Skip to main content
SpringerLink
Log in

Role of strategic cysteine residues in oxidative damage to the yeast plasma membrane H+-ATPase caused by Fe- and Cu-containing fenton reagents

  • Published:
Folia Microbiologica Aims and scope Submit manuscript

Abstract

Damage caused toSaccharomyces cerevisiae SY4 plasma membrane H+-ATPase by Fe- and Cu-Fenton reagents was determined in secretory vesicles containing enzyme in which Cys residues were replaced singly or in pairs by Ala. Cys-221 situated in a β-sheet domain between M2 and M3 segments, phosphorylation domain-located Cys-409 and Cys-532 situated at the ATP-binding site play a role in the inactivation. In the presence of all three residues the enzyme exhibited a certain basic inactivation, which did not change when Cys-532 was replaced with Ala. In mutants having intact Cys-532 but lacking one or both other cysteines, replacement of Cys-221 with Ala led to lower inactivation, suggesting that Cys-221 may serve as a target for metal-catalyzed oxidation and intact Cys-532 promotes this target role of Cys-221. In contrast, the absence of Cys-409 caused higher inactivation by Fe-Fenton. Cys-532 thus seems to serve as a target for Fe-Fenton, intact Cys-409 causing a conformational change that makes Cys-532 less accessible to oxidation. The mutant lacking both Cys-221 and Cys-409 is more sensitive to Fe-Fenton than to Cu-Fenton and the absence of both Cys residues thus seems to expose presumable extra Fe-binding sites. These data and those on protection by ATP, ADP, 1,4-dithiothreitol and deferrioxamine B point to complex interactions between individual parts of the enzyme molecule that determine its sensitivity towards Fenton reagents. ATPase fragmentation caused by the two reagents differed in that the Fe-Fenton reagent produced in Western blot “smears” whereas the Cu-Fenton reagent produced defined fragments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ambesi A., Allen K.E., Slayman C.W.: Isolation of transport-competent secretory vesicles fromSaccharomyces cerevisiae.Anal. Biochem.25, 127–129 (1997).

    Article  Google Scholar 

  • Brooker R.J., Slayman C.W.: Inhibition of the plasma membrane [H+]-ATPase ofNeurospora crassa byN-ethylmaleimide. Protection by nucleotides.J.Biol.Chem.257, 12051–12055 (1982).

    PubMed  CAS  Google Scholar 

  • Davies M.J., Dean R.T.:Radical-Mediated Protein Oxidation. From Chemistry to Medicine. Oxford University Press, New York 1997.

    Google Scholar 

  • Davies M.J., Dean R.: Stable markers of oxidant damage to proteins and their application in the study of human disease.Free Rad. Biol.Med.27, 1151–1163 (1999).

    Article  PubMed  CAS  Google Scholar 

  • Dean R.T., Fu S., Stocker R., Davies M.J.: Biochemistry and pathology of radical-mediated protein oxidation.Biochem.J.324, 1–18 (1997).

    PubMed  CAS  Google Scholar 

  • Feng Y., Forgac M.: Inhibition of vacuolar H+-ATPase by disulphide bond formation between cysteine 254 and cysteine 532 in subunit A.J.Biol.Chem.269, 13224–13230 (1994).

    PubMed  CAS  Google Scholar 

  • Fiske C.H., Subbarow Z.: The colorimetric determination of phosphorus.J.Biol.Chem.66, 375–400 (1925).

    CAS  Google Scholar 

  • Goldshleger R., Patchornik G., Shimon M.B., Tal D.M., Post R.L. Karlish S.J.: Structural organization and energy transduction mechanism of Na+,K+-ATPase studied with transition metal-catalyzed oxidative cleavage.J.Bioenerget.Biomembr.33, 387–399 (2001).

    Article  CAS  Google Scholar 

  • Guerra G., Uribe S., Pardo J.P.: Reactivity of the H+-ATPase fromKluyveromyces lactis to sulfhydryl reagents.Arch.Biochem.Biophys.321, 101–107 (1995).

    Article  PubMed  CAS  Google Scholar 

  • Khansuwan U., Kotyk A.: Effects of the Fenton reagent on transport in yeast.Folia Microbiol.45, 515–520 (2000).

    Article  CAS  Google Scholar 

  • Kotyk A., Lapathitis G., Suttajit M.: Effects of a ferrate-containing preparation on diverse metabolic processes in yeast.Folia Microbiol.45, 505–508 (2000).

    Article  CAS  Google Scholar 

  • Krasowska A., Lukaszewicz M., Oświecimska M., Witek S., Sigler K.: Spontaneous and radical-induced plasma membrane lipid peroxidation in differently oxidant-sensitive yeast species and its suppression by antioxidants.Folia Microbiol.45, 509–514 (2000).

    Article  CAS  Google Scholar 

  • Krasowska A., Chmielewska L., Gapa D., Prescha A., Váchová L., Sigler K.: Viability and formation of conjugated dienes in plasma membrane lipids ofSaccharomyces cerevisiae, Schizosaccharomyces pombe, Rhodotorula glutinis andCandida albicans exposed to hydrophilic, amphiphilic and hydrophobic pro-oxidants.Folia Microbiol.47, 145–151 (2002).

    Article  CAS  Google Scholar 

  • Laemmli U.K.: Cleavage of structural proteins during the assembly of the head bacteriophage T4.Nature227, 680–685 (1970).

    Article  PubMed  CAS  Google Scholar 

  • Mahanty S.K., Scarborough G.A.: Site-ditected mutagenesis in the Cys residues in theNeurospora crassa plasma membrane H+-ATPase.J.Biol.Chem.271, 367–371 (1996).

    Article  PubMed  CAS  Google Scholar 

  • Moreau V.H., Castilho R.F., Ferreira S.T., Carvalho-Alves P.C.: Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase at submicromolar iron concentrations: evidence for metal-catalyzed oxidation.Free Rad.Biol.Med.25, 554–560 (1998).

    Article  PubMed  CAS  Google Scholar 

  • Nakamoto R.K., Rao R., Slayman C.W.: Expression of the yeast plasma membrane [H+]-ATPase in secretory vesicles. A new strategy for directed mutagenesis.J.Biol.Chem.266, 7940–7949 (1991).

    PubMed  CAS  Google Scholar 

  • Niki E.: Free radical initiators as source of water- or lipid-soluble peroxyl radicals.Meth.Enzymol.186, 100–108 (1990).

    Article  PubMed  CAS  Google Scholar 

  • Petrov V.V., Slayman C.W.: Site-directed mutagenesis of the yeast PMAI H+-ATPase. Structural and functional role of Cys residues.J.Biol.Chem.270, 28535–28540 (1995).

    Article  PubMed  CAS  Google Scholar 

  • Petrov V.V., Pardo J.P., Slayman C.W.: Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMAI). Mapping the sites of inactivation byN-ethylmaleimide.J.Biol.Chem.272, 1688–1693 (1997a).

    Article  PubMed  CAS  Google Scholar 

  • Petrov V.V., Pardo J.P., Slayman C.W.: Yeast plasma-membrane H+-ATPase: role of Cys-409 in interaction of the enzyme with NEM and FITC.Folia Microbiol.42, 249–250 (1997b).

    Article  CAS  Google Scholar 

  • Rao R., Slayman C.W.: Mutagenesis of the yeast plasma membrane H+-ATPase. A novel expression system.Biophys.J.62, 228–234 (1996a).

    Article  Google Scholar 

  • Rao R., Slayman C.W.: Plasma-membrane and related ATPases, pp. 29–56 in R. Brambl, G.A. Marzluf (Eds):The Mycota III. Biochemistry and Molecular Biology. Springer-Verlag, Berlin-Heidelberg 1996b.

    Google Scholar 

  • Scarborough G.A.: Crystallization, structure and dynamics of the proton-translocating P-type ATPase.J.Exp.Biol.203, 147–154 (2000).

    PubMed  CAS  Google Scholar 

  • Schoneich C.: Kinetics of thiol reactions.Meth.Enzymol.251, 45–55 (1995).

    Article  PubMed  CAS  Google Scholar 

  • Shimon M.B., Goldshleger R., Karlish S.J.D.: Specific Cu2+-catalyzed oxidative cleavage of Na.K-ATPase at the extracellular surface.J.Biol.Chem.273, 34190–34195 (1998).

    Article  PubMed  CAS  Google Scholar 

  • Shin J.M., Goldshleger R., Munson K.B., Sachs G., Karlish S.J.: Selective Fe2+-catalyzed oxidative cleavage of gastric H+,K+-ATPase: implications for the energy transduction mechanism of P-type cation pumps.J.Biol.Chem.276, 48440–48450 (2001).

    PubMed  CAS  Google Scholar 

  • Sies H.: Oxidative stress: oxidants and antioxidants.Exp.Physiol.82, 291–295 (1997).

    PubMed  CAS  Google Scholar 

  • Sigler K., Gille G., Vacata V., Stadler N., Hofer M.: Inactivation of the plasma membrane ATPase ofSchizosaccharomyces pombe by hydrogen peroxide and by the Fenton reagent (Fe2+/H2O2): nonradicalvs. radical-induced oxidation.Folia Microbiol.43, 361–367 (1998).

    Article  CAS  Google Scholar 

  • Sigler K., Chaloupka J., Brozmanová J., Stadler N., Höfer M.: Oxidative stress in microorganisms — I. Microbialvs. higher cells — damage and defenses in relation to cell aging and death.Folia Microbiol.44, 587–624 (1999).

    Article  CAS  Google Scholar 

  • Soundar S., Colman R.F.: Identification of metal-isocitrate binding site of pig heart NADP-specific isocitrate dehydrogenase by affinity cleavage of the enzyme by Fe2+-isocitrate.J.Biol.Chem.268, 5264–5271 (1993).

    PubMed  CAS  Google Scholar 

  • Stadler N., Höfer M., Sigler K.: Mechanisms ofSaccharomyces cerevisiae PMAI H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents.Free Rad.Res.35, 643–653 (2001).

    Article  CAS  Google Scholar 

  • Stadtman E.R., Berlett B.S.: Reactive oxygen-mediated protein oxidation in aging and disease.Chem.Res.Toxicol.10, 485–494 (1997).

    Article  PubMed  CAS  Google Scholar 

  • Valentine J.S., Wertz D.L., Gralla E.D.: The dark side of dioxygen chemistry.Curr.Opin.Chem.Biol.2, 253–262 (1998).

    Article  PubMed  CAS  Google Scholar 

  • Walworth N.C., Novick P.: Purification and characterization of constitutive secretory vesicles from yeast.J.Cell Biol.105, 163–174 (1987).

    Article  PubMed  CAS  Google Scholar 

  • Wang Y., Floor E.: Hydrogen peroxide inhibits the vacuolar H+-ATPase in brain synaptic vesicles at micromolar concentrations.J.Neurochem.70, 646–652 (1998).

    Article  PubMed  CAS  Google Scholar 

  • Wolff S.P., Dean R.T.: Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis.Biochem.J.234, 399–403 (1986).

    PubMed  CAS  Google Scholar 

  • Zhang Z., Barlow J.N., Schoefield C.J.: Metal-catalyzed oxidation and mutagenesis on the iron(II) binding site of ACC oxidase.Biochemistry36, 5999–6007 (1997).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Heart Research Institute, Camperdown, 2050, Sydney, Australia

    N. Stadler

  2. Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20, Prague, Czechia

    L. Váchová & K. Sigler

  3. Institute of Genetics and Microbiology, Wroclaw University, 51 148, Wroclaw, Poland

    A. Krasowska

  4. Institute of Botany, University of Bonn, 53 127, Bonn, Germany

    M. Höfer

Authors
  1. N. Stadler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. Váchová
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Krasowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Höfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K. Sigler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Sigler.

Additional information

The work was supported byGrant Agency of the Academy of Sciences of the Czech Republic (grant S5020202),Ministry of Education, Youth and Sports of the Czech Republic (grant ME 577), byBundesministerium für Bildung und Forschung (grant CZE 01-032) and byInstitutional Research Concept AV 0Z 502903.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stadler, N., Váchová, L., Krasowska, A. et al. Role of strategic cysteine residues in oxidative damage to the yeast plasma membrane H+-ATPase caused by Fe- and Cu-containing fenton reagents. Folia Microbiol 48, 589–596 (2003). https://doi.org/10.1007/BF02993464

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02993464

Keywords

Search

Navigation