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

1.为了研究位于血红素远端和近端疏水簇中的高度保守的亮氨酸残基对血红素环境结构和配体结合特性的影响，制备了几种人肌红蛋白（Mb）的位点特异性突变体。利用核磁共振、红外光谱和激光光解技术研究了这些重组蛋白在不同压力下的结构和配体结合特性。亮氨酸29突变体表现出不寻常的压力依赖性的CO结合速率常数和由此产生的压力依赖性的活化体积意味着加压影响Mb的多构象的分数分布，使快速CO再结合构象在压力下增加.研究了压力对CO与人血红蛋白（Hb）α链和β链结合的重组动力学的影响。压力依赖性的活化体积变化从负值到正值的双分子CO缔合反应中观察到的两个孤立的链。这一发现归因于从键形成到配体迁移过程的速率决定步骤的改变。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、底物的分子结构等都很敏感.最后，对压力对蛋白质内电子转移反应速率的影响作了初步研究。

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.