Chlorinations catalyzed by chloroperoxidase occur via diffusible intermediate(s) and the reaction components play multiple roles in the overall process

The chlorination mechanism of the fungal enzyme chloroperoxidase (CPO) has been debated for (1) active site chlorination and (2) diffusible species mediated chlorination. Based upon the conversion of ∼ 35 different substrates belonging to different reactive groups, it was found that substrate dimensions and topography had no pronounced effect on rates of CPO chlorination reaction. Epoxidation of indene was dependent on its concentration where as chlorination was not. Also, effective conversion was seen in the chlorination mixture for substrates that could not be epoxidized or sulfoxidized. Some insoluble substrates and certain molecules that exceeded the active site dimensions were chlorinated at rates comparable to the rates required for CPO's more natural substrate, monochlorodimedone. By terminating the enzymatic reaction with an active site ligand (azide), the amount of diffusible species was correlated to CPO in the reaction mixture. The preferential utilization of a substrate, earlier attributed to the active site, is found to be due to the specificity afforded by the reaction environment. It was found that the reaction medium components of peroxide, chloride and hydronium ions affected the reaction rates through varying roles in the enzymatic and non-enzymatic process. Besides these experimental evidences, key mechanistic and kinetic arguments are presented to infer that the final chlorine transfer occurs outside the active site via a diffusible species.