You are here
Biochemical Characterization of the NifB Enzyme and NifB-cofactor
| Title: | Biochemical Characterization of the NifB Enzyme and NifB-cofactor. |
 73 views 50 downloads |
|---|---|---|
| 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 |
