The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

Andrew T. Bauman, Erik T. Yukl, Katsiaryna Alkevich, Ashley L. McCormack, Ninian J. Blackburn

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Abstract

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H 2O2, the α-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When 18O-labeled H2O2 was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with 18O and must therefore be derived from H 2O2. However, when the reaction was carried out with H216O2 in the presence of 18O 2, 60% of the product contained the 18O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the CuH center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

Original languageEnglish (US)
Pages (from-to)4190-4198
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number7
DOIs
StatePublished - Feb 17 2006

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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