The ultimate aim of this project is to better understand the chemistry of biologically driven ammonia-nitrite inter-conversion. Ammonia (a major component of fertilizer) and nitrite are two examples of "reactive nitrogen"; that is, nitrogen usable by many living organisms, as opposed to "elemental nitrogen" which makes up 78% of the air we breathe, but is directly usable by only a few bacteria. Over the last 50 years the balance between reactive and elemental nitrogen has shifted significantly towards the former, as more fertilizer was generated to produce food and (recently) biofuels. A better understanding of ammonia-nitrite inter-conversion may lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance. One to three graduate students per year are funded directly to work on this project. The project's highly interdisciplinary nature provides the students with a wide breadth of skills that make them very competitive when they go on to independent careers after graduation. In addition to graduate students, an average of three undergraduate researchers and two high school students normally work on the project during any given year. The undergraduates are typically financed through supplemental grants from NSF, or through institutional support from the University of Wisconsin Milwaukee, while the high school students are funded through the American Chemical Society's "Project SEED" program. The graduate students funded to work on this project also provide direct supervision for the more junior researchers.
The current research concentrates on the reaction mechanism of cytochrome-c nitrite reductase (ccNIR), an enzyme that allows certain bacteria to reduce nitrite to ammonia. The bacteria can extract energy from the process, which without ccNiR would be too slow for bacterial survival. The long-term aim of the project is to determine how ccNiR and hydroxylamine oxidoreductase (HAO), an enzyme that different bacteria use to extract energy from ammonia oxidation, and that has an architecture broadly similar to ccNiR's, are tailored to shepherd the ammonia-nitrite inter-conversion preferentially in one direction or the other. Intermediate states that form and decay during the reaction catalyzed by ccNiR are being investigated using a variety of methods, most notably time-resolved crystallographic techniques. X-ray crystallography is one of the few methods available for determining the 3-dimensional arrangement of atoms in a biological macromolecule. Time-resolved methods take advantage of ultrashort high intensity X-ray pulses, which are available at facilities such as BioCARS at the Advanced Photon Source, or the LCLS X-Ray free electron laser at Stanford, to create "freeze-frame" snapshots of enzyme molecules during the course of reactions. By piecing together the exposures taken at varying times after a reaction is initiated, "movies" of molecular changes are obtained. These relatively new fast crystallography techniques have enormous untapped potential, the development of which is an important complementary aim of the project.
