Functional characterization of Arabidopsis thaliana CC-type glutaredoxin ROXY9
by Pascal Mrozek
Date of Examination:2022-09-07
Date of issue:2023-07-10
Advisor:Prof. Dr. Christiane Gatz
Referee:Prof. Dr. Christiane Gatz
Referee:Prof. Dr. Ivo Feußner
Persistent Address:
http://dx.doi.org/10.53846/goediss-9989
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Abstract
English
Glutaredoxins are small proteins with a conserved thioredoxin fold. This fold allows for two functions, depending on the sequence of the active site. Class I glutaredoxins, which are characterized by a CPYC motif, show oxidoreductase activity and serve to control the redox status of reactive thiols in proteins under conditions of oxidative stress. The tripeptide glutathione serves as a cofactor for the underlying redox reactions. Class II glutaredoxins, which encode a highly conserved CGFS motif, are instrumental for the assembly and transfer of Fe-S-clusters. Here, the co-factor glutathione provides two of the four thiols that coordinate the cluster, while two cysteines of two glutaredoxins provide the other two thiols. Based on the two different functions, members of both classes do not only differ in the active site but also in their binding mode for glutathione. Because of the essential functions of glutaredoxins in the anti-oxidative system and in Fe-S-cluster biogenesis, class I and class II glutaredoxins are widely distributed from archaea to eukaryotes. Class III glutaredoxins encode a CCM/LC/S motif and are only found in land plants. Their gene family has expanded during evolution and the genome of the model plant Arabidopsis thaliana contains 21 members, which contribute to different functions in e.g. development, stress responses and adaptation to nutrient supply. Up to now, their molecular mechanism of action is unknown, but based on a number of indirect evidences, it has been speculated that they control the redox status of transcription factors of the TGA family. In this thesis, the question was addressed, whether class III glutaredoxin ROXY9 can function as an oxidoreductase or as an Fe-S-cluster binding protein. To this aim, an insect cell expression system was established which allowed to obtain mg amounts of soluble ROXY9. Class I glutaredoxin GRXC2 was used as a control in all in vitro assays. Comparison of the CD spectra of both proteins showed that ROXY9 adopts a thermostable thioredoxin fold, which is influenced by glutathione, with this influence leading to less pronounced minima as compared to GRXC2. ROXY9 did not function as a reductase in the standard HEDS assay and not as an oxidase with roGFP2 or TGA1 as substrates. A weak deglutathionylation activity was observed with the enzyme GAPDH. GRXC2 was active in these assays, with the exception of TGA1 as a substrate. In conclusion, ROXY9 rather resembles class II glutaredoxins with respect to oxidoreductase activity. Similar to class II glutaredoxins, ROXY9 was found to form dimers during in vitro reconstitution experiments with Fe and S. Dimers and monomers were separated by gel filtration and Fe and S was only detectable in the fractions containing the dimer. An interesting difference between ROXY9 and GRXC2 was observed when comparing their midpoint redox potentials. In the presence of the redox pair DTT/dithiane, both proteins were identified to have a midpoint redox potential of approximately −240 mV, with the oxidized species containing a disulphide bridge between the cysteines of the active center predominantly formed at lower redox potentials. The same midpoint redox potential was observed for ROXY9 when the redox potential was adjusted by reduced and oxidized glutathione (GSH/GSSG). Again only the reduced and oxidized species with the disulphide bridge were detected for ROXY9. In contrast, GRXC2 was already oxidized at a very low redox potential, with a glutathionylated species and the species with the disulphide bond being formed in roughly equimolar amounts. Based on these results, we postulate that the high tendency of a class I glutaredoxins to bind a glutathione moiety of GSSG or of other substrates might determine their ability to efficiently (de-)glutathionylate target molecules. We further speculate that due to a different glutathione binding mode, ROXY9 does not efficiently accept and transfer a glutathione. The highly unfavourable glutathionylation of the active site cysteine is resolved either by GSH or by the second cysteine of the active site. Thus, ROXY9 might not function as a promiscuous oxidoreductase, but could have the potential to redox modulate specific proteins.
Keywords: Glutaredoxin; Oxidoreductase; Iron Sulfur Cluster; Glutathione; TGA Transcription Factors
