Reactivity of deoxy- and oxyferrous dehaloperoxidase B from Amphitrite ornata: identification of compound II and its ferrous-hydroperoxide precursor

Abstract

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. The bifunctional nature of DHP as a globin peroxidase appears to be at odds with the traditional starting oxidation state for each individual activity. Namely, reversible oxygen binding is only mediated via a ferrous heme in globins, and peroxidase activity is initiated from ferric centers and to the exclusion of the oxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the details of our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays, stopped-flow UV-visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. We demonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and show that this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified a new species that we have preliminarily attributed to a ferrous-hydroperoxide precursor that undergoes heterolysis to generate the aforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidase competent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatory starting point for the peroxidase catalytic cycle of dehaloperoxidase. 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

(A) Stopped-flow UV-visible spectroscopic monitoring of the reaction (900 scans, 85 sec) between oxyferrous DHP B (10 μM) containing one equivalent TCP (10 uM) and a 10-fold excess of H₂O₂ at pH 8. (B) Calculated UV-visible spectra for both oxyferrous (black) and Compound II (red) are shown; the rapid-scanning data from panel A were fit to a one-step, two species sequential irreversible model using Specfit global analysis software. (C) Relative concentration profile determined from the three component fit used in (B).

Figure 2

EPR spectrum of DHP B Compound II at a freeze-quench time of 100 ms (pH 8). Data collected at 1 and 30 second quench times for DHP B Compound II similarly showed no signal. For comparative purposes, the EPR spectrum of Compound ES is also shown. Rapid freeze-quench samples were prepared from the reaction of oxyferrous DHP B (50 μM final) containing 1 equivalent of TCP with a 10-fold excess of H₂O₂ at 25 °C, and rapidly frozen in an isopentane slurry.

Figure 3

UV-visible spectroscopic monitoring (10 scans, 0.1 min interval/scan, 60 sec total) of the reaction between ferrous DHP B (10 μM) with (A) 2.5 equiv (B) 10 equiv and (C) 25 equiv H₂O₂ yielding Compound II at pH 8. Inset: Same experimental conditions as described above except that the ferrous DHP B concentration was 50 μM.

Figure 4

Comparison of the visible spectroscopic features of ferrous DHP B (10 μM, black), ferric DHP B (green), Compound II (red), and the putative ferrous-hydroperoxide intermediate (blue) that was formed prior to Compound II in 100 mM KP_i (pH 8) at 4 °C.

Scheme 1

Proposed catalytic cycle for (de)oxyferrous dehaloperoxidase B.^{a a} Adapted from reference [(26)]. The pathways forming Compounds RH and P₄₂₆, as well as monohalophenol inhibition, have been omitted for clarity.

Scheme 2

Proposed tautomerization of the distal histidine in oxyferrous DHP B upon trihalophenol binding. A direct interaction between His⁵⁵ and TXP is depicted, however binding may occur elsewhere, leading to the proposed tautomerization.