Docking site dynamics of ba3-cytochrome c oxidase from Thermus thermophilus
Docking site dynamics of ba3-cytochrome c oxidase from Thermus thermophilus
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DOI：
10.1074/jbc.m307117200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Varotsis, C
中科院分区：
文献类型：
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作者：
Koutsoupakis, C;Soulimane, T;Varotsis, C
配体的轨道被困在一个对接网站或在一个内部的空腔附近的蛋白质的活性位点是重要的问题，对阐明这种复杂的系统，其中活动需要穿梭的定向配体的反应机制和从其活性位点。本文用时间分辨步进扫描傅里叶变换红外差谱研究了室温下血红素a（3）光解CO在嗜热栖热菌ba（3）-细胞色素c氧化酶中的配体运动。在光解离时，15 - 20%的CO没有共价连接到Cu-B，而是被捕获在血红素a（3）丙酸酯的环A附近的对接位点内。观测到CO的两条轨道，分别为nu（CO）= 2131 cm（-1），t（d）= 10 - 35 μ s和nu（CO）= 2146 cm（-1），t（d）= 85 μ s。在稍后的时间（t（d）= 110亩）的对接网站重组的CO和迅速建立一个充满活力的障碍，有利于平衡的配体与蛋白质溶剂。我们观察到的CO轨迹的时间依赖性移位归因于配体周围的对接位点的构象运动。这些结果的影响，相对于对接位点的能力，以约束配体的取向和对接位点的反应动力学在本文中进行了讨论。
Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba(3)-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved step-scan Fourier transform infrared difference spectra of photodissociated CO from heme a(3) at ambient temperature. Upon photodissociation, 15 - 20% of the CO is not covalently attached to Cu-B but is trapped within a docking site near the ring A of heme a(3) propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (nu(CO) = 2131 cm(-1), t(d) = 10 - 35 mus and nu(CO) = 2146 cm(-1), t(d) = 85 mus) are observed. At later times (t(d) = 110 mus) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herein.