Oxidation of Carboxylic Acids by Horseradish Peroxidase Results in Prosthetic Heme Modification and Inactivation

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Abstract
Hemoproteins are powerful oxidative catalysts. However, despite the diversity of functions known to be susceptible to oxidation by these catalysts, it is not known whether they can oxidize carboxylic acids to carboxylic radicals. We report here that incubation of horseradish peroxidase (HRP) at acidic pH with H2O2 in acetate buffer results in rapid modification of the heme group and loss of catalytic activity. Mass spectrometry and NMR indicate that an acetoxy group is covalently bound to the δ-meso-carbon in the modified heme. A heme with a hydroxyl group on the 8-methyl is also formed as a minor product. These reactions do not occur if protein-free heme and H2O2 are co-incubated in acetate buffer, if the HRP reaction is carried out at pH 7, in the absence of H2O2, or if citrate rather than acetate buffer is used. A similar heme modification is observed in incubations with n-caproic and phenylacetic acids. A mechanism involving oxidation of the carboxyl group to a carboxylic radical followed by addition to the δ-meso-position is proposed. This demonstration of the oxidation of a carboxylic acid solidifies the proposal that a carboxylic radical mediates the normal covalent attachment of the heme to the protein in the mammalian peroxidases and CYP4 family of P450 enzymes. The hemoprotein-mediated oxidation of carboxylic acids, ubiquitous natural constituents, may play other roles in biology.