Koutsoupakis Constantinos, Soulimane Tewfik, Varotsis Constantinos
Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece.
J Biol Chem. 2003 Sep 19;278(38):36806-9. doi: 10.1074/jbc.M307117200. Epub 2003 Jul 8.
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 ba3-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 a3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to CuB but is trapped within a docking site near the ring A of heme a3 propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (vCO = 2131 cm-1, td = 10-35 micros and vCO = 2146 cm-1, td = 85 micros) are observed. At later times (td = 110 micros) 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.
被困在蛋白质活性位点附近的对接位点或内部腔体内的配体轨迹，对于阐明此类复杂系统的反应机制至关重要，在这些系统中，活性需要定向配体在其活性位点之间穿梭。通过测量嗜热栖热菌ba3-细胞色素c氧化酶在室温下光解离的一氧化碳的时间分辨步进扫描傅里叶变换红外差谱，研究了配体在其中的运动。光解离后，15%-20%的一氧化碳并非共价连接到CuB上，而是被困在血红素a3丙酸酯A环附近的对接位点内。观察到两条在光谱和动力学上有区别的一氧化碳轨迹（vCO = 2131 cm-1，td = 10-35微秒；vCO = 2146 cm-1，td = 85微秒）。在稍后的时间（td = 110微秒），对接位点围绕一氧化碳重新组织，并迅速建立一个能量屏障，促进配体与蛋白质溶剂的平衡。我们观察到的一氧化碳轨迹随时间的变化归因于围绕配体的对接位点的构象运动。本文讨论了这些结果对于对接位点限制配体取向能力和对接位点反应动力学的意义。
