Nitrogenase, femo co biosynthesis, nifb co, nifb, nitrogen fixation
The Mo-nitrogenase complex is composed of two components, Fe-protein and MoFe-protein. This complex is able to catalyze the reduction of N2 through the MgATP dependent transfer of electrons from the Fe-protein Fe4S4 cluster to the MoFe-protein P-cluster and, subsequently, to the iron-molybdenum cofactor (FeMoco). FeMo-co is a Fe7S9MoC-(R)-homocitrate cluster and has two biosynthetic precursors, NifB-co and L-cluster, of unknown structure and composition. The biosynthesis of FeMo-co is an enigmatic process that minimally requires NifB, NifEN, Fe-protein, MoO4 2- , (R)-homocitrate and S-adenolsylmethionine. A means to isolate the NifB enzyme for characterization has been developed through use of a GST-fusion tag. Double recombination of A. vinelandii strains with a constructed vector has yielded strains capable of nif promoter regulated expression of GST-NifB. Extracts of strains containing GST-NifB were shown to activate the Monitrogenase complex in biochemical complementation assays. Mass spectroscopy was then used to verify successful isolation of GST-NifB by GSH-Sepharose affinity purification. The number of NifB-co ligand binding sites and ligand types were examined by EXAFS analysis of samples containing selenol and thiol ligands. A Fe6S9C model for NifB-co was optimized to best fit the EXAFS data, where a 2-fold discrepancy in binding sites implied by thiol or selenol only ligand samples suggests Fe-(μ2S)-Fe binding in the absence of Se. Samples containing heterogeneous ligand types indicated that NifX bound NifB-co ligates to four cysteine residues and one molecule of DTT.
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Doctor of Philosophy (Ph.D.)
College of Sciences
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Dissertations, Academic -- Sciences, Sciences -- Dissertations, Academic,
Gevorkyan, Jirair, "Biochemical Characterization Of The Nifb Enzyme And Nifb-cofactor" (2013). Electronic Theses and Dissertations. 2533.