Intracellular Mn(II)-associated superoxide scavenging activity protects Cu,Zn superoxide dismutase-deficient Saccharomyces cerevisiae against dioxygen stress

Three Cu,Zn superoxide dismutase (SOD-1)-deficient Saccharomyces cerevisiae mutants do not grow in 100% O₂ in rich medium and require Met and Lys when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). We show herein that medium manganese(II) accumulated by the mutants rescues these O₂-sensitive phenotypes; 2 mM medium Mn²⁺ represented the threshold required for cell growth. The accumulation of Mn²⁺ was not oxygen-inducible since mutants grown aerobically and anearobically accumulated the same amount of Mn²⁺. Mn²⁺ accumultion is not unique to these mutants since wild type accumulated almost twice as much Mn²⁺ as did mutant. ESR spectra of the cell extracts and whole cells loaded with Mn²⁺ were typical of free Mn(II) ion. These spectra could not account quantitatively for the total cellular Mn²⁺, however. A screen for soluble antioxidant activities in the Mn²⁺-supplemented cells detected O₂^- (superoxide) scavenging activity, with no change in catalase or peroxidase activities. This O₂^- scavenging activity was CN^- and heat-resistant. No achromatic bands were revealed in nondenaturing gels of Mn²⁺-containing cell extracts stained for O₂^- scavenging activity. The Mn²⁺-dependent O₂^- scavenging activity in the cell extracts was quenched by EDTA and dialyzable. More than 60% of both the intracellular Mn²⁺ and the O₂^- scavenging activity was removed by 2-h dialysis. Dialyzed cells were not viable in air unless resupplemented with either Met or Mn²⁺. Although Mn²⁺ supported the aerobic growth of these mutants, excess Mn²⁺, which correlated with an elevated O₂^- scavenging activity, was toxic to both mutant and wild type. The results indicate that free or loosely bound Mn²⁺ ion protects the mutants against oxygen stress by providing an intracellular, presumably cytosolic, O₂^- scavenging activity whith replaces the absent SOD-1.