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 and primary metabolism. 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 resource: NIH R35GM147510 Novel cytochrome P450 enzymes in Direct Aromatic Nitration Nitroaromatics are highly valuable compounds, but their synthesis poses significant challenges. Nature offers a promising solution through a novel group of cytochrome P450 enzymes (CYPs), including TxtE and RufO, which catalyze direct aromatic nitration of aromatic amino acids to produce bioactive compounds with considerable therapeutic potential. Despite extensive studies on CYPs as a well-established enzyme family, the biological roles of these nitrating enzymes and their structure-function relationships remain elusive. Unlike typical CYPs that follow classic oxygenation pathways, TxtE and RufO require both O2 and nitric oxide (NO) for nitration, utilizing an unprecedented mechanism that diverges from known CYP processes. This project aims to uncover new mechanistic insights into this unique CYP activity. Additionally, it is rare for a single metal center (Cys-ligated heme) to activate both O2 and NO for C-H bond functionalization. Understanding how these enzymes selectively bind two gas molecules without inhibiting their function will provide valuable insights into heme-based small-molecule activation and regioselective aromatic functionalization. Our recent advancements in the structural and spectroscopic characterization of RufO have yielded exciting findings, paving the way for further investigation into the biological functions of nitrating enzymes. This project will identify structural features that dictate enzyme function, offering a blueprint for enzyme engineering. By comparing nitrating CYPs with canonical CYPs, we hope to repurpose CYPs and facilitate the development of novel biocatalysts.
