Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States.
Biochemistry. 2010 Dec 21;49(50):10606-15. doi: 10.1021/bi100927p. Epub 2010 Nov 29.
On the basis of X-ray crystal structures and electron paramagnetic resonance (EPR) measurements, it has been inferred that the O(2) binding to hemoglobin is stabilized by the hydrogen bonds between the oxygen ligands and the distal histidines. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO(2) A) in solution. Here, the NMR spectra of uniformly (15)N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH and temperature and also in the presence of an organic phosphate, inositol hexaphosphate (IHP). We have found significant effects of pH and temperature on the strength of the H-bond markers, i.e., the cross-peaks for the side chains of the two distal histidyl residues, α58His and β63His, which form H-bonds with the O(2) ligands. At lower pH and/or higher temperature, the side chains of the distal histidines appear to be more mobile, and the exchange with water molecules in the distal heme pockets is faster. These changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the O(2) affinity of Hb as a function of pH and temperature and are clearly illustrated by our NMR experiments. Our NMR results have also confirmed that this H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. IHP has only a minor effect on these H-bond markers compared to the effects of pH and temperature. These H-bonds are sensitive to mutations in the distal heme pockets but not affected directly by the mutations in the quaternary interfaces, i.e., α(1)β(1) and/or α(1)β(2) subunit interface. These findings provide new insights regarding the roles of temperature, hydrogen ion, and organic phosphate in modulating the structure and function of hemoglobin in solution.
基于 X 射线晶体结构和电子顺磁共振（EPR）测量结果，人们推断血红蛋白与氧的结合通过氧配体与远端组氨酸之间的氢键得到稳定。我们之前通过多核核磁共振（NMR）光谱学的研究首次在溶液中提供了人类正常成人氧合血红蛋白（HbO2A）中存在这种氢键的直接证据。在这项研究中，我们在各种 pH 值和温度实验条件下以及在存在有机磷酸肌醇六磷酸（IHP）的情况下，研究了均（15）N 标记的重组人血红蛋白 A（rHbA）和 5 种突变 rHb 的 NMR 谱。我们发现 pH 值和温度对氢键标记物（即与氧配体形成氢键的两个远端组氨酸残基α58His 和β63His 的侧链的交叠峰）的强度有显著影响。在较低的 pH 值和/或较高的温度下，远端组氨酸的侧链似乎更具流动性，并且与远端血红素口袋中的水分子的交换更快。这些与 pH 值和温度有关的氢键稳定性的变化与血红蛋白的 O2 亲和力随 pH 值和温度的变化一致，我们的 NMR 实验清楚地说明了这一点。我们的 NMR 结果还证实，β链中的这种氢键比α链中的弱，并且对 pH 值和温度的变化更敏感。与 pH 值和温度的影响相比，IHP 对这些氢键标记物的影响较小。这些氢键对远端血红素口袋中的突变敏感，但不受四元界面（即α1β1和/或α1β2亚基界面）突变的直接影响。这些发现为温度、氢离子和有机磷酸在调节血红蛋白在溶液中的结构和功能方面的作用提供了新的见解。
