Biological nitrogen fixation is a widespread metabolic process that converts atmospheric nitrogen into fertilizer through the biological activity of certain microorganisms. This project follows up on a newly discovered mechanism by which many organisms regulate the activity of nitrogenase, the enzyme that catalyses the biochemical reactions that result in nitrogen fixation. The mechanism involves a family of proteins called PII proteins that regulates nitrogen metabolic functions in a variety of organisms. The particular proteins to be studied in this research are the NifI proteins, which constitute a previously uncharacterized subfamily of PII proteins. Novel features of NifI and its regulation of nitrogenase activity include the formation a direct complex between NifI and the molybdenum-iron protein of nitrogenase, and formation of novel higher order structures of NifI complexed with itself. The research will involve biochemical, genetic, and protein structural studies using the model microbial species Methanococcus maripaludis, a member of the phylogenetic domain Archaea.
The broader impacts of the research are environmental, agricultural, and educational. Biological nitrogen fixation contributes to the nutrition of microorganisms and the plants with which they associate, without the adverse environmental effects associated with chemical fertilizers. The work will also result in the training of personnel in the areas of microbiology and biochemistry, including the integration of protein structure with the biology of nitrogen fixation. Finally, the work feeds into the investigator's teaching and outreach efforts, which include courses featuring microbial metabolism, participation in the University of Washington's interdisciplinary graduate program in Astrobiology, and participation in a K-12 outreach program called project Astrobio.
Biological nitrogen fixation (conversion of nitrogen gas to ammonia) is a major process for generating forms of nitrogen that are essential for all life. In agriculture, it is an environmentally benign process that can decrease the need for chemical fertilizers that are expensive and when over-used result in the production of pollutants. Organisms that fix nitrogen regulate the process rigorously because it is costly in terms of biological energy. When less costly (fixed) forms of nitrogen are available, nitrogen fixation is turned off, but when nitrogen gas from the atmosphere is the only available form, nitrogen fixation is turned on. We discovered a novel mode of regulation of nitrogen fixation, which became the subject of this research project. We found that the regulation works as follows: when the cell is starved for fixed nitrogen, the enzyme nitrogenase is free to catalyze nitrogen fixation. However, when fixed nitrogen becomes available, the level in the cell of the metabolic intermediate, 2-oxoglutarate, decreases because it combines with the fixed nitrogen to form nitrogenous cellular components. The lack of 2-oxoglutarate allows a protein called NifI to bind to nitrogenase and inhibit it, preventing a needless expenditure of cellular resources that would otherwise go into nitrogen fixation. The binding of NifI to nitrogenase appears to form a novel structure consisting of a circular arrangement of three NifI proteins and three nitrogenase proteins. Although NifI and its function were previously unknown, it turns out that NifI is widespread among diverse Bacteria and Archaea. It is an important mechanism for organisms to maintain the efficiency of the nitrogen assimilation process. This work has added to our knowledge of nitrogen fixation in fundamental ways that will enhance our ability to harness it for the benefit of agriculture. The work has resulted in the training of students in the fields of genetics and biochemistry and has generated collaborative interactions among scientists.
