Abstract
Glutaredoxins (GRXs) are thioredoxin superfamily members exhibiting thiol-disulfide oxidoreductase activity and/or iron-sulfur (Fe-S) cluster binding capacities. These properties are determined by specific structural factors. In this study, we examined the capacity of the class I Chlamydomonas reinhardtii GRX2 recombinant protein to catalyze both protein glutathionylation and deglutathionylation reactions using a redox sensitive fluorescent protein as a model protein substrate. We observed that the catalytic cysteine of the CPYC active site motif of GRX2 was sufficient for catalyzing both reactions in the presence of glutathione. Unexpectedly, spectroscopic characterization of the protein purified under anaerobiosis showed the presence of a [2Fe-2S] cluster despite having a presumably inadequate active site signature, based on past mutational analyses. The spectroscopic characterization of cysteine mutated variants together with modeling of the Fe–S cluster-bound GRX homodimer from the structure of an apo-GRX2 indicate the existence of an atypical Fe–S cluster environment and ligation mode. Overall, the results further delineate the biochemical and structural properties of conventional GRXs, pointing to the existence of multiple factors more complex than anticipated, sustaining the capacity of these proteins to bind Fe–S clusters.
Highlights
IntroductionCellular redox homeostasis is mostly controlled by oxidoreductases belonging to the thioredoxin (TRX) superfamily
In the presence of GSH or GSSG, respectively, we found that CrGRX2 can efficiently reduce and oxidize redox-sensitive green fluorescent protein 2 (roGFP2) as it was shown before for several class I GRXs from other organisms (Figure 3) [15,16,17,38]
Using the roGFP2 as a model protein substrate, we showed that CrGRX2 catalyzes both protein glutathionylation and deglutathionylation reactions in the presence of GSSG and GSH, respectively
Summary
Cellular redox homeostasis is mostly controlled by oxidoreductases belonging to the thioredoxin (TRX) superfamily. These members are characterized by the presence of a TRX fold at the structural level, which consists of a central four-stranded β-sheet surrounded by three α-helices with a βαβαββα topology [1,2]. Some of the canonical representatives in this superfamily, TRXs and glutaredoxins (GRXs), possess one or two extra α-helices at the N-terminal or C-terminal ends [3,4]. These proteins usually share a CxxC/S motif located on the loop between β1 and α1. The class II GRXs have known functions as iron–sulfur (Fe–S) cluster
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