Effect of Heme Modification on Oxygen Affinity of Myoglobin and Equilibrium of the Acid-Alkaline Transition in Metmyoglobin
Effect of Heme Modification on Oxygen Affinity of Myoglobin and Equilibrium of the Acid-Alkaline Transition in Metmyoglobin
复制标题
DOI：
10.1021/ja909891q
复制
发表时间：
2010-05-05
影响因子：
15
通讯作者：
Yamamoto, Yasuhiko
中科院分区：
文献类型：
--
作者：
Shibata, Tomokazu;Nagao, Satoshi;Yamamoto, Yasuhiko
肌红蛋白（Mb）的功能调节被认为是通过附近的氨基酸残基提供的血红素环境，以及内在血红素Fe反应性的微妙调节来实现的。我们已经进行了强吸电子的全氟甲基（CF 3）基团（S）作为血红素侧链（S）的Mb的取代，以获得血红素的电子结构的大的改变，以阐明Mb的O-2亲和力和血红素外周侧链的电子性质之间的关系。我们利用高铁肌红蛋白中“酸碱转换”的平衡常数（pK（a））来定量评估CF 3取代对蛋白质血红素Fe原子（rho（Fe））电子密度的影响。发现蛋白质的pK（a）值在引入一个CF 3基团后降低了约1个pH单位，并且通过对一些模型化合物的密度泛函理论计算证实了pK（a）值随着rho（Fe）值的降低而降低。发现Mb的O-2亲和力与pK（a）值有很好的相关性，即P-50值（达到50%氧合所需的O-2分压）随1 pK（a）单位的降低而增加2.7倍。对蛋白质的动力学研究表明，引入吸电子CF 3基团后O-2亲和力降低是由于O-2解离速率增加。由于CF 3基团取代的引入被认为防止了进一步的Fe 2 +-O-2键极化，并因此防止了蛋白质的氧形式的推定的Fe 3 +-O-2（-）-样物质的形成[麦克斯韦，J.C.; Volpe，J. A.;巴洛角H.的;考伊，W. S.生物化学生物物理学Res. Commun. 1974，58，166-171]，预期通过取代吸电子基团作为血红素侧链来增强0 -2解离。我们还发现，在形成鲜明对比的情况下的O-2结合的蛋白质，CO的关联和解离速率基本上是独立的rho（Fe）值。因此，吸电子基团的引入增强了CO与蛋白质的优先结合而不是O-2。这些发现不仅解决了长期存在的内在血红素Fe反应性的微妙调整机制的问题，而且还提供了新的见解的结构功能关系的蛋白质。
Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF3) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O-2 affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF3 substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by similar to 1 pH unit upon the introduction of one CF3 group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O-2 affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P-50 value, which is the partial pressure of O-2 required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O-2 affinity upon the introduction of an electron-withdrawing CF3 group is due to an increase in the O-2 dissociation rate. Since the introduction of a CF3 group substitution is thought to prevent further Fe2+-O-2 bond polarization and hence formation of a putative Fe3+-O-2(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O-2 dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O-2 binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O-2. These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure function relationship of the protein.