Reactions of Bovine Liver Catalase with Superoxide Radicals and Hydrogen Peroxide

Abstract

The oxidized intermediates generated upon exposure of bovine liver catalase to hydrogen peroxide (H₂O₂) and superoxide radical (OJ) fluxes were examined with UV-visible spectrophotometry. H₂O₂ and O₂⁻ were generated by means of glucose/glucose oxidase and xanthine/xanthine oxidase systems. Serial overlay of absorption spectra in the Soret (350-450 nm) and visible (450-700 nm) regions showed that three oxidized intermediates, namely Compounds I, II and III, can be observed upon exposure of catalase to enzymatically generated H₂O₂ and O₂⁻. Compound I is formed during the reaction of native enzyme with H₂O₂ and disappears in two ways: (i) via the catalytic reaction with H₂O₂ to restore native catalase and (ii) via the reaction with OJ to form Compound II. At low H₂O₂ concentrations (<4.8 × 10⁻⁹ M H₂O₂), Compound II reverts towards the native state mainly in a direct one-step reaction, whereas at higher H₂O₂ concentrations the pathway of Compound II back to the native enzyme involves Compound III. Formation of the latter from Compound II and H₂O₂ is irreversible and the rate constant of this reaction is 6.1 ± 0.2 × 10⁴M⁻¹s⁻¹. The formation of Compound III through the direct reaction of OJ with native enzyme has also been observed. Depending on the experimental conditions, the inactivation of catalase by OJ can be due to accumulation of Compound II (“slow” inhibition) or to the formation of Compound III (“rapid” inhibition) part of which leads to a dead end product. Formation of Compound III and of this dead end product are responsible for the irreversible inactivation in presence of an excess of H₂O₂.

Key Words:

• Catalase
• Superoxide
• Hydrogen peroxide
• Compound I
• Compound II
• Compound III