Structure-function relationships in metalloenzymes with multiple redox-active centers

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Intellectual Merit:Two metal-containing, multi-heme respiratory enzymes involved in the interconversion of ammonia and nitrite, an important stage in the biological nitrogen cycle, are being investigated. Cytochrome c nitrite reductase from Shewanella oneidensis catalyzes the six-electron reduction of nitrite to ammonia during anaerobic respiration. Hydroxylamine oxidoreductase from nitrosomonas europaea catalyzes the four-electron oxidation of hydroxylamine to nitrite, as part of a larger process in which ammonia is oxidized to nitrite during aerobic respiration. Under non-physiological conditions, hydroxylamine oxidoreductase can also reduce nitrite to ammonia similar to cytochrome c nitrite reductase, while cytochrome c nitrite reductase can oxidize hydroxylamine to nitrite. The primary aim of the project is to determine how the proteins, which have broadly similar architectures, are tailored to shepherd the ammonia-nitrite interconversion preferentially in one direction or the other. The immediate focus is on the mechanism of the multi-electron reduction of nitrite to ammonia by ccNiR. Intermediate states that form and decay during the reactions catalyzed by cytochrome c nitrite reductase are being investigated with the use of a variety of spectroscopic, electrochemical and structural techniques, most notably Laue X-ray crystallography and Laue-based time-resolved crystallography. These relatively new techniques have enormous untapped potential. The development of these techniques is an important complementary part of the project. The research outcome will provide a better understanding of the notoriously complicated redox chemistry of nitrogen at enzymatic metal centers. Broader impacts:The project's highly interdisciplinary nature provides the students in research training with a wide breadth of skills that would make them very competitive when they go on to independent careers after graduation. The project will involve an average of three undergraduate researchers per year, in addition to two high school students and two high school teachers who will perform summer research. The graduate students funded to work on this project will also provide direct supervision for many of the undergraduates and one high school student and teacher. The project also provides insight into the growing environmental problem of nitrogen cycle imbalance. 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. This shift is having many unintended negative consequences, which will soon have to be mitigated. A better understanding of ammonia-nitrite interconversion would lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance.