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.
Highlights
Glutaredoxins (Grxs)4 are thiol-disulfide oxidoreductases present in most prokaryotic and eukaryotic organisms and belonging to the thioredoxin (Trx) superfamily [1, 2]
Whereas classical Grxs have midpoint redox potentials between Ϫ170 and Ϫ230 mV [7, 8], Chlamydomonas Grx3 forms during its catalytic cycle an atypical intramolecular disulfide between the first active site cysteine and a second cysteine located in the C-terminal part of the protein [6]
Materials—G25 columns were purchased from GE Healthcare. 2-Hydroxyethyl disulfide (HED) and 5,5Ј-dithiobis-2-nitrobenzoic acid were from Acros Organics and Pierce, respectively
Summary
Glutaredoxin; GSH, reduced glutathione; Trx, thioredoxin; At, A. thaliana; Pt, poplar; HED, 2-hydroxyethyl disulfide; CFP, cyan fluorescent protein; DTTred, reduced DTT; DTTox, oxidized DTT; GSSG, oxidized glutathione; DHA, dehydroascorbate; Prx, peroxiredoxin; Cr, C. reinhardtii. Class III, which contains Grxs with a CCXX active site motif, is found in terrestrial plants, and class IV, which consists of multidomain proteins containing in their N-terminal part a Grx module, is present in eukaryotic photosynthetic organisms [11]. Through their disulfide reductase activity, class I Grxs are involved in the stress response, catalyzing the reduction of dehydroascorbate [12], and the regeneration of type II peroxiredoxins [13] and 1-Cys methionine sulfoxide reductase B1 [14, 15]. The putative role of this Grx in plastids of Brassicaceae is discussed in the light of other functions identified so far for other Grx members
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