Differential reactivity between two copper sites in Peptidylglycine α-hydroxylating monooxygenase

Eduardo E. Chufán, Sean T. Prigge, Xavier Siebert, Betty A. Eipper, Richard E. Mains, L. Mario Amzel

Research output: Contribution to journalArticle

Abstract

Peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the Cα of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (CuM and CuH) separated by ∼11 Å. In the resting state of the enzyme, CuM is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The CuH has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu M is identified as the metal involved in catalysis, while Cu H is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO2 -), azide (N3-), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO2, shows that nitrite anion coordinates Cu M in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind CuH, despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate CuM but not CuH in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN3 and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the CuH is also observed in the reduced form of the enzyme: CO binds CuM but not CuH in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5 mM ascorbic acid (reductant). The necessity of CuH to maintain its redox potential in a narrow range compatible with its role as an electron-transfer site seems to explain the lack of coordination of small molecules to CuH; coordination of any external ligand will certainly modify its redox potential.

Original languageEnglish (US)
Pages (from-to)15565-15572
Number of pages8
JournalJournal of the American Chemical Society
Volume132
Issue number44
DOIs
StatePublished - Nov 10 2010

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ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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