This research focuses on the biochemistry and structural biology of the key enzyme in the denitrification pathway. Denitrification is an intrinsic part of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release nitrous oxide (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 the denitrification pathway and numerous issues of substantial societal interest. Specifically, this research is concerned with the structure, mechanism, and assembly of the key enzyme in denitrification, nitrous oxide reductase. In the vast majority of denitrifying organisms, nitrous oxide reductase is the terminal enzyme in the denitrification pathway. The research will investigate the structures and reactivities of the novel copper sites in nitrous oxide reductase, and their roles in catalysis. Detailed experimental studies of proton-transfer and electron-transfer steps in the reduction of N2O will be carried out. The structure of the fully reduced form of the enzyme, which reacts directly with substrate, will also be pursued. An additional protein, named NosX, also appears to play a key role in the reduction of N2O. The hypothesis is that NosX activates the reductase in cells and also regenerates enzyme that has become inactivated. This hypothesis will be thoroughly tested by several approaches. Successful over-expression methods for nitrous oxide reductase and NosX have been developed and will be exploited to prepare proteins for study. Site-directed mutagenesis, together with spectroscopic and kinetics methods will be used to probe the structure, bonding, and reactivity of the catalytic sites in nitrous oxide reductase and NosX. Structural studies of the proteins will be carried out by nuclear magnetic resonance (NMR) or X-ray crystallography. Both undergraduate and graduate students will be directly involved in the project. The context and significance of the project is of broad public interest, and will be presented and featured in multiple venues by the Principal Investigator.
Broader Impacts. The Department of Chemistry and Biochemistry at Michigan State University is a NSF Research Experiences for Undergraduates (REU) site, and the PI is a member of the participating faculty. An important aim of the REU program is to provide research opportunities for Native American students and students from other underrepresented groups. It is anticipated that one or two undergraduates (annually) will be involved in this research as part of their participation in the REU program. In addition, this project may support the participation of students from Rocky Mountain College in Billings, Montana, an undergraduate college with extremely limited opportunities for undergraduate research. The research on denitrification, with its clear connections to agriculture and to environmental issues, has served as an effective focal point for informal presentations and discussions on the relationships and linkages between basic research and benefits to society. The clear connections between the denitrification pathway and environmental, agricultural, and energy issues are regularly emphasized in talks to prospective students, undergraduate and graduate students, community groups, and interest groups. The Principal Investigator, who also serves as the Provost and Vice President for Academic Affairs at MSU-Bozeman (Montana's leading research institution and a Carnegie "very high research activity" university), has many opportunities annually to speak to the importance of research and discovery to undergraduate education and to economic development in the state. He meets frequently with members of state government, legislators, and numerous constituencies.
This project focused on the properties of selected enzymes that are essential to the global nitrogen cycle – the process by which nitrogen is captured from the atmosphere, incorporated into organisms, and ultimately returned to the atmosphere. The global nitrogen cycle is critical to all life on the planet, and enzymes in both land-based and aquatic organisms are involved in the cycle. Over the course of this project we determined the structures, functions, and mechanisms of action of enzymes and proteins involved in the central pathway that returns nitrogen to the atmosphere. In particular, this project examined proteins and enzymes that function through the utilization of copper or iron, and investigated some of the roles that these metals play in the nitrogen-cycle pathway that was studied. Results from the research provided substantial new information regarding the processes by which a key copper-containing enzyme in the pathway was manufactured in the cell and how it functions. This enzyme, which transforms nitrous oxide (the active ingredient in "laughing gas") into nitrogen, plays an especially important role in the nitrogen cycle. Consequently, the new information generated by this research project may be relevant to understanding, and controlling, transformations of nitrogen compounds important to agriculture, water quality, and global climate change.
