Decomposition of 2,4-dihalophenols by dehaloperoxidase activity and spontaneous reaction with hydrogen peroxide.

The enzyme dehaloperoxidase (DHP) found in the marine worm Amphitrite ornata is capable of enzymatic peroxidation of 2,4-dichlorophenol (DCP) and 2,4-dibromophenol (DBP). There is also at least one parallel oxidative pathway and the major products 2-chloro-1,4-benzoquinone (2-ClQ) and 2-bromo-1,4-benzoquinone (2-BrQ) undergo aspontaneous secondary hydroxylation reaction. The oxidation and hydroxylation reactions have been monitored by UV-visible spectroscopy, High Performance Liquid Chromatography (HPLC), and mass spectrometry. Evidence from time-resolved UV-visible spectroscopy suggests that the hydroxylations of 2-ClQ and 2-BrQ in the presence of hydrogen peroxide (H2O2) are non-enzymatic spontaneous processes approximately ∼10 and âˆ¼ 5 times slower, respectively, than the enzymatic oxidation of DCP or DBP by DHP in identical solvent conditions. The products 2-ClQ and 2-BrQ have λmaxat 255 nm and 260 nm, respectively. Both substrates, DCP and DBP, react to form a parallel product peaked at 240 nm on the same time scale as the formation of 2-ClQ and 2-BrQ. The 240 nm band is not associated with the hydroxylation process, nor is it attributable to the catechol 3,5-dihalobenzene-1,3-diol observed by mass spectrometry. One possible explanation is that muconic acid is formed as a decomposition product, which could follow decomposition either the catechol or hydroxyquinone. These reactions give a more complete understanding of the biodegradation of xenobiotics by the multi-functional hemoglobin, DHP, in Amphitrite ornata. SYNOPSIS: The decomposition of 2,4-dihalophenols catalyzed by dehaloperoxidase was studied by UV-visible spectroscopy, High Performance Liquid Chromatography and Liquid Chromatography-Mass Spectrometry. Spectroscopic evidence suggests two major products, which we propose are 2-halo-1,4-benzoquinone and 2-halomuconic acid. These complementary techniques give a high-level view of the degradation of xenobiotics in marine ecosystems.

Comparative study of the binding and activation of 2,4-dichlorophenol by dehaloperoxidase A and B.

The dehaloperoxidase-hemoglobin (DHP), first isolated from the coelom of a marine terebellid polychaete, Amphitrite ornata, is an example of a multi-functional heme enzyme. Long known for its reversible oxygen (O2) binding, further studies have established DHP activity as a peroxidase, oxidase, oxygenase, and peroxygenase. The specific reactivity depends on substrate binding at various internal and external binding sites. This study focuses on comparison of the binding and reactivity of the substrate 2,4-dichlorophenol (DCP) in the isoforms DHPA and B. There is strong interest in the degradation of DCP because of its wide use in the chemical industry, presence in waste streams, and particular reactivity to form dioxins, some of the most toxic compounds known. The catalytic efficiency is 3.5 times higher for DCP oxidation in DHPB than DHPA by a peroxidase mechanism. However, DHPA and B both show self-inhibition even at modest concentrations of DCP. This phenomenon is analogous to the self-inhibition of 2,4,6-trichlorophenol (TCP) at higher concentration. The activation energies of the electron transfer steps in DCP in DHPA and DHPB are 19.3 ± 2.5 and 24.3 ± 3.2 kJ/mol, respectively, compared to 37.2 ± 6.5 kJ/mol in horseradish peroxidase (HRP), which may be a result of the more facile electron transfer of an internally bound substrate in DHPA. The x-ray crystal structure of DHPA bound with DCP determined at 1.48 Å resolution, shows tight substrate binding inside the heme pocket of DHPA (PDB 8EJN). This research contributes to the studies of DHP as a naturally occurring bioremediation enzyme capable of oxidizing a wide range of environmental pollutants.