Active Site Structure and Function of Copper Enzymes in Denitrification

这项研究的主要目的是对金属离子和蛋白质在亚硝酸盐和一氧化二氮还原酶中所起的结构和催化作用有一个基本的了解。对这些还原酶进行了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作用的含铜酶的结构/功能关系提供了新的机会。杜利博士还将回答有关反硝化途径中中间体对这些酶的可能调节的问题。此外，从斯图泽里假单胞菌突变株中分离出的一种部分耗铜的N2O还原酶将被彻底鉴定。通过变温X射线吸收光谱、圆二色谱、磁圆二色谱、共振拉曼光谱和电子顺磁共振光谱，可以利用铜(II)的光谱性质作为活性中心的内置探针。杜利博士计划开发一种N2O特定的电极活性分析方法，这将有助于N2O还原酶的动力学和机理研究。还将研究外源配体(抑制剂、反硝化中间体、底物类似物)和pH对铜还原酶光谱性质和动力学的影响。综合光谱实验和机理研究的结果，将允许建立多铜氧化酶和还原酶之间的新的结构/反应/功能相关性。了解亚硝酸还原酶和N2O还原酶之间的结构和机制之间的关系，对于制定合理的策略来控制和开发生物反硝化作用具有重要的意义。

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.