Determination of Superoxide Free Radical Ion and Hydrogen Peroxide as Products of Photosynthetic Oxygen Reduction

Abstract

Abstract Formation of Nitrite from Hydroxylamine in the presence of illuminated chloroplast lamellae is inhibited by superoxide dismutase but not by catalase, indicating that the superoxide free radical ion and not H2O2 is responsible for the oxidation of hydroxylamine. Decarboxylation of α-keto acids on the other hand is strongly inhibited by catalase but only slightly by superoxide dismutase. Light-dependent hydroxylamine oxidation and decarboxylation of α-keto acids can be used, therefor, as specific and sensitive probes for the determination of either the superoxide free radical ion or hydrogen peroxide, respectively. Photosynthetic oxygen reduction in the presence of ferredoxin, (monitored by the above method) yields both H2O2 and O2·-. The addition of an oxygen reducing factor (ORF, solubilized by heat - treatment of washed chloroplast lamellae) instead of ferredoxin, however, stimulates only the production of H2O2 , while O2·- - formation is not observed. The cooperation of ferredoxin and ORF during photosynthetic oxygen reduction by chloroplast lamellae apparently produces H2O2 not only by dismutation of O2·-, but also by a separate mechanism involving ORF.