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

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Abstract

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)–O2 2− 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)O2 2− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.

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Abbreviations

AIM:

Atoms in molecules

DFT:

Density functional theory

MM:

Molecular mechanical

oxPHM:

Oxidized form of peptidylglycine α-hydroxylating monooxygenase

oxPHMcc:

Oxidized catalytic core of peptidylglycine α-hydroxylating monooxygenase

PAM:

Peptidylglycine α-amidating monooxygenase

PAL:

Peptidyl-α-hydroxyglycine α-amidating lyase

PDB:

Protein Data Bank

PHM:

Peptidylglycine α-hydroxylating monooxygenase

PHMcc:

Catalytic core of peptidylglycine α-hydroxylating monooxygenase

QM:

Quantum mechanical

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Acknowledgments

We acknowledge Jean Jakoncic and Vivian Stojanoff (beamline X6A of the National Synchrotron Light Source, Brookhaven National Laboratory) for assistance in the collection of X-ray diffraction data. We thank Sandra Gabelli and Mario Bianchet for assistance in the purification of the protein and crystallographic experiments. This work was supported by National Science Foundation grant MCB-920288 and National Institutes of Health grant DK-32949.

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Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, 21205, USA

    Katarzyna Rudzka & L. Mario Amzel

  2. Department of Inorganic, Analytical and Physical Chemistry, University of Buenos Aires, Buenos Aires, Argentina

    Diego M. Moreno & Dario A. Estrin

  3. Department of Neuroscience and Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, 06030, USA

    Betty Eipper & Richard Mains

Authors
  1. Katarzyna Rudzka
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  2. Diego M. Moreno
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  3. Betty Eipper
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  4. Richard Mains
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  5. Dario A. Estrin
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  6. L. Mario Amzel
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Corresponding author

Correspondence to L. Mario Amzel.

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An interactive 3D complement page in Proteopedia is available at http://proteopedia.org/w/Journal:JBIC:18

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Rudzka, K., Moreno, D.M., Eipper, B. et al. Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study. J Biol Inorg Chem 18, 223–232 (2013). https://doi.org/10.1007/s00775-012-0967-z

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  • DOI: https://doi.org/10.1007/s00775-012-0967-z

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