The Oxidation of Catechols by Reduced Flavins and Dehydrogenases: AN ELECTRON SPIN RESONANCE STUDY OF THE KINETICS AND INITIAL PRODUCTS OF OXIDATION

Abstract

Abstract 1,2-Dihydroxybenzene-3,5-disulfonic acid (Tiron) has been investigated with regard to oxidation to the o-semiquinone form by both photochemical and enzymatic systems. The formation of the o-semiquinone radical can be recorded at pH 6.8 in the electron spin resonance spectrometer at room temperature. The kinetics of formation and decay of the radical has been determined, since it is relatively stable in solution. The parent catechol is not autoxidized to a significant extent below pH 7. The utility of Tiron as a model compound for catechol oxidation reactions lies in the stability of the o-semiquinone at lower pH values and the relative absence of side reactions or formation of highly oxidized pigments. The reaction between oxygen-reductant complexes and this catechol may be diagnostic of the formation of such intermediates where superoxide dismutase fails to inhibit effectively oxygen-dependent electron transfer. It has been previously reported (Massey, V., Palmer, G., and Ballou, D. (1971) in Flavins and Flavoproteins (Kamin, H., ed) p. 349, University Park Press, Baltimore) that in the photochemical system, reduced flavin mononucleotide reacts with oxygen to form a compound which then may dissociate to yield the flavin semiquinone and free superoxide anion. The equilibrium position for dissociation of the reduced flavin-oxygen compound may be shifted toward free superoxide anion at higher pH values. The flavin radical is shown to be unreactive with Tiron at pH 6.8. The disulfonated catechol which was used in the present work is chemically similar to catecholamines and other catechols of biological origin in that metal-catalyzed autoxidation to the o-semiquinone takes place readily above pH 8.5. Evidence is presented indicating that catechols and ferricytochrome c can react directly with the reduced flavin-oxygen compound by a pathway which is not susceptible to inhibition by superoxide dismutase. In contrast, the oxidation of Tiron by iron-flavoproteins is completely inhibited by superoxide dismutase. Thus, in the latter case, superoxide anion must be released from the enzyme active site before reacting with catechols or cytochrome c.