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

Sornampillai Sankarapandi, Jay L. Zweier

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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.

Original languageEnglish (US)
Pages (from-to)34576-34583
Number of pages8
JournalJournal of Biological Chemistry
Issue number49
Publication statusPublished - Dec 3 1999


ASJC Scopus subject areas

  • Biochemistry

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