Ansari A, Berendzen J, Braunstein D, Cowen B R, Frauenfelder H, Hong M K, Iben I E, Johnson J B, Ormos P, Sauke T B
Biophys Chem. 1987 May 9;26(2-3):337-55. doi: 10.1016/0301-4622(87)80034-0.
The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.
在10 - 300K的温度范围内，于多种溶剂中研究了羧基肌红蛋白（MbCO）的红外伸缩带以及光解离后CO与Mb的重新结合。四条伸缩带表明MbCO可存在于四个亚态，即A0 - A3。四条带强度的温度依赖性得出了相对结合焓和熵。这些带的积分吸光度和pH依赖性使得能够将这些亚态与X射线数据（Kuriyan等人，《分子生物学杂志》192 (1986) 133）中观察到的构象进行识别。在低pH值下，A0与His E7形成氢键。在75%甘油/水溶剂中，亚态A0 - A3在约180K以上相互转化，在缓冲水中则在270K以上相互转化。如果MbCO嵌入固体聚乙烯醇基质中，在任何温度下都未观察到主要的相互转化。这种转变对溶剂特性的依赖性被解释为从属玻璃转变。在低温下光解离后，CO位于血红素口袋B中。所得到的被鉴定为B亚态的CO伸缩带相对于A亚态的伸缩带发生蓝移。在40K时，研究了闪光光解后在Soret、近红外以及A和B亚态积分情况下的重新结合。所有数据都位于同一条重新结合曲线上，表明从至少100 ns到100 ks的时间内重新结合在时间上是非指数性的。未发现离散指数的证据。在红外区域进行监测的闪光光解显示在口袋B内有四条不同的途径通向结合亚态Ai。四条途径中每一条B→A的重新结合在时间上至少到10 ks都是非指数性的，并且在180K以下这四条途径具有不同的动力学。从重新结合的时间和温度依赖性中提取了活化焓分布g(HBA)和指前因子ABA。在约180K的转变温度以下，未观察到从一个A亚态泵浦到另一个A亚态，或从一个B亚态泵浦到另一个B亚态的情况。如果MbCO在被激光闪光完全光解之前暴露于强白光10 - 10³秒，长寿命态的振幅会增加。这种效应是根据亚态层次和亚态对称性破缺来解释的。血红素口袋中CO伸缩带和重新结合过程的特性强烈依赖于溶剂、pH和压力等外部参数。这种敏感性暗示了蛋白质反应可能的控制机制。
