Selective isotope labeling probes the chemical capacity and reaction mechanism of a heterobimetallic Mn/Fe protein

The R2-like ligand binding oxidase (R2lox) forms a novel tyrosine-valine crosslink upon O₂ activation, reflecting an overall two-electron oxidation reaction. However, the mechanism through which the crosslink is formed is under debate, as the reaction can be initiated through either tyrosine OH or valine CH bond activation. Here, we utilized selective isotopic labeling and several spectroscopic techniques to probe the mechanism of this oxidation process. The results suggest that R2lox is capable of performing CH bond activation, with both solvent and CH kinetic isotope effects of approximately 2. Signatures of a high-valent Mn^IV/Fe^IV intermediate were observed through rapid-freeze-quench EPR that resemble an analogous intermediate found in the heterobimetallic radical-initiating enzyme, class Ic ribonucleotide reductase. This study provides a lower bound on the free energies of CH bonds that can be cleaved by Mn/Fe cofactors and suggests the potential for such enzymes to functionally replace Fe/Fe cofactors in a range of reactions.