MECHANISTIC ASPECTS OF THE OXIDATION OF PHENOTHIAZINE-DERIVATIVES BY METHEMOGLOBIN IN THE PRESENCE OF HYDROGEN-PEROXIDE

Mechanistic aspects of the reaction of hydrogen peroxide with methemoglobin with respect to phenothiazine oxidation have been studied. Three phenothiazines, methoxy- (MoPZ), chlor- (CPZ) and methoxycarbonylpromazine (MaPZ), have been used. These phenothiazines differ only in substitution at the 2-position, which contributes substantially to the electron-donating properties of these compounds. Reaction with hydrogen peroxide oxidizes methemoglobin to ferrylhemoglobin, which contains iron(IV)-oxo porphyrin moiety and a protein radical. The phenothiazines are oxidized by ferrylhemoglobin in the presence of H2O2 mainly to their sulfoxides, with a radical cation as intermediate. The conversion rates (MoPZ > CPZ > MaPZ) decrease with the electron-withdrawing ability of the 2-substituent, as indicated by Hammett sigma-para values. Hydrogen peroxide consumption during the reaction is similar for the three phenothiazines. Denaturation reactions that occur upon exposure of methemoglobin to hydrogen peroxide have been investigated. For this heme-protein cross-linking was studied by means of heme retention by the protein after methyl ethyl ketone extraction. Furthermore, oxygen consumption during the reaction was assayed, which indicates formation of protein-peroxy radicals. The extent of both heme-protein cross-linking and oxygen consumption is decreased by phenothiazines in the same order as the phenothiazine conversion rate. CPZ sulfoxide is not converted by methemoglobin in the presence of hydrogen peroxide, and CPZ sulfoxide shows no effect on heme-protein cross-linking and oxygen consumption. The results are explained by electron transfer from phenothiazine to the protein radical. Stronger electron donors (MoPZ > CPZ > MaPZ) are converted faster and by reducing the protein radical they better protect hemoglobin against denaturation. A catalytic cycle, that takes into account our observation and the existing knowledge of hemoglobin oxidation states, is presented.