Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin
Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin
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DOI：
10.1074/jbc.m109.053934
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发表时间：
2010-03-19
影响因子：
4.8
通讯作者：
Olson, John S.
中科院分区：
文献类型：
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作者：
Birukou, Ivan;Schweers, Rachel L.;Olson, John S.
通过制备两种Hb亚基的His（E7）到Gly、Ala、Leu、Gln、Phe和Trp突变体，系统地重新检查远端组氨酸在调节配体与成人血红蛋白（HbA）结合中的作用。O-2，CO和NO结合的速率常数进行了测量，使用快速混合和激光光解实验设计，以尽量减少不稳定的非极性E7突变体的自氧化。用Gly、Ala、Leu或Phe替换His（E7）导致O-2从任一Hb亚基解离的速率增加20-500倍，明确地证明天然His（E7）咪唑侧链与α和β链中的结合O-2形成强氢键（Δ G（His（E7）H-键）接近-8 kJ/mol）。随着E7氨基酸的大小从Gly增加到Phe，观察到k（O2）2 ′、k（NO）2 ′和计算的CO进入的双分子速率（k（进入）2 ′）的降低。用Trp取代His（E7）导致β亚基中的k（O2）2 ′，k（NO）2 ′和k（entry）′进一步降低至1-2 μ M-1 s（-1），而在光解后配体与α Trp（E7）亚基的再结合是明显的双相的，快速的k（O2）2 ′，k（CO）2 ′，k（NO）′值约为150 μ M-1 s（-1），慢速率常数约为0.1 ~ 1 μ M-1 s（-1）。在高配体浓度下，α-色氨酸（E7）突变体光解后立即发生快速双分子重新结合到开放的α亚基构象。然而，在平衡时，闭合的α Trp（E7）侧链抑制配体结合速率> 200倍。这些数据强烈表明，E7侧链作为一个门的配体进入两个HbA亚基的功能。
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 (Delta G(His(E7)H-bond) approximate to -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 mu M-1 s(-1) in beta subunits, whereas ligand rebinding to alpha Trp(E7) subunits after photolysis is markedly biphasic, with fast k(O2)', k(CO)', and k(NO)' values approximate to 150 mu M-1 s(-1) and slow rate constants approximate to 0.1 to 1 mu M-1 s(-1). Rapid bimolecular rebinding to an open alpha subunit conformation occurs immediately after photolysis of the alpha Trp(E7) mutant at high ligand concentrations. However, at equilibrium the closed alpha Trp(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.