Probing the Oxyferrous and Catalytically Active Ferryl States of Amphitrite ornata Dehaloperoxidase by Cryoreduction and EPR/ENDOR Spectroscopy. Detection of Compound I

• Published in: Journal of the American Chemical Society Vol. 132; no. 42; pp. 14995 - 15004
• Main Authors: Davydov, Roman, Osborne, Robert L, Shanmugam, Muralidharan, Du, Jing, Dawson, John H, Hoffman, Brian M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27.10.2010
• Subjects: Animals; Base Sequence; Biocatalysis; DNA Primers; Electron Spin Resonance Spectroscopy; Kinetics; Molecular Probes; Oxidation-Reduction; Peroxidases - chemistry; Peroxidases - metabolism; Polychaeta - enzymology; Spectrophotometry, Ultraviolet
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja1059747

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O2] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O2 2−] heme species, reflecting the presence of conformational substates in the oxyferrous precursor. 1H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His NεH and heme-bound O2 in these conformers are similar to those in the β-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g ⊥ = 3.75 and g ∥ = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H2O2. However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)O)(porphyrin)/Tyr•]. Addition of the substrate analogue 2,4,6-trifluorophenol (F3PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)O]. These assumptions are also supported by the observation that F3PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F3PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.