| Popis: |
Firstly, isolated from Desulfovibrio gigas, the Orange Protein presents a unique type of mixed metal sulphur cluster composed by two different metals, molybdenum and copper. This protein forms a complex with other proteins, that has recently been proposed to be involved in anaerobic cell division of D. vulgaris Hildenborough. In this Master thesis, DVU2103 ATPase from D. vulgaris Hildenborough was purified and biochemically characterized. Since the metal cluster present in this protein has been proven to be oxygen sensitive, the purification was performed under anoxic environment. The protein was co-purified with other proteins of the orp operon, DVU2108 and possibly DVU2104, as a protein complex (heterotrimer) confirming that this complex has physiological meaning. The protein complex was purified with an average yield of 58 ± 28 μg, and presents 5.3 ± 0.3 Fe atoms/Total protein. DVU2103 complex presents a broad absorption band at 400 nm in its visible spectrum characteristic of [4Fe-4S] clusters and ICP-AES analysis confirm the presence of either one or two [4Fe4S] clusters. As-isolated protein is mainly EPR active presenting a rhombic signal, with g values of 2.06, 1.89 and 1.85, that switches to an axial signal when reduced with dithionite. Dithionite, unlike ascorbate, is responsible for the full reduction of the metallic centre. Studies to determine the apparent molecular mass of the complex revealed that ATP affects its conformational structure, making it more compact, and oxic conditions lead to the destruction of the [Fe-S] cluster, with concomitant formation of a larger oligomer. ATPase activity of “DVU2103” complex was tested under anoxic and oxic conditions, showing that the protein has a higher activity under the former. Considering that the exposure to oxygen leads to the destruction of the [Fe-S] cluster, it can be concluded that it is essential for higher activity. In this thesis the homologous expression and purification of DVU2108 in D. vulgaris Hildenborough was also performed. No Mo/Cu heterometallic cluster was detected in this protein after purification under anoxic conditions, nevertheless Fe/protein ratio of 1 was estimated for this sample. |
