This research proposal describes experiments aimed at determining the biophysical and enzymological properties of proteins which are specifically produced when the dinitrogen reduction process is derepressed in bacteria. The species examined will be Klebsiella pneumoniae, for which an accurate and reasonably complete genetic map suggests the production of seventeen proteins during the synthesis and activation of the nitrogenase enzyme which catalyzes dinitrogen reduction. Six major goals compose the heart of this proposal: (1) elucidation of the processes responsible for oxidation of pyruvate and transfer of electrons to nitrogenase; (2) characterization of the antagonistic pair of regulatory proteins coded for by nifA and nifL genes; (3) determination of the role of two accessory genes on the maturation of the nifH protein into functioning Fe protein component of nitrogenase; (4) discernment of the binding sites for metal centers on the two subunits of the nitrogenase MoFe protein; (5) characterization of the proteins of four genes whose function is as yet incompletely understood; (6) characterization of the metal uptake and metabolism by strains prepared with single gene deletions. Standard genetic techniques of strain construction will be employed, the major work proposed being in protein purification, biochemical and spectroscopic characterization of the proteins, and assessment of the catalytic and regulatory functions of these proteins in in vitro reconstitutions of the dinitrogenase reduction system. The project is expected to contribute an understanding of physiological problems to be faced in installing these genes and other multigene complexes in eucaryotic hosts.
| White, T C; Harris, G S; Orme-Johnson, W H (1992) Electrophoretic studies on the assembly of the nitrogenase molybdenum-iron protein from the Klebsiella pneumoniae nifD and nifK gene products. J Biol Chem 267:24007-16 |
| Harris, G S; White, T C; Flory, J E et al. (1990) Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli. J Biol Chem 265:15909-19 |
| Wahl, R C; Orme-Johnson, W H (1987) Clostridial pyruvate oxidoreductase and the pyruvate-oxidizing enzyme specific to nitrogen fixation in Klebsiella pneumoniae are similar enzymes. J Biol Chem 262:10489-96 |
| Howard, K S; McLean, P A; Hansen, F B et al. (1986) Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli. J Biol Chem 261:772-8 |
