Abstract
Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.
Highlights
Electrons during which the cysteines of PAPS reductase (PR) are oxidized to a disulfide
Redox Status of PAPS Reductase in Vivo—To investigate whether the limited growth of the null mutant was caused by an arrest of PR in the oxidized state, we determined the redox state of the enzyme in vivo
No oxidized PR could be detected in the null mutant transformed with the no-thiol glutaredoxin 2 (Grx2) (Fig. 2, lanes 2 and 3) or in any other strain
Summary
Grx are highly abundant proteins in E. coli [15] and contribute up to 98% of the GSH-dependent oxidoreductase activity, using the disulfide between -mercaptoethanol and GSH as a substrate (HED assay) [11]. Because of its high abundance (up to 1% of total soluble protein) and catalytic efficiency, it contributes to Ͼ80% of the cellular GSH-mixed disulfide reducing activities (in the HED assay) [17, 18]. Combined E. coli null mutants for glutathione reductase and the three glutaredoxins (gorϪgrxAϪgrxBϪgrxCϪ) barely grow on sulfate (SϩVI) but grow normally on sulfite (SϩIV) or methionine (SϪII) [22]. Because these mutants contain sufficient amounts of thioredoxin to reduce PR [15], this disturbed growth must represent some sort of inhibition of PR activity not based on the reduction of the enzyme’s disulfide that is formed upon the reduction of PAPS. Because Grx cannot reduce the disulfide of oxidized PR [4], this finding raises the possibility that the activity of PAPS reductase in vivo may be regulated by oxidized glutathione and glutaredoxins
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