C. Srinivasan et al., Yeast lacking superoxide dismutase(s) show elevated levels of "free iron" as measured by whole cell electron paramagnetic resonance, J BIOL CHEM, 275(38), 2000, pp. 29187-29192
A current hypothesis explaining the toxicity of superoxide anion in vivo is
that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes c
ausing release of iron and enzyme inactivation. The resulting increased lev
els of "free iron" catalyze deleterious oxidative reactions in the cell. In
this study, we used low temperature Fe(III) electron paramagnetic resonanc
e (EPR) spectroscopy to monitor iron status in whole cells of the unicellul
ar eukaryote, Saccharomyces cerevisiae. The experimental protocol involved
treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-speci
fic chelator, to promote oxidation of all of the "free iron" to the Fe(III)
state wherein it is EPR-detectable. Using this method, a small amount of E
PR-detectable iron was detected in the wild-type strain, whereas significan
tly elevated levels were found in strains lacking CuZn-superoxide dismutase
(CuZn-SOD) (sod1 Delta), Mn-SOD (sod2 Delta), or both SODs, throughout the
ir growth but particularly in stationary phase, The accumulation was suppre
ssed by expression of wild-type human CuZn-SOD (in the sod1 Delta mutant),
by pmr1, a genetic suppressor of the sod Delta mutant phenotype (in the sod
1 Delta sod2 Delta double knockout strain), and by anaerobic growth. In wil
d-type cells, an increase in the EPR-detectable iron pool could be induced
by treatment with paraquat, a redox-cycling drug that generates superoxide.
Cells that were not pretreated with desferrioxamine had Fe(III) EPR signal
s that were equally as strong as those from treated cells, indicating that
"free iron" accumulated in the ferric form in our strains in vivo. Our resu
lts indicate a relationship between superoxide stress and iron handling and
support the above hypothesis for superoxide-related oxidative damage.