Spectroscopic and mechanistic investigations of dehaloperoxidase B from Amphitrite ornata

Abstract

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.

Figure 1

Crystal Structure of DHP B (PDB accession code 3ixf). The location of the five residues (Leu⁹, Lys³², Asn³⁴, Ser⁸¹ and Gly⁹¹) relative to the heme active site that differ in DHP A are shown, as well as the proximal (His⁸⁹) and distal (His⁵⁵) histidines.

Figure 2

Resonance Raman spectra of halophenol complexes of DHP B (30-100 μM) at pH 7.0. (A) Trihalophenol complexes of TFP (4 mM), TCP (3 mM), and TBP (200 μM). (B) Monohalophenol complexes of 4-FP (8 mM), 4-CP (8 mM), 4-BP (8 mM), 4-IP (1 mM) and phenol (8 mM).

Figure 3

Oxidative dehalogenation of 2,4,6-trichlorophenol (125 μM) as catalyzed by DHP (2.5 μM) and hydrogen peroxide (250 μM) in the absence (A) and presence (B) of 4-bromophenol (250 μM). The formation 2,6-dichloroquinone was monitored at 276 nm (C).

Figure 4

(A) Stopped-flow UV-visible spectroscopic monitoring (900 scans, 85 sec) of the reaction between ferric DHP B (10 μM) and a 10-fold excess of H₂O₂ at pH 7. See Materials and Methods for details. (B) Calculated UV-visible spectra for ferric (black), Compound ES (red), and Compound RH (blue) DHP B are shown; the rapid-scanning data from panel A were compiled and fitted to a two-step, three species sequential irreversible model using the Specfit global analysis program. (C) Relative concentration profile determined from the three component fit used in panel B.

Figure 5

EPR spectra of the radical(s) in DHP B Compound ES at pH 7. Rapid-freeze quench samples were prepared from the reaction of ferric DHP B (50 μM final concentration) with a 10-fold molar excess of H₂O₂ at 25 °C, and rapidly frozen in an isopentane slurry. Spectra were recorded at 77 K using the spectrometer settings described in the Materials and Methods section. The cavity resonant frequency was 9.28005 GHz.

Figure 6

(A) Stopped-flow UV-visible spectroscopic monitoring (900 scans, 85 sec) of the double-mixing reaction between preformed DHP B Compound ES (10 μM; 500 ms) and a 7-fold molar excess of DCQ at pH 7. (B) Calculated UV-visible spectra for Compound ES (black) and a ferric/oxyferrous DHP B mixture (grey) are shown; the rapid-scanning data from panel A were compiled and fitted to a one-step, two species sequential irreversible model using the Specfit global analysis program.

Figure 7

(A) Stopped-flow UV-visible spectroscopic monitoring (900 scans, 800 sec) of the reaction between ferric DHP B (10 μM) and a 7-fold excess of DCQ at pH 7. (B) Calculated UV-visible spectra for ferric (black) and the ferric/oxyferrous DHP B mixture (grey) are shown; the rapid-scanning data from panel A were compiled and fitted to a one-step, two species sequential irreversible model using the Specfit global analysis program. (C) Double-reciprocal plot.

Figure 8

Spectroelectrochemical determination of the formal reduction potential of the ferric/ferrous couple in DHP B. (A) The UV-visible spectroelectrochemical plot of DHP B at various applied potentials (E_applied versus SHE) are shown. In the absence of dioxygen, ferric DHP B (λ_max = 407 nm) converted to the ferrous enzyme (λ_max = 432 nm) as the applied reduction potential was lowered from 497 mV to −130 mV. (B) The corresponding Nernst plot of the data in panel A.

Scheme 1

Proposed Catalytic Cycle for Dehaloperoxidase B.