Molecular basis for the enzymatic inactivity of class III glutaredoxins on standard glutathionylated substrates

Abstract

Glutaredoxins (GRXs) are small proteins that interact with the atypical tripeptide glutathione (GSH), a redox active metabolite that forms a disulfide (GSSG) upon oxidation. GRXs encoding variants of a CPYC motif at a conserved active site act as GSH-dependent thiol-disulfide oxidoreductases (class I). GRXs with a CGFS site (class II) bind GSH in a way that is non-permissive for thiol-disulfide oxidoreductase reactions and favours transient iron-sulfur cluster binding. The biochemical functions of CCxC/S-type (class III) GRXs, which are found only in land plants, have remained largely unexplored. In this study, we characterized the in vitro properties of one of the Arabidopsis thaliana class III GRXs, namely ROXY9. In contrast to class I GRX AtGRXC2, ROXY9 was inactive as a reductase on the small substrate bis(2-hydroxyethyl)disulfide (HEDS) and as an oxidase on the redox-sensitive fluorescent protein roGFP2. Redox titrations with different GSH/GSSG ratios revealed formation of a disulfide bond (S2) between the first and the last cysteine of the CCLC motif at -220 to -230 mV. Glutathionylation (SG) at the first reactive cysteine was not observed. In contrast, AtGRXC2 and another class I GRX, HsGRX1, were oxidized to roughly equal amounts of GRX-SG and GRX-S2 over a wide range of GSH/GSSG ratios. The different reactivity of ROXY9 towards GSSG as compared to class I GRXs is most likely due to subtle differences in the GSH binding mode and explains why ROXY9 does not function as a GSH-dependent oxidoreductase on standard substrates. This feature might have evolved to avoid overlapping functions with class I GRXs in planta.