Deciphering the Coordination Environment’s Impact on O–O Activation in Dicopper Enzymes: Computational Insights from AhyBURP Peptide Cyclase

Copper active sites are pivotal in oxygen activation processes, generating diverse copper–oxygen species that drive various biological transformations. AhyBURP, a peptide cyclase, contains a dicopper active center that is involved in the biosynthesis of lyciumin I and legumenin. In this study, we employed a combined quantum mechanics/molecular mechanics (QM/MM) approach with the TPSS functional to elucidate the catalytic mechanism of AhyBURP. To rigorously assess the accuracy of these calculations─particularly for strongly correlated systems─we benchmarked the QM/MM results against the high-level multireference NEVPT2 method and the advanced DFT methods tB4LYP and R-xDH7, with an emphasis on strong correlation effects. Unlike conventional dicopper enzymes, AhyBURP is capable of mediating direct O–O bond cleavage within the μ-η²:η²-peroxide dicopper(II) species, yielding the active bis-μ-oxo dicopper(III) intermediate. This intermediate subsequently abstracts hydrogen atoms from both tryptophan and glycine substrates, generating a biradical intermediate that facilitates subsequent intermolecular C–N coupling. Furthermore, our study reveals that the ligand coordination architecture critically determines the efficiency of O–O bond activation, providing valuable insights for the rational design of dicopper active sites with tailored reactivity.