From Thiol to Sulfonic Acid: Modeling the Oxidation Pathway of Protein

Jul 18, 2014 - Lenora K. Harper , Sonia Antony , and Craig A. Bayse. Chemical Research in Toxicology 2014 27 (12), 2119-2127. Abstract | Full Text HTM...
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From Thiol to Sulfonic Acid: Modeling the Oxidation Pathway of Protein Thiols by Hydrogen Peroxide Laura A. H. van Bergen,† Goedele Roos,†,‡,§ and Frank De Proft*,† †

General Chemistry Research Group (ALGC), Member of the QCMM VUB-UGent Alliance Research Group, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium ‡ Department of Structural Biology, VIB, B-1050 Brussels, Belgium § Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium S Supporting Information *

ABSTRACT: Hydrogen peroxide is a natural oxidant that can oxidize protein thiols (RSH) via sulfenic acid (RSOH) and sulfinic acid (RSO2H) to sulfonic acid (RSO3H). In this paper, we study the complete anionic and neutral oxidation pathway from thiol to sulfonic acid. Reaction barriers and reaction free energies for all three oxidation steps are computed, both for the isolated substrates and for the substrates in the presence of different model ligands (CH4, H2O, NH3) mimicking the enzymatic environment. We found for all three barriers that the anionic thiolate is more reactive than the neutral thiol. However, the assistance of the environment in the neutral pathway in a solvent-assisted proton-exchange (SAPE) mechanism can lower the reaction barrier noticeably. Polar ligands can decrease the reaction barriers, whereas apolar ligands do not influence the barrier heights. The same holds for the reaction energies: they decrease (become more negative) in the presence of polar ligands whereas apolar ligands do not have an influence. The consistently negative consecutive reaction energies for the oxidation in the anionic pathway when going from thiolate over sulfenic and sulfinic acid to sulfonic acid are in agreement with biological reversibility.

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INTRODUCTION Hydrogen peroxide (H2O2) is a well-known, albeit rather weak, reactive oxygen species. It is a side product of normal cell metabolism and it can act as a messenger molecule in vital cellular processes.1,2 A key event during redox signaling by H2O2 is the two-electron oxidation of the sulfur atom present in the side-chain of the amino acid Cysteine (Cys).3 Sulfur can easily be oxidized to various states, as depicted in Figure 1.4 This multitude of different oxidation states is due to the availability of low-lying d-orbitals, giving sulfur oxidation states ranging from −II to +VI. The first oxidized species is sulfenic acid (RSOH), exhibiting the +I oxidation state. This compound is chemically versatile; i.e., it exhibits both electrophillic and nucleophilllic reactivity. The best known subsequent step is the disulfide formation, which leads to disulfide bridges inside proteins. A well-known example of this process occurs in antioxidant protein peroxiredoxin (Prx). Its sulfenylated cysteine is reduced back to the active thiol form via the nucleophilic attack of a second thiol on CysSOH by which a disulfide is formed and H2O is released. RSOH can again be reversed to the thiol form by multiple cellular reductants, e.g., gluthatione and thioredoxin. Further oxidation of sulfenic acid leads to sulfinic acid (RSO2H), which is a harder electrophile than sulfenic acid, due to the increase of the partial positive charge on the sulfur atom, although sulfinate, the corresponding anionic form, can still act as a soft nucleophile. The oxidation process toward sulfinic acids is generally considered as being biologically irreversible, partly due to the low pH ( ΔG°,‡,1 − ΔGR,1 ° ,

RESULTS AND DISCUSSION For reactions 1−6, mentioned in the Introduction, we determined both the reaction and activation free energies. First, we compare the barriers of the anionic (1−3) and neutral (4−6) oxidation pathways of the reference systems without the presence of environmental ligands. Subsequently, the effects of the different ligands are investigated. 1. Orientation of H2O2 in Transition States and Reactant Complexes. We start by making a remark on the relative position (which can be either cis or trans, depicted in Figure 3) of the H2O2 molecule with respect to the substrate. In

Figure 3. Two conformations found for H2O2 during optimization for the reactant complexes. (b) is closer to the configuration in the TS states than (a).

the transition state, the H2O2 always takes a similar position; i.e., the oxygen that transfers is closer to the sulfur atom than the second oxygen, as expected. When the reactant complexes are optimized after the IRC calculations, the H2O2 can adopt two different conformations, either cis-like (Figure 3a), where the hydrogens are on the same side of the oxygens, or trans-like (Figure 3b), where the hydrogens are on opposite sites of the oxygens. When the sulfur-containing molecule is in the neutral form, the H2O2 is always in a trans conformation, similar to when it is in the TS; i.e., the oxygens have different distances to the sulfur. This conformation is consistent with previous QM/ 6080

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Figure 5. Barrier and reaction energies of the oxidation pathway of methanethiol by H2O2, without any explicit environment present. All energies are given in kcal/mol.

which is in agreement with biological reversibility. RSO− can easily be reduced to RS−, whereas for the reduction of RSO2− only one specialized enzyme is known at the moment and the RSO3− form is in proteins currently considered as irreversible. It is also important to note that the reaction barrier for the third oxidation step is halved compared to the previous two barriers. 2.2. Neutral CH3SH Oxidation Pathway. The barriers for the oxidation pathway of methanethiol are comparable for each consecutive oxidation (∼50 kcal/mol). Contrary to the anionic pathway, the neutral form has an extra barrier after the first oxidation. Figure 6 depicts the two-step mechanism of the

for the anions, the charge on the sulfur, going from CH3SH to CH3SO3H, barely changes, staying within the neutral regions (green color). This is probably due to the hydrogen atom, which has a strong positive charge in all of the different states. Because ΔG°R does change considerably for the three different oxidations, it can be either that it is more sensitive to changes in the electrostatic potential, note that the range of the values for the different forms have different end points, or that other factors play a role as well. 3. Environmental Influences. The environmental influences of H2O, NH3, and CH4 on the reaction energy and activation barriers are studied next. These ligands were chosen because they provide simple model for residues from enzymatic environments, for example, the amino acids serine (polar), leucine (apolar), the NH backbone or a water molecule (see, for example, the active site of Prx.27). After the optimization of the different sulfur-containing molecules, the environmental ligands were placed at various places around the molecules and these systems were optimized as a whole. Next, the TS’s were determined and IRC’s were preformed starting from the TS structures. The IRC’s were used to determine which of the multitude of optimized combinations of the sulfur-containing molecules and the environmental ligands were connected with the found TS structures. Every TS has two IRC’s, one reverse, leading to a reactant complex, and one forward, leading to a product complex. These reaction and product complexes can then be linked to specific free molecule orientations. In this way we made combinations of one free reactant with one TS and one free product; if this free product orientation was equal to a free reactants orientation, a complete pathway of oxidation could be made. 3.1. Anionic CH3S− Oxidation Pathway. Table 1 shows the influence of the three different environmental ligands, on ΔG°,‡

Figure 6. Hydrogen transfer from CH3S(O)H to form CH3SOH in the neutral oxidation pathway.

oxidation of methanethiol to the formation of methanesulfenic acid. After the first oxidation step, a thiol S-oxide is formed, which is a tautomer of the expected sulfenic acid. According to research by Alkorta and Elguero,26 methanesulfenic acid should be 90 kJ/mol (21.5 kcal/mol) more stable than the corresponding S-oxide. This is in agreement with our findings that the methanethiol S-oxide lies 25.9 kcal/mol higher in energy than methanesulfenic acid; however, the barrier of 39.5 kcal/mol indicates that this process is not effortless. Because the calculations to determine the transition states were done in the gas phase and solvation effects were included afterward, there is a possibility that the barrier for this transfer might disappear in solution phase. The EPS maps of the neutral forms are shown in Figure 7. It can be seen that in contrast to the EPS

Figure 7. Electrostatic potential surface (EPS) plotted on the electron isodensity surface (0.0004 au) for the neutral forms of the sulfur oxidation pathway. Due to the hydrogen atom taking most of the positive charge, the sulfur atom stays close to neutral throughout all of the oxidations (a)− (d). Color gradient: red corresponds to the most negative and blue corresponds to the most positive value. The ranges (a.u.) are (a) −2.863e−2 to 2.863e−2, (b) −5.874e−2 to 5.874e−2, (c) −6.742e−2 to 6.742e−2, and (d): −7.507e−2 to 7.507e−2. 6081

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NH3 raises this barrier by ∼3 kcal/mol. For ΔG°R,2, the difference is 1 kcal/mol or less and is negligible. The barrier for the third oxidation step to CH3SO3− is the most affected by the environment. As can be seen in Table 1, both polar and apolar ligands raise the barrier, with the latter having a 2-fold bigger influence than the former. On the contrary, the influence on ΔG°R,3 is not significant. As noted earlier, the relative position between H2O2 and NH3 in the TS states for the second and third oxidation step changes. This would suggest that, in enzymes, either the active site undergoes a strong local, conformational change, probably needing a vast amount of energy, or the H2O2 attacks from a different location in the active site. Both suggestions could be looked at with MD simulations or QM/MM calculations (work in progress). To validate the observed trends, the ΔGR were also computed on the MP2/6-31+G** level of theory (Supporting Information); indeed, similar trends were found to emerge from these data. 3.2. Neutral CH3SH Oxidation Pathway. The position of the apolar ligand (CH4) in the neutral oxidation pathway is similar to its position in the anionic oxidation pathway. When the TS for the different oxidation reactions of the neutral pathway are connected (via IRC), however, multiple possibilities remain for the polar ligands. Two possibilities emerge: one where the polar ligand does not actively participate in the oxidation of the sulfur (passive pathway) and the SAPE mechanism, where the ligand actively participates by donating and receiving a hydrogen atom (SAPE pathway). The influence of the ligands is shown in Table 2. In the passive route, the environmental ligand is close to H2O2, but somewhat away from the sulfur-containing molecule, similar to the position of the ligand in the anionic pathway (Figure 8a). With the SAPE pathway, the polar ligand is between the H2O2 and the hydrogen atom of the sulfur-containing molecule (Figure 9a).

Table 1. Comparison of the Barrier and Reaction Energies of the Anion Form Oxidation Pathway for the Reference without Ligands and the Pathways with Involvement of the Environmental Factors (All Values in kcal/mol) ligand

none

CH4

H2O

NH3

ΔG‡,1 ΔGR,1 ΔG‡,2 ΔGR,2 ΔG‡,3 ΔGR,3

28.4 −38.5 28.1 −57.0 15.8 −80.9

31.3 −38.0 31.4 −57.3 25.5 −79.9

27.0 −42.5 28.1 −55.9 19.7 −76.7

29.3 −40.1 31.2 −56.5 18.3 −79.2

and ΔG°R of the methanethiolate oxidation pathway. During the optimization, for every ligand, only a single total oxidation pathway resulted. CH4 remained close to the H2O2 and further away from the sulfur-containing molecule (Figure 8a). H2O is

Figure 8. Two different positions the environmental ligands take during the oxidation of the anionic form of sulfur. (a) is the starting position for both the apolar and polar ligands. (b) is the final position of the polar ligands.

close to H2O2 during the first barrier, at the same position as CH4, but could only be optimized away from H2O2 and close to the oxygen atoms that were already bound to the sulfur atom (Figure 8b). NH3 stays close to H2O2 for the first two barriers, the same as CH4, but like with the water molecule for the last barrier, only the position on the other side of the sulfur than the peroxide could be optimized. For the first step, an apolar environment slightly raises the energy barrier, although the difference of ∼3 kcal/mol is close to the intrinsic accuracy of the computational method. The influence of a polar environment on the barrier seems to be more complex, both changes due to H2O and NH3 are minor and the influences are opposite to each other. The results for ΔGR,1 ° are consistent with each other, with a polar environment increasing the energy (2−4 kcal/mol), whereas an apolar environment has no influence on the reaction energy. The barrier for the second oxidation step is not influenced by H2O, whereas the presence of both CH4 and

Figure 9. Position (a) and movement (b) of the atoms during the SAPE mechanism (active) pathway, due to proton shuffling.

The first oxidation step for the reference neutral pathway has two barriers to the formation of sulfenic acid, as mentioned early. When either CH4 or a single H2O is present as a (passive) environmental ligand, the formation of methanesul-

Table 2. Comparison of the Barrier and Reaction Energies of the Neutral Form Oxidation Pathway between the Reference without Ligands and the Pathways with Involvement of the Environmental Factorsa (All Values in kcal/mol) passive

SAPE

ligand

none

CH4

H2O

NH3

1 H2O

2 H2O

1 NH3

2 NH3

ΔG‡,1 ΔGR,1 ΔG‡,2 ΔGR,2 ΔG‡,3 ΔGR,3

52.8 −50.3 47.3 −43.2 49.4 −62.7

51.2 −49.3 47.9 −43.9 50.3 −62.0

43.0 −51.7 43.0 −44.5 49.8 −61.0

n.a. n.a. 43.2 −45.4 48.8 −61.6

n.a. n.a. 37.8 −44.5 45.4 −62.5

38.9 −52.5 36.7 −43.4 32.1 −66.1

31.2 −49.5 40.0 −44.7 20.4 −64.9

27.6 −54.9 n.a. n.a. n.a. n.a.

For CH4 only the influence of the connected states is shown, whereas for the two polar ligands the influence of both the “passive” and a SAPE pathway is shown. a

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neglibible, with CH4 and H2O slightly raising the barrier and NH3 slightly lowering it. The influence of the SAPE mechanism is strongly dependent on the type of environmental molecule as well as the number of these molecules involved in the mechanism. The presence of one water molecule lowers the barrier with only 4 kcal/mol, whereas adding a second one gives a barrier that is over 7 kcal/mol lower than the one with a single water. The presence of a single NH3 lowers the barrier with 29−20.4 kcal/mol, which is close to biological relevance. For the second oxidation step, a pathway involving two ammonia could not be found. As with the second barrier, our model differs from the one used by Bayse, in the same regard. In contrast to the previous step, there is a noticeable difference in our trends and those calculated by Bayse. Although Bayse found that the ΔG‡,3 is larger than the previous two barriers, in our results this is the lowest of the three barrier with the active presence of two water molecules. As a whole, it can be seen that our barriers for the SAPE two-water network oxidations are still considerably higher than the ones reported in ref 2, whereas the reaction energies are roughly comparable. It is highly probable that the difference in the model used for the sulfur-containing molecule and oxidizing agent are responsible for the difference in the SAPE mechanism for the second and third oxidation step and for the different energetics. In the contribution of Bayse, CH3OOH was used as the oxidizing agent, whereas we have used H2O2. In addition, ref 2 considers the single amino acid cysteine as the model for his calculations, whereas in the present study methanethiol was used as the sulfur substrate. The smaller size of both our substrate and oxidizing agent might thus give rise to more compact transition state structures, resulting in higher strain and higher activation barriers. For both the neutral and anionic pathways, the influence of the environment is more targeted toward the barrier energy rather than to the reaction energy. The influence on ΔG°,‡ can be significant as well as nonexisting, depending on the exact position of the specific ligand with respect to the sulfurcontaining molecule, i.e., whether a SAPE can take place. As with the anionic pathway, the trends of ΔGR were also computed with the MP2/6-31+G** level of theory, found in the Supporting Information. Again, similar trends were found to emerge from these data.

fenic acid goes through the methanethiol S-oxide intermediate. We were unable to calculate the influence of these two ligands on the hydrogen-transfer step. This intermediate step, where the methanethiol S-oxide is formed, disappears in the presence of an ammonia or two or more water molecules, which is due to their active participation in the oxidation process via the SAPE mechanism, as depicted in Figure 9b. Compared to the reference system without the ligands present, the barrier to form TS1 is lowered by ∼14 kcal/ mol in the presence of two water molecules with both waters participating in the process. The passive influence of one water on the other hand lowers the barrier ∼10 kcal/mol. For the optimization with the inclusion of an ammonia molecule, only a TS where it is actively participating could be found; the opposite holds true for the involvement of water, where at least two molecules need to be present. The active presence of a single NH3 has a drastic influence, lowering the barrier with more than 21 kcal/mol; adding a second ammonia reduces the barrier by an extra ∼3.5 kcal/mol. The influence of an apolar environment on the second oxidation step is nonexistent, both for ΔG°,‡,2 and ΔG°R,2. The passive presence of a polar environment, either H2O or NH3, decreases the barrier with ∼4 kcal/mol. For the active involvement of the polar environment, the situation is more complex. A single H2O decreases the barrier with 9.5 kcal/mol, adding a second water to this gives a decrease of ∼10.5 kcal/mol. The presence of NH3 is somewhat less influential, with a decrease of 7.3 kcal/mol. The IRC pathway for the formation of sulfinic acid with the active presence of two ammonia molecules reveals that only one these molecules actually participates in the SAPE mechanism, and it was thus decided to not pursue this pathway in more detail. It should also be mentioned that for our specific substrate model system and oxidizing agent, the SAPE mechanism at this step as well as the subsequent one slightly differs from the mechanism described by Bayse,2 as depicted in Figure 10. For the two-

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CONCLUSIONS In this work, we have studied the reaction barriers and reaction energies of the complete anionic and neutral oxidation pathway from thiol to sulfonic acid. We found that the reaction barriers for all three oxidation steps, i.e., thiol (RSH) to sulfenic (RSOH), RSOH to sulfenic (RSO2H) and the RSO2H to sulfonic (RSO3H) oxidation step, are lower in the anionic pathway than in the neutral pathway. However, assistance of the environment in the solvent-assisted proton-exchange mechanism can lower the barrier in the neutral pathway. In general, polar ligands can decrease the reaction barriers, whereas apolar ligands do not influence the barrier heights. The same holds for the reaction energies. With this study we give for the first time insight in the influence of various ligand types on the complete anionic and neutral thiol to the sulfonic oxidation pathway.

Figure 10. Difference between the SAPE mechanism for the second oxidation as proposed by Bayse (a) and the one used in this paper (b).

water assistance in our model, the second oxidation step has a ∼2 kcal/mol lower barrier then the first step, the difference in the model of Bayse being 2−3.5 kcal/mol. It should be noted that the present model only needs one specific molecule to be present, whereas the Bayse model needs at least two molecules. On the other hand, our model can only be used on the neutral pathway, due to the requirement of the hydrogen atom on the sulfur molecule, whereas Bayse also investigated the first oxidation step of the anionic pathway. The energetic gain from the environment for ΔGR,2 is small, 0.5−2 kcal/mol, and is at least 10 kcal/mol less than on the anionic pathway. For the last oxidation step, the influence of a passive environment is

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ASSOCIATED CONTENT

S Supporting Information *

Table comparing the barriers and reaction free energies (298.15 K) of the oxidation pathways of methanethiol and meth6083

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(13) Luo, D.; Smith, S. W.; Anderson, B. D. Kinetics and Mechanism of the Reaction of Cysteine and Hydrogen Peroxide in Aqueous Solution. J. Pharm. Sci. 2005, 94, 304−316. (14) Bach, R. D.; Su, M.-D.; Schlegel, H. B. Oxidation of Amines and Sulfides with Hydrogen Peroxide and Alkyl Hydrogen Peroxide. The Nature of the Oxygen-Transfer Step. J. Am. Chem. Soc. 1994, 116, 5379−5391. (15) Bayse, C. A. DFT Study of the Glutathione Peroxidase-Like Activity of Phenylselenol Incorporating Solvent-Assisted Proton Exchange. J. Phys. Chem. A 2007, 111, 9070−9075. (16) Zhao, Y.; Truhlar, D. G. The M06 Suite of Density Functionals for Main Group Thermochemistry, Thermochemical Kinetics, Noncovalent Interactions, Excited States, and Transition Elements: Two New Functionals and Systematic Testing of Four M06-Class Functionals and 12 Other Functionals. Theor. Chem. Acc. 2008, 120, 215−241. (17) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio Molecular Orbital Theory; Wiley: New York, 1986; Vol. 33. (18) Frisch, M. J.; et al. Gaussian 09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2010. (19) Nakamura, T.; Kado, Y.; Yamaguchi, T.; Matsumura, H.; Ishikawa, K.; Inoue, T. Crystal Structure of Peroxiredoxin from Aeropyrum Pernix K1 Complexed with Its Substrate, Hydrogen Peroxide. J. Biochem. 2010, 147, 109−115. (20) Fukui, K. The Path of Chemical−reactions − The IRC Approach. Acc. Chem. Res. 1981, 14, 363−368. (21) Hratchian, H. P.; Schlegel, H. B. Accurate Reaction Paths Using a Hessian Based Predictor-Corrector Integrator. J. Chem. Phys. 2004, 120, 9918−9924. (22) Hratchian, H.; Schlegel, H. Using Hessian Updating to Increase the Efficiency of a Hessian Based Predictor-Corrector Reaction Path Following Method. J. Chem. Theory Comput. 2005, 1, 61−69. (23) Frisch, M. J.; et al. Gaussian 03, Revision E.01; Gaussian, Inc.: Wallingford, CT, 2007. (24) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum Mechanical Continuum Solvation Models. Chem. Rev. 2005, 105, 2999−3094. (25) Ho, J.; Klamt, A.; Coote, M. L. Comment on the Correct Use of Continuum Solvent Models. J. Phys. Chem. A 2010, 114, 13442− 13444. (26) Alkorta, I.; Elguero, J. Classical versus Redox Tautomerism: Substituent Effects on the Keto/Enol and Sulfoxide/Sulfenic Acid Equilibria. Tetrahedron Lett. 2004, 45, 4127−4129. (27) Hall, A.; Karplus, P. A.; Poole, L. B. Typical 2-Cys Peroxiredoxins-Structures, Mechanisms and Functions. FEBS J. 2009, 276, 2469−2477.

anethiolate without model ligands present at the CCSD/6311+G** and the M06-2X/6-311+G** levels of theory. Tables containing the ΔGR calculated with the MP2/6-31+G** level of theory. The Cartesian coordinates of all structures considered in this paper. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*F. De Proft. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Frank De Proft acknowledges the Free Universiteit of Brussels (VUB) and the Research Foundation - Flanders (FWO) for continuous support to his group. Goedele Roos thanks the FWO for a postdoctoral fellowship. The authors also acknowledge financial support of the Research Foundation Flanders (FWO) through research program FWOAL622. In addition, the authors acknowledge Prof. Craig A. Bayse (Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia, United States) for providing the Cartesian coordinates of the structures reported in ref 2.

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