Biosynthesis, Structure, and Regulation of Nitrous Oxide Reductase

本研究的重点是反硝化途径中关键酶的生物化学和结构生物学。固氮、同化和反硝化的环境生物学对农业生产力和水质有重要影响。反硝化作用可能向大气释放N2O，从而导致臭氧消耗和全球变暖。因此，在反硝化的生物学和生物化学基础研究与许多重大社会利益问题之间存在着明确和直接的联系。具体而言，将对氧化亚氮还原酶的结构、机理和金属簇组装进行研究。本研究将直接解决有关新型铜位点在氧化亚氮还原酶中的组装机制及其催化作用的问题。由nos簇基因nosD， L和X（参与生物合成）和氧化亚氮还原酶（由nosZ编码）编码的周质蛋白将被纯化和表征。用纯化的氧化亚氮还原酶和其他nos蛋白进行体外聚簇研究。nos蛋白在体内的功能将通过敲除的产生和表征来探讨。位点导向诱变，结合结构、光谱和动力学方法，将用于探测电子转移催化位点的结构、键合和反应性。由于最近证明氧化亚氮还原酶具有催化活性，因此将强调氧化亚氮还原酶的还原活化形式的机理、光谱和结构研究。更广泛的影响：本科生和研究生将直接参与研究项目，包括来自本科学院的学生和在科学领域未被充分代表的群体。此外，该研究是作为密歇根州立大学博兹曼复杂生物系统IGERT项目的一部分进行的，因此可能有助于研究生教育的制度改进。鉴于全球氮循环和反硝化的重要性，将有许多机会（包括正式和非正式的）展示基础研究与广泛关注和对社会重要的问题之间的联系。

This research focuses on the biochemistry and structural biology of the key enzyme in the denitrification pathway. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N2O to the atmosphere, thereby contributing to ozone depletion and global warming. Hence there exist clear and direct linkages between basic research on the biology and biochemistry of denitrification and numerous issues of substantial societal interest. Specifically, studies of the structure, mechanism, and metal cluster assembly in nitrous oxide reductase will be conducted. This research will directly address questions regarding the mechanism of assembly of the novel copper sites in nitrous oxide reductase, and their roles in catalysis. Periplasmic proteins coded by the nos cluster genes nosD, L, and X (which are involved in the biosynthesis) and nitrous oxide reductase (coded by nosZ), will be purified and characterized. Investigation of cluster assembly in vitro will be conducted with purified nitrous oxide reductase and other nos proteins. Functions of the nos proteins in vivo will be probed by the generation and characterization of knockouts. Site-directed mutagenesis, together with structural, spectroscopic and kinetic methods will be used to probe the structure, bonding, and reactivity of the electron-transfer catalytic sites. Mechanistic, spectroscopic, and structural studies of the reductively-activated form of nitrous oxide reductase will be emphasized owing to the recent demonstration that this form is catalytically competent.Broader Impact: Undergraduate and graduate students will be directly involved in the research project, including students from baccalaureate colleges and groups underrepresented in science. Further, the research is conducted as part of MSU-Bozeman's IGERT program in Complex Biological Systems and therefore may contribute to institutional improvement in graduate education. Given the importance of the global nitrogen cycle and denitrification, there will be numerous opportunities (both formal and informal) to present the linkages between basic research and issues of broad interest and importance to society.