Cyanide binding to cytochrome c peroxidase (H52L)
Cyanide binding to cytochrome c peroxidase (H52L)
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DOI：
10.1021/bi034632k
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发表时间：
2003-09-16
期刊：
影响因子：
2.9
通讯作者：
Erman, JE
中科院分区：
文献类型：
--
作者：
Bidwai, A;Witt, M;Erman, JE
氰化物结合到细胞色素c过氧化物酶（CcP）的变体，其中远端组氨酸已被替换为亮氨酸残基，CcP（H52 L），已被研究作为pH值的函数，使用光谱，平衡和动力学方法。在pH 4和8之间，CcP（H52 L）/氰化物复合物的表观平衡解离常数变化60倍，从pH 4.7的135 μ M到pH 8.0的2.2 μ M。结合动力学是双相的，涉及两种反应物的双分子缔合，随后是酶/氰化物复合物的异构化。缔合速率常数可以确定到pH 8.9使用pH跳跃技术。在研究的pH范围内，缔合速率常数增加了近4个数量级，从pH 4时的1.8 x 10（2）M-1 s（-1）增加到pH 8.6时的9.2 x 10（5）M-1 s（-1）。与野生型CcP相比，其中HCN的结合是主要的结合途径，CcP（H52 L）优先结合氰化物阴离子。在pH 8以上，氰化物与CcP（H52 L）的结合比氰化物与野生型CcP的结合更快。氰化物从突变蛋白质中解离慢4倍，尽管CcP（H52 L）和CcP的解离速率常数的pH依赖性基本相同。在pH 4和8之间观察到CcP（H52 L）/氰化物络合物的异构化，并使络合物稳定。的异构化速率常数具有类似的幅度和pH值的依赖性作为氰化物的解离速率常数，和这两个反应耦合在低氰化物浓度。这种异构化在野生型CcP/氰化物复合物中没有对应物。
Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods. Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 muM at pH 4.7 to 2.2 muM at pH 8.0. The binding kinetics are biphasic, involving bimolecular association of the two reactants, followed by an isomerization of the enzyme/cyanide complex. The association rate constant could be determined up to pH 8.9 using pH-jump techniques. The association rate constant increases by almost 4 orders of magnitude over the pH range investigated, from 1.8 x 10(2) M-1 s(-1) at pH 4 to 9.2 x 10(5) M-1 s(-1) at pH 8.6. In contrast to wild-type CcP, where the binding of HCN is the dominant binding pathway, CcP(H52L) preferentially binds the cyanide anion. Above pH 8, cyanide binding to CcP(H52L) is faster than cyanide binding to wild-type CcP. Cyanide dissociates 4 times slower from the mutant protein although the pH dependence of the dissociation rate constant is essentially identical for CcP(H52L) and CcP. Isomerization of the CcP(H52L)/cyanide complex is observed between pH 4 and 8 and stabilizes the complex. The isomerization rate constant has a similar magnitude and pH dependence as the cyanide dissociation rate constant, and the two reactions are coupled at low cyanide concentrations. This isomerization has no counterpart in the wild-type CcP/cyanide complex.