Theory Demonstrated a Coupled Mechanism for O2 Activation and Substrate Hydroxylation by Binuclear Copper Monooxygenases

Multiscale simulations have been performed to address the longstanding issue of dioxygen activation by the binuclear copper monooxygenases (PHM and D beta M), which have been traditionally classified as noncoupled binuclear copper enzymes. Our QM/MM calculations rule out that Cu-M(II)-O-2(center dot) is an active species for H-abstraction from the substrate. In contrast, Cu-M(II)-O-2(center dot) would abstract an H atom from the cosubstrate ascorbate to form a Cu-M(II)-OOH intermediate in PHM and D beta M. Consistent with the recently reported structural features of D beta M, the umbrella sampling shows that the open conformation of the Cu-M(II)-OOH intermediate could readily transform into the closed conformation in PHM, in which we located a mixed-valent mu-hydroperoxodicopper(I,II) intermediate, (mu-OOH)Cu(I)Cu(II). The subsequent O-O cleavage and OH moiety migration to Cu-H generate the unexpected species (mu-O-center dot)(mu-OH)Cu(II)Cu(II), which is revealed to be the reactive intermediate responsible for substrate hydroxylation. We also demonstrate that the flexible Met ligand is favorable for O-O cleavage reactions, while the replacement of Met with the strongly bound His ligand would inhibit the O-O cleavage reactivity. As such, the study not only demonstrates a coupled mechanism for O-2 activation by binuclear copper monooxygenases but also deciphers the full catalytic cycle of PHM and D beta M in accord with the available experimental data. These findings of O-2 activation and substrate hydroxylation by binuclear copper monooxygenases could expand our understanding of the reactivities of the synthetic monocopper complexes.