Compartment-specific Protection of Iron-Sulfur Proteins by Superoxide Dismutase

Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, we have undertaken a genetic analysis of the interaction between iron and oxygen metabolism in Drosophila. We show that Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. We also find that exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, we find that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. We conclude that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments. Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, we have undertaken a genetic analysis of the interaction between iron and oxygen metabolism in Drosophila. We show that Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. We also find that exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, we find that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. We conclude that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments. Iron and oxygen are indispensable but potentially harmful elements of aerobic life. Individually, their reactivity has been harnessed through association with a variety of proteins and the regulation of iron and oxygen metabolism constitutes one of the major triumphs of molecular evolution (1Rouault T. Klausner R. Curr. Top. Cell Regul. 1997; 35: 1-19Crossref PubMed Scopus (213) Google Scholar). Iron sulfur cluster proteins function in electron transport during oxidative phosphorylation and metabolism, but can also serve as iron and oxygen sensors (2Beinert H. Holm R.H. Munck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1497) Google Scholar). For instance, iron regulatory protein-1 (IRP1) 1The abbreviations used are: IRP1iron regulatory protein-1IREiron responsive elementSODsuperoxide dismutase.1The abbreviations used are: IRP1iron regulatory protein-1IREiron responsive elementSODsuperoxide dismutase. exerts its dual activities through the reciprocal use or dissasembly of its cubane iron sulfur [4Fe-4S] cluster; the holoprotein functions as a cytosolic aconitase, whereas the apoprotein is an RNA-binding translational regulator (1Rouault T. Klausner R. Curr. Top. Cell Regul. 1997; 35: 1-19Crossref PubMed Scopus (213) Google Scholar, 3Haile D.J. Rouault T.A. Tang C.K. Chin J. Harford J.B. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7536-7540Crossref PubMed Scopus (215) Google Scholar). The stability and functionality of IRP1 as a translation regulator is affected not only by iron levels, but also by oxidative stress, which induces IRP1 to bind iron responsive elements (IREs) located on the 5′ and 3′ untranslated regions of target genes (4Rouault T.A. Klausner R.D. Trends Biochem. Sci. 1996; 21: 174-177Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 5Fillebeen C. Pantopoulos K. Redox Rep. 2002; 7: 15-22Crossref PubMed Scopus (38) Google Scholar). Although it is established that [4Fe-4S] cluster proteins can be specifically inactivated by superoxide (O2·¯) (6Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar, 7Flint D.H. Tuminello J.F. Emptage M.H. J. Biol. Chem. 1993; 268: 22369-22376Abstract Full Text PDF PubMed Google Scholar, 8Gardner P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), the questions of whether the IRP1 [4Fe-4S] cluster reacts with O2·¯, and the cellular origin of this O2·¯ have not yet been elucidated (9Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (294) Google Scholar, 10Bouton C. Raveau M. Drapier J.C. J. Biol. Chem. 1996; 271: 2300-2306Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 11Cairo G. Castrusini E. Minotti G. Bernelli-Zazzera A. FASEB J. 1996; 10: 1326-1335Crossref PubMed Scopus (114) Google Scholar). iron regulatory protein-1 iron responsive element superoxide dismutase. iron regulatory protein-1 iron responsive element superoxide dismutase. Studies in Saccharomyces cerevisiae have suggested an important role for cytosolic and mitochondrial superoxide dismutases (SODs) in iron metabolism (12Srinivasan C. Liba A. Imlay J.A. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 29187-29192Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13De 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 (81) Google Scholar). In addition to its function in the cytosol, SOD1 localizes in the mitochondrial intermembrane space and appears to also contribute to mitochondrial superoxide scavenging (14Sturtz L.A. Diekert K. Jensen L.T. Lill R. Culotta V.C. J. Biol. Chem. 2001; 276: 38084-38089Abstract Full Text Full Text PDF PubMed Google Scholar). Conversely, overexpression of the mitochondrial SOD2 was shown to compensate for lack of the cytosolic enzyme in a set of experiments assessing resistance to freeze-thaw stress (15Park J.I. Grant C.M. Davies M.J. Dawes I.W. J. Biol. Chem. 1998; 273: 22921-22928Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Although these results may point to some extent of functional redundancy between the two enzymes, other aspects of the Sod1Δ phenotype, such as vacuolar fragmentation, cannot be rescued by Sod2 overexpression (16Corson L.B. Folmer J. Strain J.J. Culotta V.C. Cleveland D.W. J. Biol. Chem. 1999; 274: 27590-27596Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Moreover, only recombinant bacterial FeSOD that is targeted to yeast mitochondria can rescue Sod2Δ, but it cannot rescue Sod2Δ if the mitochondrial targeting sequence is omitted and FeSOD is expressed in cytosol (17Balzan R. Bannister W.H. Hunter G.J. Bannister J.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4219-4223Crossref PubMed Scopus (29) Google Scholar, 18Balzan R. Agius D.R. Bannister W.H. Biochem. Biophys. Res. Commun. 1999; 256: 63-67Crossref PubMed Scopus (19) Google Scholar). These results suggest a functional compartmentalization of superoxide metabolism and have broad implications for both physiologic redox signaling and cellular oxidative stress (19Pani G. Bedogni B. Colavitti R. Anzevino R. Borrello S. Galeotti T. IUBMB Life. 2001; 52: 7-16Crossref PubMed Scopus (50) Google Scholar). However, the question of whether mitochondrially derived superoxide normally transfers into the cytosol (20St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. Chem. 2002; 277: 44784-44790Abstract Full Text Full Text PDF PubMed Scopus (1224) Google Scholar, 21Han D. Antunes F. Canali R.D.R Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar), or if this is an abnormality associated only with apoptosis (22Li Q.Y. Pedersen C. Day B.J. Patel M. J. Neurochem. 2001; 78: 746-755Crossref PubMed Scopus (91) Google Scholar, 23Tabuchi A. Funaji K. Nakatsubo J. Fukuchi M. Tsuchiya T. Tsuda M. J. Neurosci. Res. 2003; 71: 504-515Crossref PubMed Scopus (42) Google Scholar, 24Dussmann H. Kogel D. Rehm M. Prehn J.H.M. J. Biol. Chem. 2003; 278: 12645-12649Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) remains controversial. To address these questions, we used the cytosolic (IRP1) and mitochondrial aconitases as compartment-specific markers of O2·¯ reactivity in conjunction with genetic modulation of superoxide dismutase levels in the cytosolic and mitochondrial compartments. We present evidence that, in Drosophila, these compartments define and limit the range of O2·¯ reactivity. Drosophila Stocks—Drosophila was cultured at 25 °C on standard cornmeal agar medium. Sod1n108 and Sod1x39 represent null-activity alleles of the Sod1 gene (25Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (304) Google Scholar, 26Phillips J.P. Tainer J.A. Getzoff E.D. Boulianne G.L. Kirby K. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8574-8578Crossref PubMed Scopus (87) Google Scholar). For RNA interference studies, UAS-Sod1IR 2J. Hu, K. Kirby, A. J. Hilliker, and J. P. Phillips, unpublished results. and UAS-Sod2IR strains (27Kirby K. Hu J. Hilliker A.J. Phillips J.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16162-16167Crossref PubMed Scopus (173) Google Scholar) were crossed to daG32Gal4 (Flybase: P{GAL4-da.G32}), which provided widespread expression of the UAS-transgenes and corresponding silencing of the respective endogenous Sod genes. All RNAi experiments presented here were reproduced with different independent transgene insertions and by crossing to another ubiquitous driver line, tub-Gal4 (Flybase: P{GAL4-tubP}). For transgene-mediated overexpression of Sod2, UAS-Sod2 transformants were generated by standard embryo injection methods (28Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2330) Google Scholar) using a pUAST transformation vector (29Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar) carrying a full-length Drosophila Sod2 cDNA (30Duttaroy A. Parkes T. Emtage P. Kirby K. Boulianne G.L. Wang X. Hilliker A.J. Phillips J.P. DNA Cell Biol. 1997; 16: 391-399Crossref PubMed Scopus (38) Google Scholar) co-injected with p(Δ2–3) helper plasmid into w1 recipient embryos. Other Gal4-driver lines used in this study include D42-Gal4, which drives expression in the motorneurons (31Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (691) Google Scholar), and MHC-Gal4, which drives expression in muscle (gift from G. Boulianne, University of Toronto). Drosophila Cell Culture—Schneider II cells were maintained at 25 °C in Schneider's Drosophila medium (Invitrogen). Sixteen hours prior harvesting, either 20 and 50 μm deferoxamine or 20 and 100 μg/ml ferric ammonium citrate (Sigma) were added to the medium. RNA Mobility Shift Assay—Whole flies or cell pellets were homogenized directly in band shift buffer (40 mm KCl, 25 mm Tris HCl, pH 7.5) containing 1% Triton X-100, 5 mm dithiothreitol, and protease inhibitors. Extracts were centrifuged twice at >16000 × g on a bench-top centrifuge (4 °C), and the supernatant was immediately used for further analysis. Protein concentrations were determined using the Bradford reagent (Bio-Rad, Hercules, CA). Ten μg of total protein were added to a final volume of 12.5 μl of band shift buffer with or without 2% 2-mercaptoethanol, which activates IRP1 in vitro. The samples were incubated for 5 min at room temperature with 12.5 μl of a reaction mixture containing 20% glycerol, 0.2 units/μl Super RNAsine, 2 μg/μl yeast tRNA, 200 μm dithiothreitol, and 32P-labeled IRE from the human ferritin H chain gene (2000 counts/μl). Twenty μl of the reaction mixture were loaded onto a 10% acrylamide/Tris borate EDTA (89 mm Tris, 89 mm boric acid, 10 mm EDTA, pH 8.0 gel, run at 200 V for 2 h, then the gel was dried and exposed for autoradiography. A single band was observed, which could be competed out by addition of 10-fold excess of cold IRE probe (data not shown). Aconitase Activity Assay—Mitochondrial and cytosolic aconitase activities were assayed jointly in whole-fly extracts after electrophoretic separation. Thirty adult males were homogenized in 120 μl of extraction buffer (0.6 mm MnCl2, 2 mm citric acid, 50 mm Tris-HCl, pH 8.0) and centrifuged at 16,000 × g. Aliquots were electrophoresed on Sepraphore III membranes (Pall). Aconitase activity was detected chromogenically by incubating the membrane in 100 mm potassium phosphate, pH 6.5, 1 mm NADPH, 2 acid, mm mm 25 mm 5 All were from SOD2 Activity were homogenized in mm phosphate, pH mm The was for 10 to mitochondria and centrifuged at × g. The supernatant was incubated in mm for 1 at room temperature to SOD2 activity was then determined by the of at and °C in μl containing mm F. Biochem. PubMed Scopus Google Scholar). Protein was determined using the Bradford reagent or adult males were exposed to or 10 mm paraquat in 1% (Sigma) for or was at h, w1 flies were for aconitase and RNA shift males were 1 after in of 10 and to or Drosophila IRP1 and Aconitase in to two IRP1 with that are during M. N. D. A. P. Hentze M.W. J. Biochem. 1998; PubMed Scopus Google Scholar). These IRP1 can bind to iron responsive elements of J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and ferritin 1 chain S. K. 1998; PubMed Scopus Google Scholar) in this are to to the B.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). We if of this molecular interaction to regulation of Drosophila IRP1 by RNA shift on extracts of Drosophila cells cultured with concentrations of 1 that of iron by deferoxamine enhances of IRP1 to In addition of ferric ammonium which the [4Fe-4S] converts IRP1 to its aconitase This not between the two Drosophila IRP1 these results suggest that the [4Fe-4S] cluster that the functional between a translational regulator and a cytosolic aconitase in to iron levels is between Drosophila and IRP1 Activity in the of To whether cytosolic O2·¯ specifically reacts with the [4Fe-4S] cluster of we used a for the cytosolic Sod1 (25Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (304) Google Scholar) to a of oxidative We that this cytosolic the of IRP1 to IRE through the loss of the [4Fe-4S] cluster of IRP1 extracts of flies cytosolic Sod1 activity show IRP1 activity in to Sod1+ with an genetic IRP1 by of but then whether O2·¯ that in mitochondria with IRP1 in the for other Drosophila a of that is confined to mitochondria (30Duttaroy A. Parkes T. Emtage P. Kirby K. Boulianne G.L. Wang X. Hilliker A.J. Phillips J.P. DNA Cell Biol. 1997; 16: 391-399Crossref PubMed Scopus (38) Google Scholar). However, no Sod2 has been that could be used to a mitochondrial of oxidative stress, we strains in this that Sod2 by of RNAi (27Kirby K. Hu J. Hilliker A.J. Phillips J.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16162-16167Crossref PubMed Scopus (173) Google Scholar). For we generated strains with Hu, K. Kirby, A. J. Hilliker, and J. P. Phillips, in O2·¯ not normally membrane as suggested by its and as by experiments in cells P.R. Raineri I. Epstein L.B. White C.W. J. Biol. Chem. 1995; 270: 13399-13405Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) IRP1 is to be unaffected Sod2 expression is that IRP1 to is unaffected Sod2 is In silencing of Sod1 by the mechanism to of IRP1 in with the of IRP1 in the and by of SOD2 and established that IRP1 activity is unaffected by mitochondrial O2·¯, we if silencing Sod2 corresponding on cytosolic aconitase shown in also K. Hu J. Hilliker A.J. Phillips J.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16162-16167Crossref PubMed Scopus (173) Google Scholar) silencing of mitochondrial Sod2 has no affect on cytosolic aconitase In loss of SOD1 activity by mutation or silencing strongly but not aconitase A and the results presented in 2 and strongly for a redox in which the reactivity of O2·¯ generated within either the mitochondrial or cytosolic compartment is to protein iron-sulfur within the respective to by of of the by of SOD1 and SOD2 in the cytosolic and mitochondrial we if the two function in the of as was shown for the of Drosophila F. M. S. U. K. Phillips J.P. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, F. S. N. H. K. Hilliker A.J. Phillips J.P. H. Biol. Chem. 2003; PubMed Scopus (59) Google Scholar). To address this we UAS-Sod2 transformants that Sod2 through use of the (29Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar). the daG32Gal4 driver to Sod2 a of mitochondrial SOD2 activity We then the of Sod2 overexpression against the of a used (25Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (304) Google Scholar). In to the by of SOD1 (31Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (691) Google Scholar, I. A. Biol. 1991; PubMed Scopus Google Scholar, A.J. Parkes T.L. Kirby K. P. Boulianne G.L. Phillips J.P. Hilliker A.J. Biol. 1999; PubMed Scopus Google Scholar), of SOD2 activity provided no protection against the of paraquat This was also with of SOD2 using or muscle (data not and is with results on the of Sod2 overexpression to provide resistance to stress, and J.J. J.J. Biochem. Biophys. 1999; PubMed Scopus Google Scholar). Aconitase to IRP1 but on then the compartment-specific of paraquat exposure on the cytosolic and mitochondrial flies were exposed to the concentrations of paraquat as in but for a as to and IRP1 and aconitase activities were A in cytosolic aconitase activity and a reciprocal in IRP1 activity that, as paraquat exposure an of O2·¯ in the cytosol but not in mitochondria and The of Sod2 overexpression to protection against paraquat may from the of paraquat reactivity to the cytosol and the of SOD2 to this of SOD2 the Sod1-null we whether the of mitochondria to O2·¯ through SOD2 activity could the and of the Sod1-null A to this question the that the cytosol and mitochondria represent O2·¯ compartments in Drosophila. To this we Sod2 in the Sod1x39 genetic using or and determined the of the adults The results that, in to expression of Sod2 no on the of Sod1x39 adults it the of Sod1x39 (data not shown). We conclude that the role of SOD2 in cytosolic oxidative stress in and scavenging cytosolic O2·¯ in is These represent the in analysis in a of the of genetic modulation of cytosolic and mitochondrial SODs on the function of iron-sulfur proteins that within those respective compartments. These iron-sulfur proteins are both important cellular and of oxidative The subcellular of the cytosolic and mitochondrial aconitases and the of methods to these two activities in the provided the to investigate whether superoxide generated in the mitochondrial compartment can affect an iron-sulfur protein in the cytosol and a of genetic and methods to compartment-specific oxidative stress, we the of the cytosolic and mitochondrial The results are with the that in Drosophila, cytosolic aconitase is unaffected by superoxide generated in mitochondria and that mitochondrial aconitase is unaffected by superoxide generated in the We these results to that of O2·¯ within the compartment of its origin with the to that compartment is a of oxygen in Drosophila. this homeostatic mechanism to the that of Drosophila (25Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (304) Google Scholar, 26Phillips J.P. Tainer J.A. Getzoff E.D. Boulianne G.L. Kirby K. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8574-8578Crossref PubMed Scopus (87) Google Scholar) as with the relatively of the corresponding in R.H. Nat. Genet. 1996; PubMed Scopus Google Scholar) is at In the of iron from the reaction of O2·¯ with [4Fe-4S] clusters (12Srinivasan C. Liba A. Imlay J.A. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 29187-29192Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13De 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 (81) Google Scholar, K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), could also contribute to some of the of (25Phillips J.P. Campbell S.D. Michaud D. Charbonneau M. Hilliker A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2761-2765Crossref PubMed Scopus (304) Google Scholar). In this it is to that in iron-sulfur cluster genes have been shown to the Sod1 of S. cerevisiae J. J. S. Culotta V.C. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), that iron sulfur protein expression can O2·¯ in genes have on in Drosophila remains an The results here with a in study of mitochondria. D. Antunes F. Canali R.D.R Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar) present evidence that superoxide can be from mitochondria cultured in through between Drosophila and the use of different methods used to and superoxide and in in are to these experiments be to this These set the for of the role of Sod2, and oxidative stress in iron metabolism in Drosophila. The results presented here the of compartment in oxygen in Drosophila and could have implications in to the oxidative stress of We for and for and at Cell and for with RNA shift and for of this

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