Oxygen Activation by the Noncoupled Binuclear Copper Site in Peptidylglycine α-Hydroxylating Monooxygenase. Spectroscopic Definition of the Resting Sites and the Putative CuIIM−OOH Intermediate

Abstract

Spectroscopic methods, density functional calculations, and ligand field analyses are combined to define the geometric models and electronic structure descriptions of the Cu_M and Cu_H sites in the oxidized form of the noncoupled binuclear copper protein peptidylglycine α-hydroxylating monooxygenase (PHM). The Cu_M site has a square pyramidal geometry with a long axial Cu−methionine bond and two histidines, H₂O, and OH^- as equatorial ligands. The Cu_H site has a slightly D_2d distorted square planar geometry with three histidines and H₂O ligands. The structurally inequivalent Cu_M and Cu_H sites do not exhibit measurable differences in optical and electron paramagnetic resonance (EPR) spectra, which result from their similar ligand field transition energies and ground-state Cu covalencies. The additional axial methionine ligand interaction and associated square pyramidal distortion of the Cu_M site have the opposite effect of the strong equatorial OH^- donor ligand on the Cu d orbital splitting pattern relative to the Cu_H site leading to similar ligand field transition energies for both sites. The small molecule NO₂^- binds in different coordination modes to the Cu_M and Cu_H site because of differences in their exchangeable coordination positions resulting in these Cu^II sites being spectroscopically distinguishable. Azide binding to PHM is used as a spectroscopic and electronic structure analogue to OOH^- binding to provide a starting point for developing a geometric and electronic structural model for the putative Cu^II_M−OOH intermediate in the H-atom abstraction reaction of PHM. Possible electronic structure contributions of the Cu^II_M−OOH intermediate to reactivity are considered by correlation to the well-studied L3Cu^II−OOH model complex (L3 = [HB{3-tBu-5-iPrpz}₃]). The Met-S ligand of the Cu_M site is found to contribute to the stabilization of the Cu^II_M−oxyl species, which would be a product of Cu^II_M−OOH H-atom abstraction reaction. This Met-S contribution could have a significant effect on the energetics of a H-atom abstraction reaction by the Cu^II_M−OOH intermediate.