Characterization of Dehaloperoxidase Compound ES and Its Reactivity with Trihalophenols

Abstract

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 approximately 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.

Kinetic Analysis of a Naturally Occurring Bioremediation Enzyme: Dehaloperoxidase-Hemoglobin from Amphitrite ornata

“…We have recently shown that compound I is not observed in DHP A and that compound ES is rapidly formed. 12 We recently confirmed similar observations for DHP B. 9 Both compound I and compound ES consist of a Fe(IV)dO species and a cation radical.…”

“…We have further shown that the radical is located on one of the five tyrosines in DHP A. 12 Since compound ES formation appears to be rapid in DHP, we present the peroxidase rate scheme with a compound ES intermediate. The compound ES is reduced in two one-electron steps by a substrate, XAOH, which is 2,4,6-TCP in the present study.…”

“…The external substrate binding site in DHP is related to external binding in peroxidases 10,11 in that electron transfer from the substrate to the heme edge (or alternatively to an amino acid radical) 12 is the key step in substrate oxidation as shown in Figure 2. Similar considerations apply to myoglobin mutants that have peroxidase activity.…”

Structure of dehaloperoxidase B at 1.58 Å resolution and structural characterization of the AB dimer fromAmphitrite ornata

“…Upon the substitution of this tyrosine with asparagine in DHP B this hydrogen bond is lost, but Asn34 is capable of forming a much stronger hydrogen bond to the carbonyl O atom of Glu31 as well as a couple of very weak ionic interactions with the side chains of Glu31 and Asn96. Thus, since Tyr34 has been implicated as one possible site of radical formation in DHP B Compound ES (Feducia et al, 2009), its replacement by a redox-inactive asparagine residue may affect either the site of radical formation in DHP B Compound ES or may alter the pathway for the migration of this radical out of the active site. Furthermore, the substitution-induced differences in the distal pocket of DHP B versus DHP A directly affect both the conformations of critical is the ith measurement of I(hkl) and hI(hkl)i is the weighted mean of all measurements of I(hkl).…”

“…However, two isoforms of dehaloperoxidase, termed DHP A and DHP B, occur in A. ornata and are encoded by two separate genes (dhpA and dhpB; Han et al, 2001). Both have been shown to catalyze the oxidative dehalogenation of 2,4,6-trihalogenated phenols to the corresponding 2,6-dihalo-1,4-benzoquinones in the presence of hydrogen peroxide (Feducia et al, 2009;D'Antonio et al, 2010). Although they share 96% sequence identity (DHP B differs from DHP A at five positions: I9L, R32K, Y34N, N81S and S91G), significant spectroscopic and mechanistic differences between dehaloperoxidase isoenzymes A and B have recently been elucidated (D'Antonio et al, 2010).…”