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.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001441:Bacteroides fragilis / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D007501:Iron / Q000302:isolation & purification*; D008345:Manganese / Q000302:isolation & purification*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D001426:Bacterial Proteins; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase; D015032:Zinc