Biosynthesis, Structure, and Regulation of Nitrous Oxide Reductase

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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.