LIGAND-BINDING TO HEME-PROTEINS - CONNECTION BETWEEN DYNAMICS AND FUNCTION
LIGAND-BINDING TO HEME-PROTEINS - CONNECTION BETWEEN DYNAMICS AND FUNCTION
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DOI：
10.1021/bi00230a026
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发表时间：
1991-04-23
期刊：
影响因子：
2.9
通讯作者：
YOUNG, RD
中科院分区：
文献类型：
--
作者：
STEINBACH, PJ;ANSARI, A;YOUNG, RD
用闪光光解的方法研究了大范围的时间(100 ns-1K)和温度(10-320K)范围内的配基与血红素蛋白的结合。在约200K以下的75%甘油/水溶剂中，配基重新结合发生在血红素口袋中，并且在时间上是非指数的。动力学用高度为H的势垒在口袋和束缚态之间的分布g(H)来解释。在170K以上，再结合速度明显减慢。以前，我们将减速解释为配体在重新结合之前进入蛋白质基质所导致的“基质过程”。对带III的实验迫使我们重新解释数据。带III是一个在760 nm(几乎等于13000厘米-1)附近的非均匀加宽的电荷转移带，处于(一氧化碳)肌红蛋白的光解状态(Mb*)。在Mb*中对带III的动态烧孔测量建立了带III的均匀分量的位置与势垒H之间的关系，因为带III在Mb*中比Mb*红移了116 cm-1，这一关系意味着驰豫Mb中的势垒比Mb*中的高12kJ/mol。因此，在170K以上，再结合动力学的减慢是由Agmon和Hopfield[(1983)J.Chem]提出的松弛Mb*--&Gt；Mb引起的。太棒了。79、2042-2053]。这一结论得到了160~290K之间重新结合数据的拟合，表明整个分布g(H)发生了移动。在约200K以上，构象亚态之间的平衡涨落为配体打开了通过蛋白质基质的通道，同时也缩小了速率分布。蛋白质的驰豫和波动在时间上是非指数的，在温度上是非阿累尼乌斯的，这表明这些蛋白质运动的集体性质。松弛Mb*--&Gt；Mb基本上与溶剂粘度无关，这意味着这一运动涉及蛋白质的内部部分。然而，导致通路开放的蛋白质波动强烈依赖于溶剂粘度，这表明蛋白质的很大一部分参与其中。虽然详细的研究涉及MbCO，但关于MbO2和CO与人血红蛋白和苏黎世血红蛋白的β-链结合的数据也类似。结果表明，蛋白质的动力学对蛋白质的功能是必不可少的，在生理温度下，所有这些血红素蛋白质中从溶剂结合的结合系数都受血红素上的势垒控制。
Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (almost-equal-to 13 000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb* --> Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb* --> Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta-chains of human hemoglobin and hemoglobin Zurich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.