Radical Energies and the Regiochemistry of Addition to Heme Groups. Methylperoxy and Nitrite Radical Additions to the Heme of Horseradish Peroxidase

摘要

The heme of hemoproteins, as exemplified by horseradish peroxidase (HRP), can undergo additions at the meso carbons arid/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 NO_2~· and CH_3OO~· radicals. The NO_2~· 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)CH_2OOCH_3]. [~(18)O]-labeling studies indicate that the hydroxyl group in this adduct derives from water and the methylperoxide oxygens from O_2. Under anaerobic conditions, methyl radicals formed by fragmentation of the autocatalytically generated tert-BuO~· radical add to both the δ-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., CH_3~·) add to the meso carbon. Less energetic radicals, including NO_2~· and CH_3OO~·, 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.