Salt Effects on Ionization Equilibria of Histidines in Myoglobin Yung-Hsiang Kao, Carolyn A. Fitch, Shibani Bhattacharya, Christopher J. Sarkisian, Juliette T.J. Lecomte, Bertrand García-Moreno E.  Biophysical Journal  Volume 79, Issue 3, Pages 1637-1654 (September 2000) DOI: 10.1016/S0006-3495(00)76414-9 Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 1 Fractional charge (Z) of selected histidines in Pc-Mb versus pH* (pH uncorrected for isotope effects) at NaCl concentrations of 0.02M (○), 0.20M (□), 0.50M (♢), and 1.50M (△). Data are shown for (A) His36 (class 2 residue; see classification in text); (B) His48 (class 1); (C) His81 (class 1); and (D) His113 (class 3). The raw experimental data were fitted simultaneously to Henderson-Hasselbalch equations 1 (histidine ionization) and 2 (hemic acid dissociation). Histidine pKa values (listed in Table 1), Hill coefficients, and limiting chemical shifts obtained as fitted parameters for Eq. 1 were used to calculate the Z curves shown in the figure. Deviations from the Z curves at high pH are attributed to the formation of methydroxy Mb in the hemic acid dissociation represented by Eq. 2 and are particularly pronounced for His48 and His113 (Cocco et al., 1992). Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 2 Salt dependence of pKa values of histidines in Pc-Mb measured by 1H-NMR spectroscopy. The data are plotted over two panels for clarity. The artificial data point at the top of each panel indicates the size of the experimental error. (A) His36 (■), His116 (●), His12 (▴), His119 (○), and His48 (♢). A straight line has been fitted to the points only to guide the eye. The dashed line (– – –) represents the fit to the data for the histidine in GHG. (B) N-terminus amino group. (×), His81 (△) and His113 (□). A straight line has been fitted to the points only to guide the eye. Lines are also included for the histidines in pEHG-NH2 (– – ·· – –), and FKHLK (·····) peptides for comparison. (■), effects of salts on the pKa of a blocked histidine, calculated relative to a pKa of 7.0 at 0.02M NaCl from solvation energies obtained with FDPB. Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 3 Salt dependence of pKa values of histidines in Eq-Mb measured by 1H-NMR spectroscopy. The data are plotted over two panels for clarity. The artificial data point at the top of each panel indicates the size of the experimental error. (A) His36 (■), His116 (●), His119 (○), and His48 (♢). A straight line has been fitted to the points only to guide the eye. The dashed line (– – –) represents the fit to the data for the histidine in GHG. (B) His81 (△) and His113 (□). A straight line has been fitted to the points only to guide the eye. Lines are also included for the histidines in pEHG-NH2 (— ·· —) and FKHLK (·····) peptides for comparison. (■), effects of salts on the pKa of a blocked histidine, calculated relative to a pKa of 7.0 at 0.02M NaCl from solvation energies obtained with FDPB. Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 4 pKa values of histidine residues in model peptides measured by 1H-NMR spectroscopy. Lines represent fits to the data for GHG (♢), pEHG-NH2 (△), cyclo-GH (○), FKHLK (▴), the major component of KSHPE (■, KSHPEt), and the minor component of KSHPE (□, KSHPEc). Dotted lines join the pKa values calculated, relative to a pKa of 6.4 at 0M NaCl, with Eq. 6 (●) and from solvation energies of a blocked histidine calculated with FDPB (■). Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 5 Proton titration curve of Pc-Mb measured potentiometrically in 0.1M KCl (●) and in 1.0M KCl (○). Lines through the experimental data points represent fits with fifth-order polynomials. The solid line in the bottom section of the figure represents the difference between the two proton binding curves. The dotted line represents the difference in the proton titration behavior in 0.2M and 1.5M NaCl, calculated with the pKa values of histidines and the amino terminus listed in Table 1. Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 6 Comparison between calculated and observed salt dependence of pKa values of histidines in Pc-Mb: His12 (▴), His24 (♦), His48 (♢), His81 (△), His116 (●), and His119 (○). The lines joining the experimental data points (- - - -) and the calculated values (——) were drawn to guide the eye. (A) pKa values calculated with the SATK algorithm. (B) pKa values calculated with FDPB, using Dprot=20. The calculated pKa curve of each histidine in A and B has been individually shifted along the ordinate to force passage through the experimental pKa at 0.02M NaCl. This concentration is marked by a vertical dotted line. Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 7 Comparison between calculated and observed salt dependence of the pKa of His36 in Pc-Mb. (●), the experimental data. Solid lines join the pKa values calculated by SATK (△), and by FDPB, using Dprot=20 (□) or Dprot=4 (▿). A thin dashed line describes the salt dependence of the interaction energy between an ion pair in solution separated by 2.5Å, as predicted with Eq. 7, using DH2O=50. All calculated pKa values have been shifted along the ordinate to force coincidence with the experimental data point at 0.02M NaCl as in Fig. 6. Compared to Fig. 6, the vertical scale has been changed. A measure of the experimental error is indicated by an artificial data point at the bottom of the panel. Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 8 (A) Comparison between calculated and observed pKa values of His113 in Pc-Mb. A thin solid line represents the linear fit to the experimental values (●). ▴, The pKa values of His in the FKHLK peptide, shifted along the ordinate to force coincidence with the pKa of His113 at 0.02M NaCl. Solid lines join the pKa values calculated with SATK (△) and with FDPB with Dprot=20 (□). pKa values calculated with FDPB have been shifted to force coincidence with the pKa of His113 at 0.02M NaCl. ▿, pKa values calculated with a modification of the SATK algorithms that includes the effects of a chloride ion bound between His113 and Arg31. This set of pKa values has been shifted to force coincidence with the experimental values at 0.02M NaCl. (B) Occupancy of the chloride binding site at pH 7 (——) and pH 4 (·····), calculated with values for Kint in Eq. 4 of 1.0 (●), 0.5 (○), and 0.01 (■). Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 9 Change in calculated pKa resulting from increasing salt concentration. (A) His12. (B) His36. (C) His113. (D) His116. The total effect obtained by the FDPB method using Dprot=20 (▴) is decomposed into Coulombic (○), Born (□), and background (△) energy terms. Note that the vertical scale in each of the panels spans a different range of values. The total salt-induced ΔpKa estimated from solvation energies of a blocked histidine calculated by FDPB is shown in each panel (■) for scaling purposes. In all curves, the value at 1μM salt concentration was subtracted from each data point to achieve a ΔpKa at 0 in the limit of low salt concentration. Biophysical Journal 2000 79, 1637-1654DOI: (10.1016/S0006-3495(00)76414-9) Copyright © 2000 The Biophysical Society Terms and Conditions