Exploration of the chemical and mechanistic diversity of mixed-valent iron oxygenases and oxidases

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With the support of the Chemistry of Life Sciences Program in the Division of Chemistry, Professors Carsten Krebs, J. Martin Bollinger, Jr., and Alexey Silakov at Penn State University will use analytical, kinetic, and spectroscopic methods to understand how a new functional class of iron enzymes use molecular oxygen (O2) to carry out complex biochemical transformations in nutrient-acquisition and biosynthetic pathways in bacteria. Such enzymes catalyze reactions that generally exceed the current capabilities of synthetic chemists and could be used directly or purposefully evolved to make high-value chemicals including drugs. Most of these enzymes have cofactors consisting of one or two iron ions in the reduced iron(II) form. In 2006 the investigators identified a new functional class of enzymes that use mixed-valent diiron cofactors, i.e. cofactors that contain one reduced iron(II) and one oxidized iron(III) ion to catalyze complex four-electron oxidations of diverse substrates. How this first subfamily of mixed-valent diiron enzymes catalyze their reactions is only partly understood. The research will seek to reveal key principles of the catalytic cycles of the enzymes by direct structural interrogation of reactive intermediates. Together with collaborators, the scientists will also study multinuclear iron enzymes that have an architecture called "TIM-barrel." These enzymes catalyze remarkable rearrangement of peptides to make copper chaperones and antibiotics and also use mixed-valent di- or tri-iron cofactors. The project will be one of several used to reveal general principles and approaches in metallobiochemistry within the Penn State group's biennial bioinorganic-chemistry training program, which trains undergraduate and Ph.D. students from around the world via recorded and publicly available lectures and hands-on practical training sessions on more than 20 techniques central for bioinorganic chemistry and related research areas.In the first aim of the project, the nature of the related diiron(III)-superoxide intermediates that abstract hydrogen atoms to initiate oxidative carbon-carbon and carbon-phosphorus bond cleavage reactions in three different enzymes will be probed by isotope-labeling and pulse electron paramagnetic resonance (EPR) methods. Analytical methods to trace the path of the oxygen atoms from O2 and H2O into the products will help explain puzzling preliminary observations and clarify their mechanisms. In the second aim, the nature of the cofactors in the new heterodimeric TIM-barrel, mixed-valent iron enzymes will be defined by Mössbauer and EPR spectroscopies. The crucial questions are whether there are two or three iron ions in the cofactors and whether they form superoxo-Fe2-3 complexes that abstract hydrogen atoms to initiate the complex rearrangements of cysteine-containing peptides that make the copper chaperones known as methanobactins and the antibiotic L-thiaglutamate. The understanding that will emerge from these studies will inform discovery and directed evolution of new biocatalytic reactions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.