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Papers In Press, published online ahead of print July 5, 2000
Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095-1569
Corresponding Author: egralla{at}chem.ucla.edu
A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] causing release of iron and enzyme inactivation. The resulting "free iron" catalyzes deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant iron chelator, to promote oxidation of all the "free iron" to the Fe(III) state and enable detection by EPR. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZnSOD (sod1*), MnSOD (sod2*), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZnSOD, by pmr1 (a genetic suppressor of the sod* mutant phenotype),and by anaerobic growth. In wild type cells, an increase in EPR-detectable iron was induced by treatment with paraquat, a superoxide generating drug. Cells that were not pretreated with desferrioxamine had similar Fe(III) EPR signals to those from treated cells, indicating that "free iron" accumulated in the ferric form in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.
J. Biol. Chem, 10.1074/jbc.M004239200
Submitted on May 17, 2000
Revised on June 30, 2000
Accepted on July 5, 2000
Yeast lacking superoxide dismutase show elevated levels of "free iron" as measured by whole-cell EPR
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