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.

Heart Research Institute Camperdown 2050 Sydney Australia

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Nature. 1970 Aug 15;227(5259):680-5 PubMed

J Bioenerg Biomembr. 2001 Oct;33(5):387-99 PubMed

Biochemistry. 1997 Dec 16;36(50):15999-6007 PubMed

J Biol Chem. 1993 Mar 5;268(7):5264-71 PubMed

Folia Microbiol (Praha). 1997;42(3):249-50 PubMed

J Cell Biol. 1987 Jul;105(1):163-74 PubMed

Folia Microbiol (Praha). 2000;45(6):509-14 PubMed

J Biol Chem. 1998 Dec 18;273(51):34190-5 PubMed

Biochem J. 1986 Mar 1;234(2):399-403 PubMed

Chem Res Toxicol. 1997 May;10(5):485-94 PubMed

Folia Microbiol (Praha). 2002;47(2):145-51 PubMed

J Exp Biol. 2000 Jan;203(Pt 1):147-54 PubMed

Anal Biochem. 1997 Aug 15;251(1):127-9 PubMed

Methods Enzymol. 1995;251:45-55 PubMed

Free Radic Res. 2001 Dec;35(6):643-53 PubMed

Methods Enzymol. 1990;186:100-8 PubMed

Arch Biochem Biophys. 1995 Aug 1;321(1):101-7 PubMed

J Biol Chem. 1997 Jan 17;272(3):1688-93 PubMed

Folia Microbiol (Praha). 1999;44(6):587-624 PubMed

J Neurochem. 1998 Feb;70(2):646-52 PubMed

Exp Physiol. 1997 Mar;82(2):291-5 PubMed

J Biol Chem. 1995 Dec 1;270(48):28535-40 PubMed

J Biol Chem. 1996 Jan 5;271(1):367-71 PubMed

Free Radic Biol Med. 1999 Dec;27(11-12):1151-63 PubMed

J Biol Chem. 1982 Oct 25;257(20):12051-5 PubMed

Biophys J. 1992 Apr;62(1):228-34; discussion 235-7 PubMed

J Biol Chem. 1994 May 6;269(18):13224-30 PubMed

Folia Microbiol (Praha). 1998;43(4):361-7 PubMed

Biochem J. 1997 May 15;324 ( Pt 1):1-18 PubMed

J Biol Chem. 2001 Dec 21;276(51):48440-50 PubMed

Folia Microbiol (Praha). 2000;45(6):505-7 PubMed

J Biol Chem. 1991 Apr 25;266(12):7940-9 PubMed

Folia Microbiol (Praha). 2000;45(6):515-20 PubMed

Curr Opin Chem Biol. 1998 Apr;2(2):253-62 PubMed

Free Radic Biol Med. 1998 Sep;25(4-5):554-60 PubMed

Removal of copper ions from dilute solutions by Streptomyces noursei mycelium. Comparison with yeast biomass