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The peroxiredoxuis (Prxs) are a ubiquitous family of antioxidant enzymes that regulate intracellular levels of H2O2 where they are implicated in both tissue protection against oxidative stress and H2O2-niediated signalling pathways (Wood et al., 2003). This thesis describe our results on the structure-function studies of PrxIII, a mitochondrial typical 2-Cys peroxiredoxin. Bovine PrxIII was cloned previously in our laboratory; however PrxIII requires its cognate partners, mammalian mitochondrial thioredoxin (Trx2) and mammalian mitochondrial thioredoxin reductase (TRR2), to reconstitute the complete antioxidant defence system. To establish a direct in vitro assay for PrxIII, Trx2 and TRR2 were cloned, overexpressed and purified in this study. As TRR2 is a selenocysteine (SeCys) protein, a suitable selenocysteine insert sequence (SECIS) for the translation of its penultimate SeCys codon was introduced by incorporating it into the reverse primer for PCR. A combination of different approaches was used for the successful overexpression of active TRR2. Overexpression in modified rich LB media at 15°C in the presence of low IPTG concentrations gave good overexpression of soluble enzyme. Moreover, the addition of the SECIS at the C-terminal of the insert, in the presence of 1 nM Na2SeO3 and co-expression of the SelABC plasmid ensured an optimal supply of the relevant tRNA, tRNA synthase and elongation factor for translation of the UGA SeCys codon. Assays showed that NADPH-linked oxidation needed the presence of all three enzymes to reduce H2O2. PrxIII was also shown to reduce other organic peroxides, although with lower activity. Cys47 and Cysl68, but not Cys66, proved to be crucial for peroxiredoxin activity. Interestingly, at high H2O2 concentrations in the non- physiological range, TRR2 also had the capacity to reduce H2O2 directly in an NADPH-dependent manner. PrxIII is also shown to be susceptible to overoxidation and loses peroxidase activity at increased H2O2 levels in the range 50μM to 1mM. This was monitored by SDS-PAGE analysis of partially or fully overoxidised forms of H2O2-mediated PrxIII and PrxIII pathway assays. Gel filtration chromatography was used to determine under which conditions the PrxIII dodecamer would dissociate into dimers. The results show that redox state, protein concentration and the N-terminal His-tag all affect the oligomerization of PrxIII. The crystal structure of the PrxIII C168S mutant from bovine mitochondria has been determined at a resolution of 3.3 angstroms. The structure reveals that the toroid is composed of 12 (not 10) monomers with a 6(2,2) symmetry. Each ring has an external diameter of 150 angstroms and encompasses a central cavity 70 angstroms in width. Surprisingly, two PrxIII rings are mechanically interlocked in the crystal to form a protein catenane. Interestingly, the catenated form represents only a small proportion (3-5%) of the total PrxIII population, as observed by electronic microscopy studies at dilute concentration (10-50μg/ml). Preliminary analytical ultracentrifugation data suggest that 2-ring catenane formation is concentration dependent. A general model illustrating catenane formation arising from polar contacts between two basic dimeric units is described. It is not clear whether the catenated form of PrxIII has any physiological function. However, the observation that Prxs can protect cells from heat shock in a peroxidase-independent process might provide new insights into possible novel functions.