Active Site Structure and Function of Copper Enzymes in Denitrification

提出的研究的主要目标是对亚硝酸盐和氧化亚氮还原酶中金属离子和蛋白质部分的结构和催化作用的基本理解。建议对这些还原酶进行广泛的光谱和化学研究。这类多铜氧化还原酶为研究参与反硝化的含铜酶的结构/功能关系提供了新的机会。Dooley博士还将讨论反硝化途径中中间体对这些酶的可能调节。此外，从stutzeri假单胞菌突变株中分离出的部分铜耗尽形式的N2O还原酶将被彻底表征。Cu（II）的光谱特性将通过变温x射线吸收光谱、圆二色光谱、磁圆二色共振拉曼光谱和EPR光谱来作为活性位点的内置探针。Dooley博士计划开发一种N2O特异性电极测定活性，这将促进N2O还原酶的动力学和机制研究。外源配体（抑制剂、反硝化中间体、底物类似物）和pH对铜还原酶的光谱性质和动力学的影响也将被研究。综合光谱实验和机理研究的结果将允许在多种铜氧化酶和还原酶之间建立新的结构/反应性/功能相关性。了解亚硝酸盐和N2O还原酶的结构和机制之间的关系，有望在制定合理的控制和利用生物反硝化策略方面发挥重要作用。

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.