electron transfer from the T1 to © 2016 American Chemical Society

Figure 1. T1 and T2 Cu sites with surrounding residues. We substituted Cys135 for Ala in the structure of the C135A mutant of GtNiR complexed with nitrite (PDB ID 3WKP).26

the T2 Cu site has been shown by pulse radiolysis,21 electrochemical,22,23 and laser photoexcitation studies.24 We have investigated the reaction mechanism of nitrite reduction at the T2 Cu site in thermophilic Geobacillus CuNiR from Geobacillus thermodenitrificans NG80-2 (GtNiR) by density functional theory (DFT) calculations.25 GtNiR is one of a genus of extremophilic Gram-positive bacteria and has a complete set of all genes needed for absolute denitrification.26 We concluded that the reduction mechanism of nitrite on T2 Cu site is as shown in Scheme 1.25 The conformation of the Received: May 2, 2016 Revised: July 20, 2016 Published: July 26, 2016 4697

DOI: 10.1021/acs.biochem.6b00423 Biochemistry 2016, 55, 4697−4707

Article

Biochemistry Scheme 1. Nitrite Reduction Mechanism for CuNiRs Proposed in Our Previous Study25

possible pathways by using their new method to estimate the pKa values and redox potentials. In this study, we investigated the entire enzymatic turnover in GtNiR, including the proton and electron transfers, by DFT calculations. We examined the reformation of the resting state, in which the water molecule is coordinated at the T2 Cu site after the protonation of nitrite and the departure of NO. After NO leaves, protonation of HO− at the T2 Cu site is the most favorable way to reproduce the resting state, and it seems that the proton is not supplied via catalytic residues such as Asp98 and His 244 but rather by the proton channel. We examined the dependence of the reduction potential on the protonation states of Asp98, His244, and nitrite to show the order of nitrite binding, electron transfer, and protonation of nitrite. In our mechanism, protonation of His244 to build the favorable hydrogen-bond network for the reduction of nitrite occurs after reduction of the T2 Cu site, which is the same as the result from our previous study.25 Before the favorable hydrogen-bond network is achieved, there are two possible and competitive pathways that differ in regard to the order of Cu reduction. Our results show that the enzyme reaction of CuNiR proceeds by way of the random-sequential mechanism rather than the ordered mechanism, in which nitrite binding is followed by T2 Cu reduction.

hydrogen-bond network composed by the nitrite, catalytic residues, and two water molecules also changes and shows a dependence on both the Cu oxidation state and the protonation state of the catalytic residues.25 The protonation state of Nε2-His244 has a significant effect not only to stabilize the bidentate form of nitrite, as has been suggested by the experimental results,7 but also to make the proton transfer from Asp98 to nitrite more efficient by reducing the distance between HOδ2 of Asp98 and the O atom of nitrite.25 The protonation of Nε2-His244 followed by the proton transfer from Asp98 to nitrite occurs after reduction of the T2 Cu site.25 The nitrite reduction occurs at the T2 Cu(I) site, and then the nitric oxide is released to the solvent to form the OH− coordinated on the Cu(II) site.25 All CuNiRs contain a so-called “proton channel” to supply protons to the catalytic site. CuNiR of Alcaligenes xylosoxidans (AxNiR) has two putative proton channels.9,27 The first channel connects the Asp92 catalytic residue to the protein surface via Asn90 and several other residues and water molecules. The second one extends along the monomer− monomer interface and connects the catalytic residue to the protein surface via His254, which is proposed to regulate the proton flow. The first channel via Asn90 is proposed to be the main source of protons to the active site, and Asn90 has a very important role in sustaining the hydrogen-bond network. In the first channel of GtNiR, the water network is maintained by Phe109 and Gly136 instead of Ser96 at the same position as Asn90 of AxNiR.26 However, there is the hydrophobic Val249 in the middle of the second channel of GtNiR, and Val249 blocks the proton flow from the proton surface. The electronic and geometrical structures of the ligandbound T2 Cu site28−33 and the mechanism of nitrite reduction at the T2 Cu site have been investigated by the theoretical methods.12,34−36 Theoretical studies of the mechanism of heme-containing NiR have also been reported.37−39 Maekawa and co-workers studied the entire catalytic nitrite reduction pathway, including the proton and electron transfers, using DFT with the cluster model of the T2 Cu site coordinated to three His residues.40 They showed that there are various

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MATERIALS AND METHODS Initial coordinates of the nitrite complex model of GtNiR, in which the nitrite is bound to the T2 Cu site in η1-O end-on form, were derived from the X-ray structure of the C135A mutant complexed with nitrite (PDB ID 3WKP).26 Hydrogen atoms were added to each residue in the standard manner by assuming a standard protonation state under physiological pH conditions, as shown in Figure 2. The model included the T2 Cu site, the nitrite, three His residues coordinated to the T2 Cu site, and the second sphere residues Asp98, His244, and Val246. Additionally, two water molecules were included: the one labeled WAT1 occupies an intermediate position between Asp98 and His244, and the other one, labeled WAT2, seems to 4698


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and AH, respectively. The values of ΔGsol(A−) and ΔGsol(AH) were calculated using the PCM. The values of ΔGsol(H+) estimated by experimental and computational methods are in the range between −254 and −270 kcal/mol.38,39,42−48 We used the value −260 kcal/mol for ΔGsol(H+), which was adopted from the literature.42−45 A value of −6.32 kcal/mol was used for the gas-phase free energy of the proton at 298 K and standard pressure.49 We first tested our calculation’s reliability by calculating the pKa values for the deprotonations of free His and Asp residues in water. The obtained pKa values for His and Asp were 6.1 and 5.0, respectively, which are qualitatively in good agreement with the standard values of 6.5 and 3.9, respectively.50 Although the Born−Haber thermodynamic cycle has been used for the estimation of pKa values, the ab initio prediction of pKa values is still very difficult. An error of 1 kcal/mol in the Gibbs free energy difference translates to an error of approximately 0.73 in the estimated pKa. The error in the estimation of Gibbs free energy by DFT has been reported as 2−3 kcal/mol.51 The size of our models also affects the estimation of pKa values because of the localization of charge on amino residues, the hydrogen bonding, and so on. However, the calculated values of pKa based on the Born− Haber cycle have shown reasonable qualitative results compared with experimental results in the literature.38,39,42−45 Therefore, we think the comparison of pKa values among our computational models is qualitatively correct. We also calculated the redox potential using a Born−Haber thermodynamic cycle. The Gibbs free energy of one-electron reduction is given by the following equation:

Figure 2. Model used for DFT calculations on nitrite bound to the T2 Cu site of GtNiR.

interact with Asp98 in the initial coordinates of the X-ray structure. In most CuNiRs, the Nδ1 proton of the catalytic histidine residue is proposed to interact with glutamine or threonine residues to prevent reorientation of the catalytic histidine plane.11,26 We supposed that Nδ1 of His244 is protonated throughout the nitrite reduction. The B3LYP functional was used for the geometry optimization and single-point calculations. The 6-311G(d) basis set for Cu, N, and O atoms and the 6-31G(d) basis set for C and H atoms were used in the geometry optimizations. The geometries of the primary chain were fixed during the geometry optimization, while those of side chains, waters, nitrite, and the T2 Cu site were fully optimized. Single-point calculations were performed using these optimized structures with the 6311+G(d) basis set. We used the polarizable continuum model (PCM) with ε = 78.39 as an implicit solvent model.41 We calculated Gibbs free energy, pKa, and reduction potential values using smaller models in which the imidazole and formate groups were substituted with His and Asp amino residues, respectively, and Val246 was removed from the model shown in Figure 2 to reduce the computational costs. The geometries optimized with the larger models, as shown in Figure 2, were used for free energy calculations at the B3LYP/ 6-311G+(d) level. We estimated the pKa values from the Gibbs free energy difference ΔG for the deprotonation by using the following equations: AH → A− + H+

(1)

ΔG = −RT ln K a

(2)

ΔGRP = ΔGgRP − ΔGsol(Ox) + ΔGsol (Red)

(4)

ΔGRP g

where is the free energy difference for the one-electron reduction in the gas phase and ΔGsol(Ox) and ΔGsol(Red) are the solvation free energies for the oxidized and reduced forms, respectively. The values of ΔGsol(Ox) and ΔGsol(Red) were calculated using the PCM. The standard redox potential E0 is connected to the Gibbs free energy ΔGRP by the Faraday g constant F (equal to 23.06 kcal·mol−1·V−1), as shown in the following equation: E0 =

−ΔGRP − 4.44 V F

(5)

where the last term is obtained from the free energy change of −4.44 eV associated with the reference normal hydrogen electrode (NHE).52 An error of 1 kcal/mol in the Gibbs free energy difference translates to an error of approximately 43 mV in the redox potential estimated using the Born−Haber cycle. However, the calculation of redox potentials based on the Born−Haber cycle has provided reasonable qualitative results compared with experimental results in the literature.39,42,53 We think that this method reduces the computational costs, while the resulting accuracy is comparable. All of the calculations were carried out using Gaussian 09.54 Facio was used for visualization of the DFT calculation results.55

where AH and A− represent the protonated and deprotonated states, respectively. The deprotonation free energy difference was estimated using a Born−Haber thermodynamic cycle, as shown in eq 3:

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RESULTS AND DISCUSSION Reproduction of the Resting State after the Reduction of Nitrite. We first investigated the reproduction of the resting state, in which the water molecule binds at the T2 Cu site, by the protonation of structure V (Cu(II)−OH−, His244Im-Nε2, and Asp98-CγOOH) in Scheme 1. Protonated Asp98

ΔG = ΔGg + ΔGsol(A−) + ΔGsol(H+) − ΔGsol(AH) (3)

where ΔGg is the free energy difference for the deprotonation of AH in the gas phase and ΔGsol(A−), ΔGsol(H+), and ΔGsol(AH) are the solvation free energies of A−, the proton, 4699


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Biochemistry Scheme 2. Supposed Reaction Pathways To Reproduce the Resting State

Figure 3. (a, b) Optimized geometries of (a) structure V(Ox) and (b) structure VI(Ox). The hydroxide ion and water bind to the T2 Cu(II) site. Val246 is not shown. The dashed red lines denote the hydrogen-bond network. (c, d) Optimized geometries of (c) structure V(Red) and (d) structure VI(Red). The hydroxide ion and water bind to the T2 Cu(I) site. Asp98, His244, Val246, WAT1, and WAT2 have been omitted for clarity. The yellow lines show the triangle plane formed by the three His ligands.

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Scheme 3. Supposed Reaction Pathways To Exchange the Ligand and Form the Hydrogen-Bond Network Required for Nitrite Reductiona

a

The Gibbs free energy differences are shown in kcal/mol.

Asp98 and WAT2, as shown in Figure 3a,b. The Cu−O(ligand) distance increases from 1.83 to 1.98 Å by the protonation of structure V. The hydrogen-bond distances between the H(ligand) and Oδ-Asp98 are 1.94 and 1.89 Å for structures V and VI, respectively. The hydrogen-bond distances between the ligand and WAT2 are 1.79 and 1.68 Å for structures V and VI, respectively. Exchange of Ligand from Water to Nitrite and Construction of the Hydrogen-Bond Network Required for Nitrite Reduction. We examined the exchange of the ligand from the resting state (structure VI in Scheme 3) to the state with nitrite bound to the T2 Cu site (structure X in Scheme 3). The nitrite bound to the T2 Cu site is more stable than the resting state by 8.6 kcal/mol in Gibbs free energy. The exchange of ligand from HO− bound to the T2 Cu site (from structures V to X) seems not to occur, as shown in Scheme 3. The Gibbs free energy difference between structures V and X is 22.4 kcal/mol. We have reported that both of the catalytic amino acid residues Asp98 and His244 should be protonated to build the hydrogen-bond network, which is required for the reduction of nitrite.25 There is no excess proton to transfer to nitrite in structure X (NO2−−Cu(II), His244-Im-Nε2, and Asp98CγOOH), in which the proton of Asp98 is stabilized by composing the hydrogen-bond network with WAT1 and His244. One other proton needs to be supplied to form structure III (NO2−−Cu(II), His244-Im-Nε2H+, and Asp98CγOOH). We assumed that the proton is supplied via the proton channel connected to Asp98 and that the protonation of

constitutes the hydrogen-bond network with WAT1 and His244 in structure V, and there is no excess proton to transfer to HO− around the T2 Cu site. We supposed that the proton is supplied from the protein surface via the main proton channel connecting to Asp98. WAT2 forms the hydrogen-bond network with the other water molecules in the main proton channel and exists near Asp98. We supposed that the proton from the protein surface is supplied to the T2 Cu site via WAT2. When the protonated WAT2 approaches structure V, as shown in structure V′ of Scheme 2, the proton moves not to Asp98 but instead to the hydroxide ion bound to the T2 Cu site. Structure VII, in which the proton is transferred to His244 via Asp98, is unstable, while structure VI, which is the resting state, is stable. The pKa of the water molecule bound to the Cu in structure VI (H2O−Cu(II), His244-Im-Nε2, and Asp98CγOOH) is 7.0. We next examined the possibility of the proton being supplied from the protein surface before the NO leaves. When the protonated WAT2 approaches structure IV (HONO− Cu(I), His244-Im-Nε2, and Asp98-CγOOH), as shown in structure IV′ in Scheme 2, the proton does not move to either Asp98 or HONO bound to the T2 Cu site: structure VIII is unstable, and the pKa of protonated Asp98 in structure IX has a negative value. Reproduction of the resting state occurs by proton supply from protein surface after NO leaves, as shown in Scheme 2 structures V to VI. The hydroxide ion and water bound to the T2 Cu site are involved in the hydrogen-bond network with 4701


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Scheme 4. Supposed Reaction Pathways To Form the Nitrite-Bound State at the T2 Cu Site with Protonated Asp98 and Protonated His244a

a

The Gibbs free energy differences are shown in kcal/mol. The values under the arrows denote the reduction potentials.

Reduction Potential and the Formation of the Hydrogen-Bond Network. We next examined the reduction potentials of relevant protonations and ligand binding states. The reduction potential of the T2 Cu site in the resting state before nitrite binding (structure VI) is 0.32 V, as shown in Scheme 4, while that in the nitrite-bound state (structure X) is −0.094 V. Structure X(Ox) (NO2−−Cu(II), His244-Im-Nε2, and Asp98-CγOOH) is stabilized by a Gibbs free energy of 8.6 kcal/mol upon binding of nitrite and departure of the water molecule from structure VI(Ox) (H2O−Cu(II), His244-ImNε2, and Asp98-CγOOH), while structure X(Red) (NO2−− Cu(I), His244-Im-Nε2, and Asp98-CγOOH) is 8.3 kcal/mol less stable compared with structure VI(Red) (H2O−Cu(I), His244Im-Nε2, and Asp98-CγOOH). Nitrite binding occurs easily before the reduction of the T2 Cu site rather than after the reduction. The reduction of the T2 Cu site of structure VI (H2O− Cu(II), His244-Im-Nε2, and Asp98-CγOOH) induces the conformational change as shown in Figure 3d. Structure

Asp98 is followed by transfer of the proton to His244 via WAT1 to form structure III. The proton transfer between His244 and Asp98 via WAT1 can occur during the protonation of nitrite according to our previous study.25 The excess proton from the proton channel initially attaches to Asp98, and then the proton transfer to His244 occurs via WAT1 to form structure III from structure X as shown in Scheme 3. Since the value of pKa is 0.6, the formation of structure III by protonation of structure X is difficult. We next examined the formation of structure III via structure XI (H2O−Cu(II), His244-Im-Nε2H+, and Asp98-CγOOH) obtained by protonation of structure VI. The protonation of structure VI seems not to occur because of the negative pKa value of structure XI. Nitrite binding from the resting state, in which both of the catalytic residues are neutral, occurs easily and is stabilized. The formation of the hydrogen-bond network, in which both Asp98 and His244 are protonated, does not occur in the resting state and before reduction of the T2 Cu site. 4702


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Scheme 5. Summary of the Enzymatic Turnover Pathways for Nitrite Reduction at the T2 Cu Site from the Resting Statea

a

In route A, reduction of the T2 Cu site is followed by nitrite binding. In route B, nitrite binding occurs before reduction of the T2 Cu site. The Gibbs free energy differences are shown in kcal/mol. The values under the arrows denote the reduction potentials.

III(Red) (NO2−−Cu(I), His244-Im-Nε2H +, and Asp98CγOOH), respectively, as shown in Scheme 4. Since the value of pKa for structure XI(Red) is negative, as is that for structure XI(Ox) as shown in Scheme 3, protonation of structure VI before or after reduction is difficult. The values of pKa for structures III(Ox) and III(Red) are 0.6 and 6.6, respectively. The reduction potential to go from structure III(Ox) to structure III(Red) is 0.059 V, which is larger than that for structure X. We translated the redox potential difference between structures III and X into a Gibbs free energy difference, and the estimated value is 3.8 kcal/mol. Therefore, we think that in the path from structure X(Ox) to structure III(Red), reduction of the T2 Cu site occurs before protonation of His244. We summarize the sequence events of the nitrite reduction at the T2 Cu site as shown in Scheme 5. We have reported that reduction of the T2 Cu site and protonation of both catalytic residues Asp98 and His244 are required for the nitrite reduction.25 There are two possible pathways to achieve structure III (NO2−−Cu(II), His244-Im-Nε2H+, and Asp98CγOOH) from the resting state, structure VI(Ox) (H2O− Cu(II), His244-Im-Nε2, and Asp98-CγOOH) in Scheme 5. In route A, reduction of the T2 Cu site is followed by nitrite binding, while nitrite binding occurs before reduction of the T2 Cu site in route B (as shown in Scheme 5). The reduction potential of structure VI(Ox) is 0.32 V. The reduction potentials of the T1 and T2 Cu sites have been reported as 255 and 244 mV at pH 7.0 for AxNiR.59 Although the value of 0.32 V for the T2 Cu site is slightly overestimated compared with the experimental value, we can discuss our results qualitatively. The energy barrier for nitrite binding is 8.3 kcal/mol after reduction of the T2 Cu site in route A, while structure X(Ox) (NO2−−Cu(II), His244-Im-Nε2, and Asp98CγOOH) is stabilized by 8.6 kcal/mol upon the nitrite binding in route B. The intramolecular electron transfer from the T1 to

VI(Red) is highly symmetric tetrahedral compared with structure VI(Ox). The distance between the water and Cu in structure VI(Red) is 2.42 Å, which is longer than that in structure VI(Ox). The distance between the Cu atom and the centroid of the triangle formed by the three Nε atoms of the His ligands decreases from 0.64 to 0.37 Å (Δ = −0.27 Å) upon reduction of the T2 Cu site in structure VI. Fukuda and coworkers have reported that the T2 Cu atom before nitrite binding sank toward a “ligand plane” composed of three Nε atoms of His ligands during the photoreduction by X-ray dose, and the distance between the Cu atom and the ligand plane changed from 0.88 to 0.64 Å (Δ = −0.24 Å).56 Our results show a good agreement with the experimental data, although the values are slightly smaller. Since the value of pKa for structure VI(Ox) is 7.0, there is an equilibrium between structures V(Ox) (Cu(II)−OH−, His244Im-Nε2, and Asp98-CγOOH) and VI(Ox) at the slightly high pH. We next investigated the reduction of the T2 Cu site in structure V. The reduction of the T2 Cu site in structure V(Ox) induces a conformational change of His100, as shown in Figure 3c. The T2 Cu site, the Nε atoms of two His ligands, and the HO− in structure V(Red) are in-plane, and the two Nε−Cu and O−Cu bond distances are 1.88, 2.44, and 1.86 Å, respectively. Before the reduction, those distances are 2.02, 2.05, and 1.86 Å, respectively. The reduction of the T2 Cu site increases the distance between the Nε atoms of His100 and the Cu atom from 2.07 to 2.85 Å. Reduction of the T2 Cu site before nitrite binding has been reported to induce the three-coordinate inactive form.57,58 However, the reduction potential of structure V(Ox) is −0.44 V, so the reduction of this structure may be difficult. The formation of the hydrogen-bond network required for nitrite reduction was examined by adding a proton to structures VI(Red) and X(Red) to produce the structures XI(Red) (H2O−Cu(I), His244-Im-Nε2H+, and Asp98-CγOOH) and 4703


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Figure 4. Conformational changes along the catalytic cycle in Scheme 5. In each step, the structures before and after reaction are denoted by red and green sticks, respectively. The hydroxide ion and water molecule bind to the T2 Cu site in structures V and VI, respectively. In both structures, Asp98 is protonated and His244 is neutral. Nitrite binds to the T2 Cu site in structures X and III. In structure X, Asp98 is protonated and His244 is neutral, while in structure III, both catalytic residues are protonated.

We suggested in our previous study that the reduction of the T2 Cu site occurs in structure I in Scheme 1, which is more stable than structure X(Ox) in Scheme 4.25 However, considering the continuity of the reaction mechanism, structure I in Scheme 1 does not appear in the reaction mechanism. In this study, structure X appears instead of structure I. Maekawa and co-workers reported the redox potential of the T2 Cu site during the catalytic reaction calculated using DFT with a cluster model of the T2 Cu site coordinated to three His residues.40 They used tris(pyrazolyl)methane (TPM) and hydrotris(pyrazolyl)borate (TPB) as the cluster model for the catalytic site and reported that the value of the redox potential calculated with TPB was lower than that with TPM because of the less positive charge of TPB.40 Second-sphere residues and water molecules interacting with the His residues coordinating to the T2 Cu site may affect the value of the redox potential of the T2 Cu site. We used the PCM with ε = 78.39 as an implicit water solvent model for the free energy calculations. Since the T2 Cu site is located at the boundary of two monomers and is connected to the proton channel, there are several water molecules around the catalytic site. However, the environment around the catalytic site is different from the bulk water solvent. Therefore, we examined the influence of the dielectric constant of the medium in the energy calculations. We compared the values in Scheme 5 calculated with ε = 4.0, 10.0, 20.0, and 78.39. The values for the ligand exchange from water to nitrite at the Cu(II) site (from structure VI(Ox) to X(Ox) in Scheme 5) are −22.0, −13.3, −9.82, and −8.6 kcal/mol with ε = 4.0, 10.0, 20.0 and 78.39, respectively. This tendency is due to the more positively charged structure VI compared with structure X. Likewise, the values for the ligand exchange at the Cu(I) site (from structure VI(Red) to X(Red)) are 12.1, 11.1, 8.7, and 8.3 kcal/mol with ε = 4.0, 10.0, 20.0 and 78.39, respectively. The

the T2 Cu site becomes the rate-determining step in route B because of the low reduction potential. If the nitrite concentration is high enough for binding to the T2 Cu site, the exchange of ligand from structure VI(Ox) to structure X(Ox) occurs before reduction of the T2 Cu site. This result is in good agreement with the experimental results that the presence of nitrite lowers the rate of intramolecular electron transfer.13,21,57,60,61 We assume the random-sequential mechanism rather than the ordered mechanism, in which nitrite binding is followed by T2 Cu reduction. A random-sequential mechanism for CuNiR has been assumed from the results of kinetic and redox cycling studies.62,63 Wijma and co-workers reported the pH dependence of the nitrite reduction, and route B is faster than route A above pH 6.5.62 This dependence is due to the conversion of H2O to OH− in the resting state.62 In our results, the OH− bound at the T2 Cu site appears at high pH, as shown in structure V(Ox) (Cu(II)−OH−, His244-Im-Nε2, and Asp98-CγOOH) in Scheme 4, and the reduction of structure V(Ox) is very slow compared with those of the H2O- and nitrite-bound structures because of its low reduction potential. EXAFS and X-ray crystallography results showed that reduction of the T2 Cu site lowers the affinity of the T2 Cu site for the ligand, even nitrite.10,16,58 It is concluded that the reduced T2 Cu site is incapable of binding the solvent or the nitrite.10,16,58 In the ordered mechanism, the nitrite first binds to the oxidized T2 Cu site, followed by reduction of the T2 Cu site. However, our results show that the reduced T2 Cu site is capable of binding the water molecule, structure VI(Red) (H2O−Cu(I), His244-Im-Nε2, and Asp98-CγOOH). The exchange of the ligand from water to nitrite is very slow because of the 8.3 kcal/ mol energy difference. Wijma et al.64 concluded by protein film voltammetry experiments that the reduced T2 Cu site is capable of binding the water molecule, and they proposed the random-sequential mechanism. 4704


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outer protein occurs. This loop is called the sensor loop, and it is suggested to play an important role in transferring information on the reduction.57,58 His134 connects the T1 Cu site via Cys135, as shown in Figure 1. Our results on the conformational changes resulting from both events, nitrite binding and reduction, may connect to these experimental results. The conformation changes induced by nitrite binding are more significant those induced by reduction of the T2 Cu site, as shown in Figure 4, and His100 moves upon nitrite binding rather than reduction of the T2 Cu site or protonation of catalytic residues. This may indicate that His100 plays an important role in transmitting information on nitrite binding. In contrast, His244 moves only upon protonation of catalytic residues. In view of the results of the X-ray photoreduction study,56 when the hydrogen-bond network around the T2 Cu site required for the transfer of the proton from Asp98 to nitrite is formed, the conformation of His244 changes to adjust the proton relay via the hydrogen-bond network.

values of the reduction potentials for structure VI(Ox), with water bound to Cu(II), are 1.21, 0.62, 0.35, and 0.32 V with ε = 4.0, 10.0, 20.0 and 78.39, respectively, while those for structure X(Ox), with nitrite bound on Cu(II), are 0.098, 0.011, −0.061, and −0.094 V, respectively. Therefore, the reduction of the water-bound state occurs more easily than that of the nitritebound state in any medium. The pKa values for the deprotonation of His244-Im-Nε2H+ in structure III are 5.2, 5.1, 6.1, and 6.6 with ε = 4.0, 10.0, 20.0, and 78.39, respectively. There is a dependence of the free energy calculation results on the value of the dielectric constant, but the qualitative results are not affected. Conformational Changes upon Nitrite Binding and Reduction of the T2 Cu Site. The conformational changes along the catalytic cycle of Scheme 5 are shown in Figure 4. The formation of the resting state, from structure V(Ox) (Cu(II)−OH−, His244-Im-Nε2, and Asp98-CγOOH) to structure VI(Ox) (H2O−Cu(II), His244-Im-Nε2, and Asp98CγOOH) does not show any conformational changes. The hydrogen-bond network including catalytic residues, WAT1, and ligands does not change upon protonation of the OH− bound to the T2 Cu site, as shown in Figures 3 and 4. In the first step in route A, reduction of the T2 Cu site occurs, as shown in Scheme 5 from structure VI(Ox) to structure VI(Red) (H2O−Cu(I), His244-Im-Nε2, and Asp98CγOOH). Reduction of the T2 site causes the conformational change of His134 as shown in Figure 4. The Cu−Nε−Cδ−Cχ dihedral angle of His134 changes from −169.5° to −153.0°. Then the nitrite binds to the T2 Cu site in the next step in route A, as shown in Scheme 5, from structure VI(Red) to X(Red) (NO2−−Cu(I), His244-Im-Nε2, and Asp98-CγOOH). The nitrite binding returns the conformation of His134 back to that in structure VI(Ox), as the Cu−Nε−Cδ−Cχ dihedral angle of His134 changes from −153.0° to −162.4°. At the same time, the conformations of His100 and His294 are also changed. The Cu−Nε−Cδ−Cχ dihedral angles of His100 and His294 change from −179.9° and −169.7° to −162.7° and −140.9°, respectively. In route B, nitrite binding occurs prior to reduction of the T2 Cu site, as shown in Scheme 5. Nitrite binding causes the conformational changes of His100 and His134 as shown in Figure 4. The Cu−Nε−Cδ−Cχ dihedral angle of His100 changes from −171.7° to −162.2°, and that of His134 changes from −169.5° to −137.5°. In the following reduction step, the conformation of His134 returns into that in structure VI(Ox), as the Cu−Nε−Cδ−Cχ dihedral angle of His134 changes from −137.5° to −162.4°. The conformation of His134 becomes very close to that in structure VI(Ox) upon addition of the proton to structure X(Red) to form structure III(Red) (NO2−−Cu(I), His244-ImNε2H+, and Asp98-CγOOH), as the Cu−Nε−Cδ−Cχ dihedral angle of His134 changes from −162.4° to −172.0°. The conformation of His244 also changes in going from structure X(Red) to structure III(Red), as the Cu−Nε−Cδ−Cχ dihedral angle changes from 60.1° to 78.0°. It has recently been reported that the structure of His244 in GtNiR was changed by X-ray photoreduction.56 The imidazole ring of His244 rotates by 10° upon reduction of the T2 Cu site.56 The results suggest the importance of two hydrogen bonds of His244 with Thr268 and Gln267, and the rotation of His244 may play a role as a switch for proton relay.56 Moreover, His100 is located at the end of the loop connecting to the protein surface where the electron transfer from the

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CONCLUSIONS We have investigated the entire enzyme reaction mechanism using DFT. Our results indicate the random-sequential mechanism rather than the ordered-mechanism. In our suggested mechanism, there are two possible routes, A and B. In route A, reduction of Cu occurs prior to nitrite binding, whereas nitrite binding is followed by reduction of Cu in route B. Since the pKa value of the resting state (in which water binds to the T2 Cu site) is 7.0, the water-bound state is in equilibrium with the OH−-bound state. At high pH, this equilibrium lowers the reaction rate of route A. Route B becomes faster with a higher concentration of nitrite because of the affinity for nitrite. Reduction of the T2 Cu site in route B is the rate-determining step because of the low reduction potential. Our results are good agreement with the experimental results,13,21,57,60,61 and the random-sequential mechanism has been assumed from the results of kinetic and redox cycling studies.62,63 The conformation of His244 changes upon protonation of catalytic residues, and this conformation change is in good agreement with the results of an X-ray-photoreduction study, in which the rotation of His244 has been assumed to play a role as a switch for the proton relay by changing the interaction with Thr268 or Gln267.56 The conformations of His100 and His134 change upon nitrite binding and Cu reduction. The conformational change of His100 located at the end of the sensor loop between the T1 and T2 sites is more significant with the event of nitrite binding compared with reduction of Cu. His100 may play an important role in transmitting information on nitrite binding. The random-sequential mechanism might be inconsistent with the existence of the sensor loop. Because we did not consider the second-sphere residues for the calculation models, except for two catalytic residues (Asp98 and His244), it is not possible to clarify the function of the second-sphere residues. The substitution of a surface residue that exists at the entrance of the channel and far from the T2 Cu site has been reported to change the conformations of second-sphere residues around the T2 Cu site.65 Those conformational changes in the second sphere affect the intramolecular electron transfer, substrate binding, and catalytic activity.65 Investigations using larger models that include the second-sphere amino residues and water molecules are expected to reveal more details of the CuNiR mechanism. 4705


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nitrite reductase from Alcaligenes faecalis S-6: structural model of a transient catalytic intermediate. Biochemistry 40, 9132−9141. (17) Beaumont, H. J., Lens, S. I., Reijnders, W. N., Westerhoff, H. V., and Van Spanning, R. J. (2004) Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Mol. Microbiol. 54, 148−158. (18) Abraham, Z. H., Smith, B. E., Howes, B. D., Lowe, D. J., and Eady, R. R. (1997) pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans. Biochem. J. 324, 511−516. (19) Jacobson, F., Pistorius, A., Farkas, D., De Grip, W., Hansson, Ö ., Sjölin, L., and Neutze, R. (2007) pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase. J. Biol. Chem. 282, 6347−6355. (20) Olesen, K., Veselov, A., Zhao, Y., Wang, Y., Danner, B., Scholes, C. P., and Shapleigh, J. P. (1998) Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wildtype and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3. Biochemistry 37, 6086−6094. (21) Kobayashi, K., Tagawa, S., Deligeer, and Suzuki, S. (1999) The pH-dependent changes of intramolecular electron transfer on coppercontaining nitrite reductase. J. Biochem. 126, 408−412. (22) Krzemiński, Ł., Ndamba, L., Canters, G. W., Aartsma, T. J., Evans, S. D., and Jeuken, L. J. (2011) Spectroelectrochemical investigation of intramolecular and interfacial electron-transfer rates reveals differences between nitrite reductase at rest and during turnover. J. Am. Chem. Soc. 133, 15085−15093. (23) Wijma, H. J., MacPherson, I., Farver, O., Tocheva, E. I., Pecht, I., Verbeet, M. P., Murphy, M. E., and Canters, G. W. (2007) Effect of the methionine ligand on the reorganization energy of the type-1 copper site of nitrite reductase. J. Am. Chem. Soc. 129, 519−525. (24) Brenner, S., Heyes, D. J., Hay, S., Hough, M. A., Eady, R. R., Hasnain, S. S., and Scrutton, N. S. (2009) Demonstration of protoncoupled electron transfer in the copper-containing nitrite reductases. J. Biol. Chem. 284, 25973−25983. (25) Lintuluoto, M., and Lintuluoto, J. M. (2016) DFT study on nitrite reduction mechanism in copper-containing nitrite reductase. Biochemistry 55, 210−223. (26) Fukuda, Y., Tse, K. M., Lintuluoto, M., Fukunishi, Y., Mizohata, E., Matsumura, H., Takami, H., Nojiri, M., and Inoue, T. (2014) Structural insights into the function of a thermostable coppercontaining nitrite reductase. J. Biochem. 155, 123−135. (27) Hough, M. A., Eady, R. R., and Hasnain, S. S. (2008) Identification of the proton channel to the active site type 2 Cu center of nitrite reductase: structural and enzymatic properties of the His254Phe and Asn90Ser mutants. Biochemistry 47, 13547−13553. (28) Kujime, M., Izumi, C., Tomura, M., Hada, M., and Fujii, H. (2008) Effect of a tridentate ligand on the structure, electronic structure, and reactivity of the copper (I) nitrite complex: Role of the conserved three-histidine ligand environment of the type-2 copper site in copper-containing nitrite reductases. J. Am. Chem. Soc. 130, 6088− 6098. (29) Periyasamy, G., Sundararajan, M., Hillier, I. H., Burton, N. A., and McDouall, J. J. (2007) The binding of nitric oxide at the Cu (I) site of copper nitrite reductase and of inorganic models: DFT calculations of the energetics and EPR parameters of side-on and endon structures. Phys. Chem. Chem. Phys. 9, 2498−2506. (30) Fujisawa, K., Tateda, A., Miyashita, Y., Okamoto, K.-i., Paulat, F., Praneeth, V., Merkle, A., and Lehnert, N. (2008) Structural and spectroscopic characterization of mononuclear copper (I) nitrosyl complexes: end-on versus side-on coordination of NO to copper (I). J. Am. Chem. Soc. 130, 1205−1213. (31) Merkle, A. C., and Lehnert, N. (2009) The side-on copper (I) nitrosyl geometry in copper nitrite reductase is due to steric interactions with isoleucine-257. Inorg. Chem. 48, 11504−11506. (32) Lehnert, N., Cornelissen, U., Neese, F., Ono, T., Noguchi, Y., Okamoto, K.-i., and Fujisawa, K. (2007) Synthesis and spectroscopic characterization of copper (II)-nitrito complexes with hydrotris (pyrazolyl) borate and related coligands. Inorg. Chem. 46, 3916−3933.

AUTHOR INFORMATION

Corresponding Author

*Tel: +81-75-703-5445. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The computations in this work were performed using the Research Center for Computational Science, Okazaki, Japan. REFERENCES

(1) Zumft, W. G. (1997) Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61 (4), 533−616. (2) Gruber, N., and Galloway, J. N. (2008) An Earth-system perspective of the global nitrogen cycle. Nature 451, 293−296. (3) Galloway, J. N., Townsend, A. R., Erisman, J. W., Bekunda, M., Cai, Z., Freney, J. R., Martinelli, L. A., Seitzinger, S. P., and Sutton, M. A. (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889−892. (4) Suzuki, S., Kataoka, K., Yamaguchi, K., Inoue, T., and Kai, Y. (1999) Structure−function relationships of copper-containing nitrite reductases. Coord. Chem. Rev. 190−192, 245−265. (5) Jones, C. M., Stres, B., Rosenquist, M., and Hallin, S. (2008) Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification. Mol. Biol. Evol. 25, 1955−1966. (6) Kim, S. W., Fushinobu, S., Zhou, S., Wakagi, T., and Shoun, H. (2009) Eukaryotic nirK genes encoding copper-containing nitrite reductase: originating from the protomitochondrion? Appl. Environ. Microbiol. 75, 2652−2658. (7) Suzuki, S., Kataoka, K., and Yamaguchi, K. (2000) Metal coordination and mechanism of multicopper nitrite reductase. Acc. Chem. Res. 33, 728−735. (8) Antonyuk, S. V., Strange, R. W., Sawers, G., Eady, R. R., and Hasnain, S. S. (2005) Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism. Proc. Natl. Acad. Sci. U. S. A. 102, 12041− 12046. (9) Leferink, N. G. H., Han, C., Antonyuk, S. V., Heyes, D. J., Rigby, S. E. J., Hough, M. A., Eady, R. R., Scrutton, N. S., and Hasnain, S. S. (2011) Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase. Biochemistry 50, 4121−4131. (10) Murphy, M. E. P., Turley, S., and Adman, E. T. (1997) Structure of nitrite bound to copper-containing nitrite reductase from Alcaligenes faecalis. J. Biol. Chem. 272, 28455−28460. (11) Adman, E. T., Godden, J. W., and Turley, S. (1995) The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values, with NO2− bound and with type II copper depleted. J. Biol. Chem. 270, 27458−27474. (12) Ghosh, S., Dey, A., Sun, Y., Scholes, C. P., and Solomon, E. I. (2009) Spectroscopic and computational studies of nitrite reductase: proton induced electron transfer and backbonding contributions to reactivity. J. Am. Chem. Soc. 131, 277−288. (13) Zhao, Y., Lukoyanov, D. A., Toropov, Y. V., Wu, K., Shapleigh, J. P., and Scholes, C. P. (2002) Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence, conserved residues, and proton hyperfine structure. Biochemistry 41, 7464−7474. (14) Tocheva, E. I., Rosell, F. I., Mauk, A. G., and Murphy, M. E. P. (2004) Side-On Copper-Nitrosyl Coordination by Nitrite Reductase. Science 304, 867−870. (15) Merkle, A. C., and Lehnert, N. (2012) Binding and activation of nitrite and nitric oxide by copper nitrite reductase and corresponding model complexes. Dalton Trans. 41, 3355−3368. (16) Boulanger, M. J., and Murphy, M. E. P. (2001) Alternate substrate binding modes to two mutant (D98N and H255N) forms of 4706


Article

Biochemistry

oxidation catalyst with terminal water ligands. J. Chem. Theory Comput. 6, 2395−2401. (54) Frisch, M. J., et al. (2009) Gaussian 09, revision D.01, Gaussian, Inc., Wallingford, CT. (55) Suenaga, M. (2008) Development of gui for gamess/fmo calculation. J. Comput. Chem., Jpn. 7, 33−53. (56) Fukuda, Y., Tse, K. M., Suzuki, M., Diederichs, K., Hirata, K., Nakane, T., Sugahara, M., Nango, E., Tono, K., Joti, Y., et al. (2016) Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography. J. Biochem. 159, 527− 538. (57) Hough, M. A., Ellis, M. J., Antonyuk, S., Strange, R. W., Sawers, G., Eady, R. R., and Hasnain, S. S. (2005) High resolution structural studies of mutants provide insights into catalysis and electron transfer processes in copper nitrite reductase. J. Mol. Biol. 350, 300−309. (58) Strange, R. W., Murphy, L. M., Dodd, F. E., Abraham, Z. H., Eady, R. R., Smith, B. E., and Hasnain, S. S. (1999) Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase. J. Mol. Biol. 287, 1001−1009. (59) Leferink, N. G., Pudney, C. R., Brenner, S., Heyes, D. J., Eady, R. R., Hasnain, S. S., Hay, S., Rigby, S. E., and Scrutton, N. S. (2012) Gating mechanisms for biological electron transfer: Integrating structure with biophysics reveals the nature of redox control in cytochrome P450 reductase and copper-dependent nitrite reductase. FEBS Lett. 586, 578−584. (60) Suzuki, S., Deligeer, Yamaguchi, K., Kataoka, K., Kobayashi, K., Tagawa, S., Kohzuma, T., Shidara, S., and Iwasaki, H. (1997) Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases. JBIC, J. Biol. Inorg. Chem. 2, 265−274. (61) Deligeer, Fukunaga, R., Kataoka, K., Yamaguchi, K., Kobayashi, K., Tagawa, S., and Suzuki, S. (2002) Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrif icans A3151. J. Inorg. Biochem. 91, 132−138. (62) Wijma, H. J., Jeuken, L. J., Verbeet, M. P., Armstrong, F. A., and Canters, G. W. (2006) A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase. J. Biol. Chem. 281, 16340−16346. (63) Goldsmith, R. H., Tabares, L. C., Kostrz, D., Dennison, C., Aartsma, T. J., Canters, G. W., and Moerner, W. (2011) Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase. Proc. Natl. Acad. Sci. U. S. A. 108, 17269−17274. (64) Wijma, H. J., Jeuken, L. J., Verbeet, M. P., Armstrong, F. A., and Canters, G. W. (2007) Protein film voltammetry of copper-containing nitrite reductase reveals reversible inactivation. J. Am. Chem. Soc. 129, 8557−8565. (65) Leferink, N. G. H., Antonyuk, S. V., Houwman, J. A., Scrutton, N. S., Eady, R. R., and Hasnain, S. S. (2014) Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase. Nat. Commun. 5, 4395.

(33) Silaghi-Dumitrescu, R. (2006) Copper-containing nitrite reductase: a DFT study of nitrite and nitric oxide adducts. J. Inorg. Biochem. 100, 396−402. (34) Sundararajan, M., Hillier, I. H., and Burton, N. A. (2007) Mechanism of nitrite reduction at T2Cu centers: Electronic structure calculations of catalysis by copper nitrite reductase and by synthetic model compounds. J. Phys. Chem. B 111, 5511−5517. (35) De Marothy, S., Blomberg, M. R., and Siegbahn, P. E. (2007) Elucidating the mechanism for the reduction of nitrite by copper nitrite reductase - A contribution from quantum chemical studies. J. Comput. Chem. 28, 528−539. (36) Li, Y., Hodak, M., and Bernholc, J. (2015) Enzymatic mechanism of copper-containing nitrite reductase. Biochemistry 54, 1233−1242. (37) Martí, M. A., Crespo, A., Bari, S. E., Doctorovich, F. A., and Estrin, D. A. (2004) QM-MM study of nitrite reduction by nitrite reductase of Pseudomonas aeruginosa. J. Phys. Chem. B 108, 18073− 18080. (38) Bykov, D., and Neese, F. (2011) Substrate binding and activation in the active site of cytochrome c nitrite reductase: a density functional study. JBIC, J. Biol. Inorg. Chem. 16, 417−430. (39) Bykov, D., and Neese, F. (2012) Reductive activation of the heme iron-nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study. JBIC, J. Biol. Inorg. Chem. 17, 741−760. (40) Maekawa, S., Matsui, T., Hirao, K., and Shigeta, Y. (2015) Theoretical study on reaction mechanisms of nitrite reduction by copper nitrite complexes: toward understanding and controlling possible mechanisms of copper nitrite reductase. J. Phys. Chem. B 119, 5392−5403. (41) Simonson, T., and Brooks, C. L. (1996) Charge screening and the dielectric constant of proteins: insights from molecular dynamics. J. Am. Chem. Soc. 118, 8452−8458. (42) Wang, T., Brudvig, G., and Batista, V. S. (2010) Characterization of proton coupled electron transfer in a biomimetic oxomanganese complex: Evaluation of the DFT B3LYP level of theory. J. Chem. Theory Comput. 6, 755−760. (43) Hwang, S.-G., and Chung, D.-S. (2005) Calculation of the solvation free energy of the proton in methanol. Bull. Korean Chem. Soc. 26, 589−593. (44) Reiss, H., and Heller, A. (1985) The absolute potential of the standard hydrogen electrode: a new estimate. J. Phys. Chem. 89, 4207− 4213. (45) Jang, Y. H., Sowers, L. C., Caǧin, T., and Goddard, W. A. (2001) First principles calculation of pKa values for 5-substituted uracils. J. Phys. Chem. A 105, 274−280. (46) Lim, C., Bashford, D., and Karplus, M. (1991) Absolute pKa calculations with continuum dielectric methods. J. Phys. Chem. 95, 5610−5620. (47) Tawa, G., Topol, I., Burt, S., Caldwell, R., and Rashin, A. (1998) Calculation of the aqueous solvation free energy of the proton. J. Chem. Phys. 109, 4852−4863. (48) Tissandier, M. D., Cowen, K. A., Feng, W. Y., Gundlach, E., Cohen, M. H., Earhart, A. D., Coe, J. V., and Tuttle, T. R. (1998) The proton’s absolute aqueous enthalpy and Gibbs free energy of solvation from cluster-ion solvation data. J. Phys. Chem. A 102, 7787−7794. (49) Fifen, J. J., Dhaouadi, Z., and Nsangou, M. (2014) Revision of the thermodynamics of the proton in gas phase. J. Phys. Chem. A 118, 11090−11097. (50) Pace, C. N., Grimsley, G. R., and Scholtz, J. M. (2009) Protein ionizable groups: pKa values and their contribution to protein stability and solubility. J. Biol. Chem. 284, 13285−13289. (51) Neese, F. (2006) A critical evaluation of DFT, including timedependent DFT, applied to bioinorganic chemistry. JBIC, J. Biol. Inorg. Chem. 11, 702−711. (52) Trasatti, S. (1987) Interfacial behaviour of non-aqueous solvents. Electrochim. Acta 32, 843−850. (53) Wang, T., Brudvig, G. W., and Batista, V. S. (2010) Study of proton coupled electron transfer in a biomimetic dimanganese water 4707


DFT Study on Enzyme Turnover Including Proton and Electron

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