Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide

Prior spin trapping studies reported that H₂O₂ is metabolized by copper,zinc-superoxide dismutase (SOD) to form ·OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free ·OH or ·OH scavenged off the Cu²⁺ catalytic site. To distinguish whether ·OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different ·OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging ·OH as the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging ·OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free ·OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free ·OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free ·OH. EPR studies of the copper center demonstrated that while high H₂O₂ concentrations induce release of Cu²⁺, the magnitude of spin adducts produced by free Cu²⁺ was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H₂O₂ and SOD. Thus, SOD and H₂O₂ oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound ·OH not release of ·OH from the enzyme.