Cu,Zn-superoxide dismutase (SOD1) acts as a peroxidase in the presence of H2O2 at high pH (pH > 9). The high pH species of H2O2, HO2/-, was previously implicated as the reactive species. However, recent EPR studies of the enzyme performed in the physiological pH range 7.4-7.6 with the spin trap 5,5'-dimethyl-1-pyrolline-Noxide attributed the intense EPR signal of 5,5'- dimethyl-1-pyrolline-N-oxide-OH obtained from SOD1 and H2O2 to the peroxidase activity of the enzyme. The present study establishes that this intense signal is obtained only in the presence of bicarbonate. To explore the critical role of HC3/-, a comprehensive EPR investigation of the radical production and redox state of the active site copper was performed. The results indicate that HC3/- competes with other anions for the anion- binding site of SOD1 (Arg141) but does not bind directly to the copper. Structurally different anions that bind to Arg141 did not stimulate, but rather blocked, peroxidase function, ruling out an effect due to mere anion binding. However, the structurally similar anions HSeO3/- and HSO3/- mimic HCO3/- in stimulating peroxidase function. These data suggest that HCO3/- hound to Arg141 anchors the neutral H2O2 molecule at the active site copper, enabling its redox cleavage. Thus, SOD1 acquires peroxidase activity at physiological pH only in the presence of HCO3/- or structurally similar anions. Alterations in pH that shift the HCO3/-/CO2 equilibrium as occur in disease processes such as ischemia, sepsis, or shock would modulate the peroxidase function of SOD1.
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