Key words: superoxide dismutase, eatalase, cytochrome c peroxidase, iron, hydrogen peroxide, yeast mutants,.

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1 Vl. 44, N. 3, March 1998 Pages PROTECTIVE ROLE OF SUPEROXIDE DISMUTASE IN IRON TOXICITY IN YEAST Renata Wi~nicka 1, Anna Krzepi/k 1, Jarstaw Wawryn 1, Zdzislawa Krawiec ~ and Tmasz Biliflski l'z. 1Zam~6 Cllege f Agriculture, Agricultural University f Lublin Zam~6 ul. Szczebrzeska Pedaggical University f Rzeszw Rzeszw ul. Rejtana 16A POLAND Received July 21, 1997 Received after revisin December 2, 1997 Summary It has been fund that yeast mutants deficient in cytslic superxide dismutase CuZnSOD are hypersensitive t ferrus irn. In cntrast mutants that are deficient in catalases and cytchrme c perxidase d nt differ frm the standard strain in this respect. These findings suggest that irn txicity may depend n the redx status f the cell. They als shed light n the rle f superxide dismutases in preventing the txic effects f xygen. Key wrds: superxide dismutase, eatalase, cytchrme c perxidase, irn, hydrgen perxide, yeast mutants,. Intrductin The simultaneus presence f ferrus salts and mlecular xygen in an envirnment may result in the dangerus generatin f hydrxyl radicals O1t by the fllwing reactins. 2Fe ~u) I-I +... > 2Fe ~ + H22 (Equatin l) Fe ~n) + H22... > Fe ~m) + OH +-OH (Equatin2) When taken up, ferrus catins can als be respnsible fr similar damage t intracellular cmpnents. It is f nte that H22 can als be generated by varius metablic surces(l), and thus further increase hydrxyl radical frmatin. Therefre rganisms that rived during the perid f the xidative transfrmatin f the Earth's atmsphere, as well as thse which currently thrive in waters rich in ferrus irn, had t evlve a means f preventing the mass prductin f hydrxyl radicals within the cell and cell membranes(2). One may therefre anticipate that the amunt f irn taken up has t be strictly regulated. Because mst txic xidative efl~cts are ascribed t hydrxyl radicals it is imprtant t establish the mechanisms f preventing OH frmatin in varius This research was supprted by Grant and 6PO4A frm the Plish Crrix~ttee fr Scientific Research. The authrs are als indebted t the Plish Science Fundatin fr supplying us with the scientific equipment used in this research. 2 Crrespnding authr /98/ / Cpyright by Academic Press Australia. All rights f repraductin in anyjrm reserved.

2 rganisms. Accrding t equatin 2, tw basic strategies are pssible. Organisms may either strictly cntrl the presence f hydrgen perxide within the cell r maintain ferrus irn at the lwest pssible level. Ferric irn frmed in this reactin may be repetedly cycled t the ferrus frm. Thus, the efficiency f the cellular mechanisms f irn xidatin and reductin may have an imprtant rle in the regulatin f irn txicity. Besides enzymatic mechanisms f irn reductin sme lw mlecular weight cellular reductants can als react with ferric irn. Tw f these, ascrbate and superxide radical, are cnsidered t have the mst paranlunt rle (3) Yeast cells d nt prduce ascrbate and thus nly the rle f superxide is investgated. Recent studies f the reductive irn uptake systems in yeast (4) shwed that it is cupled with ferrxidative activity f the permease cmplex. This means that althugh irn has t be reduced (5) befre its uptake, it finally enters the cell in the ferric frm. It is therefre reasnable t expect that irn txicity depends nt nly n the amunt f the metal taken up by the cell but als n the redx status f the cell. The superxide radical -O2, which is generated in cellular systems, may influence the redx status f cellular irn as shwn in Equatin 3 Fe ~ + O2... > Fe ~ + 2 (Equatin 3) It is expected that the accumulatin f superxide radicals in yeast cells deficient in superxide dismutases (6) shuld increase the steady state cncentratin f ferrus irn. It is f nte that the level f superxide within nrmal E. cil cells is 2xl-1~ which may be t lw t play an imprtant rle in irn reductin. Its level was estimated t be 6.7x1 ~ M (7) in the superxide deficient mutant. It is shwn (8) that yeast cells grwn in media cntaining glucse as a carbn surce are killed by ferrus irn in the presence f xygen. Their sensitivity t irn is dependent n the phase f grwth. The mst sensitive are cells in the expnential phase f grwth. Yeast grwn n ethanl as a carbn surce are mre tlerant f irn. Respiratry deficiency and antimycin A prevent txic effects f ferrus ins in the late expnential phase f grwth. Uncuplers such as CCCP and DNP cmpletely prevented txicity. In (8) it is shwn that irn txicity is mainly crrelated with the amunt f irn taken up by the cells and t a lesser extent n the prductin f reactive xygen species in mitchndria. The aim f this investigatin was t test the rles f cytslic superxide dismutase CuZnSOD and f the enzymes respnsible fr hydrgen perxide remval in the preventin f irn txicity in yeast under cnditins f ferrus irn verlad. 636

3 BIOCHEMISTRYnd MOLECULAR BIOLOGY INTERNATIONAL Methds Yeast strains. Strain SP-4 Mat a leul arg4 was used as a standard strain (9) Strains DSCDI-IC Mata leul arg4 sdl and A5 a leul arg4 cttl ctal were islated as described in (6) (1). All f these yeast strains have an isgenic backgrund. Strains GA74-1A Mat a leu2 ura3 his3 trpl ade8 ctal-2 CTTI:LACZ and 998 Mata LEU2 URA3 his3 trpl ade8 ctal-2 CTTI:LACZ cttl ccpl were cnstructed by Dr. G. Adam and were kindly prvided by Dr. H. Ruis, University f Vienna were als used. These tw strains differed nly in being deficient catalase T and cytchrme c perxidase. Chemicals Difc Yeast Extract, Bact Peptne and Agar were used t prepare media. The remdming chemicals were f analytical grade. Culture media A cmplete YPG medium cntaining 1% Yeast Extract, 1% Bact Peptne and 2% glucse was used. The YPG medium used fr viability tests was slidified by 2% fdifc Agar. An anaerbic medium was supplemented with.5% Tween 8 an.25% ergsterl. Grwth cnditins Cells were grwn in a standard liquid medium n a rtatry shaker (New Bnmswick G-24 at 28~ 15 rev/min) under aerbic cnditins and harvested at the expnential phase f grwth. Irn treatment Expnential cultures were centrifuged and suspended in fresh YPGlucse medium adjusted t ph 6., ptimal fr ferrus irn txicity. Ferrus salt FeSOs was added t the medium t a final cncentratin f 2 mm, and the samples were incubated aerbically r anaerbically n the rtatry shaker at t 8 rev/min. Aliquts frm all samples were taken after varius intervals and plated n a YPGlucse medium slidified with 2% Difc Agar in rder t establish the number f survivrs. Results are presented as a percentage f the nunaber f clnies at zer time. Anaerbic experiments Cells were grwn r incubated in special bttles with hydraulic valves clsed with mercury. The bttles were als equipped with a side arm t enable the additin f irn in anxia. Cultures were flushed with high purity nitrgen fr tw hurs after inculatin. Samples were remved thrugh an adjustable stpcck. Irn uptake was established accrding t (11) The adsrptin f irn by the cell wall creates serius technical prblems. Therefre, the fllwing cntrl experiments were perfrmed: cntrl withut irn, cntrl f adsrptin f ferrus irn at zer time and cntrl f ferric irn adsrptin after 2 hurs. The data presented in the tables represent results btained after 2 hurs f incubatin minus zer time cntrl. The experiments were carried ut in the apprpriate culture media as described in the sectin,,irn treatment" Hydrgen perxide decmpsitin was measured accrding t (12). Results and Discussin As shwn in equatin 2, the txic effects f ferrus irn may be influenced by changes in the level f en~mes respnsible fr hydrgen perxide remval. It was anticipated that deficiencies in catalases and cytchrme c perxidase may lead t hypersensitivity f the mutant cells t irn The results f the experiments were negative, i.e. the strain deficient in catalases A and T and cytchrme c perxidase did nt differ in sensitivity t irn frm the strain deficient nly in catalase A (nt shwn). The rle f catalase A, hwever, is unimprtant under the experimental cnditins, 637

4 BIOCHEMISTRYnd MOLECULAR BIOLOGY INTERNATIONAL because it is absent in yeast cells grwn expnentially n glucse(t3, 14). The level f cytchrme c perxidase is very lw under the experimental cnditins (15). N differences were fund between the sensitivity t irn f the standard SP-4 strain and that f its catalase deficient cunterpart. One f the explanatins fr these negative results is that these enzymes d nt play a crucial rle in the remval f hydrgen perxide. Such a rle culd be played by glutathine perxidases, whse presence was reprted in yeast (16). T test this pssibility, studies were perfrmed n the rles f catalases and cytchrme c perxidase fr the remval f extracellular hydrgen perxide. Figure 1 shws that catalases played the mst imprtant rle in hydrgen perxide remval even at lw levels f substrates, which are nt txic fr yeast cells(17). Thus, the rle f glutathine perxidase is prbably negligible. The results led t the cnclusin that 1.6 E t"- r-'-.) t- " x ' % % -3 "3.\ 9 %% % 9 %% Q. r-- Ur~.32 r-. ' ' ~'%' I I I time (min) Figure 1. Remval f hydrgen perxide by varius yeast strains. Suspensins f late expnential cells (2x19 cells/ml) pregrwn in the YPGlucse medium were treated with 2 mm hydrgen perxide. The samples were taken at time and after 2,4,6,8 and 1 minutes f incubatin. The cncentratin f H22 in the samples was determined. At the end f the experiment the viability f the cells was determined. In each case all the cells survived the experimental cnditins. diamnds- strain deficient in eatalases A, T and cytchrme e perxidase (cp 1, etal, ctt 1) triangles - strain deficient in catalases A and T (ctal, ctt 1) circles - strain deficient in catalase A (CCP1, ctal, CTT1) crsses - standard strain (CCP1, CTA1, CTT1) 638

5 intracellular presence f hydrgen perxide des nt have an imprtant rle in ferrus irn txicity. This suggests that autxidatin f ferrus irn within the cell (Equatin 1) may be an imprtant surce f hydrgen perxide fr the Fentn reactin (Equatin 2) and that it takes place in clse prximity t the cell membrane, where lcal cncentratins f ferrus irn may be high and where the scavenging enzymes cannt play their ptimum rle. In rder t study the pssible rle f superxide in irn txicity in yeast, the DSCDI-IC mutant with the sdl mutatin was used. Figure 2 shws that grwth f this mutant is inhibited by lwer cncentratins f irn in the presence f xygen than the grwth f the standard strain under these cnditins. Surprisingly, ferrus irn tlerance was much higher in the presence f xygen than in anxia. This, hwever, may be the result f rapid autxidatin f ferrus salts in 2.~a a ~ Ob,,.. \ c- O (..) (1) c'- 3>., "5 c- "s s "-5 ~,2 t ~+ ~ ~ t " t~. '='r" #' = ' ~ ~....f.,, 4-,,,'=,,'=,,'=, ~, ~ ~ ~,,~,=,=.~ Figure 2. irn cncentratin (mm) Cell densities f the standard and superxide dismutase deficient strain cultures after their grwth in the presence f varius cncentratins f ferrus irn under aerbic and anaerbic cnditins. Cells were harvested after 18 hurs. Cell densities are expressed as % f the density f the samples nt treated with irn. Anaerbic cultures were flushed with nitrgen in bttles equipped with hydraulic valves fr 2 hurs prir t the experiment. diamnds- standard strain under aerbic cnditins triangles- superxide dismutase deficient strain under aerbic cnditins crsses- superxide dismutase deficient strain under anaerbic cnditins circles- standard strain under anaerbic cnditins. 639

6 VL 44, N. 3, 1998 the neutral media. The hypersensitivity f sdl mutants t irn may result either frm an increased Fe uptake r ~m an increase in the irn reducing capacity f the mutant cell. Studies n irn uptake in mutants bearing the sdl mutatin shwed that the mutant cells accumulated much less irn than the standard cells (TaMe 1). The results als shwed that in the wild type cells irn uptake was nt strngly influenced by the remval f xygen, whereas in the mutant cells irn uptake is dramatically decreased in the presence f xygen. Thus it may be cncluded that the bserved hypersensitivity f the sdl mutant cells t irn des nt result frm an excessive irn uptake. Mrever, it may result frm an increased rate f ferric irn reductin within the cell. The results suggest that ferrus catins underg cycfic xidatin reductin prcesses, and that the rle f superxide dismutase in preventing irn txicity may be imprtant. Thus, it is difficult t evaluate an effective cncentratin f ferrus irn within the cell exclusively n the basis f irn accumulatin within the cell, The results lead t the fllwing cnclusins: an essential rle f superxide dismutases in the preventin f xygen txicity r xygen dependent ferrus irn txicity results Irn uptake by yeast. Table I Strain Gentype Cnditins f experiment Irn uptake during 2 hur perid nml/l 6 cells SP-4 a leul arg4 aerbic wild type anaerbic DSCDI-IC aleul arg4 sdl aerbic.296 CuZnSOD deficient anaerbic Irn uptake was determined as described in the Methds Sectin. Mean values btained in the cntrl experiments were: Cntrl withut irn nm/1 6 cells. Cntrl incubated fr 2 hurs in the presence f ferric irn -.3 nm/1 6 cells Values fr the zer time cntrl ranged frm.22 -,3 nm/16 cells 64

7 frm the ability f the enzymes t assure maintanance f an ptimal redx ptential f the cell. Thus these enzymes prevent xidatin f cellular sulphhydryl grups and at the same time may prevent reductin f ferric irn t its reactive ferrus frm. References 1. Fndvich, L (1984) In Methds in Enzymlgy 15 (S.P. Clwick, N.O. Kaplan, and L. Packer Eds.), pp Academic Press, Orland 2. Bilifiski, T. (1991) BiSystems 24, HalliweH, B. (1981) In Age Pigments (E.S. Shal Ed. pp Elsevier, Nrth- Hlland, Amsterdam 4. Stearman, R., Yuan, D.S., Yamaguchi-Iwai, Y., Klausner, R.D. and Dancis, A. (1996) Science 271, Lessuisse, E., Crichtn, R.R. and Labbe, P. (199) Bichim. Biphys. Acta 138, Bilifiski, T., Krawiec, Z., Liczmafiski, A. and Litwifiska, J. (1985) Bichem Biphys Res. Cmmun. 13, Imlay, J.A. and Fridvich, I. (1991) J. Bil. Chem. 266, 695% Wi~nicka, R., Krzepilk, A., Wawryn, J. and Bilifiski, T. (1997) Acta Micrbil. Pln. submitted, 9. Rytka, J., Sledziewski, A., Litwifiska, J. and Bilifiski, T. (1976) Mlec.Gen.Genet. 145, Traczyk, A., Bilifiski, T., Litwifiska, J., Skneczny, M. and Rytka, J. (1985) Acta Micrbil. Pln. 34, Tangeras, A., Fiatmark, T., B~ickstrfim, D. and Ehrenberg, A. (198) Bichim. Biphys. Acta 89, Pifferi, P.G., Lanzarini, G. and Biancani, M. (1981) Annali di Chimica Rytka, J., Sledziewski, A., Lukaszkiewicz, J. and Bilifiski, T. (1978) Mlec. Gen. Genet. 16, Crss, H.S. and Ruis, H. (1978) Mlec.Gen.Genet. 166, Sels, A.A. and Crciamnt, C. (1968) Bichem. Biphys. Res. Cmmun. 32, Galiazz, F., Schiesser, A. and Rtili, G. (1987) Bichem.Biphys.Res.Cmmun. 147, Bilifiski, T., Krawiec, Z., Litwifiska, J. and Liczmafiski, A. (1988) In Oxy-Radicals in Mlecular Bilgy and Pathlgy pp Alan R. Liss, Inc., 641

Module 3. Chapter 5 Microbial Metabolism. Catabolic and Anabolic Reactions Metabolism Two general types of metabolic reactions: o :

Module 3. Chapter 5 Microbial Metabolism. Catabolic and Anabolic Reactions Metabolism Two general types of metabolic reactions: o : Mdule 3 Chapter 5 Micrbial Metablism Catablic and Anablic Reactins Metablism Tw general types f metablic reactins: : : Recall frm Chapter 2: Energy can be when bnds Energy can be when bnds Catablism Purpse