Oxygen activation by the noncoupled binuclear copper site in peptidylglycine α-hydroxylating monooxygenase.: Reaction mechanism and role of the noncoupled nature of the active site

Reaction thermodynamics and potential energy surfaces are calculated using density functional methods to investigate possible reactive Cu/O-2 species for H-atom abstraction in peptidylglycine a-hydroxylating monooxygenase (PHM), which has a noncoupled binuclear Cu active site. Two possible mononuclear Cu/O-2 species have been evaluated, the 2-electron reduced Cu-M(II)-OOH intermediate and the 1-electron reduced side-on Cu-M(II)-superoxo intermediate, which could form with comparable thermodynamics at the catalytic Cum site. The substrate H-atom abstraction reaction by the Cu-M(II)-OOH intermediate is found to be thermodynamically accessible due to the contribution of the methionine ligand, but with a high activation barrier (similar to37 kcal/mol, at a 3.0-Angstrom active site/substrate distance), arguing against the Cu-M(II)-OOH species as the reactive Cu/O-2 intermediate in PHM. In contrast, H-atom abstraction from substrate by the side-on Cu-M(II)-superoxo intermediate is a nearly isoenergetic process with a low reaction barrier at a comparable active site/substrate distance (similar to14 kcal/mol), suggesting that side-on Cu-M(II)-superoxo is the reactive species in PHM. The differential reactivities of the Cu-M(II)-OOH and Cu-M(II)-superoxo species correlate to their different frontier molecular orbitals involved in the H-atom abstraction reaction. After the H-atom abstraction, a reasonable pathway for substrate hydroxylation involves a "water-assisted" direct OH transfer to the substrate radical, which generates a high-energy Cu-M(II)-oxyl species. This provides the necessary driving force for intramolecular electron transfer from the Cu-H site to complete the reaction in PHM. The differential reactivity pattern between the Cu-M(II)-OOH and Cu-M(II)-superoxo intermediates provides insight into the role of the noncoupled nature of PHM and dopamine beta-monooxygenase active sites, as compared to the coupled binuclear Cu active sites in hemocyanin, tyrosinase, and catechol oxidase, in O-2 activation.