Bicarbonate is required for the peroxidase function of Cu,Zn-superoxide dismutase at physiological pH

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.