Purification of superoxide dismutase from fungi and characterization of the reaction of the enzyme with catechols by electron spin resonance spectroscopy.

Abstract

Superoxide dismutase catalyzes the conversion of the single electron reduced species of molecular oxygen to hydrogen peroxide and oxygen. The widely distributed copper enzyme has been purified from two fungi. Identical chromatographic and electrophoretic behavior of the enzyme isolated from different sources indicates great similarity in the molecular properties of the enzyme from eucaryotic organisms. A photosensitized recording assay procedure for the enzyme was developed which elminates the use of a second enzyme system for generating the substrate, superoxide anion. Kinetic data indicate that the reaction between enzyme and superoxide anion shows a logarithmic dependence on concentration under the conditions of the method.The fungal enzyme contains 2 mol of zinc and 2 mol of copper per mole of holoenzyme. The reaction between the enzyme-bound copper and several catechol derivatives has been examined through the use of electron spin resonance spectroscopy. It was concluded from these studies that these compounds reduce a portion of the copper to the cuprous form and also may form a complex with enzyme copper. The substituted catechols are oxidized noncatalytically by the enzyme to the semiquinone forms in the presence of oxygen. Much higher concentrations of the semiquinones are formed in the presence of the enzyme than are possible with oxygen alone at pH 8. Catechols also react readily with superoxide anion. Because of the complex relationships between catechols, superoxide anion, and superoxide dismutase, it is difficult to assess the effect of catechols on the catalytic activity of the enzyme.