Structural Regulation Mechanism for Reactivity in Metalloproteins

本研究的主要结果如下：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催化活性中起着关键作用。2)解析了对鲁米诺具有较高反应活性的ARP的晶体结构，提出了底物结合部位。利用时间分辨共振拉曼光谱检测了反应中间产物ARP，并与辣根过氧化物酶进行了比较。3)跟踪了细胞色素c氧化酶与过氧化氢的反应，鉴定了反应中的一些关键中间产物。4)利用突变体研究了亚硝酸盐还原酶中末端氧化酶的调控机制。5)用脉冲辐解方法研究了亚硝酸还原酶细胞色素CD_1中的电子转移，提出了该酶的还原机理。6)铁氧化还蛋白的系统突变显示了亚硝酸根还原酶与亚硫酸盐还原酶之间的特异性相互作用部位。7)我们解析了另一种亚硝酸盐还原酶P450 NOR，P450 NOR的晶体结构并探讨了该酶还原亚硝酸根的分子机理。8)用EPR研究了细胞色素P450 cam与其电子供体Putidaredoxin之间的相互作用。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.