Department of Biochemistry and Cell Biology, W M Keck Center for Computational Biology, Rice University, Houston, Texas 77005, USA.
J Biol Chem. 2010 Mar 19;285(12):8840-54. doi: 10.1074/jbc.M109.053934. Epub 2010 Jan 15.
The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O(2), CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20-500-fold increases in the rates of O(2) dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O(2) in both the alpha and beta chains (DeltaG(His(E7)H-bond) approximately -8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k(O2)', k(NO)', and calculated bimolecular rates of CO entry (k(entry)') are observed. Replacing His(E7) with Trp causes further decreases in k(O2)', k(NO)', and k(entry)' to 1-2 microM(-1) s(-1) in beta subunits, whereas ligand rebinding to alphaTrp(E7) subunits after photolysis is markedly biphasic, with fast k(O2)', k(CO)', and k(NO)' values approximately 150 microM(-1) s(-1) and slow rate constants approximately 0.1 to 1 microM(-1) s(-1). Rapid bimolecular rebinding to an open alpha subunit conformation occurs immediately after photolysis of the alphaTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed alphaTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.
重新系统地检查了远端组氨酸在调节成人血红蛋白 (HbA) 与配体结合中的作用，方法是制备 Hb 亚基的 His(E7) 至 Gly、Ala、Leu、Gln、Phe 和 Trp 突变体。使用快速混合和激光光解实验测量 O(2)、CO 和 NO 结合的速率常数，这些实验的设计旨在最小化不稳定的非极性 E7 突变体的自动氧化。用 Gly、Ala、Leu 或 Phe 取代 His(E7)会导致 O(2)从任一 Hb 亚基解离的速率增加 20-500 倍，这明确表明天然 His(E7)咪唑侧链在α和β链中与结合的 O(2)形成强氢键（DeltaG(His(E7)H-bond) 约为 -8 kJ/mol）。随着 E7 氨基酸的大小从 Gly 增加到 Phe，观察到 k(O2) '、k(NO) '和计算的 CO 进入的双分子速率 (k(entry) ') 降低。用 Trp 取代 His(E7)会导致β亚基中的 k(O2) '、k(NO) '和 k(entry) '进一步降低至 1-2 microM(-1) s(-1)，而光解后 alphaTrp(E7)亚基的配体再结合明显呈双相，快速 k(O2) '、k(CO) '和 k(NO) '值约为 150 microM(-1) s(-1)，缓慢的速率常数约为 0.1 至 1 microM(-1) s(-1)。在高配体浓度下，光解 alphaTrp(E7)突变体后，立即发生快速的双分子再结合到开放的 alpha 亚基构象。然而，在平衡时，封闭的 alphaTrp(E7)侧链抑制配体结合的速率 >200 倍。这些数据强烈表明，E7 侧链在 HbA 亚基中作为配体进入的门。
