Conduits and Control of KatG Intramolecular Electron Transfer: Formation and Operation of a Novel Cofactor

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This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes Program in the Division of Chemistry.This project investigates an enzyme (KatG) at the center of the chemical warfare that rages between pathogenic microorganisms and their plant/animal hosts. Hosts produce hydrogen peroxide as part of an elaborate microbial killing system, and many pathogens produce this enzyme to defend themselves. KatG-producing pathogens range from the rice-blast fungus, which annually costs billions of dollars in crop losses, to tuberculosis, which kills millions of people each year. Clearly, it is important to understand how KatG works. Such knowledge may provide a foundation for future interventions that make these pathogens more susceptible to their hosts' own defenses. This project provides research training for graduate and undergraduate students. Most importantly, it contributes to increasing success of at-risk students from underrepresented groups, minimizing their attrition from STEM majors and maximizing the likelihood they will complete their degrees. Finally, this research helps generate new STEM learning opportunities for Alabama prisoners by contributing material that excites interest and engages future participation of prisoners in STEM learning. Intramolecular electron transfer is central to all aspects of KatG catalysis, including formation of its methionine-tyrosine-tryptophan (MYW) cofactor, the use of the cofactor for catalysis, the accumulation of inactive intermediates, and return of those intermediates to activity by exogenous electron donors. However, the intermediates of these processes and their mechanisms of control are poorly defined. To address these unknowns, this project is divided into two parts. The first evaluates KatG already containing its MYW cofactor, and the second evaluates KatG prior to cofactor formation. The former will elucidate the intermediates necessary for active catalysis as well as those that lead to inactivation, whereas the latter will address the intermediates that lead to and detract from de novo MYW cofactor synthesis. In both cases, the mechanisms by which the KatG protein itself controls the routes of electron transfer will be addressed, as will the ability of exogenous electron donors to restore inactive intermediates. As such, a broad suite of paired kinetic and spectroscopic methods, from chemical-quench peptide mapping to freeze-quench Mossbauer spectroscopy, will be applied to KatG variants targeting the proximal tryptophan and/or the arginine switch.