Mechanism of hydrogen cyanide binding to myoglobin
Mechanism of hydrogen cyanide binding to myoglobin
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DOI：
10.1021/bi9600299
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发表时间：
1996-06-04
期刊：
影响因子：
2.9
通讯作者：
IkedaSaito, M
中科院分区：
文献类型：
--
作者：
Dou, Y;Olson, JS;IkedaSaito, M
氰化物与肌红蛋白的结合速度比其他铁和亚铁配体慢得多，表明远端口袋内的键形成和破坏限制了速率。这一解释得到了两个关键实验观察的支持。首先，His64（E7）突变为Gly和Ala，这打开了从溶剂到铁原子的直接通道，Phe46（CD4）突变为Leu、Ile和瓦尔，这增加了远端组氨酸的迁移率，对氰化物结合的缔合速率常数几乎没有影响。相反，这些突变导致叠氮化物结合的速率常数增加100 - 1000倍，令人信服地表明这种配体的结合受到其移动到蛋白质中的速率的限制。第二，氰化物解离的速率常数不受改变系列Gly、瓦尔、Leu、Ile、Phe中64位（E7）残基大小的影响，而从Gly 64到Phe 64高铁肌红蛋白的叠氮化物解离速率降低2000倍。氰化物亲和力的主要决定因素是高铁肌红蛋白中三价铁原子的水置换容易程度、蛋白质内部HCN的酸解离常数（K *（a））以及第六配位位置处的空间位阻和静电相互作用。与远端组氨酸的直接氢键似乎在稳定结合的氰化物方面没有发挥重要作用，相反，远端口袋的一般极性及其对K *（a）的影响是生理条件下调节氰化物亲和力的关键因素。
Cyanide binding to myoglobin is much slower than that of other ferric and ferrous ligands, suggesting rate limitation by bond formation and disruption within the distal pocket. This interpretation is supported by two key experimental observations. First, His64(E7) to Gly and Ala mutations, which open a direct channel from the solvent to the iron atom, and Phe46(CD4) to Leu, Ile, and Val mutations, which increase the mobility of the distal histidine, have little effect on the association rate constant for cyanide binding. In contrast, these mutations cause 100-1000-fold increases in the rate constant for azide binding, showing convincingly that the binding of this ligand is limited by the rate of its movement into the protein. Second, the rate constant for cyanide dissociation is unaffected by changing the size of the residue at position 64(E7) in the series Gly, Val, Leu, Ile, Phe, whereas there is a 2000-fold decrease in the rate of azide dissociation in going from Gly64 to Phe64 metmyoglobin. The major determinants of the cyanide affinity are the ease of water displacement from the ferric iron atom in metmyoglobin, the acid dissociation constant of HCN inside the protein (K*(a)), and steric hindrance and electrostatic interactions at the sixth coordination position. Direct hydrogen bonding to the distal histidine does not appear to play an important role in stabilizing bound cyanide, Instead, the general polarity of the distal pocket and its effect on K*(a) are the key factors regulating cyanide affinity under physiological conditions.