EPR Spin-Trapping of a Myeloperoxidase Protein Radical Olivier M. Lardinois and Paul R. Ortiz de Montellano 1 Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446 Received February 15, 2000 Incubation of myeloperoxidase (MPO) with H 2 O 2 in the presence of the spin trap DBNBS (3,5-dibromo-4- nitrosobenzenesulfonic acid) results in the EPR- detectable formation of a partially immobilized pro- tein radical. The radical was only formed in the presence of both MPO and H 2 O 2 , indicating that cata- lytic turnover of the protein is required. The changes in the EPR spectrum of the adduct upon treatment with pronase confirm that the spin trap is bound to a protein residue. These results establish that MPO, like lactoperoxidase [Lardinois, O. M., Medzihradszky, K. F., and Ortiz de Montellano, P. R. (1999) J. Biol. Chem. 274, 35441–35448], reacts with H 2 O 2 to give a protein radical intermediate. The protein radical may have a catalytic role, may be related to covalent bind- ing of the prosthetic heme group to the protein, or may reflect a process that leads to inactivation of the enzyme. © 2000 Academic Press Key Words: myeloperoxidase; peroxidases; spin trap- ping; protein radical. Myeloperoxidase (MPO) is a member of the mamma- lian family of hemoprotein peroxidases that includes lactoperoxidase (LPO), eosinophil peroxidase, and thy- roid peroxidase (1, 2). All of these enzymes, with the exception of thyroid peroxidase, are primarily involved in defense of the host against pathogenic infections. Two distinct catalytic activities of the mammalian per- oxidases support their biological function: (a) their ability to oxidize halides to reactive hypohalides, and (b) their ability to catalyze the one-electron oxidation of phenols and other readily oxidized substrates to free radical species (3). The first step in both of these cata- lytic processes is reaction of the ferric peroxidase with H 2 O 2 to give a Compound I species in which the iron is oxidized to a ferryl species and either the porphyrin or the protein (P stands for both in Eq. [1]) is oxidized to a radical cation (Eq. [1]): P Fe III  + H 2 O 2 3 P •+  Fe IV AO + H 2 O [1] P •+  Fe IV AO + Cl - + H + 3 P Fe III  + HOCl [2] The Compound I with a porphyrin radical cation is thought to be responsible for the oxidation of halides to hypohalides (Eq. [2]) (4, 5). MPO is unique in that it readily oxidizes chloride ion to hypochlorous acid, but the other mammalian peroxidases oxidize iodide, bro- mide, and pseudohalides such as thiocyanate (3, 5). The second catalytic activity of the peroxidases, one- electron oxidation of susceptible substrates such as phenol (PhOH in the equations), can be catalyzed by either Compound I species (Eq. [3]) or by Compound II (Eq. [4]), the intermediate in which the Fe IV AO species remains intact but the porphyrin or amino acid radical has been quenched: P •+  Fe IV AO + PhOH 3 P Fe IV AO + PhO • + H + [3] P Fe IV AO + PhOH + H + 3 P Fe III  + PhO • + H 2 O [4] It is likely that the Compound I species with a protein radical is formed in all cases by decay of the initially formed porphyrin radical cation. Evidence from the plant and fungal peroxidases indicates that, in some instances, the protein radical is important for substrate oxidation. Thus, the oxidation of cytochrome c by cytochrome c per- oxidase appears to be mediated by the protein radical (6), and recent work shows that the oxidation of veratryl alcohol by lignin peroxidase is mediated by a tryptophan radical at the protein surface (7). One of the primary features that distinguishes the mammalian peroxidases from the plant and fungal enzymes is the fact that the prosthetic heme group in the mammalian enzymes is cross-linked to the protein. In LPO, the heme is bound to the protein through ester Abbreviations used: LPO, lactoperoxidase; MPO, myeloperoxidase; heme, iron protoporphyrin IX regardless of the oxidation and ligation states; DBNBS, 3,5-dibromo-4-nitroso-benzenesulfonic acid; EPR, electron paramagnetic resonance. 1 To whom correspondence should be addressed. Fax: (415) 476- 0688. E-mail: ortiz@cgl.ucsf.edu. Biochemical and Biophysical Research Communications 270, 199 –202 (2000) doi:10.1006/bbrc.2000.2396, available online at http://www.idealibrary.com on 199 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.