Stavrov Solomon S, Wright Wayne W, Vanderkooi Jane M, Fidy Judit, Kaposi Andras D
Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, P.O. Box 39040, 69978, Israel.
Biopolymers. 2002;67(4-5):255-8. doi: 10.1002/bip.10103.
The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.
在10至300 K的温度区间内，于不同溶剂中监测了有无底物类似物苯甲羟肟酸（BHA）时辣根过氧化物酶的光谱。Q(0,0)光吸收带的热展宽主要源于电子π→π跃迁与血红素振动的相互作用。相比之下，与血红素结合的CO的红外吸收带宽度由CO跃迁矩与蛋白质基质电场的耦合控制。甘油/H₂O溶剂中无底物酶的红外带宽在玻璃态基质中几乎不变，而在加热到玻璃化转变温度以上时会大幅增加。在海藻糖/H₂O玻璃中加热相同的酶会使该带显著展宽。底物的结合极大地减小了CO带的温度展宽。这一结果与BHA强烈降低血红素口袋环境振动幅度的观点一致。玻璃化转变温度以上CO带异常强烈的热展宽被解释为是由一个非常灵活的激发构象亚态的热占据引起的。海藻糖玻璃中同一带的热展宽是由每个构象亚态中蛋白质振动幅度的增加导致的，它们的占据情况与玻璃态基质中的温度无关。
