Structure-Function Relationships in Metalloenzymes with Multiple Redox-active Centers

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Intellectual MeritResearch will be undertaken on three metal-containing proteins that allow particular bacteria to interconvert ammonia and nitrite. Without these proteins, such an interconversion would be too slow for bacterial survival. This project involves the development and application of time-resolved X-ray crystallography and Laue crystallography methods in an effort to obtain structural information on three multi-heme respiratory enzymes; cytochrome c nitrile reductase, octaheme tetrahtionate reductase and hydroxylamine oxidoreductase, enzymes which have broadly similar architectures. The goal of the project is to elucidate the mechanism of the multi-electron interconversion of ammonium and nitrite. The investigative techniques are relatively new and have enormous untapped potential, the development of which is an important complementary aim of the project. During data collection in conventional crystallography the entire crystal is exposed to X-rays for several hours. However, exposing a protein to X-rays often results in changes on the time scale of the conventional experiment. This is especially true for metal-containing proteins such as the ones of interest in this project. As a result, the structure one obtains is often not that of the original molecule, reflecting instead the average of the original structure and those of the protein after modification by the X-rays. By contrast, a Laue pattern is obtained in 100 picoseconds, too short a time for radiation damage to appear. As a consequence, the Laue method is ideally suited to the investigation of proteins that are susceptible to X-ray damage. The Laue method can also be used to make a "movie" of molecular changes during a reaction, by piecing together exposures taken at varying times after reaction initiation (time resolved crystallography). Broader impactThree graduate students per year will work on this project. The project's highly interdisciplinary nature provides the students with a wide breadth of skills that will make them highly competitive when they go on to independent careers after graduation. The project will sponsor 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 supported on this project also provide direct supervision for many of the undergraduates as well as 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 89% 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 in balance is having many unintended negative consequences, which will soon have to be mitigated. A better understanding of ammonia-nitrite interconversion may lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance.This project is receiving co-funding from the Chemistry of Life Processes program in the Chemistry Division