Yeast Lacking Superoxide Dismutase(s) Show Elevated Levels of “Free Iron” as Measured by Whole Cell Electron Paramagnetic Resonance

Abstract

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of "free iron" catalyze 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 whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific 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 EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1⌬), Mn-SOD (sod2⌬), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1⌬ mutant), by pmr1, a genetic suppressor of the sod⌬ mutant phenotype (in the sod1⌬sod2⌬ double knockout strain), and by anaerobic growth. In wild-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 signals 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 results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.Superoxide dismutases are antioxidant enzymes that disproportionate superoxide (O 2 . ) (1) to hydrogen peroxide (H 2 O 2 ) and dioxygen (2-5). This class of enzyme is found in almost all aerobic organisms (6). Eukaryotes, including Saccharomyces cerevisiae, contain Mn-SOD 1 (product of the SOD2 gene) in the matrix of the mitochondria, and CuZn-SOD (product of the SOD1 gene) elsewhere in the cell, most notably in the cytoplasm, nucleus and lysosomes. Yeast cells lacking either SOD gene are viable but compromised in various ways. Yeast sod1⌬ mutant strains grow poorly in air, are very sensitive to redoxcycling drugs such as paraquat or menadione, die quickly in stationary phase, and exhibit lysine and methionine auxotrophies when grown aerobically. sod2⌬ mutants are oxygen-sensitive and, when required to respire, grow poorly and are particularly sensitive to paraquat. The double sod1⌬sod2⌬ mutant is more severely compromised, exhibiting essentially a summation of the single mutant phenotypes (7-9).We have been searching for explanations for the severity of the sod1 mutant phenotype in yeast, and thus for the toxicity of superoxide in vivo. Superoxide is more selective in its chemical reactions than most other reactive oxygen species and thus less likely to exert its toxicity throug...

The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival

“…cerevisiae and E. coli SOD mutants are more prone to mutations (Farr et al, 1986;Huang et al, 2003) and contain high levels of intracellular free iron, which is evidenced by their marked Fenton-dependent DNA damage (Srinivasan et al, 2000). Consistent with these observations, we showed that SOD mutants of C. glabrata exhibited not only high levels of O 2 $2 , but also sensitivity to DNA damage agents and a high mutation rate (Fig.…”

Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

“…For this reason, free iron is poised to participate in Fenton reactions and thereby contribute to mutation rates. Interestingly, this is not true of Saccharomyces cerevisiae, whose pools of free iron are predominantly oxidized (64). It is possible that in yeast this iron is localized in a compartment that lacks univalent reductants, thereby minimizing the impact of the iron upon oxidative DNA damage.…”

Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans

“…There are previous reports that deletion of CuZn and/or manganese SOD genes leads to accumulation of free iron in S. cerevisiae cells (70,71). Thus it appears that Cap2-mediated repression of SOD gene expression could additionally mobilize iron in vivo.…”