No abstract

Myeloperoxidase (MPO), eosinophil peroxidase (EPO), and chloroperoxidase can oxidize iodide, bromide, and chloride, but most peroxidases, including the prototypical horseradish peroxidase (HRP), reportedly only oxidize iodide and, in some cases, bromide. We report here that incubation of HRP with Br(-) and H(2)O(2) at acidic pH results in both bromination of monochlorodimedone and modification of the heme group. Mass spectrometry indicates that the heme 2- and 4-vinyl groups are modified by either replacement of a vinyl hydrogen by a bromide or addition of HOBr to give a bromohydrin. These reactions do not occur if protein-free heme and Br(-) are co-incubated with H(2)O(2) or if the HRP reaction is carried out at pH 7. Surprisingly, similar prosthetic heme modifications occur in incubations of HRP with H(2)O(2) and Cl(-). A mechanism is proposed involving oxidation of Br(-) or Cl(-) to give HOBr or HOCl, respectively, followed by addition to a vinyl group. In the reaction with Cl(-), a meso-chloro heme adduct is also formed. This first demonstration of Cl(-) oxidation by HRP, and the finding that prosthetic heme modification occurs when Br(-) or Cl(-) is oxidized in the absence of a cosubstrate, show that only modest tuning is required to achieve the unique chloride oxidation activity of MPO and EPO. The results raise the question of how the prosthetic hemes of MPO and EPO, whose function is to produce oxidized halide species, escape modification.

Oxidation of SCN؊ , Br ؊ , and Cl ؊ (X ؊ ) by horseradish peroxidase (HRP) and other plant and fungal peroxidases results in the addition of HOX to the heme vinyl group. This reaction is not observed with lactoperoxidase (LPO), in which the heme is covalently bound to the protein via two ester bonds between carboxylic side chains and heme methyl groups. To test the hypothesis that the heme of LPO and other mammalian peroxidases is protected from vinyl group modification by the hemeprotein covalent bonds, we prepared the F41E mutant of HRP in which the heme is attached to the protein via a covalent bond between Glu 41 and the heme 3-methyl. We also examined the E375D mutant of LPO in which only one of the two normal covalent heme links is retained. The prosthetic heme groups of F41E HRP and E375D LPO are essentially not modified by the HOBr produced by these enzymes. The double E375D/D225E mutant of LPO that can form no covalent bonds is inactive and could not be examined. These results unambiguously demonstrate that a single heme-protein link is sufficient to protect the heme from vinyl group modification even in a protein (HRP) that is normally highly susceptible to this reaction. The results directly establish that one function of the covalent heme-protein bonds in mammalian peroxidases is to protect their prosthetic group from their highly reactive metabolic products.The mammalian enzymes lactoperoxidase (LPO), 2 myeloperoxidase (MPO), and eosinophil peroxidase efficiently oxidize iodide, bromide, thiocyanide, and, at least in the case of myeloperoxidase, chloride ions (1-3). Indeed, the antimicrobial and other roles of the mammalian peroxidases depend on their oxidation of halide and/or pseudohalide ions. In contrast, the substrates of most plant and fungal peroxidases are low oxidation potential compounds such as phenols, but the enzymes can also oxidize iodide, thiocyanide, bromide, and, albeit very poorly, chloride ions (4 -6). Apart from these differences in their normal substrates, the most notable difference between the mammalian and plant/fungal peroxidases is the presence, in the mammalian enzymes, of two (or in MPO three) covalent bonds between the heme group and active site residues. In LPO, Glu 375 and Asp 225 form covalent ester bonds with the 1-and 5-methyl groups, respectively, of the heme (7-9). In MPO, in addition to the two ester bonds common to all the mammalian peroxidases, the 2-vinyl is attached via an unusual vinyl sulfonium link to Met 243 (10,11). No such covalent links have been detected in native plant or fungal peroxidases.We have demonstrated that HRP can oxidize thiocyanide, bromide, and chloride ions and that these reactions result in the addition of HOX (where X ϭ SCN, Br, or Cl) to the prosthetic heme 2-and/or 4-vinyl groups (6, 12). These results were recently extended to the Arthromyces ramosus and soybean peroxidases (13), confirming that modification of the heme vinyl groups is a common property in the oxidation of halides and pseudohalides by plant and fungal peroxidases...

No abstract

The heme of hemoproteins, as exemplified by horseradish peroxidase (HRP), can undergo additions at the meso carbons and/or vinyl groups of the electrophilic or radical species generated in the catalytic oxidation of halides, pseudohalides, carboxylic acids, aryl and alkyl hydrazines, and other substrates. The determinants of the regiospecificity of these reactions, however, are unclear. We report here modification of the heme of HRP by autocatalytically generated, low-energy NO2* and CH3OO* radicals. The NO2* radical adds regioselectively to the 4- over the 2-vinyl group but does not add to the meso positions. Reaction of HRP with tert-BuOOH does not lead to heme modification; however, reaction with the F152M mutant, in which the heme vinyls are more sterically accessible, results in conversion of the heme 2-vinyl into a 1-hydroxy-2-(methylperoxy)ethyl group [-CH(OH)CH2OOCH3]. [18O]-labeling studies indicate that the hydroxyl group in this adduct derives from water and the methylperoxide oxygens from O2. Under anaerobic conditions, methyl radicals formed by fragmentation of the autocatalytically generated tert-BuO* radical add to both the delta-meso carbon and the 2-vinyl group. The regiochemistry of these and the other known additions to the heme indicate that only high-energy radicals (e.g., CH3*) add to the meso carbon. Less energetic radicals, including NO2* and CH3OO*, add to heme vinyl groups if they are small enough but do not add to the meso carbons. Electrophilic species such as HOBr, HOCl, and HOSCN add to vinyl groups but do not react with the meso carbons. This meso- versus vinyl-reactivity paradigm, which appears to be general for autocatalytic additions to heme prosthetic groups, suggests that meso hydroxylation of the heme by heme oxygenase occurs by a controlled radical reaction rather than by electrophilic addition.