Our lab utilizes an integrated approach that combines structural biology, spectroscopy, transient kinetics, and various biochemical techniques to investigate the catalytic mechanisms and structure-function relationships of metalloenzymes, with a particular focus on those involved in natural product biosynthesis. Contribution of Ligand Sets to Oxygen Activation in Iron-dependent Biocatalysts Due to its spin-forbidden nature, molecular oxygen faces a kinetic barrier when reacting with ground-state singlet molecules. To overcome this limitation and regulate the production of reactive oxidative species, aerobic organisms utilize metalloenzymes to activate oxygen and facilitate biomolecular transformations. Among these metalloenzymes, heme and non-heme iron enzymes are among the most powerful and widespread natural catalysts. While many well-studied systems have shed light on their mechanisms, the catalytic pathways of iron-dependent oxygenases with less common ligand sets remain largely unexplored. Our project aims to compare biomedically significant heme and non-heme oxygenases that feature iron centers coordinated exclusively by nitrogen-donating ligands, such as 4His-ligated non-heme oxygenases and His-ligated heme oxygenases. By investigating the catalytic mechanisms and structure-function relationships of these enzymes, we seek to understand how these unique ligand environments drive unusual biochemical transformations and how the presence or absence of a porphyrin ring affects oxygen activation and intermediate reactivity. Our research aims to advance the understanding of iron-oxygen chemistry, inspire the development of biomimetic complexes and engineered biocatalysts, and contribute to new therapeutic strategies for pathological conditions. Funding support: NIH R35GM147510 To Be Nitrated or To Be Crosslinked: Illuminating Unusual Cytochrome P450 Enzymes in RiPP Biosynthesis Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding class of natural products with remarkable structural diversity and significant therapeutic potential. Cytochrome P450 enzymes (CYPs) play important roles in RiPP maturation by introducing selective chemical modifications that diversify peptide structures and biological activities. While CYPs are best known for oxygenation chemistry, an emerging family of RiPP-associated CYPs catalyzes unusual transformations on structurally minimal pentapeptides, including direct aromatic nitration and aromatic crosslinking reactions. How closely related CYPs recognize similar peptide substrates yet direct them toward fundamentally different chemical outcomes remains poorly understood. Our lab seeks to uncover the molecular determinants of enzyme specificity and catalytic selectivity in these unusual CYP systems. One major focus is understanding RufO-catalyzed aromatic nitration using molecular oxygen and nitric oxide. In parallel, we investigate a closely related enzyme P450Blt that acts on the same peptide substrate but catalyzes Tyr–His crosslinking. Structural and functional comparisons of these enzymes will allow us to identify subtle differences in active-site architecture that control reaction outcome and to explore whether their catalytic activities can be rationally reprogrammed. Beyond these model systems, we are exploring the broader diversity of peptide-modifying P450 enzymes that catalyze diverse coupling reactions, providing an exciting platform for discovering new enzymatic chemistry. Together, this research expands our fundamental understanding of heme-based catalysis and natural product biosynthesis while uncovering new enzymatic tools for selective peptide and aromatic functionalization. Funding support: NSF 2543564
