A signature of the T ---> R transition in human hemoglobin

Abstract

Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are His alpha 103(G10) and His alpha 122(H5) in each alpha subunit of hemoglobin. They are located at the alpha(1)beta(1) and alpha(2)beta(2) interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the beta chains. We have measured the solvent exchange rates of side chain protons of His alpha 103(G10) and His alpha 122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by approximately 1.5 kcal/(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric alpha(1)beta(1) and alpha(2)beta(2) interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.

Figure 1

Exchangeable proton NMR resonances of Hb A in deoxy form (Lower) and CO form (Upper) in 0.1 M Bis-Tris buffer containing 0.18 M chloride ions at pH 7.1 and at 37°C.

Figure 2

Dependence of the exchange rate of Hisα103(G10) N_ɛ2H proton on pH (A), and on the concentration of H⁺ ions in the pH range from 5.6 to 7.6 (B). (♦, ⋄) deoxy Hb; (●, ○) HbCO; (▾, ▿) oxy Hb; (□) CN-met Hb; (▵) azido-met Hb. Filled symbols: 0.1 M Bis-Tris with 0.18 M chloride ions. Open symbols: 0.1 M Tris with 0.18 M chloride ions. The solid lines correspond to nonlinear least-squares fits to Eq. 3 with the following fitted parameters: k_op = (105 ± 16) s⁻¹, k_OH/k_H = (6.0 ± 1.1) 10⁻³, k_w/k_H = (1.2 ± 0.4) 10⁻⁷ M and (k_op + k_cl)/k_H = (6.2 ± 2.6) 10⁻⁷ M for deoxy Hb A, and k_op = (76 ± 5) s⁻¹, k_OH/k_H = 0, k_w/k_H = (9.4 ± 1.4) 10⁻⁸ M and (k_op + k_cl)/k_H = (1.6 ± 0.2) 10⁻⁶ M for ligated Hb A.

Figure 3

Dependence of the exchange rate of Hisα122(H5) N_ɛ2H proton on pH (A) and on concentration of OH⁻ ions (B). (♦, ⋄) deoxy Hb; (●, ○) HbCO; (▾, ▿) oxy Hb; (□) CN-met Hb; (▵) azido-met Hb. Filled symbols: 0.1 M Bis-Tris with 0.18 M chloride ions. Open symbols: 0.1 M Tris with 0.18 M chloride ions. Solid lines correspond to nonlinear least-squares fits to Eq. 3 with k_H = 0 and the following fitted parameters: k_op = (63 ± 2) s⁻¹, k_w/k_OH = (5.7 ± 0.8) 10⁻⁷ M and (k_op + k_cl)/k_OH = (7.2 ± 0.8) 10⁻⁶ M for deoxy Hb A, and k_op = (19 ± 3) s⁻¹, k_w/k_OH = (1.6 ± 0.6) 10⁻⁶ M and (k_op+ k_cl)/k_OH = (6.6 ± 4.5) 10⁻⁶ M for ligated Hb A.