Untersuchung der Expression und Aktivität des Molybdän-Eisen Protein in Wolinella succinogenes

Abstract

In nature, the ability to fix nitrogen is restricted to some prokaryotes that possess the enzyme nitrogenase. It was possible to simulate this important process and achieve industrial nitrogen fixation through the invention of Haber and Bosch. Although this is the sole industrial process available to provide fixed nitrogen, it does bring along many problems with respect to pollution and energy consumption. More efficient catalysts modeled on the working principles of the enzyme nitrogenase would therefore be highly desirable. Nitrogenase, the machine that is responsible for converting dinitrogen to ammonium, comprises two oxygen-labile protein components, iron protein or dinitrogenase reductase and molybdenum iron protein or dinitrogenase. Over the last two decades, several structures of nitrogenase components from different microorganisms have been solved and lately, Einsle, et al. reported a new model of the active site, at an improved resolution of 1.16 Å, which includes an internal hexa-coordinate light atom within the FeMo-cofactor. However, the mechanistic details of how the dinitrogen triple bond is broken at the unique metal center of nitrogenase remain to be elucidated. Therefore, studying the nitrogenase system in different species of bacteria may contribute in uncovering its enigmatic features. In the course of genome projects, many species of bacteria, which were not formerly known to be able to fix dinitrogen, have been found to possess the entire set of genes required for the production of a functional nitrogenase system. Such bacteria include the epsilon-proteobacterium Wolinella succinogenes, an enteric bacterium isolated from rumen fluid of cattle. Initial experiments on W. succinogenes showed that it is not able to grow diazotrophically but it does have nitrogenase activity at nitrogen-limiting conditions. The main task of this work was to investigate the nitrogenase system in Wolinella succinogenes. Activity assays were employed in order to establish cultivation conditions that allow for the purification and characterization of nitrogenase components. As a well-studied diazotroph model, Azotobacter vinelandii was used as a standard and a control for comparison at each stage of the work. Acetylene reduction the ethene, the typical assay for nitrogenase activity, was used in combination with gas chromatography. In addition, other analytic methods were be employed, such as SDS-PAGE and mass spectrometry, for unequivocal identification of the enzyme. Detection of nitrogenase activity in W. succinogenes is not sufficient to prove its expression because the conditions in which nitrogenase is expressed and the regulation of nitrogen status are completely unknown in this case. Therefore, further biochemical assays, in parallel with the results of Azotobacter vinelandii, were applied for identification of the enzyme in W. succinogenes.