Knapp James E, Bonham Michele A, Gibson Quentin H, Nichols Jeffry C, Royer William E
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Biochemistry. 2005 Nov 8;44(44):14419-30. doi: 10.1021/bi051052+.
Residue F4 (Phe 97) undergoes the most dramatic ligand-linked transition in Scapharca dimeric hemoglobin, with its packing in the heme pocket in the unliganded (T) state suggested to be a primary determinant of its low affinity. Mutation of Phe 97 to Leu (previously reported), Val, and Tyr increases oxygen affinity from 8- to 100-fold over that of the wild type. The crystal structures of F97L and F97V show side chain packing in the heme pocket for both R and T state structures. In contrast, in the highest-affinity mutation, F97Y, the tyrosine side chain remains in the interface (high-affinity conformation) even in the unliganded state. Comparison of these mutations reveals a correlation between side chain packing in the heme pocket and oxygen affinity, indicating that greater mass in the heme pocket lowers oxygen affinity due to impaired movement of the heme iron into the heme plane. The results indicate that a key hydrogen bond, previously hypothesized to have a central role in regulation of oxygen affinity, plays at most only a small role in dictating ligand affinity. Equivalent mutations in sperm whale myoglobin alter ligand affinity by only 5-fold. The dramatically different responses to mutations at the F4 position result from subtle, but functionally critical, stereochemical differences. In myoglobin, an eclipsed orientation of the proximal His relative to the A and C pyrrole nitrogen atoms provides a significant barrier for high-affinity ligand binding. In contrast, the staggered orientation of the proximal histidine found in liganded HbI renders its ligand affinity much more susceptible to packing contacts between F4 and the heme group. These results highlight very different strategies used by cooperative hemoglobins in molluscs and mammals to control ligand affinity by modulation of the stereochemistry on the proximal side of the heme.
在紫贻贝二聚体血红蛋白中，残基F4（苯丙氨酸97）经历了最显著的配体连接转变，其在未结合配体（T）状态下位于血红素口袋中的堆积被认为是其低亲和力的主要决定因素。将苯丙氨酸97突变为亮氨酸（先前已报道）、缬氨酸和酪氨酸后，氧亲和力比野生型提高了8至100倍。F97L和F97V的晶体结构显示，R态和T态结构在血红素口袋中均有侧链堆积。相比之下，在亲和力最高的突变体F97Y中，即使在未结合配体的状态下，酪氨酸侧链仍保留在界面处（高亲和力构象）。对这些突变的比较揭示了血红素口袋中的侧链堆积与氧亲和力之间的相关性，表明由于血红素铁向血红素平面的移动受损，血红素口袋中更大的质量会降低氧亲和力。结果表明，先前假设在氧亲和力调节中起核心作用的一个关键氢键，在决定配体亲和力方面至多只起很小的作用。抹香鲸肌红蛋白中的等效突变仅使配体亲和力改变5倍。对F4位置突变的显著不同反应源于细微但功能关键的立体化学差异。在肌红蛋白中，近端组氨酸相对于A和C吡咯氮原子的重叠取向为高亲和力配体结合提供了显著障碍。相比之下，在结合配体的HbI中发现的近端组氨酸的交错取向使其配体亲和力更容易受到F4与血红素基团之间堆积接触的影响。这些结果突出了软体动物和哺乳动物中协同血红蛋白通过调节血红素近端侧的立体化学来控制配体亲和力所采用的非常不同的策略。
