Svetlana Tsogoeva (@svetlanatsogoeva.bsky.social)

Mechanism of O2 Activation and Cysteine Oxidation by the Unusual Mononuclear Cu(I) Active Site of the Formylglycine-Generating Enzyme | #ACSCentralScience #DiamondOpenAccess @pubs.acs.org pubs.acs.org/doi/10.1021/...

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Mechanism of O<sub>2</sub> 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 Cβ–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 Streptomyces coelicolor. Our results define the reaction coordinate of FGE, which involves H-atom abstraction from the Cβ–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. https://pubs.acs.org/doi/10.1021/acscentsci.5c00183