Specific protein cysteine residues can serve as structural components, catalysts, or redox switches. In peroxiredoxins (Prx), cysteines facilitate the enzymes ability to reduce peroxides by forming intra- or intermolecular disulfide bridges and transient mixed disulfides. Within the framework of my thesis, I aimed to further understand the underlying molecular processes. To characterize the role of specific, catalytically active cysteines during the recycling step of the catalytic cycle of Prxs, a surface plasmon resonance-based method was developed. With this new approach, which shows particular advantages over other methods investigating protein-protein interactions (PPIs), the regioselectivity of Plasmodium falciparum (Pf) thioredoxin (Trx) to recycle disulfides at active site cysteines of PfPrxs was characterized in molecular detail. The data generated were confirmed with electrophoretic mobility shift assays on a selected, representative interaction couple. To compare the SPR-based approach with other methods currently used for studying PPI, isothermal titration calorimetry and microscale thermophoresis were conducted with selected PPI partners. Comparing these three methods revealed the SPR-based approach as clearly preferable to ITC and MST in answering the leading question. Additionally, within this thesis the thiol-dependent interactomes of PfPrx1a and PfPrx1m were revealed using a pull-down assay. In the applied assay, 2-Cys Prxs and their mutants lacking the resolving or peroxidatic cysteines were used as bait to identify potential interacting partners. With this, 127 proteins were found to interact with PfPrx1a and 20 proteins with PfPrx1m via a mechanism involving disulfide bound formation. Based on bioinformatic and bibliographic analyses, the proteins identified components of various metabolic pathways such as carbohydrate metabolism, protein folding, the translational machinery, S-adenosylmethionine metabolism, signal transduction, and others. These results provide new insights into the regulatory mechanism of Prx-mediated redox biology in Plasmodium falciparum and many new candidate targets for oxidation signal transduction by PfPrxs. Furthermore, proteins caught with resolving cysteine mutants may allow the innovative pronouncement of an additional function of Prxs as proteins that are capable of reducing oxidized proteins. This follows a catalytic process similar to peroxide reduction using the second active site cysteine to resolve the transient mixed disulfide between Prx and the targeted protein. In most cells, the thioredoxin (Trx) and glutathione systems are essentially involved in maintaining redox homeostasis in a thiol-dependent mechanism. The selenoprotein thioredoxin glutathione reductase (TGR) is a hybrid enzyme in which a glutaredoxin (Grx) domain is linked to a thioredoxin reductase (TrxR) and which is also capable of reducing glutathione disulfide (GSSG), thus representing an important link between the two redox systems. In this thesis, human TGR (hTGR wild type) was recombinantly produced by fusing its open reading frame with a bacterial SECIS element and co-expressing the construct in E. coli together with the selA, selB, and selC genes. Additionally, the Sec→Cys mutant (hTGRU642C) of the full-length protein as well as the isolated TrxR domain (hTGR151-642) and the Grx domain containing a monothiol active site (hTGR1-150) were produced and purified. All four proteins were kinetically characterized in direct comparison using Trx, DTNB, HEDS, or GSSG as oxidizing substrates. Interestingly, the HEDS reduction activity was Sec independent and comparable in the full-length protein and the isolated Grx domain, whereas the TrxR and the glutathione reductase(GR) reactions were clearly Sec-dependent, with the GR reaction requiring the Grx domain. Site-directed mutagenesis studies revealed novel insights into the mechanism of GSSG reduction. Furthermore, several glutathionylation sites in hTGR were identified with an inhibitory effect of these modifications on enzyme activity. In contrast to other TGRs, e.g. from platyhelminth parasites, hTGR did not exhibit hysteretic behavior. These findings provide new insights into the reaction mechanism and regulation of monothiol Grx containing TGRs.
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