Characterization of Dehaloperoxidase Compound ES and Its Reactivity with Trihalophenols

Dehaloperoxidase (DHP), the oxygen transport hemoglobin from the terebellid polychaete Amphitrite ornata, is the first globin identified to possess a biologically relevant peroxidase activity. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. Herein, we demonstrate that the first detectable intermediate following the addition of hydrogen peroxide to ferric DHP contains both a ferryl heme and a tyrosyl radical, analogous to Compound ES of cytochrome c peroxidase. Furthermore, we provide a detailed kinetic description for the reaction of preformed DHP Compound ES with the substrate 2,4,6-trichlorophenol and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a g approximate to 2.0058 signal confirming the presence of a protein radical in DHP Compound ES. In the absence of substrate, DHP Compound ES evolves to a new species, Compound RH, which is functionally unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between peroxidase and oxygen transport activities in this bifunctional protein.