STUDY OF VANADIUM-CONTAINING NITROGENASE

点击翻译按钮获取中文摘要

Nitrogen fixation, the conversion of dinitrogen from the air into ammonia, is catalyzed by the enzyme nitrogenase. In spite of the large volume of research that has been done on this enzyme during the past twenty years, the mechanism of nitrogen fixation is still unclear. The purpose of this research is to gain a better understanding of this mechanism by studying a new alternative vanadium-containing nitrogenase enzyme recently isolated in our laboratory from the bacterium Azotobacter vinelandii. Specifically, the proposed research will include projects on the enzymology of V-nitrogenase. The substrate (Km) and inhibitor (Ki) properties of N2, C2H2, HCN, CH3NC, cyclopropene and CO will be studies as will their dependency on temperature and pH. The amino acid sequences of the enzyme's different polypeptides will be determined and used, along with isolated antibodies, to synthesize oligolnucleotide probes for nif genes corresponding to the alternative enzyme. The techniques of ESR, ENDOR, MCD, Mossbauer and x-ray absorption spectroscopy will be used to elucidate the physical and chemical properties of the different metal clusters in the alternative enzyme. In these experiments, the enzyme will be studied in different oxidation states and in the presence and absence of different substrates and inhibitors. Furthermore, using established procedures, the VFe cofactor cluster will be isolated and characterized with regard to elemental analysis and electronic and magnetic properties. This cofactor will be used to reconstitute activity in apo-component 1 of both the conventional and alternative enzymes. Finally, purification will be undertaken of both conventional and alternative nitrogenase containing mixed metal clusters.