Defining the involvement of HOCl or Cl sub(2) as enzyme-generated intermediates in chloroperoxidase-catalyzed reactions

Peroxidatic substrates, catechol (CAT) and 2,4,6-trimethylphenol (TMP) were used as probes of the chloride-dependent reactions catalyzed by chloroperoxidase (CPO). All data are consistent with a mechanism in which chloride competes with CAT for binding to both CPO compound I and the CPO chlorinating intermediate (EOCl). Chloride binding to CPO compound I leads to the formation of EOCl and initiates the CPO chloride-dependent pathway. When CAT binds to either compound I or EOCl, it is directly oxidized to product. When chloride binds to EOCl, it either induces release of HOCl or reacts with EOCl to produce Cl sub(2), which is released from the enzyme. TMP and CAT compete for reaction with the free oxidized halogen species. This is the first direct evidence for kinetically significant involvement of a free oxidized halogen species as an intermediate in any CPO-catalyzed reaction.