We have examined the H2O2-dependent oxidative dehalogenation of 2,4,6-trihalophenols and p-halophenols catalyzed by Caldariomyces fumago chloroperoxidase (CCPO). CCPO is significantly more robust than other peroxidases and can function under harsher reaction conditions, and so its ability to dehalogenate halophenols could lead to its use as a bioremediation catalyst for aromatic dehalogenation reactions. Optimal catalysis occurred under acidic conditions (100 mM potassium phosphate solution, pH 3.0). UV-visible absorption spectroscopy, high-performance liquid chromatography, and gas chromatography/mass spectrometry clearly identified the oxidized reaction product for CCPO-catalyzed dehalogenation of 2,4,6-trihalophenols as the corresponding 2,6-dihalo-1,4-benzoquinones. This reaction has previously been reported for two His-ligated heme-containing peroxidases (see Osborne, R. L.; Taylor, L. O.; Han, K. P.; Ely, B.; Dawson, J. H. Biochem. Biophys. Res. Commun. 2004, 324, 1194-1198), but this is the first example of a Cys-ligated heme-containing peroxidase functioning as a dehaloperoxidase. The relative catalytic efficiency (turnover number) of CCPO reported herein is comparable to that of horseradish peroxidase (Ferrari, R. P.; Laurenti, E.; Trotta, F. J. Biol. Inorg. Chem. 1965, 4, 232-237). The mechanism of dehalogenation has been probed using p-halophenols as substrates. Here the major product is a dimer with 1,4-benzoquinone as the minor product. An electron-transfer mechanism is proposed that accounts for the products formed from both the 2,4,6-trihalo- and p-halophenols. Finally, we note that this is the first case of a peroxidase known primarily for its halogenation ability being shown to also dehalogenate substrates.

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The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.