JBIC Journal of Biological Inorganic Chemistry

, Volume 18, Issue 2, pp 223–232

Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study

  • Katarzyna Rudzka
  • Diego M. Moreno
  • Betty Eipper
  • Richard Mains
  • Dario A. Estrin
  • L. Mario Amzel
Original Paper

DOI: 10.1007/s00775-012-0967-z

Cite this article as:
Rudzka, K., Moreno, D.M., Eipper, B. et al. J Biol Inorg Chem (2013) 18: 223. doi:10.1007/s00775-012-0967-z


Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction—the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (CuH and CuM). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-Å-resolution structure (hydro)peroxide binds solely to CuM in a slightly asymmetric side-on mode. The O–O interatomic distance of the copper-bound ligand is 1.5 Å, characteristic of peroxide/hydroperoxide species, and the Cu–O distances are 2.0 and 2.1 Å. Density functional theory calculations using the first coordination sphere of the CuM active site as a model system show that the computed energies of the side-on L3CuM(II)–O22− species and its isomeric, end-on structure L3CuM(I)–O2·− are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O22− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.


Peptidylglycine α-hydroxylating monooxygenase Peroxide Amidation of peptides Copper-containing proteins 



Atoms in molecules


Density functional theory


Molecular mechanical


Oxidized form of peptidylglycine α-hydroxylating monooxygenase


Oxidized catalytic core of peptidylglycine α-hydroxylating monooxygenase


Peptidylglycine α-amidating monooxygenase


Peptidyl-α-hydroxyglycine α-amidating lyase


Protein Data Bank


Peptidylglycine α-hydroxylating monooxygenase


Catalytic core of peptidylglycine α-hydroxylating monooxygenase


Quantum mechanical

Supplementary material

775_2012_967_MOESM1_ESM.pdf (103 kb)
Supplementary material 1 (PDF 102 kb)

Copyright information

© SBIC 2012

Authors and Affiliations

  • Katarzyna Rudzka
    • 1
  • Diego M. Moreno
    • 2
  • Betty Eipper
    • 3
  • Richard Mains
    • 3
  • Dario A. Estrin
    • 2
  • L. Mario Amzel
    • 1
  1. 1.Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of MedicineJohns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Inorganic, Analytical and Physical ChemistryUniversity of Buenos AiresBuenos AiresArgentina
  3. 3.Department of Neuroscience and Molecular, Microbial and Structural BiologyUniversity of Connecticut Health CenterFarmingtonUSA

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