Reactivity of 2,4-dichlorophenol in hemoglobin-dehaloperoxidase A & B.

Abstract

The multi-functional protein dehaloperoxidase-hemoglobin (DHP), from the coelom of a marine terebellid polychaete Amphitrite ornata, has diverse functions, which depend on the heme Fe oxidation state (ferric or ferrous) and on substrate binding at various internal and external binding sites. Long known for its reversible oxygen (O2) binding, further studies have established DHP as the first example of hemoglobin to have activity as a peroxidase (halophenols and guaiacols), oxidase (5-bromo-3-oxindole), oxygenase (2,3-dimethylindole and 2,3-dimethylpyrrole), and peroxygenase (nitrophenol, pyrroles and indoles). We focused on 2,4-dichlorophenol (DCP) because it is an example of a substrate that is apparently oxidized by two separate reactions, peroxidase and peroxygenase. The related molecule, 2,4-dibromophenol is a naturally occurring xenobiotic that is significantly more prevalent in marine ecosystems than 2,4,6-tribromophenol. Thus, it is a mechanistic example of novel structure/function relationships that further expands the versatility of DHP for a biological protective function in its host organism. The catalytic efficiency is 2 times higher for DCP oxidation in DHPB than DHPA. DCP shows self-inhibition even at modest concentration analogous to 2,4,6-trichlorophenol at higher concentration. The rate of DCP oxidation by DHPA of approximately 19.3 kJ/mol compared to 37.2 kJ/mol in HRP. The product 2-chloro-1,4-benzoquinone (2-CLQ) can also be hydroxylated to 2-chloro-3-hydroxy-1,4-benzoquinone (2-ClQOH) at a 10 times slower rate than the primary oxidation process. Hydroxylation of 2-ClQ in the presence DHP is not catalyzed by DHP since only H2O2 is required. The x-ray crystal structure of DHPA bound with DCP (1.48 Å) shows tight substrate binding inside the heme pocket of DHPA. Based on the electron density, we proposed two different conformations, DCPA and DCPB. This research suggests that DHP can be used as bioremediation to neutralize/oxidize a wide range of environmental pollutants.