title: Caldariomyces fumago chloroperoxidase catalyzes the oxidative dehalogenation of chlorophenols by a mechanism involving two one-electron steps.
abstract: We have employed rapid scan stopped-flow spectroscopy to examine whether the mechanism of oxidative dehalogenation catalyzed by C. fumago chloroperoxidase (CCPO) involves two consecutive one-electron steps or a single two-electron oxidation. First, we optimized the formation of CCPO compound I (CCPO-I) [Fe(IV)=O/porphyrin radical] and CCPO compound II (CCPO-II) [Fe(IV)=O] for use in double mixing rapid scan stopped-flow experiments. Reaction of CCPO-I with 2,4,6-trichlorophenol (TCP) quickly yielded CCPO-II. Reaction of CCPO-II, a one-electron oxidant, with TCP rapidly regenerated the ferric resting state of the enzyme. The rates of the reaction of both CCPO-I and -II with TCP are first-order with respect to [TCP]. In the absence of organic substrate, CCPO-I is slowly reduced to CCPO-II and then the ferric state. The ability of both CCPO-I and -II to carry out the oxidative dehalogenation reaction is consistent with a mechanism involving two consecutive one-electron oxidations. In contrast, reaction of CCPO-I with thioanisole generated the ferric enzyme with no evidence of CCPO-II, consistent with a single two-electron oxidation by insertion of an oxygen atom. The relative stability of CCPO-I and -II has allowed us to differentiate between one- and two-electron substrate oxidations using rapid scan stopped-flow techniques.
authors.affiliation: Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Osborne
authors.forename: Michael K
authors.initials: MK
authors.lastname: Coggins
authors.forename: James
authors.initials: J
authors.lastname: Terner
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
mesh_terms: D002384:Catalysis; D002711:Chloride Peroxidase / Q000378:metabolism*; D002733:Chlorophenols / Q000737:chemistry* / Q000378:metabolism*; D004583:Electrons; D005658:Fungi / Q000201:enzymology*; D054879:Halogenation; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013053:Spectrophotometry
chemical_list: D002733:Chlorophenols; D002711:Chloride Peroxidase