Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate:: A mechanism for the opening of axial imidazole rings
Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate:: A mechanism for the opening of axial imidazole rings
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DOI：
10.1093/jb/mvj189
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发表时间：
2006-10-01
影响因子：
2.7
通讯作者：
Tsubaki, Motonari
中科院分区：
文献类型：
--
作者：
Nakanishi, Nobuyuki;Takeuchi, Fusako;Tsubaki, Motonari
我们使用细胞色素B的可溶性结构域研究了血红素配位咪唑与焦碳酸二乙酯的反应性（5）。利用包括MALDI-TOF-MS在内的各种光谱方法的分析表明，细胞色素B（5）的两个轴向His残基（His 44和His 68）受到几种因素的保护，即，轴向咪唑对溶剂的有限空间暴露、Fe-N-NH 2配位键以及通过与其紧邻环境形成氢键而使N δ（1）位置质子化。然而，一旦在较高浓度的焦碳酸二乙酯的情况下，轴向His残基的N-epsilon 2位置发生N-乙氧羰基化，蛋白质部分中的血红素辅基的置换就会继续。同时，它促进了第二个N-乙氧羰基化发生在同一咪唑环的N-C1 - 1位，导致双-N-乙氧羰基化衍生物，并进一步导致开环衍生物。一个类似的机制似乎在一个非轴向的His残基（His 85），其中的N-δ 1原子作为一个强氢键的氢受体和其他N-δ 2原子是质子化的形式，导致开环衍生物的形成后，用较高浓度的焦碳酸二乙酯处理。这些结果表明，使用焦碳酸二乙酯的MALDI-TOF-MS分析可能提供一个独特的方法来表征的质子化状态的His残基和它们的氢键在酶的活性位点的强度。
We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N-epsilon 2 coordination bond, and protonation of the N delta(1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N-epsilon 2 position of the axial His residues occurred with a higher concentration of diethylpyrocarbonate, displacement of heme prosthetic group from the protein moiety continued. Simultaneously, it facilitated the second N-carbethoxylation to take place at the N-epsilon 1 position of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. A similar mechanism seemed in operation for one non-axial His residue (His85), in which the N-delta 1 atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N-epsilon 2 atom is in a protonated form, resulting in a formation of the ring-opened derivative upon treatment with a higher concentration of diethylpyrocarbonate. These results suggested that the use of diethylpyrocarbonate for MALDI-TOF-MS analysis might provide a unique method to characterize the protonation state of His residues and the strength of their hydrogen-bondings at the active site of enzymes.