Structural Regulation Mechanism for Reactivity in Metalloproteins

本课题的主要研究结果如下：1)辣根过氧化物酶的广泛突变揭示了远端高度保守的氢网络在过氧化物酶的催化活性中起关键作用。详细讨论了过氧化物酶反应的分子机理。2)解析了ARP对鲁米诺具有特异高反应活性的晶体结构，并确定了底物结合位点。利用时间分辨共振拉曼光谱分析了ARP反应的中间产物，并与辣根过氧化物酶的中间产物进行了比较。3)对细胞色素c氧化酶与过氧化氢的反应进行了跟踪研究，确定了反应中的关键中间体。4)利用突变体研究了大肠杆菌末端氧化酶的调控机制。5)用脉冲辐射法研究了亚硝酸盐还原酶细胞色素cd_1的电子转移，并提出了该酶的还原机理。6)铁氧还蛋白系统突变显示了铁氧还蛋白与亚硫酸盐还原酶的特异性相互作用位点。7)解析了另一种亚硝酸盐还原酶P450nor的晶体结构，并探讨了该酶还原亚硝酸盐的分子机理。8)利用EPR研究细胞色素P450cam与其电子给体putidaredoxin的相互作用。阐明了一些特定的相互作用。

The Primary results in this research project are as follows :1)The extensive mutation in horseradish peroxidase has revealed that the highly conserved hydrogen network in the distal site plays a key role in catalytic activity in peroxidases. The detailed molecular mechanism in peroxidase reaction was discussed.2)The crystal structure of ARP,which shows specific high reactivity for luminol, was resolved and the substrate binding site was proposed. By using time-resolved resonance Raman spectroscopy, we examined the intermediate species in the reaction ARP and compared them with those of horseradish peroxidase.3)We followed the reaction of cytochrome c oxidase with hydrogen peroxide and idetified some key inter mediates in the reaction.4)The Regulation mechanism for the terminal oxidase in Escherichia coli was investigated by using the mutants.5)The electron transfer in nitrite reductase, cytochrome cd_1, was examined by pulse radiolysis and reduction mechanism for the enzyme was proposed.6)The systematic mutation in ferredoxin showed the specific interaction sites between ferredoxin and sulfite reductase.7)We resolved crystal structure of another nitrite reductase, P450nor, and discussed the molecular mechanism for reduction of nitrite by the enzyme.8)EPR was utilized for the investigation of the interaction between cytochrome P450cam and its electron donor, putidaredoxin. Some specific interactions were elucidated.