Project Summary The central theme of this project is dioxygen activation for C-H/C-C bond functionalization, which is fundamental for aerobic life. Molecular oxygen is a powerful oxidant, but the reaction with ground-state singlet molecules is kinetically unfavorable due to the spin-forbidden nature. To harness the oxidizing power of oxygen and circumvent the unregulated production of reactive oxidative species, metalloenzymes are frequently employed by aerobic organisms to activate oxygen and manipulate biomolecules. Heme and non-heme iron enzymes are two of the most ubiquitous and potent natural catalysts. Representative systems have been well characterized with near-complete mechanistic delineation. However, catalytic pathways of iron-dependent oxygenases with less common ligand sets lack description, and studies emphasizing an individual system do not often consider the intrinsic differences between heme and non-heme coordination. A holistic picture to systematically compare these two systems regarding oxygen activation and reactivity is deficient in the field. This project desires to fill the knowledge gap by investigating how the ligand sets contribute to reaction outcomes and why a particular system exploits one coordination over the other for a specific reaction. An apparent challenge of direct comparison is the massive variability between systems: the selection of ligand donors, active site environment, substrate-binding mode, and overall protein scaffold. To simplify the coordination environment for an unbiased comparison, two unique multifunctional oxygenases are chosen, each with an iron center ligated only by nitrogen-donors. One is a His-ligated heme- dependent enzyme responsible for pyrrolnitrin production, promoting remarkable rearrangement of a tryptophan derivative. The other is a non-heme iron-dependent enzyme crucial for the visual cycle, which catalyzes oxidative alkene cleavage and isomerization using a 4-His ligated ferrous iron. The catalytic mechanisms and structure-function correlations of both enzymes are poorly understood and the ligand sets are rarely found to mediate oxygenation reactions. Over the five-year funding period, we will comparatively investigate the heme and non-heme systems to decode how the atypical ligand sets promote the unusual biotransformations and how oxygen activation is tuned by the presence or absence of a porphyrin ring. Ultimately, the proposed research will leverage the understanding of iron-oxygen chemistry, inspire the design of biomimetic complexes and engineered biocatalysts, and advance the potential for biomedical treatments for fungal infection and impaired vision.