Chemical modification of iron- and manganese-containing superoxide dismutases from Escherichia coli

The manganese-containing (MnSOD) and iron-containing (FeSOD) superoxide dismutases from Escherichia coli are extensively (>95%) inactivated by treatment with phenylglyoxal. The relatively high concentrations of phenylglyoxal and high pH required for optimal inactivation suggest that inactivation may be due to modification of an arginine with a “normal” elevated p K a , i.e., one not in an active site cavity where the p K a is likely to be lowered because of lower solvent accessibility and decreased polarity of the local environment. Treatment of either enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2-hydroxy-5-nitrobenzyl bromide, m-chloroperoxybenzoate, or tetranitromethane causes no inactivation, while 2,4,6-trinitrobenzenesulfonate, N-acetylimidazole, or diethyl pyrocarbonate cause 55–75% inactivation of each enzyme. Failure of hydroxylamine to reverse inactivation by the latter two suggests that in each instance loss of activity is due to lysine modification. The previously reported inactivation of FeSOD by H 2O 2 was further investigated, and no evidence was found for an affinity mechanism, i.e., a reversible binding of peroxide that precedes inactivation.