Biosynthesis, Structure, Function and Regulation of Nitrous Oxide Reductase

该项目解决了关于一氧化二氮还原酶中新型铜位点组装机制及其在催化中的作用的基本问题。 由nos簇基因nosD、F、Y、L编码的蛋白质，其是一氧化二氮还原酶的生物合成所需的，将被纯化和表征。 研究者的假设是，一个或多个Nos蛋白参与组装新的铜簇，这是一氧化二氮还原酶的催化位点。 一氧化二氮还原酶和其他nos蛋白的过表达方法已经被开发出来。 定点诱变，连同光谱和动力学方法，将被用来探测的结构，键合和反应性的电子转移（铜）和催化位点。 计划对蛋白质进行结构研究（通过NMR或X射线晶体学）。 这些结果将适用于现代生物化学中的几个主要主题，如金属蛋白质的结构和功能，金属离子簇在蛋白质中的组装，以及代谢途径中动力学惰性小分子的识别和激活。 研究的重点是细菌反硝化途径中的末端酶。 反硝化途径是全球氮循环的一部分，通过将固定氮返回大气来平衡循环。 全球农业生产力和水质直接受到固氮、生物同化和反硝化等生物过程的影响。 此外，在某些情况下，脱氮可能会向大气中释放一氧化二氮，从而导致臭氧消耗和全球变暖。 因此，准确了解生物体如何进行反硝化以及如何控制这一过程是非常重要的。

The project addresses basic questions regarding the mechanism of assembly of the novel copper sites in the enzyme nitrous oxide reductase, and their roles in catalysis. Proteins encoded by the nos cluster genes nosD,F,Y,L, which are required for the biosynthesis of nitrous oxide reductase, will be purified and characterized. The investigator's hypothesis is that one or more of the Nos proteins are involved in the assembly of the novel copper cluster that is the catalytic site in nitrous oxide reductase. Over-expression methods for nitrous oxide reductase and other nos proteins have been developed. Site-directed mutagenesis, together with spectroscopic and kinetics methods, will be used to probe the structure, bonding, and reactivity of the electron-transfer (CuA) and catalytic sites. Structural studies (by NMR or x-ray crystallography) of the proteins are planned. The results should be applicable to several major themes in modern biochemistry such as metalloprotein structure and function, the assembly of metal ion clusters in proteins, and the recognition and activation of kinetically-inert small molecules in metabolic pathways. The enzyme that is the focus of the research is the terminal enzyme in the denitrification pathway of bacteria. The denitrification pathway is a part of the global nitrogen cycle and balances the cycle by returning fixed nitrogen to the atmosphere. Global agricultural productivity and water quality are directly affected by the biological processes of nitrogen fixation, assimilation by organisms, and denitrification. Furthermore, under some circumstances denitrification may release nitrous oxide to the atmosphere, thereby contributing to ozone depletion and global warming. Consequently, it is very important to understand exactly how organisms carry out denitrification, and how to control this process.