title: Mechanisms of Saccharomyces cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents.
abstract: Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H+-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H2O2, Fe2+, Fe- and Cu-Fenton reagents. Inactivation by Fe2+ required the presence of oxygen and hence involved auto-oxidation of Fe2+ to Fe3+. The highest Fe2- (100 microM) and H2O2 (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe2+ content than on H2O2 concentration, occurred only when Fe2+ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN3, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDS, bipyridine, 1,10-phenanthroline). Inactivation by Fe- and Cu-Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH*. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H+-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe2+ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe2+, blocked H+-ATPase inactivation by Fe2+ and the Fenton reagent but not that caused by H2O2, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H+-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.
authors.affiliation: Institute of Botany, University of Bonn, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Stadler
authors.forename: M
authors.initials: M
authors.lastname: Höfer
authors.forename: K
authors.initials: K
authors.lastname: Sigler
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000494:pharmacology*; D015227:Lipid Peroxidation; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D022142:Secretory Vesicles / Q000201:enzymology; D013997:Time Factors
chemical_list: D002614:Chelating Agents; C045076:Fenton's reagent; D016166:Free Radical Scavengers; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D007501:Iron; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases