Mechanism of O₂ Activation and Cysteine Oxidation by the Unusual Mononuclear Cu(I) Active Site of the Formylglycine-Generating Enzyme

The formylglycine-generating enzyme (FGE) catalyzes the selective oxidation of a peptidyl-cysteine to form formylglycine, a critical cotranslational modification for type I sulfatase activation and a useful bioconjugation handle. Previous studies have shown that the substrate peptidyl-cysteine binds to the linear bis-thiolate Cu-(I) site of FGE to form a trigonal planar tris-thiolate Cu-(I) structure that activates O₂ for the oxidation of the C_β-H of the cysteine substrate via an unknown mechanism. Here, we employed a combination of stopped-flow kinetic, spectroscopic (UV-vis absorption, XAS, and EPR), and computational (DFT/TD-DFT calculations) methods to observe and characterize the key intermediates in this reaction for FGE from <i>Streptomyces coelicolor</i> _. Our results define the reaction coordinate of FGE, which involves H-atom abstraction from the C_β-H bond of the cysteine substrate by a reactive Cu-(II)-O₂ ^•- species to form the now experimentally observed Cu-(I)-OOH intermediate bound to a peptidyl-thioaldehyde, which proceeds to oxidize one of the protein-derived cysteine residues to a sulfenate that is end-on O-coordinated to Cu-(I). These results provide fundamental insights into how the unusual mononuclear Cu-(I) site of FGE activates O₂ for cysteine oxidation and stores oxidizing equivalents during catalysis by employing a Cu-(I)-sulfenate intermediate with an end-on O-coordination that is unprecedented in biology.