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Studies on Multi-nuclear Copper Enzymes and their Model Complexes

多核铜酶及其模型复合物的研究

基本信息

批准号：
11440198
负责人：
SUZUKI Shinnichiro
金额：
$ 7.74万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11440198/
关键词：
Multi-nuclear Copper Enzymes denitrifying bacteria nitrite reductase type 1 Cu type 2 Cu pulse radiolysis nitrous oxide reductase intramolecular electron transfer process 銅酵素タンパク質内電子移動銅タンパク質変異体亜硝酸還元酸素

项目摘要

The purpose of this project is to clarify the structure and function of Cu-containing nitrite reductase and nitrous oxide reductase.1) In order to shed light on the His255 and Asp98 residues around the type 2 Cu center in blue Achromobacter xylosoxidans nitrite reductase, two mutants were expressed in E. coil.The Asp98Ala and His255Ala mutants show the very low enzyme activities compared with the wild enzyme at pH 5.5, suggesting that these residues play a role of proton donation for the formation of water molecule.The Asp and His255 residues are also very important for the control of the intramolecular electron transfer rate from type 1 Cu to type 2.2) The nitrite reductase from Hyphomicrobium sp. A3151 has the two type 1 Cu's and one type 2 Cu, which is different from the other nitrite reductases containing type 1 Cu : type 2 Cu = 1 : 1. One of type 1 Cu(II) ions is blue and the other is green.3) The nitrous oxide reductase from Hyphomicrobium sp. A3151 is aerobically stable, and there are two forms, purple oxidized and blue reduced forms.4) The type 2 Cu(I) and (II) model complexes were prepared. The Cu(I) complex binding nitrite with nitrogen atom stoichiometrically gives NO gas in the presence of proton.

本课题的目的是阐明含铜亚硝酸盐还原酶和氧化亚氮还原酶的结构和功能。1)为了揭示蓝色无色杆菌亚硝酸盐还原酶2型Cu中心附近的His255和Asp98残基，在E. coil中表达了两个突变体。在pH为5.5时，Asp98Ala和His255Ala突变体的酶活性与野生酶相比非常低，表明这些残基对水分子的形成起质子捐赠作用。Asp和His255残基对于控制从1型Cu到2.2型的分子内电子转移速率也非常重要。菌丝微生物sp. A3151的亚硝酸盐还原酶具有2个1型Cu和1个2型Cu，这与其他含有1型Cu的亚硝酸盐还原酶不同：2型Cu = 1:1。1型Cu（II）离子中的一个是蓝色的，另一个是绿色的。3)菌丝菌A3151氧化亚氮还原酶具有好氧稳定性，有紫色氧化型和蓝色还原型两种形式。4)制备了2型Cu(I)和（II）模型配合物。Cu(I)配合物将亚硝酸盐与氮原子结合，在质子存在下产生NO气体。