Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

Osborne, Robert L.; Sumithran, Suganya; Coggins, Michael K.; Chen, Yung-Pin; Lincoln, David E.; Dawson, John H.

doi:10.1016/j.jinorgbio.2006.02.008

Cited by 32 publications

(40 citation statements)

References 43 publications

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“…The concentration of ferric enzyme (DHP A or mutants) was in the range of 100 – 200 µM. Soret molar absorptivity values used to calculate concentrations were determined by the pyridine hemochrome assay (42). Such values were recorded for the protein in 100 mM KPB (pH 7.0); ε 407 for DHP A is 116.4 mM −1 cm −1 (42); ε 412 for M86E is 126.6 mM −1 cm −1 , ε 413 for M86D is 127.0 mM −1 cm −1 , ε 410 for M86A is 116.5 mM −1 cm −1 , ε 409 for HHMb is 185.0 mM −1 cm −1 , and ε 403 for HRP-type I is 102.0 mM −1 cm −1 (35).…”

Section: Methodsmentioning

confidence: 99%

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

et al. 2011

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The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine-86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D (PDB 3MYN) and M86E (PDB 3MYM) mutants reveals the structural origins of a stable catalytic triad in DHP A. A decrease in rate of H2O2 activation, as well as a lowered reduction potential than the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine to bind to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is 5-coordinate and this resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a 6-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a 6-coordinate low-spin adduct, which inhibits the ligation and activation of H2O2 as required for peroxidase activity. The results suggest that the role of the proximal charge relay in peroxidases regulates the redox potential of the heme Fe, but that the trans effect is a carefully balanced property that can both activate H2O2 and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, three mutants in the M86 position, M86A, M86D, and M86E were studied by spectroelectrochemistry and NMR spectroscopy of 13C15N -labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the Nδ hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.

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Section: Methodsmentioning

confidence: 99%

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

et al. 2011

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“…This coelomic hemoglobin was originally named dehaloperoxidase (DHP) due to its ability to catalyze the oxidative dehalogenation of 2,4,6-trihalophenols to their corresponding 2,6-dihaloquinones. − More recently, it has been shown that both known isoenzymes of DHP (A and B) exhibit peroxygenase activity when haloindoles are employed as substrates . These activities are believed to have arisen from the evolutionary pressure to overcome high levels of volatile brominated secondary metabolites (e.g., halophenols and haloindoles) that are secreted as repellents by other infaunal marine organisms that coinhabit the benthic ecosystems within which A. ornata is found. , Mechanistic studies − have shown that DHP appears to function via a Poulos-Kraut type mechanism in which H 2 O 2 reacts with a ferric heme to form DHP Compound I, , the iron(IV)-oxo (ferryl) porphyrin π-cation radical species that is common to both the peroxidase and P450 cycles. Given its activity against naturally occurring halogenated phenols and indoles, the ability for DHP to degrade similar compounds of anthropogenic origin may shed light on how infaunal organisms may impact POPs in previously unreported ways.…”

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Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin from Amphitrite ornata

et al. 2016

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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.

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“…Several recent studies have focused on the characterization of DHP, as well as elucidating the mechanism of this reaction (18, 20-34). Using stopped-flow UV-visible and rapid freeze-quench EPR spectroscopic methods, we have previously demonstrated that ferric DHP reacts with hydrogen peroxide to yield Compound ES, an iron(IV)-oxo heme center with an amino acid radical (20).…”

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Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata

et al. 2010

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Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UVvisible, resonance Raman and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. 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 SD1); UVvisible spectra of the (tri)halophenol complexes of DHP B ( Figure SD2); UV-visible spectroscopic monitoring of the oxidative dehalogenation of trihalophenols as catalyzed by DHP B in the presence of hydrogen peroxide ( Figure SD3); dependence of k obs for the reaction between ferric DHP B with hydrogen peroxide (2.5 -25 equivalents) at pH 7 yielding Compound ES ( Figure SD4); stopped-flow UV-visible spectroscopic monitoring of ( Figure SD5), and DCQ product formation and TCP co-substrate loss for ( Figure SD6), the double-mixing reaction between preformed DHP B Compound ES and TCP at pH 7; stopped-flow UV-visible spectroscopic monitoring of ( Figure SD7), and DCQ product formation and TCP co-substrate loss for ( Figure SD8), the doublemixing reaction between ferric DHP B pre-incubated with TCP for 500 ms prior to its reaction with a 10-fold excess of H 2 O 2 (in situ generated Compound ES) at pH 7; stopped-flow UV-visible spectroscopic monitoring of the double-mixing reaction between ferric DHP B pre-incubated with a 7-fold molar excess of DCQ for 500 ms prior to its reaction with a 2.5-fold excess of H 2 O 2 (in situ generated Compound ES) ( Figure SD9); stopped-flow UV-visible spectroscopic monitoring of the reaction between ferric DHP B and a 7-fold excess of DCQ at pH 7 ( Figure SD10); reduction of Compound RH yielding Compound P 426 ( Figure SD11), reduction of Compound RH yielding oxyferrous DHP B ( Figure SD12). This m...

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Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

Cited by 32 publications

References 43 publications

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin from Amphitrite ornata

Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata

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