PUB - Publikationen an der Universität Bielefeld

Peroxiredoxins (Prx), a ubiquitous superfamily of peroxidases, are an important component of the antioxidant network of the cell, with members of the subgroups of 2-Cys Prx and type II Prx being in focus of the present work. A comparative characterisation of cytosolic PrxII B and PrxII C was performed in order to get insight into the functional stability and the biochemical differences between these highly homologous isoforms. In comparison to other Prxs, both cytosolic isoforms, especially PrxII C, exhibited remarkably high reaction velocities for conversion of peroxides with dithiothreitol as artificial electron donor. The relative turnover of various substrates revealed preference towards small peroxides, a characteristic that was more pronounced for PrxII B with exclusive conversion of hydrogen peroxide and tertiary butylhydroperoxide. The employment of different regeneration systems resulted in activities of PrxII B and PrxII C in presence of thiredoxin (Trx) and glutaredoxin (Grx). In vitro, Trx enabled higher activity of PrxII B than of PrxII C, whereas Grx-dependent peroxidase function was more enhanced with PrxII C. The cytosolic Grx CxxC4 allowed for the regeneration of both Prxs but not the cytosolic Grx CxxC10 or two chloroplastidic Grxs. A bioinformatic analysis also indicated a relationship between both components of the thiol-disulfide redox network since PrxII B and PrxII C/D transcripts were co-regulated with those of specific members of these protein families. Moreover, the differentiation of both cytosolic isoforms could be substantiated by means of an additional in silico analysis of publicly available expression data. The comparison of transcript regulation during different developmental states, in various organs and tissues as well as in dependence of a multitude of exogenous stimuli revealed differential regulation patterns for PrxII B and PrxII C/D. The impairment of functional stability, which was observed during concentration-dependent measurements of the cytosolic type II Prx activities, might tentatively be interpreted to indicate a function of PrxII B and PrxII C in signal transduction. The inhibition of catalytic activity is hypothesised to be due to overoxidation and provides for a regulatory mechanism to control the high peroxidatic potential of PrxII B and PrxII C. Consequently, this redox-dependent modulation might allow for a function as peroxide sensor and therefore facilitates signals via influencing levels of ROS. Besides a functional characterisation of Prxs, it is important for a mechanistic understanding of catalytic functions to determine structural properties with a special focus on analysis of conformational dynamics and involved transition steps. In this context dynamical imaging via diffracted x-ray tracking (DXT) provides a new tool to investigate the complex conformational changes with high spatial and time resolution. The chloroplastidic 2-Cys Prx undergoes the necessary structural alterations that are important for its proper functionality. To establish a protein for dynamical imaging via x-ray dependent techniques, various prerequisites have to be fulfilled. Their analysis was the subject of the present work. Particular attention was given to the characterisation of functional stability of 2-Cys Prx upon x-ray treatment. In this context, amino acid variants of 2-Cys Prx were generated by site-directed mutagenesis. The modified but still functional proteins on the one hand enabled the stable immobilisation on a Ni-NTA surface by its N-terminally fused His-tag as visualised by surface plasmon resonance (SPR). On the other hand, a stable interaction of gold-nanocrystals with introduced thiol-groups of the variants was investigated as another necessity to visualise conformational changes via DXT. The specificity of binding events could be considerably increased by supplementing the binding reaction buffer with additives. The supplements minimised the unspecific hydrophobic interactions significantly. As another very important aspect, irradiation impacts on different biochemical properties of 2-Cys Prx were characterised. Besides a dose-dependency of the occurring impairments, two different amino acids could be tentatively assigned as primary sites of damage. Tyrosine residues can be hypothesised to be responsible for structural impairments that were manifested by formation of higher molecular aggregates at the expense of monomeric 2-Cys Prx. In contrast, impacts on functional features as peroxidase activity and DNA protection from ROS-mediated cleavage were likely due to modification of cysteinyl residues. Overall, the 2-Cys Prx exhibited a remarkably high stability to x-ray treatment. These characteristics underlined its suitability as candidate to be used for dynamical imaging via DXT. Furthermore, radiation-mediated damaging effects could be alleviated by adding scavenging molecules.