A hybrid superoxide dismutase containing both functional iron and manganese.
A hybrid superoxide dismutase containing functional Mn and Fe has been isolated from Escherichia coli. Streptomycin, which binds tightly to both the Mn- and the Fe-containing superoxide dismutases, had the expected effect on the electrophoretic and chromatographic behavior of the hybrid. Treatment of the hybrid with H2O2, which selectively inactivates the Fe-containing enzyme, resulted in partial inactivation accompanied by a resegregation of subunits, with the formation of active Mn-enzyme and inactive Fe-enzyme. A similar resegregation of subunits was observed when the hybrid was exposed to 2.5 M guanidinium chloride. Hybrids containing Mn or Fe could be generated in vitro by mixing the Mn-enzyme with the Fe-enzyme, removing metals with 8-hydroxyquinoline in the presence of 2.5 M guanidinium chloride, and then dialyzing against Mn(II) or Fe(II) salts. Ten per cent of the activity of the Fe-superoxide dismutases is resistant to H2O2, which correlates with its content of Mn. Since the activity remaining after exhaustive treatment with H2O2 exhibited the electrophoretic mobility of the Fe-enzyme, we concluded that some of the active sites of the Fe-enzyme were actually occupied by Mn. It should be noted, however, that for purposes of metal reconstitution experiments, a definite specificity was demonstrated. The Mn-enzyme was reconstituted with Mn(II), whereas the Fe-enzyme activity was recovered using only Fe(II). We propose that the Fe-superoxide dismutase may be heterogeneous and that 10% of its activity is actually due to a Mn-containing variant with the same electrophoretic mobility. Only the apohybrid enzyme regained enzymatic activity using both Mn(II) and Fe(II).
D004926:Escherichia coli / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000032:analysis*; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000032:analysis*; D055503:Protein Multimerization; D013482:Superoxide Dismutase / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D008345:Manganese; D006861:Hydrogen Peroxide; D007501:Iron; D013482:Superoxide Dismutase