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Structural and Functional Analysis of Hemoproteins Under High Pressure by Las Photolysis Measurements

通过激光光解测量对高压下血红素蛋白进行结构和功能分析

基本信息

批准号：
03453009
负责人：
MORISHIMA Isao
金额：
$ 4.48万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03453009/
关键词：
Myoglobin Hemoglobin High-Pressure Kinetics Laser Photolysis Activation Volume Ligand Binding Electron Transfer CO Rebinding

项目摘要

1. Several site-specific mutants of human myoglobin(Mb) have been prepared in order to examine the influence of highly conserved leucine residues located in the hydrophobic clusters in the heme distal and proximal site on the heme environmental structures and ligand binding properties. Structural and ligand binding characterization of these recombinant proteins were studied by NMR, IR and laser photolysis measurements under different pressures. The leucine 29 mutants exhibited unusual pressure dependence of CO binding rate constant and the resulting pressure dependence of the activation volume implies that pressurization affects the fractional distributions of multi-conformers of Mb so that the fast CO rebinding conformers is increased under pressure.2. The effects of pressure on the recombination kinetics of CO binding to the isolated alpha and beta chains of human hemoglobin(Hb) were studied. Pressure dependent activation volume change from negative to positive values in the bimolecular CO association reaction was observed for both isolated chains. This finding was attributed to a change in the rate-determining step from the bond formation to the ligand migration process.3. We have also studied pressure effects on the CO association reaction for cytochrome P-450. It was found that the activation volume is quite sensitive to the presence or absence of the substrate, the molecular structure of the substrate.4. Finally, we have made a preliminary study on the pressure effect on the intraprotein electron transfer reaction rate.

1.制备了几种人肌红蛋白(Mb)的位点特异性突变体，以考察位于血红素远端和近端疏水簇上的高度保守的亮氨酸残基对血红素环境结构和配体结合性质的影响。用核磁共振、红外光谱和激光光解等方法研究了不同压力下重组蛋白的结构和配体结合特性。29个亮氨酸突变体表现出异常的CO结合速率常数的压力依赖性，由此得到的激活体积的压力依赖性表明，加压影响了Mb的多构象的分数分布，从而使快速CO结合构象在压力下增加。研究了压力对CO与分离的人血红蛋白(Hb)α链和β链结合的复合动力学的影响。在双分子CO缔合反应中，观察到两个孤立链的活化体积随压力变化由负值变为正值。这一发现归因于速率决定步骤从成键过程到配体迁移过程的变化。我们还研究了压力对细胞色素P-450的CO缔合反应的影响。研究发现，活化体积对底物的存在与否、底物的分子结构都非常敏感。最后，我们对压力对蛋白质内电子转移反应速率的影响进行了初步研究。