Spectroscopic Probes of the Reactive Intermediates of Dehaloperoxidase from Amphitrite ornata

Abstract

The enzyme intermediates of dehaloperoxidase (DHP) from the marine worm Amphitrite ornata are unique within both the globin and cytochrome c peroxidase superfamilies. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. We show that the initially formed heme intermediate in this reaction is not Compound I as is often the case in peroxidases, but rather is a combination of Compound II and a tyrosyl radical that has similarity to the Compound ES intermediate of cytochrome c peroxidase. Using stopped-flow UV-visible spectroscopy, we provide a detailed kinetic description for the reaction of pre-formed DHP Compound II and tyrosyl radical with the substrate 2,4,6-trichlorophenol, and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Furthermore, using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a signal at g ≈ 2.0058, confirming the presence of a protein radical in DHP Compound II, and assign it as a tyrosyl radical based upon mutagenesis studies and structural arguments. In the absence of a halophenol substrate, the DHP Compound II + tyrosyl radical intermediate decomposes to a new and significantly less active species, termed Compound RH, which is 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 the peroxidase and oxygen-transport activities in this bifunctional protein.