The chemical transformations catalyzed by proteins rely on the variability provided by the twenty amino acids. Despite the diversity available, the types of reactions are still limited. With the addition of metals, biology greatly expands the chemistry of life while working within the twenty amino acid limits. The catalytic reactions of metalloenzymes are some of life’s most important transformations, including nitrogen fixation by nitrogenase, oxygen formation by photosystem II, oxygen utilization to drive energy production in cytochrome c oxidase, and drug metabolism by cytochrome P450 enzymes. The metals contained within these enzymes are supported by the amino acids of the peptide and organic ligands that poise the metal for stereo- and regio- selective reactions required by life. The peptide binding pockets of these enzymes allow flexibility during a catalytic cycle while maintaining rigidity in the overall energy landscape to ensure rapid catalysis and favorable catalytic outcomes.  The protonation state, oxidation state, and bonding angles are all elements that can modulate the poised catalytic cycle and buffer the effects of changes in the primary coordination sphere of the metal or changes in the substrates and extended cofactors during catalysis.

The Wilcoxen lab focuses on two of these metals, iron and molybdenum, with the goal of understanding how the peptide environment tunes the metals, their organic cofactors, and reaction intermediates for successful catalytic outcomes while preventing unwanted side reactions that can be destructive to the protein and cellular environment resulting in disease.  We use a multidisciplinary approach encompassing chemical biology, biochemistry, inorganic and physical chemistry to understand the role of the peptide environment on catalysis. Our goals are to understand a) how the hydrogen bonding network and aromatic residues tune the reaction landscape and molybdopterin cofactor, and b) prevent unwanted side reactions and support positive catalytic outcomes in radical SAM enzymes involving complex and multicomponent reactions. A long-term goal is to apply what we learn to synthetic models, artificial metalloenzymes, and leverage our results and outcomes from these studies to new enzyme systems.

Projects:

Radical SAM Enzymes:

Radical SAM enzymes are a going superfamily of enzymes with over 100k members, making it the largest superfamily of enzymes. Despite the abundance of sequence information little is known of the chemistry these enzymes catalyze. With only ~80 reactions identified, there is much more chemistry to be discovered and reaction mechanisms elucidated. What is most striking with this superfamily is the diversity in the reactions catalyzed from ring contractions and expansions, sulfuration, methylations, to complete carbon skeleton rearagnement. We are particularly interested in radical SAM enzymes with auxiliary domains containing additional co-factors and co-substrates. Our primary goal is to understand how the protein environment guides the reaction path, from generating a primary carbon radical to subsequent substrate radical intermediates and product while preventing unwanted side reactions and dangerous side products.

Molybdenum Enzymes:

Molybdenum enzymes are present in all kingdoms of life catalyzing oxygen atom transfers between substrate and water. Importantly, these enzymes play important roles in the global nitrogen, carbon, and sulfur cycles.

We are interested in how the protein environment tunes the molybdenum center through direct ligation to the molybdenum or through non-covalent interactions with the organic cofactor that coordinates molybdenum in the protein active site. We approach this project in two ways, the first is to understand the structure and function of the enzymes as present in biology by studying highly purified enzyme. We examine the kinetics and electronic structure to gain insight into the mechanism of the enzyme, utilizing the differences observed in variants of the enzyme to guide out hypotheses. A second approach is to generate tools to study the enzyme that can help guide our understanding of the biological proteins.