Peroxygenase and oxidase activities of dehaloperoxidase-hemoglobin from Amphitrite ornata

Abstract

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.

Figure 1

Reaction of DHP with trihalogenated phenols and hydrogen peroxide yields quinone products.

Figure 2

(A) HPLC chromatogram of the reaction of 5-Br-indole (500 μM) with DHP B (10 μM) in the presence of H₂O₂ (500 μM) at 25 °C (5% MeOH in 100 mM KP_i, pH 7). The reaction was quenched upon addition of catalase and subjected to HPLC analysis as described in the text. (B) Product distribution for the reaction described in panel A under variable [H₂O₂].

Figure 3

ESI-MS total ion chromatograms obtained for the reaction products 5-Br-2-oxindole (A: H₂¹⁸O, H₂¹⁶O₂; C: H₂¹⁶O, H₂¹⁸O₂) and 5-Br-3-oxindolenine (B: H₂¹⁸O, H₂¹⁶O₂; D: H₂¹⁶O, H₂¹⁸O₂). Reaction conditions: [haloindole] = [H₂O₂] = 500 μM, [enzyme] = 10 μM, 5% MeOH in 100 mM KP_i (pH 7), 25 °C.

Figure 4

(A) Resonance Raman spectra of 5-X-indole (500 μM; X = F, Cl, Br, I) complexes of DHP B (50 μM) in 10% MeOH/100 mM KP_i (v/v) at pH 7. (B) Geometry optimized structure for 5-Br-indole. The 5-bromoindole model was obtained by replacement of 4-bromophenol in the PDB 3LB2 structure. In this model, the bromine atom is located in the Xe binding site as observed in the 4-bromophenol structure. (C) Geometry optimized structure for 7-Br-indole. The native substrate 2,4,6-tribromophenol (2,4,6-TBP) in the structure PDB 4HF6(40) was substituted with 7-bromoindole. Since diatomic O₂ was observed bound to the heme Fe in that structure, the bound O₂ was preserved in this model.

Figure 5

Kinetic data obtained by optical spectroscopy for the reaction of preformed Compound ES with 5-Br-indole. (A) Stopped-flow UV–vis spectra of the double-mixing reaction of preformed DHP B Compound ES (10 μM), itself formed in an initial mixing step from ferric DHP reacted with a 10-fold excess of H₂O₂ in an aging line for 350 ms, with a 10-fold excess of 5-Br-indole at pH 7.0 (800 scans over 83 s). Inset: The single wavelength (418 nm) dependence on time obtained from the raw spectra and its fit with a superposition of the calculated spectral components. (B) Calculated spectra of the four reaction components derived from the SVD analysis: Compound ES (black), ferric DHP B in the presence of excess 5-Br-indole (blue), ferryl DHP in the presence of 5-Br-indole (red), and a mixture of oxyferrous and ferric DHP B (purple). (C) Time dependences of the relative concentrations for the four components shown in the middle panel as determined from the fitting of the spectra in the top panel.

Figure 6

Kinetic data obtained by optical spectroscopy for the reaction of oxyferrous DHP B with 5-Br-indole and hydrogen peroxide. (A) Stopped-flow UV–vis spectra of the single-mixing reaction between oxyferrous DHP B (10 μM) preincubated with 2.5 equiv 5-Br-indole and a 5-fold excess of H₂O₂ at pH 7.0 (800 scans over 83 s). Inset: The single wavelength (418 nm) dependence on time obtained from the raw spectra and its fit with a superposition of the calculated spectral components. (B) Calculated spectra of the three reaction components derived from the SVD analysis: oxyferrous DHP B (black), a mixture of oxyferrous DHP with 5-Br-indole (blue), and DHP B Compound II (red). (C) Time dependences of the relative concentrations for the three components shown in the middle panel as determined from the fitting of the spectra in the top panel.

Figure 7

Kinetic data obtained by optical spectroscopy for the reaction of preformed DHP B Compound II with 5-Br-indole. (A) Stopped-flow UV–vis spectra of the double-mixing reaction between preformed DHP B Compound II (10 μM) and 25 equiv 5-Br-indole at pH 8.0 (800 scans over 83 s). DHP B Compound II was itself formed from an initial reaction between oxyferrous DHP B preincubated with 1 equiv trichlorophenol and 10 equiv H₂O₂ and reacted for 85 s prior to the second mix with 5-Br-indole. (B) Experimentally obtained spectra for Compound II derived from TCP (black, t = 2.5 ms), and Compound II observed in the presence of 5-Br-indole (t = 30 s).

Figure 8

(A) UV–vis spectra obtained under anaerobic conditions of 5-Br-3-oxindole after addition of liver esterase to a solution of 5-Br-3-acetoxyindole (∼250 μM) in 5% MeOH in 100 mM KP_i (pH 7) scanned at times indicated (aerobic). Inset: Spectral region showing minimal 5,5′-Br₂-indigo formation. (B) UV–vis spectra obtained under aerobic conditions of 5-Br-3-acetoxyindole (∼250 μM) and ferric DHP B (10 μM) in 5% MeOH in 100 mM KP_i (pH 7) prior to (black spectrum) and 0–5 min after the addition of liver esterase. (C) UV–vis spectra obtained under anaerobic conditions of 5-Br-3-oxindole (∼250 μM) and ferric DHP B (10 μM) from 3 to 12 min after the addition of liver esterase. Inset: Spectral region showing 5,5′-Br₂-indigo formation.

Figure 9

Kinetic data obtained by optical spectroscopy for the reaction of ferric DHP B with 5-Br-3-oxindole. (A) Stopped-flow UV–vis spectra of the double-mixing reaction of ferric DHP B (10 μM) with a 25-fold excess of 5-Br-3-oxindole at pH 7.0 (800 scans over 83 s). 5-Br-3-oxindole was itself formed from an initial reaction between 5-Br-3-acetoxyindole and liver esterase in an aging line prior to the second mix with ferric DHP B. (B) Experimentally obtained spectra for ferric DHP B (black, t = 2.5 ms), oxyferrous DHP B (blue, t = 8 s), and a mixture of oxyferrous DHP B and 5,5′-Br₂-indigo (red, t = 83 s).

Scheme 1. Proposed Peroxygenase Cycle for Ferric and Oxyferrous Dehaloperoxidase B