Peroxiredoxins (Prx) are antioxidant proteins capable of decompose hydrogen peroxide, organic peroxides and peroxynitrite, using highly reactive cysteines present in their active sites to achieve reductions. Most of the Prx are found on the obligatory dimer form and after peroxide reduction, the Prx cysteines form a disulfide bond between two monomers (intermolecular), wich needs to be reduced so the protein can perform a new catalysis. Most of the Prx have their cysteines reduced by Thioredoxins (Trx), which accomplish the transfer of reducing equivalents by pairs of cysteines present in their active sites. It already has been shown that in the process of electron transfer between Trx-Prx, the formation of a transient intermolecular disulfide occurs, so the substitution of a cysteine residue for a serine could result in the formation of a complex held together by stable disulfide bond between Trx and Prx. The yeast Saccharomyces cerevisiae has five Prx isoforms, three of those are found in the cytoplasm (Tsa1, Tsa2 and Ahp1), one in mitochondria (mTpx) and another in the nucleus (Dot5). Among the cytosolic isoforms, Tsa1 and Tsa2 have constant of second order to H2O2 extremely high (2 × 10e7 M-1s-1), they share a huge similarity on its primary structure (86% identity and 96% similarity), and, in addition to the peroxidase function, under certain conditions, these enzymes may associate with themselves, forming decamer structures (±2), or structures of bigger molecular weight, wich have molecular chaperone activity. On the other hand, Ahp1 has a lower ration on the sequence of amino acids (~ 28% identity and 47% similarity) with Tsa1 and Tsa2 and does not have molecular chaperone activity. However, although few studies have indicated that Ahp1 have affinity for organic peroxides 20 x bigger than Tsa1 or Tsa2, Ahp1 is the least understood of cytosolic yeast, constants of second order to H2O2 or organic peroxides have not been determinated yet and data about its oligomeric state are really controversial. Despite the differences between Ahp1 and Tsa1/Tsa2, they are efficiently reduced by yeast cytosolic thioredoxin 1 (Trx1), an enzyme of huge biological relevance that in addition to the reduction of cytosolic Prx are involved in several other biological processes such as cell growth, apoptosis inhibition, transcription activation and DNA synthesis. However, the transfer of reducing equivalents with its substrates is still poorly understood. In previous projects developed by our research group, mutants were constructed and methodologies were developed witch made it possible to obtain protein complexes Tsa1-Trx1C33S and Ahp1-Trx1C33S, which were validated by mass spectrometry. This project aims to identify and confirm in which Ahp1 cysteine is formed the mixed disulfide with Trx1, using mass spectrometry, investigate the level of oligomeric Ahp1 when in complex with Trx1 by molecular exclusion chromatography (SEC) and dynamic light scattering (DLS), lastly, determine the constants of second order to H2O2 and organic peroxides of Ahp1 by competitive kinetic with HRP and experiments involving changes in the intrinsic fluorescence of tryptophan residues related to structural changes during the redox cycle.
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