Functional Switching of Amphitrite ornata Dehaloperoxidase from O₂-Binding Globin to Peroxidase Enzyme Facilitated by Halophenol Substrate and H₂O₂

Abstract

Amphitrite ornata dehaloperoxidase (DHP) is the first heme-containing globin possessing a native peroxidase enzymatic activity. DHP catalyzes the H(2)O(2)-dependent dehalogenation of halophenols. By possessing this detoxifying enzymatic activity, these organisms are able to thrive in an environment contaminated with toxic haloaromatics. It has been proposed that DHP evolved from a dioxygen carrier globin protein and therefore possesses dual physiological roles of O(2) carrier and dehaloperoxidase. Although DHP is isolated in the catalytically inactive oxyferrous state (oxy-DHP), we find that the combination of H(2)O(2) and the substrate 2,4,6-trichlorophenol (TCP) brings about facile switching of oxy-DHP to the enzymatically active ferric state via a process likely involving substrate radicals (TCP*). In contrast, in the absence of TCP, H(2)O(2) alone converts oxy-DHP to an inactive state (compound RH) instead of oxidizing the enzyme to the ferric state. Further, although the rate of autoxidation of oxy-DHP is somewhat enhanced by the presence of TCP, the effect is too small to be the functional switch. Instead, both substrate and H(2)O(2) are needed to convert oxy-DHP to the catalytically active ferric state. These observations provide a physiological link between the O(2) carrier role of the ferrous protein and the peroxidase activity of the ferric enzyme in this bifunctional protein.

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

“…On the basis of the results from this study for the activation of oxy-DHP as well as our previous findings, 25 shown in Scheme 2, we illustrate the mechanism for the H 2 O 2 -mediated oxidation of oxy-DHP to the ferric state in the presence of substrate TCP (as proposed previously 25 ) or ferrocyanide (newly found in this study). In the first phase, oxyferrous DHP is oxidized to ferric DHP by TCP • or ferricyanide (with bimolecular rate constant k 1 or k 1 app ), where k 1 or k 1 app denotes the rate constants for ferrous DHP in the absence or presence of O 2 , respectively (i.e., k 1 app is an O 2 concentration-dependent value).…”

“…MS detection was in the electron impact (EI) ionization mode, and a VG70S mass spectrometer scanning from 50 to 450 amu was used for product identification. 25 During the conversion, an absorption spectral change with a single set of isosbestic points (412, 528, and 592 nm) was observed, suggesting that no detectable intermediates are involved. To further probe the conversion mechanism, we have performed more detailed investigations of this study with an oxy-DHP:H 2 O 2 :substrate concentration ratio of 1:1:1.…”

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

“…The catalytic competency of Compound ES for oxidizing substrates has been demonstrated (22,32,36,37). In the absence of a reducing 2,4,6-trihalophenol co-substrate, the formation of a species termed Compound RH, which has not been found in any other globin, was observed (21,22,37).…”

“…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.…”