Enzyme function of copper, zinc superoxide dismutase as a free radical generator

The peroxidative activity of Cu,Zn-containing superoxide dismutase (Cu, Zn-SOD) was studied by using a chromogen, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) which reacts with .OH radicals to form ABTS+, and the spin traps, N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The formation of ABTS+. in this study required both active Cu,Zn-SOD and H2O2 and followed first order kinetics with respect to SOD and H2O2. However, it showed a binding isotherm with ABTS that yielded a dissociation constant of ABTS-enzyme as Kd = 7.1 +/- 0.5 microM. The Kd values for DMPO and PBN were obtained as 0.63 and 11 mM, respectively, by competition reactions. A radical scavenger, formate anion, inhibits the formation of ABTS+., whereas ethanol does not. The results together indicate that DMPO and anionic scavengers have easy access inside the positively charged active channel of Cu,Zn-SOD and are thus in a position to intercept the newly formed .OH radicals. PBN and ethanol, however, stay outside of the channel and are not able to compete with ABTS for .OH radicals. We trapped free radicals, which were produced in the presence of free radical scavengers, with PBN. The formation curve of PBN-hydroxyethyl radical adduct observed in the presence of ethanol indicated that the enzyme became inactivated in a relatively short period. In contrast, in the presence of anionic scavenger formate, formyl radicals were produced catalytically, and the enzyme activity was protected by the formate against H2O2 inactivation. Thus Cu,Zn-SOD behaves as an enzyme that catalyzes the formation of free radicals using anionic scavengers and H2O2 as substrates.