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Biochemical Characterization of the NifB Enzyme and NifB-cofactor

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Date Issued:
2013
Abstract/Description:
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 (FeMo-co). 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, MoO42-, (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 Mo-nitrogenase 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-(?2S)-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.
Title: Biochemical Characterization of the NifB Enzyme and NifB-cofactor.
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Name(s): Gevorkyan, Jirair, Author
Igarashi, Robert, Committee Chair
Belfield, Kevin, Committee Member
Hernandez, Florencio, Committee Member
Kuebler, Stephen, Committee Member
Vonkalm, Laurence, Committee Member
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2013
Publisher: University of Central Florida
Language(s): English
Abstract/Description: 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 (FeMo-co). 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, MoO42-, (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 Mo-nitrogenase 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-(?2S)-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.
Identifier: CFE0004682 (IID), ucf:49865 (fedora)
Note(s): 2013-05-01
Ph.D.
Sciences, Chemistry
Doctoral
This record was generated from author submitted information.
Subject(s): Nitrogenase -- FeMo-co Biosynthesis -- NifB-co -- NifB -- Nitrogen Fixation
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFE0004682
Restrictions on Access: public 2013-05-15
Host Institution: UCF

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