Studies of the chlorinating intermediate of horseradish peroxidase

The chlorinating intermediate of horseradish peroxidase, Compound X, is the only know stable enzymatic halogenating intermediate known. The optical spectrum of Compound X is virtually identical to the optical spectrum of Compound II. Titration of Compound X by a single electron donor, potassium ferrocyanide reduces the Compound II type spectrum to a native type spectrum. Incubation of these titrated solutions with the chlorine acceptor, monochlorodimedone, MCD, shows that titrated Compound X retains a chlorine. The chlorinating properties of Compound X is therefore independent of the oxidation state of the iron. Furthermore, the optical spectra indicate that the chlorine moiety in Compound X is not associated with the heme prosthetic group. The location of the chlorine moiety in Compound X must therefore be associated with an amino acid residue. The identification of this modified residue as ASP-43 is proposed based on spectroscopic and kinetic evidence.The kinetic data presented here merges the peroxidatic and chlorinating cycles of horseradish peroxidase. Compound X is the first intermediate formed in the reaction between horseradish peroxidase and sodium chlorite at all pH values. Previous investigators reported that the first product formed in the reaction between horseradish and chlorite was Compound I. In contrast to these reports this thesis shows that Compound X is the first product. At acidic pH, Compound X is unstable and decays to Compound I in the millisecond time range. A mechanism for the formation of Compound X and subsequent decay to Compound I is proposed.U of I OnlyETDs are only available to UIUC Users without author permissio