Inactivation and destruction of conserved Trp159 of Fe-superoxide dismutase from Porphyromonas gingivalis by hydrogen peroxide.

Abstract

The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD-encoding gene. The purified protein contained both iron and a small amount of manganese. Iron- and manganese-reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe-reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5% of its original activity after a 15 min incubation at 25 degrees C at pH 7.8, whereas the Mn-reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe-reconstituted enzyme. Amino acid analysis of the inactivated Fe-reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron-reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse-phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2-inactivated SOD to 20% of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe-SODs and Mn-SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe-SODs and Mn-SODs may be caused by the difference in the identity of the active site metal in the Fe-SODs and Mn-SODs and a tuning of the properties of the iron center in the Fe-SODs.