The Reaction of Chlorite with Horseradish Peroxidase and Chloroperoxidase: ENZYMATIC CHLORINATION AND SPECTRAL INTERMEDIATES

Abstract

Chloroperoxidase and horseradish peroxidase use NaClO2 as both the oxidant and the halogen donor for the peroxidative chlorination of monochlorodimedone. Previous studies have shown that both horseradish peroxidase and chloroperoxidase can catalyze iodination reactions with hydrogen peroxide as the oxidant; however, only chloroperoxidase catalyzes chlorination reactions under these conditions. The chlorite chlorination reactions obey normal Michaelis-Menten kinetics with respect to chlorite. Free chloride ion is not required for the chlorite-promoted chlorination reaction and has no effect on the Km values for chlorite or the Vmax values for either enzyme. The Vmax for the chlorination of monochlorodimedone by chloroperoxidase when chlorite is the substrate is approximately 3 times greater than when hydrogen peroxide is the oxidant, suggesting that chlorite is an exceptionally good substrate. The pH optimum for the chlorination reaction with horseradish peroxidase as catalyst is 4.1 and with chloroperoxidase a broad optimum from pH 2.25 to 3.0 is observed. Chloroperoxidase can also utilize chlorite as the oxidant for the oxidation of classical peroxidase substrates such as guaiacol, pyrogallol, and thiourea. Horseradish peroxidase reacts with chlorite to form a relatively stable product (half-life greater than 20 min) which is spectrally indistinguishable from Compound I, the first detectable intermediate formed upon the reaction of horseradish peroxidase with peroxides. The reaction yielding the horseradish peroxidase-Compound I spectrum has a precise stoichiometry. The addition of increments of chlorite to horseradish peroxidase results in the generation of a family of spectra exhibiting sharp isosbestic points at 420, 450, and 540 nm. One mole of chlorite converts 2 moles of horseradish peroxidase to the intermediate product or products having the Compound I spectrum. The addition of reducing agents such as potassium ferrocyanide to the Compound I-type intermediate results in the rapid decomposition of the intermediate and regeneration of the spectrum of the native enzyme. Incorporation studies using 36Cl indicate that the chlorine atom derived from chlorite is incorporated directly into the substrate acceptor molecule during chlorination and does not undergo exchange with free chloride ion, even in the presence of exceedingly high levels of free halide. This suggests that the reaction of chlorite with both horseradish peroxidase and chloroperoxidase results in the formation of an enzyme-bound activated halogenating intermediate. These results, in conjunction with our previous studies on the chemical nature of Compound I, suggest that the activated halogenating intermediate may be represented as an enzyme-bound halogenium ion (—X+) or hypohalite ion (—OX+) coordinated to the heme prosthetic group.