Active Site Structure and Function of Copper Enzymes in Denitrification

拟议研究的主要目标是一个基本的 理解的结构和催化作用所发挥的作用， 亚硝酸盐和氧化亚氮中的金属离子和蛋白质部分 还原酶 广泛的光谱和化学研究， 提出了这些还原酶。 这些多铜类 氧化还原酶提供了新的研究机会 含铜酶的结构/功能关系 参与反硝化作用的微生物。 杜利博士也将 解决有关可能对论文进行管理的问题 酶通过反硝化途径中的中间体。 在 添加部分铜耗尽形式的N2 O还原酶， 分离自施氏假单胞菌的突变菌株，将 彻底定性。 Cu（II）的光谱性质 将被用作活性位点的内置探针， 变温X射线吸收光谱循环 二色性、磁圆二色性共振拉曼和EPR 谱 杜利博士计划开发一种专门针对N2 O的 用于活性的电极测定，其将促进动力学， N_2O还原酶的机理研究 外源性配体的作用 （抑制剂、脱氮中间体、底物类似物） 和pH值对铜的光谱性质和动力学的影响 还将研究还原酶。 整合来自 光谱实验和机理研究将允许新的 多铜化合物结构/反应性/功能相关性 氧化酶和还原酶。 了解 亚硝酸盐的结构与作用机理的关系 和N2 O还原酶预计将发挥重要作用， 制定合理的战略来控制和利用生物 脱氮作用

The principal goal of the proposed research is a fundamental understanding of the structural and catalytic roles played by the metal ions and protein moieties in nitrite and nitrous oxide reductases. Extensive spectroscopic and chemical studies on these reductases are proposed. These classes of multicopper redox enzymes provide new opportunities to investigate structure/function relationships in copper-containing enzymes that are involved in denitrification. Dr. Dooley will also address questions concerning the possible regulation of theses enzymes by intermediates in the denitrification pathway. In addition a partially copper-depleted form of N2O reductase, isolated from a mutant strain of Pseudomonas stutzeri, will be thoroughly characterized. The spectroscopic properties of Cu(II) will be exploited as a built-in probe of the active site by variable temperature X-ray absorption spectroscopy circular dichroism, magnetic circular dichroism resonance Raman, and EPR spectroscopy. Dr. Dooley plans to develop a N2O-specific electrode assay for activity which would facilitate kinetics and mechanism studies of N2O reductase. Effects of exogenous ligands (inhibitors, denitrification intermediates, substrate analogs) and pH on the spectral properties and kinetics of the copper reductases will also be investigated. Integrating results from spectroscopic experiments and mechanistic studies will permit new structure/reactivity/function correlations among multicopper oxidases and reductases to be formulated. Understanding the relationships between the structures and mechanisms of nitrite and N2O reductases is expected to play an essential role in developing rational strategies to control and exploit biological denitrification.