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J. Biol. Chem., Vol. 281, Issue 7, 4190-4198, February 17, 2006

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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, and Ninian J. Blackburn¹

From the Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering at Oregon Health and Sciences University, Beaverton, Oregon 97006-8921 and the Mass Spectrometry Laboratory, Vaccine and Gene Therapy Institute, Oregon Health and Sciences University, Beaverton, Oregon 97006-3448

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 H₂O₂, the -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 H₂O₂. However, when the reaction was carried out with H ¹⁶₂O₂ in the presence of ¹⁸O₂, 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 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.

Received for publication, October 14, 2005 , and in revised form, December 1, 2005.

^* This work was supported by National Institutes of Health Grant NS-27583 (to N. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Environmental and Biomolecular Systems, OGI School of Science and Engineering at OHSU, 20000 NW Walker Rd., Beaverton, OR 97006-8921. Tel.: 503-748-1384; Fax: 503-748-1464; E-mail: ninian{at}ebs.ogi.edu.

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