Primary product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol

Peroxidases are a class of enzymes that catalyze the oxidation of various phenolic substrates by hydrogen peroxide. They are common enzymes in soil and are also available commercially, so that they have been proposed as agents of phenolic pollutant transformation both in the environment and in engineered systems. Previous research on the peroxidase-catalyzed oxidation of pentachlorophenol (PCP) has suggested that tetrachloro-p-benzoquinone (chloranil) is the principal product and that a considerable fraction of the PCP added to reaction mixtures appears to be resistant to oxidation. In experiments employing alternative methods of product separation and analysis, we found that both of these observations a re artifacts of extraction and analytical methods used in previous studies. The major product of the horseradish peroxidase-catalyzed oxidation of pentachlorophenol from pH 4-7 was 2,3,4,5,6-pentachloro-4-pentachlorophenoxy-2,5-cyclohexadienone (PPCHD), (PPCHD), which is formed by the coupling of two pentachlorophenoxyl radicals. The yield of chloranil and other soluble products was negligible. PPCHD is insoluble and unreactive in aqueous media but is reactive when dissolved in various organic solvents. Substantial amounts of chloranil were formed when PPCHD was dissolved in benzene, ethyl acetate, or methanol; less was formed in hexane and acetonitrile; and negligible amounts were formed in dimethylformamide. High-pressure liquid chromatography (HPLC) analysis of PPCHD indicated that it is capable of undergoing dissociation and reduction to pentachlorophenol under typical reversed-phase HPLC conditions. Chlorinated oligomeric products are formed when PPCHD is stored in acetonitrile, either alone or with added pentachlorophenol. Our results demonstrate that the removal of PCP in peroxidase-catalyzed reactions can be much higher than indicated in previous work, as long as the initial product is separated by filtration or other physical means.