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Abstract
Glutaredoxins (Grxs) are small oxidoreductases particularly specialized in the reduction of protein-glutathione adducts. Compared to other eukaryotic organisms, higher plants present an increased diversity of Grxs which are organized into four classes. This work presents a thorough comparative analysis of the biochemical and catalytic properties of dithiol class I Grxs from poplar, namely GrxC1, GrxC2, GrxC3, and GrxC4. By evaluating the in vitro oxidoreductase activity of wild type and cysteine mutated variants and by determining their dithiol-disulfide redox potentials, pKa values of the catalytic cysteine, redox state changes in response to oxidative treatments, two subgroups can be distinguished. In accordance with their probable quite recent duplication, GrxC1 and GrxC2 are less efficient catalysts for the reduction of dehydroascorbate and hydroxyethyldisulfide compared to GrxC3 and GrxC4, and they can form covalent dimers owing to the presence of an additional C-terminal cysteine (CysC). Interestingly, the second active site cysteine (CysB) influences the reactivity of the catalytic cysteine (CysA) in GrxC1 and GrxC2 as already observed with GrxC5 (restricted to A. thaliana), but not in GrxC3 and C4. However, all proteins can form an intramolecular disulfide between the two active site cysteines (CysA-CysB) which could represent either a protective mechanism considering that this second cysteine is dispensable for deglutathionylation reaction or a true catalytic intermediate occurring during the reduction of particular disulfide substrates or in specific conditions or compartments where glutathione levels are insufficient to support Grx regeneration. Overall, in addition to their different sub-cellular localization and expression pattern, the duplication and maintenance along evolution of several class I Grxs in higher plants can be explained by the existence of differential biochemical and catalytic properties.
Highlights
Glutaredoxins (Grxs) are oxidoreductases sharing a conserved 3D structure with members of the thioredoxin (Trx) superfamily
All proteins can form an intramolecular disulfide between the two active site cysteines (CysA-CysB) which could represent either a protective mechanism considering that this second cysteine is dispensable for deglutathionylation reaction or a true catalytic intermediate occurring during the reduction of particular disulfide substrates or in specific conditions or compartments where glutathione levels are insufficient to support Grx regeneration
The primary function of Grxs was long thought to be the reduction of disulfide bonds and more those formed between reduced glutathione (GSH) and a protein cysteinyl residue, a process known as glutathionylation, but as explained below, specific Grx members could rather serve as iron-sulfur (FeS) cluster transfer proteins (Rouhier, 2010)
Summary
Glutaredoxins (Grxs) are oxidoreductases sharing a conserved 3D structure with members of the thioredoxin (Trx) superfamily. A few additional proteins, as some specific thioredoxins, can catalyze deglutathionylation reactions, Grxs are likely the major deglutathionylation system, at least in plants (Bedhomme et al, 2012; Chibani et al, 2012) They are usually regenerated via a NADPH/glutathione reductase (GR)/GSH system, but a few Grxs can be reduced by ferredoxin- or NADPH-dependent thioredoxin reductases (Johansson et al, 2004; Zaffagnini et al, 2008). The monothiol mechanism is used for the reduction of glutathionylated proteins and requires a priori only the catalytic cysteine (the first or more N-terminal of the two active site cysteines, referred to as CysA) It performs a nucleophilic attack on the proteinglutathione adduct, the Grx becoming glutathionylated. If the target protein is glutathionylated, the first step is similar to the monothiol mechanism, but the glutathione-adduct formed on www.frontiersin.org
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