The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5′-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5′-Br2-indigo, and together suggest a newly identified oxidase activity for DHP.

The marine hemoglobin dehaloperoxidase (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of nitrophenols, an unprecedented nonmicrobial degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol (4-NP) as a representative substrate, the major monooxygenated product was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that the O atom incorporated was derived exclusively from H2O2, indicative of a peroxygenase mechanism for 4-NP oxidation. Accordingly, X-ray crystal structures of 4-NP (1.87 Å) and 4-NC (1.98 Å) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of persistent organic pollutants such as nitrophenols in the environment.

The use of oxidoreductases as biocatalysts in the syntheses of functionalized, monomeric pyrroles has been a challenge owing to, among a number of factors, undesired polypyrrole formation. Here, we have investigated the ability of dehaloperoxidase (DHP), the coelomic hemoglobin from the terebellid polychaete Amphitrite ornata, to catalyze the H2O2-dependent oxidation of pyrroles as a new class of substrate for this enzyme. Substrate oxidation was observed for all compounds employed (pyrrole, N-methylpyrrole, 2-methylpyrrole, 3-methylpyrrole and 2,5-dimethylpyrrole) under both aerobic and anaerobic conditions. Using pyrrole as a representative substrate, only a single oxidation product, 4-pyrrolin-2-one, was observed, and notably without formation of polypyrrole. Reactivity could be initiated from all three biologically relevant oxidation states for this catalytic globin: ferric, ferrous and oxyferrous. Isotope labeling studies determined that the O-atom incorporated into the 4-pyrrolin-2-one product was derived exclusively from H2O2, indicative of a peroxygenase mechanism. Consistent with this observation, single- and double-mixing stopped-flow UV-visible spectroscopic studies supported Compound I, but not Compounds ES or II, as the catalytically-relevant ferryl intermediate involved in pyrrole oxidation. Electrophilic addition of the ferryl oxygen to pyrrole is proposed as the mechanism of O-atom transfer. The results demonstrate the breadth of chemical reactivity afforded by dehaloperoxidase, and provide further evidence for establishing DHP as a multifunctional globin with practical applications as a biocatalyst.

The toxicities of azole pollutants that have widespread agricultural and industrial uses are either poorly understood or unknown, particularly with respect to how infaunal organisms are impacted by this class of persistent organic pollutant. To identify a molecular basis by which azole compounds may have unforeseen toxicity on marine annelids, we examine here their impact on the multifunctional dehaloperoxidase (DHP) hemoglobin from the terebellid polychaete Amphitrite ornata. Ultraviolet-visible and resonance Raman spectroscopic studies showed an increase in the six-coordinate low-spin heme population in DHP isoenzyme B upon binding of imidazole, benzotriazole, and benzimidazole (K values of 52, 82, and 110 μM, respectively), suggestive of their direct binding to the heme-Fe. Accordingly, atomic-resolution X-ray crystal structures, supported by computational studies, of the DHP B complexes of benzotriazole (1.14 Å), benzimidazole (1.08 Å), imidazole (1.08 Å), and indazole (1.12 Å) revealed two ligand binding motifs, one with direct ligand binding to the heme-Fe, and another in which the ligand binds in the hydrophobic distal pocket without coordinating the heme-Fe. Taken together, the results demonstrate a new mechanism by which azole pollutants can potentially disrupt hemoglobin function, thereby improving our understanding of their impact on infaunal organisms in marine and aquatic environments.

Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K = 2960 ± 940 μM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCP, suggesting that the other site, TCP, remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCP binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.