Mechanism of the chlorination reaction catalyzed by horseradish peroxidase with chlorite.

Horseradish peroxidase and chlorite, NaC102, are able to catalyze chlorination of monochlorodimedone to form dichlorodimedone. Catalytic amounts of horseradish peroxidase act to disproportionate chlorite forming chlorine dioxide and chloride ion. The chlorine dioxide thus formed is responsible for the chlorination of monochlorodimedone. It was previously thought (Chiang, R., Rand-Meir, T., Makino, R., and Hager, L. P. (1976) J. Biol. Chem 251, 6340-6346) that the initial transient intermediate formed between horseradish peroxidase and chlorite was a halogenating intermediate and was called Compound X. However, complete transient state spectra of the enzyme species formed directly from horseradish peroxidase with chlorite shows this intermediate to be Compound II. This result is the first recognized example of direct oxidation of native horseradish peroxidase to Compound II. The disproportionation of chlorite by horseradish peroxidase was investigated in the absence of a halogen acceptor. At pH 5.05, 5 mol of chlorite are disproportionated to 4 mol of chlorine dioxide and 1 mol of chloride ion. Chlorine dioxide yields are limited by the inactivation of the enzyme by chlorine dioxide. A maximum yield of 2.5 x lo3 nmol of chlorine dioxide/nmol of horseradish peroxidase was obtained by optimizing the pH and optimizing the enzyme/chlorite ratio. Titration of horseradish peroxidase with chlorite showed a 1:l molar stoichiometry for the formation of Compound I. The reaction between the enzyme and excess chlorite failed to produce measurable quantities of hydrogen peroxide. The reaction of chlorine dioxide with monochlorodimedone was also studied in the absence of horseradish peroxidase and chlorite. Chlorine dioxide and monochlorodimedone react with a 1:l molar stoichiometry to form 1 mol of protons, 0.4 mol of dichlorodimedone, and 0.6 mol of chlorite.