Project Summary This research program will develop accurate, detailed models of enzymatic heme degradation. In biological systems, heme oxygenases degrade heme to non-heme iron and oxygenated organic products, such as: biliverdin, staphylobilin, and mycobilin. In eukaryotes, the liberated non-heme iron is ultimately recycled into iron-dependent proteins. In prokaryotes, heme oxygenases are often part of iron acquisition pathways whereby pathogenic organisms acquire iron from host-derived heme. Despite a general understanding of the overall reactions catalyzed by heme oxygenases, the mechanistic details of how these enzymes catalyze the insertion of two to three oxygen atoms into a heme substrate remain poorly understood. This mechanistic knowledge is needed to selectively target heme oxygenase without disrupting other heme-dependent proteins. Recently, we developed new optical assays to accurately measure heme binding constants and elucidate the partitioning of heme between heme oxygenases and other heme-dependent proteins. We have also discovered an unprecedented, dynamic out-of-plane distortion of heme within some heme oxygenase active sites, which is correlated with enzymatic activity. Finally, we have revealed that an active site hydrogen bond promotes heme degradation in staphylobilin-producing heme oxygenases by stabilizing a resonance structure with a cationic radical at the carbon site of oxygenation. In the next five years, we will employ a combined spectroscopic and computational approach to elucidate the mechanism of heme monooxygenation to meso-hydroxyheme by mycobilin-producing heme oxygenases, and the mechanisms of meso-hydroxyheme oxygenation by biliverdin- and staphylobilin-producing heme oxygenases. In general, we will employ a variety of spectroscopic techniques to characterize analogues of key enzymatic intermediates. These experimental data will be used to develop accurate computational models of the enzymatic reactions. Ultimately, this program will provide detailed insight into a fascinating chapter of heme biochemistry, namely, self-oxygenation.