A signature of the T → R transition in human hemoglobin

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 Hisα103(G10) and Hisα122(H5) in each α subunit of hemoglobin. They are located at the α(1)β(1) and α(2)β(2) interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the β chains. We have measured the solvent exchange rates of side chain protons of Hisα103(G10) and Hisα122(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 ≈1.5 kcal/(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric α(1)β(1) and α(2)β(2) interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.

血红蛋白（hemoglobin）的变构效应（allosteric effects）源于该分子至少两种能量状态之间的平衡：紧张态（tense state，简称T态）与松弛态（relaxed state，简称R态）。两种状态的差异体现在血红蛋白亚基间相互作用的数量与能量上。在T态中，亚基间的约束会阻碍配体结合所引发的结构变化；而在R态中，此类约束被解除，从而提升了配体结合亲和力。本研究报道了血红蛋白中四个相对于T态在R态下结构更稳定的位点，分别为每个α亚基中的组氨酸（histidine）α103(G10)与组氨酸（histidine）α122(H5)。这些位点位于血红蛋白四聚体的α(1)β(1)与α(2)β(2)界面处，此处的组氨酸侧链可与β链的特定氨基酸残基形成氢键。本研究通过核磁共振波谱法（NMR spectroscopy）测定了脱氧与结合配体两种状态下，上述两个组氨酸位点侧链质子的溶剂交换速率。实验发现，脱氧-T态中的交换速率高于结合配体的-R态。基于局部解折叠模型对交换速率进行分析后可知，相较于T态，R态下这两个组氨酸位点的结构稳定自由能平均高出约1.5千卡/(摩尔四聚体)。这两个组氨酸位于二聚体内的α(1)β(1)与α(2)β(2)界面处，这一位置也提示此类界面在血红蛋白的变构平衡中发挥了一定作用。