Reactivity of Deoxy- and Oxyferrous Dehaloperoxidase B from Amphitrite ornata: Identification of Compound II and Its Ferrous–Hydroperoxide Precursor

摘要

Dehaloperoxidase (DHP) from the terebellidnpolychaete Amphitrite ornata is a bifunctional enzyme thatnpossesses both hemoglobin and peroxidase activities. Thenbifunctional nature of DHP as a globin peroxidase appears tonbe at odds with the traditional starting oxidation state for eachnindividual activity. Namely, reversible oxygen binding is onlynmediated via a ferrous heme in globins, and peroxidase activity isninitiated from ferric centers and to the exclusion of thenoxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the detailsnof our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays,nstopped-flow UVu0001visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. Wendemonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and shownthat this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified annew species that we have preliminarily attributed to a ferrousu0001hydroperoxide precursor that undergoes heterolysis to generate thenaforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidasencompetent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatorynstarting point for the peroxidase catalytic cycle of dehaloperoxidase. The data presented herein provide a link between the peroxidasenand oxygen transport activities, which furthers our understanding of how this bifunctional enzyme is able to unite its two inherentnfunctions in one system.