Bonaventura C, Cashon R, Bonaventura J, Perutz M, Fermi G, Shih D T
Marine Biomedical Center, Duke University Marine Laboratory, Beaufort, North Carolina 28516.
J Biol Chem. 1991 Dec 5;266(34):23033-40.
Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.
血红蛋白（Hb）奇科（β链第66位赖氨酸（Lysβ66）在E10位点突变为苏氨酸（Thr））具有降低的氧亲和力（施，D.T.-b.，琼斯，R.T.，施，M.F.-C.，琼斯，M.B.，科勒，R.D.，和霍华德，J.（1987年）《血红蛋白》11，453 - 464）。我们的研究表明，其P50约为Hb A的两倍，并且在pH 7至8之间其协同性、阴离子效应和波尔效应是正常的。pH 8以上的波尔效应有所降低，表明由Lysβ66形成的离子键在碱性波尔效应中有微小但此前未被记录的作用。由于α-血红素的氧亲和力可能正常，四聚体中β-血红素的氧亲和力可能在结合能上相当于每个β-血红素降低了1.2千卡/摩尔。值得注意的是，在所有检测条件下，与Hb A相比，Hb奇科与氧结合的初始和最终阶段的亲和力均降低。分离出的β链也显示出氧亲和力降低。在T态Hb A中，Lys（E10β）与其中一个血红素丙酸酯形成盐桥，但与其他血红蛋白变体的比较表明，该桥的断裂并非氧亲和力低的原因。现在对脱氧结构的X射线分析表明，Thrβ66要么通过一个桥连水分子向Hisβ63提供氢键，要么从Hisβ63接受氢键。这给配体与T态的结合引入了额外的空间位阻，导致配体结合速率减慢。我们测量了O2/CO分配系数以及氧解离和一氧化碳结合的动力学，发现Hb奇科的R态四聚体和分离出的β链也表现出O2和CO亲和力降低。
