Kinetic hole burning, hole filling, and conformational relaxation in heme proteins: Direct evidence for the functional significance of a hierarchy of dynamical processes
Kinetic hole burning, hole filling, and conformational relaxation in heme proteins: Direct evidence for the functional significance of a hierarchy of dynamical processes
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DOI：
10.1021/bi9700274
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发表时间：
1997-11-25
期刊：
影响因子：
2.9
通讯作者：
Friedman, JM
中科院分区：
文献类型：
--
作者：
Huang, J;Ridsdale, A;Friedman, JM
波段 III 是平衡和非平衡五配位亚铁高自旋血红素的无序和构象敏感的近红外（类似于 760 nm）电荷转移吸收带特征，六配位亚铁血红蛋白或肌红蛋白光解后该吸收带的时间演化可以提供有关构象弛豫的详细信息，包括由此产生的热驱动波动 在从非均质到均质配体重新结合动力学的转变中，由于低温下的样品恢复时间较长，因此在一定温度范围内进行这种时间分辨测量是困难的。一种新的光栅技术允许大型光学低温恒温器快速移动，与纳秒时间分辨近红外吸收光谱结合使用，为成人血红蛋白 (COHbA) 的一氧化碳衍生物以及在更有限的程度上马肌红蛋白 (COMb) 的光产物生成带 III 作为时间的函数。测量在较宽的温度范围内进行，从远低于溶剂（75% 甘油：水）玻璃化转变温度（接近 180 K）到环境温度。观察到三种与温度和/或粘度相关的现象。在最高温度下，75% 甘油样品仅观察到构象弛豫。在非常高的粘度（大于或等于 400 cp）下，构象松弛急剧减慢，并且观察到动力学空穴燃烧和“空穴”填充（动态空穴填充）。随着温度降低，构象松弛减慢并最终停止。在低至 140 K 的温度下观察到动力学烧孔和动态孔填充以及带 III 的额外展宽。动态烧孔 (KHB) 的观察表明样品在配体重新结合的时间尺度上不均匀，从而产生 kHB。空穴填充的开始是导致 KHB 的构象亚态初始不均匀分布的热均匀化的直接表现。观察到的动力学用于解释与高于类似于 180 K 的成对重新结合的非阿伦尼乌斯减慢相关的逆温度效应。逆温度效应似乎不仅源于构象弛豫的开始，而且还源于构象亚态的初始不均匀分布的热平均速率的增加。
Band III is a disorder and conformation-sensitive near-infrared (similar to 760 nm) charge transfer absorption band characteristic of equilibrium and nonequilibrium five coordinate ferrous high-spin hemes, The time evolution of this absorption band subsequent to photodissociation of six coordinate ferrous hemoglobin or myoglobin can provide detailed information regarding conformational relaxation, including the thermally driven fluctuations that result in the transition from inhomogeneous to homogeneous ligand rebinding kinetic, Such time-resolved measurements over a range of temperatures are difficult due to long sample recovery times at cryogenic temperatures. A new rastoring technique that allows for the rapid movement of a large optically accessible cryostat is used in combination with nanosecond time-resolved near-infrared absorption spectroscopy to generate band III as a function of time for the Photoproducts of the carbon monoxide derivative of adult human hemoglobin (COHbA) and, to a more limited extent, horse myoglobin (COMb). The measurements are made over a wide range of temperatures extending from well below the solvent (75% glycerol:water) glass transition at similar to 180 K to ambient temperatures. Three temperature-and/or viscosity-dependent phenomena are observed. At the highest temperatures, only conformational relaxation is observed for the 75% glycerol sample. At very high viscosity (greater than or equal to 400 cp), conformational relaxation slows dramatically, and both kinetic hole burning followed by the filling in of the ''hole'' (dynamic hole filling) are observed. As the temperature is lowered, conformational relaxation slows and finally ceases. Kinetic hole burning and dynamic hole filling as well as additional broadening of band III are observed down to 140 K. The observation of kinetic hole burning (KHB) is indicative of the sample being inhomogeneous on the time scale of the ligand rebinding giving rise to kHB. The onset of hole filling is a direct manifesation of the thermal homogenization of the initial inhomogeneous distribution of conformational substates responsible for KHB. The observed dynamics are used to explain the inverse temperature effect associated with the non-Arrhenius slow down of geminate rebinding above similar to 180 K. The inverse temperature effect appears to arise not only from the onset of conformational relaxation but also from the increase in the rate on thermal averaging of the initial inhomogeneous distribution of conformational substates.