Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

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

The work presented in this thesis was oriented to the study of two different types of proteins isolated from sulphate reducing bacteria. One of them is the mononuclear molybdenum containing enzyme Formate dehydrogenase from D. desulfuricans ATCC 27774 (Dd Fdh). Formate dehydrogenases are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide. Dd Fdh belongs to the dimethylsulphoxide reductase family and is closely related to other Fdhs isolated from both sulphate reducing organisms and E. coli. The crystal structures of three Fdhs belonging to this family have been reported to date: the Mo-containing Fdh-H and Fdh-N from E coli; and the Wcontaining Fdh from D. gigas. The catalytic subunit of these three proteins contains the active site and one [4Fe-4S] cluster, and presents an identical fold. The oxidized active site comprises a [Mo/W(VI)] ion coordinated to four sulphurs from two pterins, a Se atom provided by the Seleno-Cysteine, and a sixth ligand whose nature is still controversial. This ligand has been identified as a hydroxyl group in Dd and E. coli Fdhs. However, the X-ray data obtained from the D. gigas enzyme suggest a sulphur atom. Despite all these enzymes catalyze a unique reaction, their physiological roles are variable, which could arise from their different cellular localization, physiological partners, and subunit and cofactor composition. The work performed was oriented to understand the gene organization, and the kinetic and EPR properties of Dd Fdh. The genes codifying the Fdh enzyme are organized as a fdhABEC cluster similar to the D. vulgaris Hildenborough Fdh-3 operon. Two gaps containing ORFs without homology with any annotated sequences were identified upstream and downstream from fdhE. Kinetic studies using deuteroformate as substrate revealed a primary isotope effect indicating that the break of the C-H bond is the rate limiting-step in the formate oxidation. Inhibition studies showed that nitrate is a competitive inhibitor whereas azide and cyanide are mixed inhibitors. The reduction of Dd Fdh with sodium formate yields a different signal from the dithionite reduced enzyme. The EPR signal obtained on formate reduction is a rhombic signal and is split by two interacting I=1/2 nuclei. One of them is not solvent exchangeable and according to the proposed structure for the Mo site is located on the β-methylene carbon of the Se-Cysteine. In contrast, the second interacting nucleus is exchangeable with solvent and could be produced by protons of a solvent molecule bound to the Mo. EPR studies in inhibiting conditions yields a signal called 2.094 which was proposed to be produced by an intermediate of the catalytic reaction in the E coli enzyme. The relevance of these paramagnetic species in catalysis as well as the nature of the six ligand of the Mo site is discussed. The second type of proteins corresponds to two proteins belonging to a new family of metalloproteins that receives the generic name of Blue proteins. These proteins were isolated from several bacteria of the Desulfovibrio genus and their function in the cell is still unknown. There are few reports on these proteins. The first ones, which date from 1978 and 1986, describe the isolation of two novel Mo-Fe containing proteins from D. africanus and D. salexigens, respectively. More recently, a homologous protein, containing Mo, Fe and Cu, was isolated from D. gigas. In this work, the Blue proteins isolated from Desulfovibrio aminophilus and Desulfovibrio alaskensis are reported. The function of both proteins is also unknown and the work was oriented to perform their primary characterization. These Blue proteins are multimeric proteins sharing low aminoacid sequence homology. The sequencing of the open reading frame showed to contain a signal peptide which suggests a periplasmic location. The high pI calculated from the deduced amino acid sequences is in agreement with the behaviour showed through anionic chromatographic columns. Metal analysis detected Mo and Fe for D. alaskensis and Cu and Fe for the D. aminophilus. X-ray adsorption spectroscopy studies suggest multinuclear clusters with potential differences in the coordination around metal ions, and which has not been observed in Mo-containing proteins.