title: The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate.
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(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O 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 Cu(H) 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.
authors.affiliation: Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering at Oregon Health and Sciences University, Beaverton, 97006-8921, USA.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Bauman
authors.forename: Erik T
authors.initials: ET
authors.lastname: Yukl
authors.forename: Katsiaryna
authors.initials: K
authors.lastname: Alkevich
authors.forename: Ashley L
authors.initials: AL
authors.lastname: McCormack
authors.forename: Ninian J
authors.initials: NJ
authors.lastname: Blackburn
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D009097:Multienzyme Complexes; D013481:Superoxides; D003300:Copper; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase