Abstract
Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.
Highlights
Oxygen is an essential element of life, but it is at the origin of the generation of damaging reactive oxygen species (ROS)1 that are produced by cellular metabolism and mitochondrial respiration [1, 2]
The inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress, the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive
The Tsa1p null mutant is very sensitive to H2O2, whereas the Ahp1p null mutant is very sensitive to tert-butyl hydroperoxide (t-BOOH), an alkyl hydroperoxide [15]
Summary
Oxygen is an essential element of life, but it is at the origin of the generation of damaging reactive oxygen species (ROS)1 that are produced by cellular metabolism and mitochondrial respiration [1, 2]. Modification of Ahp1p in Oxidatively Stressed Cells—We carefully compared two-dimensional electrophoresis gels of total soluble proteins extracted from cells lacking superoxide dismutase activities (⌬sod1 or ⌬lys7) with those for wild type cells.
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