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

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.

Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules

“…The same conclusion can be drawn for an oxidative (42) or for the production of NO 2 -Tyr on a plant Hb (46). In these two cases, the nitrating agent is proposed to be the NO 2 • radical based on the peroxidase activity (pathway 3) of the hemoproteins (42,46). In fact, in these and our own studies, the possibility that H 2 O 2 might be generated inside the heme pocket cannot be entirely ruled out.…”

“…We can exclude a direct involvement of ONOOH formed outside the protein (pathway 1) because SIN-1 did not nitrate the Lb heme and superoxide dismutase and catalase did not prevent nitration. The same conclusion can be drawn for an oxidative (42) or for the production of NO 2 -Tyr on a plant Hb (46). In these two cases, the nitrating agent is proposed to be the NO 2 • radical based on the peroxidase activity (pathway 3) of the hemoproteins (42,46).…”

Distinguishing Nitro vs Nitrito Coordination in Cytochrome c′ Using Vibrational Spectroscopy and Density Functional Theory

“…It has been shown that these conditions lead to nitration of the porphyrin 2-vinyl group (Mb) or the 4-vinyl group (HRP). 54,55 This so-called “nitriheme” is the basis of the green coloration in cured meat upon prolonged exposure to excess nitrite under acidic conditions. Mb incubated with 50 mM nitrite leads to the formation of a 6cHS Fe(III) heme nitrito complex ( λ max 410 nm) and, upon prolonged incubation,to a 6cLS heme nitrito/nitrovinyl complex ( λ max 450 nm).…”

The broad diversity of heme-protein cross-links: An overview

“…It is interesting to observe that the formation of sulfonium ion bond and thioether bond was under oxidative and reducing conditions, respectively, whereas each of Met-heme and Cys-heme cross-links could be formed within the same protein scaffold of APX [44]. It should be noted that although the construction of a sulfonium ion bond in APX was succeeded, attempts to construct the bond in other peroxidases such as LPO [54] and horseradish peroxidase (HRP) [55] were unsuccessful. It suggests that the formation of a sulfonium ion bond requires stringent constraints in a protein scaffold, which might limit the application of this bond in designing artificial heme proteins.…”