Metalloenzymes are crucial for many biological processes, such as photosynthesis, redox reactions in the gas cycles, and metabolism. The ferritin-like protein superfamily (FLP) represents a large and diverse group of metalloproteins. This group of proteins is involved in both Fe and O<sub>2</sub> transport and storage, radical generation, and C-H bond functionalization, among other things, and is characterized by a common four-helix bundle as well as an Fe/Fe active site. One subset of proteins in the FLP are the ribonucleotide reductases (RNRs), which catalyze <i>de novo</i> DNA synthesis. Canonically, these enzymes possess the Fe/Fe active site common to the other members of the FLP; however, subclasses have also been identified that require either a Mn/Mn or a Mn/Fe active site. In addition to the nontraditional RNRs, a new class of proteins was discovered that also binds a heterobimetallic Mn/Fe site. Due to its structural similarity to RNR, this group has been named R2-like ligand-binding oxidases, or R2lox. In addition to the unique metal selection, R2lox also binds an exogenous lipid that coordinates to each metal. Interestingly, the metal binding properties of the active site display site- and sequence-selectivity; furthermore, upon metal binding, R2lox binds oxygen to oxidize a protein-derived C – H bond to produce a Tyr-Val ether crosslink. We are using a state-of-the-art resonance Raman system to investigate the structure of the active site during assembly and probe differences induced by changes in metallation. Through this study, we can gain a deeper understanding of cofactor assembly processes within metalloenzymes.

Using resonance Raman spectroscopy to probe cofactor assembly in R2lox, a novel heterobimetallic oxidase

251st National Meeting (2016)

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