Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: reconstitution as a Mn-containing enzyme.

Superoxide dismutase from the anaerobe Bacteroides fragilis has been purified to apparent homogeneity. The protein, Mr 42,000, is a dimer of equally sized subunits joined by noncovalent interactions. Metal analysis of the native enzyme revealed 1.8-1.9 g-atoms Fe, 0.2 g-atoms Zn, and less than 0.05 g-atoms Mn per mole dimer in a preparation whose specific activity was 1200 U/mg. Exposure of the enzyme to guanidinium chloride plus 8-hydroxyquinoline (T. Kirby, J. Blum, I. Kahane, and I. Fridovich, 1980, Arch. Biochem. Biophys. 201, 551-555) resulted in complete loss of enzymatic activity. Activity could be restored by dialysis of the denatured apoprotein against Tris buffer containing either ferrous ammonium sulfate or manganous chloride. The Fe-reconstituted enzyme was inhibited by 1 mM azide and inactivated by H2O2 in a manner similar to the native enzyme. Mn-reconstituted enzyme was inhibited by azide but resisted inactivation by H2O2 comparable to other purified manganese-containing superoxide dismutases. The manganese reconstituted protein contained approximately 1 gm-atom Mn/mol dimer. Zn ion potently inhibited reconstitution of the denatured apoprotein by either Mn or Fe and bound to the protein with a stoichiometry of 2-3 g-atoms/mol dimer.