Calculated Microscopic Stability Constants for Deprotonation of the Zwitterions H2cys and H2pen

Supporting Information

Theoretical and Experimental Sulfur K-edge X-ray Absorption Spectroscopic Study of Cysteine, Cystine, Homocysteine, Penicillamine, Methionine and Methionine Sulfoxide

Emiliana Damian Risberg,1 Farideh Jalilehvand,2 Bonnie O. Leung,2 Lars G.M. Pettersson3 and Magnus Sandström1*

1 Department of Physical, Inorganic and Structural Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden. 2 Department of Chemistry, University of Calgary, Calgary, AB T2N 1N4, Canada. 3 Department of Physics, Alba Nova, Stockholm University, SE-106 91 Stockholm, Sweden.

Supporting Information Available: Tables S1, S3: calculated microscopic stability constants for deprotonation of the zwitterions H2Cys and H2Pen. Tables S2, S4, S5: calculated energies and intensities for the numbered transitions in Figs. S6, 17 and S16 for the Cys2-, 2- [Ni(Cys)2] and Hg((HCys)S)2 species, respectively. Fig. S1 displays the fractions of the major cysteine species at different pH values. Figs. S2; S3; S4; S6; S7; S8; S9; S11; S12; S13; S14; S16 show MO contours for the transitions marked in the calculated XANES spectra for + - - - the following (hydrated) species: H3Cys , H2Cys, HCys : thiol (HCys )N and thiolate (HCys )S, 2- - - - 2- Cys , H2Pen, HPen : thiol (HPen )N and thiolate (HPen )S; Pen , homocysteine HS-CH2-CH2- + - + - CH(NH3) COO , methionine CH3S(CH2)2CH(NH3 )COO , methionine sulfoxide CH3-SO- + - - + + - (CH2)2-CH( NH3)-COO , cystine OOC-CH(NH3 )CH2S-SCH2CH(NH3 )COO , and the trans-Hg((HCys)S)2 complex. Fig. S5 displays the experimental S K-edge XANES spectra for reduced and oxidized glutathione in the solid state and in solutions at different pH values, and Figs. S10 and S15 the spectra for homocysteine and cystine, respectively.

1 TABLE S1. Cysteine: Calculation of the microscopic stability constants for the deprotonation of the zwitterion H2Cys. Relative amounts (%) from fitting two components consisting of, 1: acidic (pH = 4.9) and 2: alkaline (pH = 11.4) cysteine spectra, to the spectra of 0.3 mol·dm-3 cysteine solutions (Photon Factory, beamline 9A). Component 1 represents the sum of the - - 2- thiols, H2Cys and (HCys )N, and 2 represents the sum of the thiolates, (HCys )S and Cys . The reported macroscopic stability constants (pKa1 = 1.71, pKa2 = 8.36, and pKa3 = 10.75) are used 2- to estimate the relative amount of the H2Cys and Cys species (%). The resulting amounts of - - the (HCys )N and (HCys )S ions are then used to derive the microscopic stability constants for the deprotonation of the zwitterion H2Cys: from the amino group:

N - pKa2 = pH – log{[(HCys )N]/[H2Cys]}, and from the thiol group:

S - pKa2 = pH – log{[(HCys )S]/[H2Cys]}.

2- - - N S pH 1 2 H2Cys Cys (HCys )N (HCys )S pKa2 pKa2 4.94 100 0 100 0 0 0 7.78 83.5 16.5 79 0.0 4.5 16.5 9.02 8.46 8.07 72.3 27.7 66 0 6.3 27.5 9.09 8.45 8.37 61.1 38.9 49 1 12.1 37.9 8.98 8.48 8.69 49.6 50.4 32 1.0 17.6 49.4 8.95 8.50 8.95 39.6 60.4 20 1.4 19.6 59 8.96 8.48 9.00 36.0 64.0 19 2 17 62 9.05 8.49 9.35 31.9 68.1 9 4 22.9 64 8.94 8.50 9.87 24.8 75.2 3 12 21.8 63.2 9.01 8.55 11.4 100 0 81 ~4 15 Mean: 8.98±0.05 8.49±0.05

TABLE S2. Deprotonated cysteine anion, Cys2-: Changes induced by 15 hydrating water molecules on the energy and intensity of the numbered transitions in Figs. S6.

Energy (eV) Oscillator strength Transition (Mbarn) No. Cys2- Solvated Cys2- Solvated Cys2- Cys2- 1 2469.5 2469.3 0.0109 0.0045 2 --- 2469.6 --- 0.0057

2 TABLE S3. Calculation of the microscopic stability constants for the deprotonation of the zwitterion H2Pen. Relative amounts (%) from fitting two components consisting of, 1: acidic (pH = 4.94) and 2: alkaline (pH = 11.17) penicillamine spectra, to the spectra of ca. 0.05 -3 - mol·dm solutions. Component 1 represents the sum of the thiols, H2Pen and (HPen )N, and 2 - 2- represents the sum of the thiolates, (HPen )S and Pen . The relative amount of the H2Pen and 2- Pen species (%) are estimated from the reported macroscopic stability constants (pKa1 = 1.94, pKa2 = 7.93, and pKa3 = 10.39, ionic strength = 0.3 (Ref. 35)). The resulting amounts of - - the (HPen )N and (HPen )S ions are then used to derive the microscopic stability constants for the deprotonation of the zwitterion H2Pen: from the amino group

N - pKa2 = pH – log{[(HPen )N]/[H2Pen]}, and from the thiol group

S - pKa2 = pH – log{[(HPen )T]/[H2Pen]}.

2- - - N S pH 1 2 H2Pen Pen (HPen )N (HPen )S pKa2 pKa2 4.94 100 0 100 0 0 0 8.07 59.3 40.7 52 0.0 7.3 49 8.92 8.10 8.39 46.5 53.5 34 0.5 12.5 53 8.82 8.20 8.55 38.5 61.5 26 1 12.5 60.5 8.87 8.18

9.24 28.9 71.1 7 3 21.9 68.1 8.74 8.25

11.17 100 0 100

Mean: 8.87±0.10 8.2±0.1

TABLE S4. Energy and intensity of the calculated transitions (Fig. 17) for the gas phase 2- [Ni(Cys)2] species (trans-conformation) and the energies for the first two experimental peaks for the solid K2[Ni(Cys)2] compound.

2- [Ni(Cys)2] Experimental peak Transition energies (eV)

No. Solid K2[Ni(Cys)2] Oscillator Energy strength 1 2

(eV) (Mbarn) 1 2468.2 0.0278 2 2470.7 0.0089 2468.26 2469.91 3 2470.8 0.0131 4 2470.9 0.0115

TABLE S5. Energy and intensity of the calculated transitions (Fig. S16) for the Hg((HCys)S)2 complex (trans-conformation, gas phase) and the energies for the first two experimental peaks of the solid Hg(HCys) compound.

Hg((HCys)S)2 Experimental peak Transition energies (eV) No. Solid Hg(HCys)2 Oscillator Energy strength 1 2

(eV) (Mbarn) 1 2469.5 0.0542 2 2470.2 0.0048 3 2471.0 0.0125 4 2471.0 0.0151 2469.55 2470.25 5 2471.3 0.0318 6 2471.8 0.0454

2− [Cys ]TOT = 50.00 mM 2− + H2Cys Cys 1. 0 H3Cys HCys− 0. 8

H - + OOC C NH3 0. 6 H2C 0. 4 SH

Fr act i on H2Cys 0. 2

0. 0 02468101214 pH

Figure S1.

Figure S1. Fraction diagram showing the major cysteine species at different pH values, calculated for 0.05 mol⋅dm-3 cysteine solution and ionic strength I = 0 (macroscopic acid dissociation constants: pK1 = 10.75, pK2 = 8.36 and pK3 1.71, Ref. 17) by means of the MEDUSA Chemical Equilibrium Software, Royal Institute of Technology, Department of Chemistry, Stockholm, 2004; http://www.kemi.kth.se/medusa/, author I. Puigdomenech.

5 Figure S2. Protonated + cysteine, H3Cys : Comparison of calculated spectra for the protonated + cysteine cation, H3Cys , solvated by five water molecules (cf. Fig. 3a), in panel A, and without water in panel B, with the experimental spectrum of a solution at pH = 0. The electron density contours of the MOs corresponding to the numbered transitions in the theoretical spectra are illustrated below.

H3Cyszwcomparison.ti A

Figure S2.

Figure S3. Cysteine zwitterion, H2Cys: Comparison of calculated spectra for the cysteine zwitterion, H2Cys, solvated by five water molecules (cf. Fig. 3b), in panel A, and without water in panel B, with the spectrum of cysteine in solution at pH = 4.94. The electron density contours of the MOs corresponding to the numbered transitions in the theoretical spectra are illustrated below.

H2Cyscomparison.tif A

Figure S3.

7 A Figure S4. Cysteine thiol - and thiolate HCys : Comparison of spectra calculated for the two solvated HCys- species, - the thiol (HCys )N = - HSCH2CH(NH2)COO - and thiolate (HCys )S = - + - SCH2CH(NH3 )COO (cf. Figs. 3c and 3d) in panels A and B, with experimental spectra at pH 8.1 and 11.3, respectively. The electron density contours of the MOs corresponding to the numbered transitions in the theoretical spectra are illustrated .

Figure S4.

Figure S5.

Figure S5. Experimental S K-edge XANES spectra of the tripeptide glutathione, γ- glutamylcysteinylglycine, in its reduced (left) and oxidized (right) zwitterionic forms in the solid state, and in solutions at different pH values. The peak energies (maxima from 2nd derivatives) of the main spectral peaks are shown. For reduced glutathione the deprotonation of the thiol group is evident in the spectrum for pH = 12. Note the shift in the energy scale (+2.82 eV) in this figure due to the calibration used for these experimental spectra by setting the lowest energy peak of sodium thiosulfate (Na2S2O3·5H2O) to 2472.02 eV. The molecular formula above (from Wikipedia) shows reduced glutathione in its unionized form.

Figure S6. Cysteine anion, 2- Cys : Comparisons of calculated 2- spectra for Cys , solvated by fifteen water molecules (cf. Figure 3e) in panel A, and without water in panel B, compared with the experimental XANES spectrum for cysteine solution at pH = 13 (dot- dashed line). The vertical bars for both models represent the calculated transition energies and cross-sections, convoluted with 0.9 eV FWHM Gaussians below 2469.3 eV, linearly increasing to 8 eV FWHM at 2489.3 eV and 8 eV above. The electron density contours of the MOs for the numbered transitions in the theoretical spectra are illustrated. A

Figure S6.

Figure S7.

Figure S7. Penicillamine zwitterion, H2Pen: The calculated spectrum for the penicillamine zwitterion, H2Pen, solvated by six water molecules (cf. Figure 3f) compared to the experimental XANES spectrum for penicillamine in aqueous solution at pH = 4.94. The electron density contours of the MOs corresponding to the transitions denoted in the theoretical spectrum are illustrated below the spectra.

Figure S8. Penicillamine - thiol and thiolate, HPen : Comparison of spectra calculated for the solvated HPen- species, the thiol, - HSC(CH3)2CH(NH2)COO - (HPen )N, and the thiolate, - + - SC(CH3)2CH(NH3) COO - (HPen )S, (cf. Figs. 3g and 3f) in panels A and B, with experimental spectra at pH 8.07 and 9.24, respectively. The electron density contours of the MO:s corresponding to the numbered transitions in the theoretical spectra are illustrated below.

Figure S9.

Figure S9. Calculated S K-edge XANES spectrum (solid line) of the deprotonated hydrated Pen2- ion solvated by five water molecules (cf. Figure 3i) compared to the experimental XANES spectrum for penicillamine in aqueous solution at pH = 11.17 (dot-dashed line). The vertical bars represent the calculated transition energies and cross-sections, convoluted with 0.8 eV FWHM Gaussians below 2469.3 eV, linearly increasing to 8 eV FWHM at 2489.3 eV, and 8 eV above. The electron density contours of the MOs corresponding to the transitions denoted in the theoretical spectrum are illustrated below the spectra.

Figure S10.

Figure S10. Homocysteine: Experimental sulfur K-edge spectra of homocysteine in the solid state and in solution at pH= 5.1 and 12.0, respectively. Note the shift in the energy scale (+2.82 eV) in this figure due to the calibration used for these experimental spectra by setting the lowest energy peak of sodium thiosulfate (Na2S2O3·5H2O) to 2472.02 eV.

Figure S11.

Figure S11. Homocysteine, zwitterion: Computed S K-edge XANES spectrum (solid line) of the hydrated zwitterion of homocysteine solvated by six water molecules (cf. Figure 3j) compared to the experimental XANES spectrum of homocysteine in aqueous solution at pH = 5.1 (dot-dashed line). The electron density contours of the MOs corresponding to the transitions denoted in the theoretical spectrum are illustrated below the spectra.

Figure S12.

Figure S12. Methionine zwitterion, HMet: Comparison of calculated spectra for the + - methionine zwitterion, CH3S(CH2)2CH(NH3 )COO , solvated by six (cf. Figure 3k) and five water molecules, illustrated in panel A and B, respectively, and without water in panel C. The electron density contours of the MOs corresponding to the numbered transitions in the theoretical spectra are displayed below the spectra.

17 Figure S13

18 Figure S13, contd.

Figure S13. Methionine sulfoxide zwitterion: Comparison of calculated spectra for the + - methionine sulfoxide zwitterion (CH3-SO-(CH2)2-CH(NH3 )-COO ), solvated by six (cf. Figure 3l), and one water molecule, illustrated in panels A and B, respectively, and without water in panel C. The electron density contours of the MOs corresponding to the numbered transitions in their theoretical spectra are displayed below the spectra.

20 Figure S14. Cystine: Comparison of calculated spectra for the cystine (disulfide) zwitterion: - + ( OOC-CH(NH3 )-CH2-S- + - S-CH2-CH(NH3 )-COO ), solvated by eight (cf. Figure 3m) water molecules in panel A,. and without water in panel B. The electron density contours of the MOs corresponding to the numbered transitions in the theoretical spectra are displayed below the spectra.

Figure S15.

Figure S15. Experimental spectra of cystine in the solid state and in solution (saturated solution at pH=7.1). The peak energies (from 2nd derivatives) are given in Table 8.

Figure S16. Calculated (gas-phase) S K-edge XANES spectrum (solid line) of the Hg((HCys)S)2 complex in trans-conformation, (cf. the model beside the spectrum) compared to the experimental XANES spectrum of solid Hg(HCys)2 (dot-dashed line). The vertical bars represent the calculated transition energies and cross-sections, convoluted with 0.8 eV FWHM Gaussians below 2475.1 eV, linearly increasing to 8 eV FWHM at 2495.1 eV, and 8 eV above. The electron density contours of the MOs corresponding to the transitions denoted in the theoretical spectrum are illustrated below the spectra.