TY  - THES
AB  - According to the current knowledge, the Mo storage protein and its tungstate containing analogue, both intensively studied in the present work, are the only proteins in nature binding polyoxomolybdate or polyoxotungstate clusters, respectively. Therefore this storage system combines the fields of biomacromolecules and inorganic supramolecules. The Mo storage protein is a part of the nitrogen fixation process in A. vinelandii accumulating large amounts of Mo inside the cell and providing, if required, Mo for the nitrogenase system. The high-capacity of Mo storage protein in combination with the high-affinity Mo transport proteins (Mo uptake into the cell) represent a system that supply Mo to the organism in sufficient amounts even at temporarily lack of Mo which thus guarantee A. vinelandii a significant competitive advantage towards other diazotrophic bacteria.
Biochemical studies indicated that the release of Mo (as molybdate) from the Mo storage protein (MoSto) is strongly influenced by temperature and pH value and other factors like incubation time, protein concentration and degree of purity. A detailed pH titration (from slightly acidic to alkaline region) revealed that for the Mo release process three different Mo release steps can be distinguished. Each release step appears to be accompanied by a deprotononation of a amino acid (histidine) and/or the decomposition of polymolybdate clusters which become distinctly unstable upon increasing the high OH- concentration. The Mo release process does not require ATP but the cosubstrate prevents the Mo release or at least reduces the degree of the Mo release, respectively. Consequently, in addition to the pH value ATP plays an important role in intracellular control of Mo release. The presence of both ATP and MgCl2 is the essential prerequisite for the binding of molybdate and tungstate to the Mo storage protein respectively. The reconstitution of the holoprotein indicates a possible allosteric dependence on ATP. A corresponding tungsten-containing storage protein ("WSto") could be synthesized in-vivo as well as constructed in-vitro by a metal-ion exchange procedure based on experiments with the isolated MoSto protein. The high W content of WSto and the low yield of tungstate release strongly suggests that the W-oxide-based clusters are tighter bound to the protein than the polyoxomolybdate compounds.
On the contrary to the octameric subunit composition described in an earlier publication, x-ray structure analysis of WSto revealed a heterohexamer ([alpha]3[beta]3) with a size of about 100x100x70 Å cubed 3 which can be subdivided into a trimer of [alpha][beta]-dimers adopting a threefold pseudosymmetry. Additional research by analytical ultracentrifugation and electrophoretic trials imply also higher oligomeric states in the soluble state of the protein. Furthermore, five different W(VI)-cluster types which are embedded into pockets at the surface of the protein, were identified and structurally characterized. An accurate description of these clusters is hampered by the fact that the cluster formation mostly not completely occupied and only partially synthesized so that the strong scattering of tungsten partly mask the oxygen in the electron density map.
DA  - 2006
KW  - , Röntgenstrukturanalyse , Metall-Speicherprotein , Polyoxometallat-Cluster , Bioanorganische Chemie , X-ray structure analysis , Metal storage protein , Polyoxometalate cluster , Bioanorganic chemistry
LA  - ger
PY  - 2006
TI  - Biochemische Charakterisierung und Untersuchungen der Röntgenstruktur am Molybdänspeicherprotein aus Azotobacter vinelandii
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-10303
Y2  - 2024-11-22T04:31:26
ER  -