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J Biol Chem. 2011 Aug 5;286(31):27515-27. doi: 10.1074/jbc.M111.228726. Epub 2011 Jun 1.

Arabidopsis chloroplastic glutaredoxin C5 as a model to explore molecular determinants for iron-sulfur cluster binding into glutaredoxins.

Author information

1
Unité Mixte de Recherches 1136, Institut National de la Recherche Agronomique-Nancy Université, Interactions Arbres Microorganismes, Institut Fédératif de Recherche 110 Ecosystèmes Forestiers, Agroressources, Biomolécule et Alimentation, 54506 Vandoeuvre-lès-Nancy Cedex, France.

Abstract

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

PMID:
21632542
PMCID:
PMC3149344
DOI:
10.1074/jbc.M111.228726
[Indexed for MEDLINE]
Free PMC Article

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