Oxygen activation by the noncoupled binuclear copper site in peptidylglycine alpha-hydroxylating monooxygenase. Spectroscopic definition of the resting sites and the putative CuIIM-OOH intermediate

Biochemistry. 2004 May 18;43(19):5735-47. doi: 10.1021/bi0362830.


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 alpha-hydroxylating monooxygenase (PHM). The Cu(M) site has a square pyramidal geometry with a long axial Cu-methionine bond and two histidines, H(2)O, and OH(-) as equatorial ligands. The Cu(H) site has a slightly D(2)(d) distorted square planar geometry with three histidines and H(2)O 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 NO(2)(-) 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Catalytic Domain
  • Circular Dichroism
  • Copper / chemistry*
  • Copper / metabolism*
  • Electron Spin Resonance Spectroscopy
  • Hydroxylation
  • Ligands
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / metabolism*
  • Models, Chemical
  • Models, Molecular
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism*
  • Nitrites / chemistry
  • Nitrites / metabolism
  • Oxygen / metabolism*
  • Thermodynamics
  • Water / chemistry


  • Ligands
  • Multienzyme Complexes
  • Nitrites
  • Water
  • Copper
  • Mixed Function Oxygenases
  • peptidylglycine monooxygenase
  • Oxygen