Role of strategic cysteine residues in oxidative damage to the yeast plasma membrane H(+)-ATPase caused by Fe- and Cu-containing Fenton reagents
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
14976714
DOI
10.1007/bf02993464
Knihovny.cz E-resources
- MeSH
- Cell Membrane drug effects enzymology MeSH
- Cysteine metabolism MeSH
- Copper MeSH
- Mutation MeSH
- Oxidative Stress drug effects MeSH
- Hydrogen Peroxide pharmacology MeSH
- Proton-Translocating ATPases chemistry genetics metabolism MeSH
- Saccharomyces cerevisiae chemistry drug effects enzymology MeSH
- Amino Acid Substitution MeSH
- Free Radicals MeSH
- Structure-Activity Relationship MeSH
- Iron pharmacology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Cysteine MeSH
- Fenton's reagent MeSH Browser
- Copper MeSH
- Hydrogen Peroxide MeSH
- Proton-Translocating ATPases MeSH
- Free Radicals MeSH
- Iron MeSH
Damage caused to Saccharomyces 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 beta-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.
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