Studies on Multi-nuclear Copper Enzymes and their Model Complexes

本研究的目的是阐明含铜亚硝酸还原酶和一氧化二氮还原酶的结构和功能。1）为了阐明蓝色木糖氧化无色杆菌亚硝酸还原酶2型铜中心附近的His 255和Asp 98残基，在大肠杆菌中表达了两个突变体。Asp 98 Ala和His 255 Ala突变体在pH5.5时与野生型酶相比表现出非常低的酶活性，表明这些残基对水分子的形成起着质子贡献的作用，Asp和His 255残基对控制1型Cu到2型Cu的分子内电子转移速率也很重要。丝微菌A3151的亚硝酸还原酶具有两个1型Cu和一个2型Cu，这与其它含有1型Cu：2型Cu = 1：1的亚硝酸还原酶不同。3）丝微菌A3151的一氧化二氮还原酶在有氧条件下稳定，有紫色氧化态和蓝色还原态两种形式; 4）制备了2型Cu（I）和（II）模型配合物。在质子存在下，亚硝酸根与氮原子化学计量结合的Cu（I）络合物产生NO气体。

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.