Caldariomyces fumago chloroperoxidase catalyzes the oxidative dehalogenation of chlorophenols by a mechanism involving two one-electron steps.

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.