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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033965-06
Application #
3284221
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1985-09-05
Project End
1993-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Louisiana State University A&M Col Baton Rouge
Department
Type
Schools of Arts and Sciences
DUNS #
075050765
City
Baton Rouge
State
LA
Country
United States
Zip Code
70803
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Onate, Y A; Finnegan, M G; Hales, B J et al. (1993) Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S]+ synthetic analog clusters. Biochim Biophys Acta 1164:113-23
Oliver, M E; Hales, B J (1993) Using dysprosium complexes to probe the nitrogenase paramagnetic centers. Biochemistry 32:6058-64
Hales, B J; Case, E E (1987) Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium. J Biol Chem 262:16205-11
Morningstar, J E; Johnson, M K; Case, E E et al. (1987) Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy. Biochemistry 26:1795-800
Hales, B J; Case, E E; Morningstar, J E et al. (1986) Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry 25:7251-5
Hales, B J; Langosch, D J; Case, E E (1986) Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem 261:15301-6