Dissertation presented to obtain the Master Degree in Molecular Genetics and BiomedicineThe following work aims to contribute to a better understanding of systems involved in resistance to oxidative stress species, namely hydrogen peroxide. The work is focused in one protein from the pathogen Campylobacter jejuni: desulforubrerythrin. Desulforubrerythrin is a non-heme iron protein in which the catalytic centre harbours a diiron cluster. Besides, the protein has a desulforedoxin domain at the N-terminal and a rubredoxin domain at the C-terminal. With the objective of understanding the protein catalytic mechanism three site-directed mutants, as well the wild type protein, were over expressed in Escherichia coli, purified and studied through biochemical and spectroscopic techniques. The amino acid residues selected for mutations are two tyrosines near the catalytic centre (residues 59 and 127). These residues are strictly conserved in rubrerythrins; moreover in diiron centres containing proteins tyrosines play a role in dissipating oxidizing species of iron (IV) by forming a tyrosil radical. The selected residues were replaced by a phenalanine residue which gave rise to three mutants: Y59F, Y127F and Y59F Y127F. These were characterized having as reference the wild type protein. All proteins have a molecular mass of 24 kDa and are tetramers in solution. The EPR and UV-visible techniques confirmed the presence of the three metallic domains in the wild type and Y59F mutant. The Y127F mutant was successfully used to test a protocol for diiron centre reconstitution in desulforubrerythrin.
Finally, crystals of the wild type and, for the first time, of the Y59F and double mutants were obtained. The X-ray data for the mutants were collected with a resolution of 1.9 Å and its structure will be determined
