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 absence of trihalophenol. Prior to Compound II formation, we have identified a new species which 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.

Evidence of the Direct Involvement of the Substrate TCP Radical in Functional Switching from Oxyferrous O2 Carrier to Ferric Peroxidase in the Dual-Function Hemoglobin/Dehaloperoxidase from Amphitrite ornata

“…34 An upper estimate for k 5 for oxidation of deoxyferrous DHP by H 2 O 2 is used on the basis of the value (1.60 × 10 3 M −1 s −1 ) determined for DHP B at room temperature and pH 8.0 by D'Antonio and Ghiladi. 26 The k 5 value should change depending on O 2 concentration and will be denoted as k 5 app under nonanaerobic conditions. As shown in Scheme 2, ferric DHP ligands (Im and CN − ) effectively inhibit the functional switch by blocking the in Scheme 2), ferric DHP with H 2 O 2 , and DHP Cpd II with TCP, 4-BP, or ferrocyanide, respectively.…”

“…In particular, oxidation of deoxyferrous DHP to ferryl enzyme by H 2 O 2 is a possible pathway for the functional conversion for DHP as recently proposed by D'Antonio and Ghiladi. 26 In fact, such a reaction of deoxyferrous heme proteins (such as Mb, 41 leghemoglobin, 42 Table 2 and Figure 4A). Therefore, these estimated k When the O 2 concentration is much lower (as low as <5% of the normal value of ∼260 μM at ∼20°C) and the O 2 binding equilibrium is largely shifted toward the deoxyferrous form, pathway B could become competitive with the radical mechanism.…”

“…26 The latter mechanism is built on the experimental observation that Cpd II can be formed directly from deoxyferrous DHP by reaction with H 2 O 2 alone. 26 However, such a reaction has only been demonstrated under anaerobic conditions. With O 2 bound to the ferrous heme, Cpd II DHP can not be formed upon addition of H 2 O 2 .…”

Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: Toward a functional halophenol bioremediation catalyst

“…Detailed studies have been performed regarding the oxidation of chlorophenols by lactoperoxidase [16], lignin peroxidase (LiP) [17], horseradish peroxidase (HRP) [18][19][20], dehaloperoxidase [21][22][23][24][25][26][27][28][29][30][31][32] and Caldariomyces fumago chloroperoxidase [33,34]. Among these peroxidases, HRP is an especially promising candidate for use as a bioremediation catalyst because of its (a) stability resulting in easier storage and handling, (b) ability to oxidize a large number of chlorophenols, (c) flexibility to function at wide ranges of temperature and pH, (d) and, most importantly, ready availability and relatively low cost [13,14,[35][36][37][38][39][40][41].…”

Tyrosyl Radicals in Dehaloperoxidase

“…Yet DHP is itself distinct from other globins; it has little sequence homology with other hemoglobins, possesses a crystallographically characterized internal substrate binding site for 2,4,6-trihalophenols (16,17), and has been shown to utilize hydrogen peroxide to oxidize a range of substrates such as mono-, di-, and trisubstituted halophenols with bromine, chlorine, or fluorine as substituents (18,19). Moreover, the DHP peroxidase cycle can be initiated from both the ferric and ferrous (or oxyferrous) oxidation states (20,21). Thus, as a bifunctional globin-peroxidase capable of functioning from the reduced form, DHP may represent a new emerging class of peroxidases that shares neither sequence nor structural homology with any of the known peroxidases.…”