Geometric and Electronic Structure Contributions to O-O Cleavage and the Resultant Intermediate Generated in...

This study investigates the mechanism of O-O bond cleavage in heme-copper oxidase (HCO) enzymes, combining experimental and computational insights from enzyme intermediates and synthetic models. It is determined that HCOs undergo a proton-initiated O-O cleavage mechanism where a single water molecule in the active site enables proton transfer (PT) from the cross-linked tyrosine to a peroxo ligand bridging the heme Fe^III and Cu^II, and multiple H-bonding interactions lower the tyrosine p K_a. Due to sterics within the active site, the proton must either transfer initially to the O(Fe) (a high-energy intermediate), or from another residue over a ∼10 Å distance to reach the O(Cu) atom directly. While the distance between the H⁺ donor (Tyr) and acceptor (O(Cu)) results in a barrier to PT, this separation is critical for the low barrier to O-O cleavage as it enhances backbonding from Fe into the O₂^2- σ* orbital. Thus, PT from Tyr precedes O-O elongation and is rate-limiting, consistent with available kinetic data. The electron transfers from tyrosinate after the barrier via a superexchange pathway provided by the cross-link, generating intermediate P_M. P_M is evaluated using available experimental data. The geometric structure contains an Fe^IV═O that is H-bonded to the Cu^II-OH. The electronic structure is a singlet, where the Fe^IV and Cu^II are antiferromagnetically coupled through the H-bond between the oxo(Fe) and hydroxo(Cu) ligands, while the Cu^II and Tyr^• are ferromagnetically coupled due their delocalization into orthogonal magnetic orbitals on the cross-linked His residue. These findings provide critical insights into the mechanism of efficient O₂ reduction in HCOs, and the nature of the P_M intermediate that couples this reaction to proton pumping.