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

Abstract

The coelomic O2-binding hemoglobin dehaloperoxidase (DHP) from the sea worm Amphitrite ornata is a dual-function heme protein that also possesses a peroxidase activity. Two different starting oxidation states are required for reversible O2 binding (ferrous) and peroxidase (ferric) activity, bringing into question how DHP manages the two functions. In our previous study, the copresence of substrate 2,4,6-trichlorophenol (TCP) and H2O2 was found to be essential for the conversion of oxy-DHP to enzymatically active ferric DHP. On the basis of that study, a functional switching mechanism involving substrate radicals (TCP(•)) was proposed. To further support this mechanism, herein we report details of our investigations into the H2O2-mediated conversion of oxy-DHP to the ferric or ferryl ([TCP] < [H2O2]) state triggered by both biologically relevant [TCP and 4-bromophenol (4-BP)] and nonrelevant (ferrocyanide) compounds. At <50 μM H2O2, all of these conversion reactions are completely inhibited by ferric heme ligands (KCN and imidazole), indicating the involvement of ferric DHP. Furthermore, the spin-trapping reagent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) effectively inhibits the TCP/4-BP (but not ferrocyanide)-triggered conversion of oxy-DHP to ferric DHP. These results and O2 concentration-dependent conversion rates observed in this study demonstrate that substrate TCP triggers the conversion of oxy-DHP to a peroxidase by TCP(•) oxidation of the deoxyferrous state. TCP(•) is progressively generated, by increasingly produced amounts of ferric DHP, upon H2O2 oxidation of TCP catalyzed initially by trace amounts of ferric enzyme present in the oxy-DHP sample. The data presented herein further address the mechanism of how the halophenolic substrate triggers the conversion of hemoglobin DHP into a peroxidase.

Oxidation of pyrrole by dehaloperoxidase-hemoglobin: chemoenzymatic synthesis of pyrrolin-2-ones

“…Alternatively, it has also been proposed that substrate radicals, themselves formed from trace amounts of the ferric enzyme reacting with substrate and hydrogen peroxide, oxidize oxyferrous DHP to the catalytically-active ferric form. 59,60 Here, the slow reactivity of Compounds ES/II with pyrrole, leading to the pyrrole radical cation, would also be consistent with the substrate radical activation of oxyferrous DHP in a manner that would lead to the observed differential reactivity when compared to the ferric enzyme.…”

“…Previous studies have shown that oxyferrous DHP can only be activated by H 2 O 2 in the presence of substrate (TCP, 59,60 5-bromophenol, 38 or 4-nitrophenol 39 ), leading to Compound II formation, whereas its activation in the absence of a substrate leads to the bleaching of the Soret band and/or slow conversion to Compound RH without the formation of any observable fast timescale reactive intermediates. 58 Here, we investigated the reactivity of preformed DHP Compound II with pyrrole using sequential double-mixing stopped-flow methods: oxyferrous DHP B containing 1 equivalent TCP was reacted with 10 molar equivalents of H 2 O 2 at pH 6, incubated in an aging line for 83 s to allow for the maximum accumulation of Compound II [419 (Soret), 545, 585 (sh) nm, black], 58 and subsequently mixed with an additional 50 equiv of pyrrole (Figure S11C).…”

“…It is known that DHP from both of these oxidation states forms Compound II 58 when activated with H 2 O 2 in the presence of either trihalophenol, 59,60 nitrophenol, 39 or haloindole substrates, 38 and is therefore able to initiate a peroxygenase (or peroxidase) catalytic cycle. In the present work, Compound II formation was also observed when employing pyrrole and starting from oxyferrous DHP, yet is excluded from Scheme 1 on the basis of the slow reactivity observed for this intermediate.…”

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

“…However, since the amino acid sequence defines the unique three‐dimensional structure of a protein, conversion of the function of a protein without modifying its amino acid sequence is limited. An example of a native bifunctional heme protein is dehaloperoxidase, which has been shown to convert with its substrate radical from an O 2 ‐binding hemoglobin to a peroxidase …”

“…An example of a native bifunctional heme protein is dehaloperoxidase, which has been shown to convert with its substrate radical from an O 2 -binding hemoglobin to a peroxidase. 18,19 Proteins in the cytochrome c (cyt c) protein family have been studied extensively in terms of protein folding and stability. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] Met80 and His18 are coordinated to the heme iron in cyt c, creating a relatively high redox potential for electron transfer.…”