Oxidation of carboxylic acids by horseradish peroxidase results in prosthetic heme modification and inactivation.
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 H(2)O(2) 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 delta-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 H(2)O(2) are co-incubated in acetate buffer, if the HRP reaction is carried out at pH 7, in the absence of H(2)O(2), 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 delta-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.
Contribution from the Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94143-2280, USA.
Liusheng
L
Huang
Christophe
C
Colas
Paul R
PR
Ortiz de Montellano
D019342:Acetic Acid / Q000737:chemistry / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006418:Heme / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction
D002264:Carboxylic Acids; D006418:Heme; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D019342:Acetic Acid