Catalytic oxidation of chlorinated and brominated phenols by peroxidases is well known. [[1], [2], [3], [4], [5]] Dehaloperoxidase (DHP), a multi-functional hemoglobin-enzyme found in the terebellid polychaete Amphitrite ornata, is known to catalyze the oxidative dehalogenation of halophenols into their corresponding haloquinones. [6,7] There are two isozymes known as DHP A and DHP B, which both have peroxidase activity. DHP-B has significant activity as a peroxygenase and other oxidative functions as well. [[6], [7], [8]] Previously, oxidative dehalogenation of 2,4,6-tribromophenol (TBP) and 2,4-dibromophenol (DBP) have been observed to form products that include the benzoquinones, 2,6-dibromo-1,4-benzoquinone (2,6-DBQ) and 2-bromo-1,4-benzoquinone (2-BrQ), respectively. [9] The chlorinated analogs have been studied more often because of their greater solubility. Oxidative dehalogenation of 2,4,6-trichlorophenol (TCP) to 2,6-dichloro-1,4-benzoquinone (2,6-DCQ) and 2,4-dichlorophenol (DCP) to 2-chloro-1,4-benzoquinone (2-ClQ) by DHP have both been shown to occur by a peroxidase mechanism. [2,10] The chlorinated compounds are common industrial pollutants. The naturally-occurring brominated analogs are found in marine sediments. [[11], [12], [13], [14]] The brominated aromatic compounds found in benthic ecosystems are often synthesized as repellents by organisms such as Notomastus lobatus. [13,14] These halogenated compounds are toxins to A. ornata, which is a tube worm found in shallow coastal mud flats. Oxidation of DCP/DBPs is important because of their potential to form highly toxic dioxins. In this paper several techniques were combined to examine the degradation of these toxic compounds by DHP, but also by spontaneous processes that appear to fortuitously aid their decomposition.

Peroxidases oxidize phenols to quinones by cleaving the OO bond of co-substrate H2O2 to activate the heme for electron transfer from the substrate. [[1], [2], [3],5,15] For many years the evidence that substrates are bound at the heme edge had led to the assumption that this is a universal feature of phenolic oxidations by peroxidases. [16,17] Horseradish peroxidase (HRP) is the most widely investigated in the peroxidase class for its ability to oxidize phenols. [[16], [17], [18], [19]] The structural difference of DHP with respect to HRP and other known peroxidases has opened a new possibility that substrates may be bound internally and oxidized by two subsequent single-electron transfer reactions.

Although both have a heme active site, the amino acids surrounding the active site in DHP differ significantly from HRP. Moreover, there is strong evidence for formation of protein radicals in the tyrosines of DHP, Tyr38, Tyr28, Tyr16, and Ty107. In DHP A there is an additional Tyr34 that appears to play a major role in the differences in DHP A and B reactivity. [20] One can compare this radical formation to cytochrome c peroxidase (CcP), which sequentially oxidizes the heme of each of two cytochrome c proteins by one-electron oxidation steps involving charge hopping to Trp191 to form an amino acid radical. A peroxidase intermediate comprising an amino acid radical is called compound ES. [21,22] Thus, peroxidase activation and electron transfer steps of DHP are analogous to HRP, with a compound ES intermediate. One important distinction between DHP and CcP is that a pool of up to five tyrosyl radicals in DHP acts as a collective oxidative intermediate rather than the single tryptophan in CcP. [22] The mechanism of activation of H2O2 in both involves Poulos-Kraut “push-pull” mechanism. [23,24], [20]

DHP peroxidase activity was first reported for the substrate 2,4,6-tribromophenol that is oxidized to 2,6-dibromoquinone in the presence of H2O2 as the oxidant. [9] The oxygen atom added to the para position of the phenol is derived from solvent water. Similar activity is observed for 2,4,6-trichlorophenol (TCP) and 2,4,6-trifluorophenol (TFP). Peroxidase activity has been established in previous studies of mono-, di-, tri-halophenols and guaiacols. [8,25,26]. DHP B also has oxygenase, oxidase and peroxygenase activity. Nitrophenols, [6] pyrroles [27] and indoles [7] are oxidized via peroxygenase function. 5-bromo-3-oxindole, one of the products of 5-bromoindole has been found to possess an oxidase mechanism. [7] DHP A has limited activity for these other types of oxidations.

Comparison with other heme proteins, such as cytochromes P450, would suggest that internal binding is one requirement for oxidation via a peroxygenase mechanism. [30] However, internal binding in DHP does not exclude a peroxidase mechanism where substrates can be oxidized by sequential one-electron steps in situ. Hence, both mechanisms are theoretically possible for a single substrate. One hypothesis to explain these various reactions carried out by a single enzyme is that the binding of a particular substrate acts like a switch that activates the enzyme to carry out the appropriate oxidative reaction. A previous study has shown that some substrates can play role in functional switch between the peroxidase and peroxygenase function in DHP B. [8] Recent studies also reported that DHP B can oxidize the substrates DBP and DCP by sequential peroxidase and peroxygenase reactions. [28] However, the extremely tight binding of DCP and especially DBP leads to an apparent contradiction because DHP is also inhibited by these substrates. [29] Self-inhibition has been observed previously in DHP. [5] Herein, a combination of methods was used to study the mechanism of the double oxidations of DCP and DBP, which have been reported to include peroxidation and peroxygenation. [28] UV–vis spectroscopy provides information on the most abundant species, whereas mass spectrometry reveals many species that may have low yields. While quinones are the major products of phenol oxidation there are many other oxidized species present in lesser quantities.

Research on marine organisms which has led to the identification of many novel metabolites, including more than 100 quinones. [31] Quinones are intermediates on a degradation pathway for halogenated aromatic xenobiotics. Although many quinones are toxic they appear to be degraded relatively rapidly. There is still missing information due to their low concentration and relative lack of stability. [32] Aqueous photoreactions form hydroquinones and hydroxyquinones. [33] Only a few quinones have non-photochemical hydroxylation reactions, which is of major importance for DHP. Catechols formed by peroxygenation of halophenols and hydroxyquinones can lead to ortho-quinone formation, which is a degradation pathway that forms muconic acid by ring opening. [34] In this study focus was on the spontaneous hydroxylation by H2O2 in bifunctional oxidations by DHP. While the primary peroxidase reaction of both DCP and DBP dominates we find spectral evidence for a parallel degradation pathway in both substrates and both isozymes.