Steinbach P J, Ansari A, Berendzen J, Braunstein D, Chu K, Cowen B R, Ehrenstein D, Frauenfelder H, Johnson J B, Lamb D C
Department of Physics, University of Illinois, Urbana-Champaign 61801.
Biochemistry. 1991 Apr 23;30(16):3988-4001. doi: 10.1021/bi00230a026.
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 (approximately 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 Zürich. 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.
通过在很宽的时间范围（100纳秒至1千秒）和温度范围（10至320开尔文）内使用闪光光解来研究配体与血红素蛋白的结合。在75%甘油/水溶剂中低于约200开尔文时，配体从血红素口袋重新结合，且时间上是非指数性的。动力学由口袋与结合态之间高度为H的焓垒分布g(H)来解释。高于170开尔文时重新结合明显减慢。此前我们将这种减慢解释为配体在重新结合前进入蛋白质基质导致的“基质过程”。对（碳氧合）肌红蛋白（MbCO）光解态（Mb*）中760纳米（约13,000厘米⁻¹）附近不均匀展宽的电荷转移带III进行的实验，迫使我们重新解释这些数据。对Mb中带III的动力学空穴烧蚀测量建立了带III均匀成分位置与势垒H之间的关系。由于与Mb相比，Mb中带III红移了116厘米⁻¹，该关系意味着弛豫态Mb中的势垒比Mb中的高12千焦/摩尔。因此，高于170开尔文时重新结合动力学的减慢是由弛豫Mb→Mb引起的，正如阿蒙和霍普菲尔德所提出的[(1983) J. Chem. Phys. 79, 2042 - 2053]。这一结论得到了对160至290开尔文之间重新结合数据的拟合支持，该拟合表明整个分布g(H)发生了移动。高于约200开尔文时，构象亚态之间的平衡涨落为配体打开了穿过蛋白质基质的通道，也使速率分布变窄。蛋白质的弛豫和涨落在时间上是非指数性的，在温度上是非阿累尼乌斯的，这表明这些蛋白质运动具有集体性质。弛豫Mb*→Mb基本上与溶剂粘度无关，这意味着这种运动涉及蛋白质的内部部分。然而，负责通道打开的蛋白质涨落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。虽然详细研究涉及MbCO，但对于MbO₂以及CO与人血红蛋白和苏黎世血红蛋白β链的结合也获得了类似的数据。结果表明蛋白质动力学对蛋白质功能至关重要，并且在生理温度下所有这些血红素蛋白中从溶剂中结合的缔合系数由血红素处的势垒决定。
