Periplasmic nitrate reductases: structural and spectroscopic studies

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica, especialidade Bioquímica-Física, pela Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa

Nitrate reduction occurs in the cell in order to incorporate nitrogen into biomolecules(assimilatory ammonification), as the final electron acceptor when bacteria are grown in anaerobic conditions (denitrification/dissimilatory ammonification) and to eliminate energy excess generated by the cell metabolism (dissimilatory ammonification). Nitrate reductases are enzimes that catalize the conversion of nitrate to nitrite. Most nitrate reductases are mononuclear molybdenum-containing enzymes that have, besides the Mo-pterin cofactor, additional metallic centers such as iron-sulfur clusters and b- or c-type hemes that mediate electron transfer reactions between the electron donor and the nitrate. Nitrate reductases have been classified into four groups: (1) eukaryotic nitrate reductases (Euk-NR), (2) assimilatory nitrate reductases (Nas), (3) respiratory nitrate reductases (Nar) and (4) periplasmic nitrate reductases (Nap). Naps isolated from Desulfovibrio desulfuricans (Dd), Paracoccus pantotrophus (Pp) and Rhodobacter sphaeroides (Rs) constitute the best-characterized Naps,so far. The 3D X-ray structure of Dd NapA in its oxidized form was the first reported structure for these enzymes. The active site in the oxidized state is made up by a distorted Mo6+ ion hexacoordinated by four sulfurs from the two pterin cofactors, the γ-sulfur from a cysteine (Cys140), and a hydroxo/water ligand. This thesis deals with the study of the nitrate reductases isolated from Desulfovibrio desulfuricans ATCC 27774 and Cupriavidus necator H16. Both enzymes were purified up to electrophoretic homogeinity. Nap purified from D. desulfuricans ATCC 27774 cells grown in anaerobeosis and in the presence of nitrate was isolated as a soluble monomeric protein of ~80 kDa. Kinetic studies show that, in contrast to dimeric Naps, this enzyme presents substrate promiscuity since it is able to reduce not only nitrate but also other anions of similar structure. EPRmonitored redox titrations, carried out with and without nitrate in the potential range from 200 to -500 mV (vs. NHE), and EPR studies in both catalytic and inhibiting conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site shows a high coordination flexibility. These studies show that nitrate modulates the redox properties of the active site, but not those of the iron-sulfur cluster. Nap purified from Cn H16 cells grown in aerobic conditions in the absence of nitrate was isolated as a soluble heterodimeric protein with large and small subunits of ~90 and ~15 kDa, respectively. Preliminary results are reported for this protein. The biochemical, structural, and spectroscopic properties of both enzymes are discussed in comparison with those of other closely related proteins.