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) (sod1Δ), Mn-SOD (sod2Δ), 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 sod1Δ mutant), by pmr1, a genetic suppressor of the sodΔ mutant phenotype (in thesod1Δsod2Δ 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. 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) (sod1Δ), Mn-SOD (sod2Δ), 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 sod1Δ mutant), by pmr1, a genetic suppressor of the sodΔ mutant phenotype (in thesod1Δsod2Δ 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 dismutase mutant yeast strains lacking either or both SODs electron paramagnetic resonance wildtype Superoxide dismutases are antioxidant enzymes that disproportionate superoxide (O⨪2) (1Sawyer D.T. Valentine J.S. Acc. Chem. Res. 1981; 14: 393-400Crossref Scopus (1203) Google Scholar) to hydrogen peroxide (H2O2) and dioxygen (2Fridovich I. Science. 1978; 201: 875-880Crossref PubMed Scopus (2771) Google Scholar, 3Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2732) Google Scholar, 4Valentine J.S. Wertz D.L. Lyons T.J. Liou L.-L. Goto J.J. Gralla E.B. Curr. Opin. Chem. Biol. 1998; 2: 253-262Crossref PubMed Scopus (183) Google Scholar, 5Beyer W. Imlay J. Fridovich I. Prog. Nucleic Acids Res. Mol. Biol. 1991; 40: 221-253Crossref PubMed Scopus (344) Google Scholar). This class of enzyme is found in almost all aerobic organisms (6McCord J.M. Keele Jr., B.B. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1024-1027Crossref PubMed Scopus (786) Google Scholar). Eukaryotes, including Saccharomyces cerevisiae, contain Mn-SOD1 (product of theSOD2 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. Yeastsod1Δ mutant strains grow poorly in air, are very sensitive to redox-cycling drugs such as paraquat or menadione, die quickly in stationary phase, and exhibit lysine and methionine auxotrophies when grown aerobically. sod2Δ mutants are oxygen-sensitive and, when required to respire, grow poorly and are particularly sensitive to paraquat. The doublesod1Δsod2Δ mutant is more severely compromised, exhibiting essentially a summation of the single mutant phenotypes (7Gralla E.B. Kosman D.J. Adv. Genet. 1992; 30: 251-319Crossref PubMed Scopus (138) Google Scholar, 8Gralla E.B. Scandalios J.G. Cold Spring Harbor Monograph Series, No. 34: Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 495-525Google Scholar, 9Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar). We have been searching for explanations for the severity of thesod1 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 through generalized oxidative damage reactions of the type characteristic of hydroxyl radicals, for example. Recently a particularly attractive mechanism of superoxide toxicity was proposed (10Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1994; 16: 29-33Crossref PubMed Scopus (365) Google Scholar), based on the observation that superoxide can very specifically oxidize exposed [4Fe-4S] clusters in certain enzymes, causing release of iron from the cluster and inactivation of the enzyme (3Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2732) Google Scholar, 11Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar, 12Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 1478-1483Abstract Full Text PDF PubMed Google Scholar, 13Gardner P.R. Fridovich I. J. Biol. Chem. 1992; 267: 8757-8763Abstract Full Text PDF PubMed Google Scholar, 14Gardner P.R. Fridovich I. Arch. Biochem. Biophys. 1993; 301: 98-102Crossref PubMed Scopus (45) Google Scholar, 15Liochev S.I. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5892-5896Crossref PubMed Scopus (189) Google Scholar, 16Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1993; 301: 379-384Crossref PubMed Scopus (62) Google Scholar, 17Murakami K. Yoshino M. Biochem. Mol. Biol. Int. 1997; 41: 481-486PubMed Google Scholar, 18Flint D.H. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. Brown O.R. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar). This process leads to further oxidative damage of other cellular components, as “free iron” 2It should be noted that there is no such chemical entity as “free iron” in an intracellular environment—it would most certainly exist only in complexes with other as yet undefined cellular components. We use “free iron” in quotes to indicate this fact. can promote, via the Fenton reaction, the formation of hydroxyl radical (⋅OH) (2Fridovich I. Science. 1978; 201: 875-880Crossref PubMed Scopus (2771) Google Scholar, 10Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1994; 16: 29-33Crossref PubMed Scopus (365) Google Scholar,19Meneghini R. Free Radic. Biol. Med. 1997; 23: 783-792Crossref PubMed Scopus (502) Google Scholar, 20Keyer K. Gort A.S. Imlay J.A. J. Bacteriol. 1995; 177: 6782-6790Crossref PubMed Scopus (188) Google Scholar, 21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar). Thus, in this scenario, superoxide is involved in the Fenton reaction by providing the necessary iron catalyst, and SOD both protects the [4Fe-4S] enzymes from inactivation and prevents the accumulation of excess intracellular iron. Support for this hypothesis has come from two directions. First, there is a demonstrated class of enzymes that contain exposed, superoxide-sensitive [4Fe-4S] cluster, the best-studied example of which is mitochondrial aconitase, which catalyzes the conversion of citrate to isocitrate in the citric acid cycle. Other such enzymes include homoaconitase (involved in lysine biosynthesis), isopropyl malate isomerase (Leu1p) (in the leucine biosynthetic pathway), and IRP1 (involved in iron sensing and response in many eukaryotes, but not yeast) (22Flint D.H. Allen R.M. Chem. Rev. 1996; 96: 2315-2334Crossref PubMed Scopus (225) Google Scholar, 23Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (588) Google Scholar, 24Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, New York1999Google Scholar). The sensitivity of aconitase activity to superoxide has been exploited as a method to measure steady state levels of superoxide in vivo (25Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 26Gardner P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (153) Google Scholar). Second, altered iron status has been demonstrated in organisms lacking SOD (21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar, 27Gort A.S. Imlay J.A. J. Bacteriol. 1998; 180: 1402-1410Crossref PubMed Google Scholar, 28De Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Escherichia coli cells lacking SOD have been to have elevated levels of iron that can be vivo by the desferrioxamine and detected by whole Fe(III) EPR (21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar). This pool of iron or “free iron” is from the of cellular iron that is to and not to the In E. the “free iron” is as of desferrioxamine the is and to which is EPR-detectable. This pool of was to damage (21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar). In we have found that a process in yeast lacking SOD have an increased for iron in aerobic growth and increased iron and accumulation certain growth Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the study, we have this to and the in vivo whole Fe(III) EPR to the iron status of yeast lacking either or both of the Our that superoxide yeast sodΔ mutants or have elevated levels of EPR-detectable which is in the Fe(III) The yeast strains used in this are in Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, I. Gralla E.B. Valentine J.S. P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, S. Gralla E.B. R. Valentine J.S. S. P. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). as and were from Yeast were either in yeast and with either or or in with as C. S. A. in Yeast Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar) that the and were increased were grown from a single in the type of that was used for further growth. of in a was a of and the were with and for paraquat were of in with or paraquat and the cells were grown for anaerobic was and were as above and grown for a strains used in this Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google sod2Δ as I. Gralla E.B. Valentine J.S. P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google with human S. Gralla E.B. R. Valentine J.S. S. P. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google in a The for EPR was from and Imlay (21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar). of the of the was and of the was for The was in of growth or The cells were with for and and with of The was in of of this was an EPR and the was on and EPR were The of the of the and the of EPR to of the to the EPR were for EPR were either a or a were the of the a with used for low temperature Fe(III) EPR were as conversion of EPR was either or the The was the is is the is the and is the resonance for our Fe(III) EPR was by double were and with of iron the of which were on the as the EPR The EPR from an Fe(III) with the intracellular yeast of and G. R. Scholar) and the of cells used for EPR were used to the iron levels yeast are of two Cells were grown as from stationary were by and in of and in of The and of the on the were The was in of acid and for or was to in and to a yeast and the sod1Δ mutant were grown to stationary in and treated with desferrioxamine to “free iron” to the Fe(III) low temperature Fe(III) EPR were and are in A. A was for the sod1Δ strain, but the of either yeast or human CuZn-SOD was to its by double to of iron on the that the yeast wild-type had a of EPR-detectable iron of whereas the yeast sod1Δ had a that was of the human CuZn-SOD in the sod1Δ which is to that to a wild-type the of EPR-detectable iron was The Fe(III) EPR is characteristic of ferric iron in a for of iron not to an EPR this A. and New Scholar), and as has been in E. desferrioxamine treatment not iron from (21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar). that this is the in S. from the that our wild-type has a very Fe(III) EPR when with the CuZn-SOD knockout strain, both contain of iron. The EPR was not by in the of iron in the used for desferrioxamine We in low in and very low in the of cells with desferrioxamine and no in the Fe(III) EPR with of not the that this was to or growth Fe(III) EPR were on from of the grown in either or In phase, cells lacking SOD had in EPR-detectable iron. to the for for for stationary and the in the EPR-detectable iron levels between the wild-type and the the growth were not to those with the were when cells were grown for not but the most were in we with those of the of the yeast sodΔ mutants have been Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, I. Gralla E.B. Valentine J.S. P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, Mol. Biol. 1995; PubMed Google Scholar, Mol. Biol. 1996; 16: PubMed Scopus Google Scholar, J. J. S. D.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the most is in a that a in have been to S. that SOD by causing the accumulation of which can superoxide Mol. Biol. 1995; PubMed Google Scholar). elevated EPR-detectable iron is to the phenotype we that this suppressor would iron levels to We the of the on iron accumulation in the yeast double knockout the EPR for yeast strains grown for and treated with The EPR-detectable iron in the was more than than that of the on the the for wild-type was and for the it was The of in the the EPR-detectable iron to than in thesod1Δsod2Δ strain, but than the in the wild-type This that the can the elevated EPR-detectable iron phenotype strain, which with the that the not the other phenotypes of thesod1Δsod2Δ of either CuZn-SOD or Mn-SOD in EPR-detectable iron. a of all our for mutants grown for The had in EPR-detectable the sod2Δ mutant had and thesod1Δsod2Δ double knockout had but from to iron increased growth as was for a growth of Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and this increase in iron can be by the increase in “free In the wild-type and single the “free iron” pool a small of the for wild-type and for the single the increased of iron the EPR-detectable pool in the single mutants is for by the increase in iron iron is in wild-type and the single the other in the double the EPR-detectable pool of the and the cells are to that the amount of iron is in thesod1Δsod2Δ mutant than it is in the increase in EPR-detectable iron in yeast lacking SOD is with elevated it should be to the in wild-type cells by their we treated wild-type cells with paraquat and EPR-detectable iron. is a redox-cycling drug that generates superoxide in vivo. very low levels of paraquat are mutant yeast, wild-type strains to growth is very paraquat cells in for had a increase in EPR-detectable iron with cells A increase in EPR-detectable iron was with paraquat, a which the growth was only not support the hypothesis that the excess EPR-detectable iron is to the deleterious of superoxide of the phenotype yeast strains is that it only aerobic In to the elevated EPR-detectable iron in sodΔ mutants is to the of aerobic wild-type and strains were in and for aerobic growth thesod1Δsod2Δ double mutant had EPR-detectable iron than the whereas anaerobic its of EPR-detectable iron was not significantly from that of the wild-type indicate that the elevated EPR-detectable iron in mutant is on the of In the we used desferrioxamine to all or “free iron” the cells and to Fe(III) that it would be by Fe(III) Fe(III) EPR signals are very and to In to the of iron as we Fe(III) EPR on with and desferrioxamine our the EPR signals in and of all the strains were results were for the wild-type and the mutant when was to not indicate that in yeast, in E. not of the EPR-detectable iron is in the Fe(III) In this study, we the iron status of yeast lacking CuZn-SOD Mn-SOD an EPR method that or “free as to iron to this method had been to E. coli lacking This method is particularly for the of in vivo as is and there is no for This method can be used to monitor the cellular oxidative status as as to a pool of iron. of EPR-detectable iron that increase superoxide levels (in mutants lacking and in wild-type yeast treated with and was not that superoxide levels growth or expression of wild-type The that increased the EPR-detectable iron and genetic of human or the of a the EPR-detectable iron are that the Fe(III) EPR signals were to as to is to that the single sod1Δ mutant yeast are to for of iron to the EPR-detectable pool by their iron 28De Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google and in that the of iron is to that of The doublesod1Δsod2Δ on the other is not to of its iron to the EPR-detectable pool and a of iron to This observation the severity of the double mutant The EPR method used for E. coli on a to all “free iron” to the Fe(III) which is by a In E. of the was indicating that in the cell, the iron was as (21Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (694) Google Scholar). A EPR method has been used to measure a “free iron” pool in this pool has been as A. A. A. E. A. 1996; PubMed Scopus Google Scholar). In yeast, on the the “free iron” to be chelator, indicating that in our it is in the Fe(III) We were by this of the results and the intracellular is very to that a pool of or iron would be in the The EPR-detectable iron pool could from an process iron by superoxide stress is by are which is and an and in an the could from a iron that an EPR and the of which is increased superoxide stress for of this pool could include a of of iron from this pool or a superoxide-sensitive in the with and from the that no iron has been in yeast, it that there is a to iron in an form that it not catalyze further damage. can thus be by a process of of iron from cluster enzymes of the in which the iron from the clusters in a form that is to the biosynthetic and iron be for use in the for of this hypothesis has been First, superoxide can oxidize certain enzymes with exposed causing the release of iron and inactivation of the enzyme D.H. Smyk-Randall E. Tuminello J.F. Draczynska-Lusiak B. Brown O.R. J. Biol. Chem. 1993; 268: 25547-25552Abstract Full Text PDF PubMed Google Scholar, D.H. Allen R.M. Chem. Rev. 1996; 96: 2315-2334Crossref PubMed Scopus (225) Google Scholar). The activity has been used to superoxide in organisms P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (153) Google Scholar). Second, superoxide stress iron in yeast Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and in E. iron from clusters is not used for K. Imlay J.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and in both yeast and E. mutants are levels of iron Freitas J.M. Liba A. Meneghini R. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 11645-11649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, Fridovich I. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Imlay J.A. J. 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A. 1999; 96: PubMed Scopus Google Scholar) and in vivo oxidative stress B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, New York1999Google Scholar). We our to and for the use of their EPR and their and We and for their with EPR the EPR and for on