Previous studies revealed that β-95-Cys provides an essential ligand to one of the Fe atoms on the P cluster within the MoFe protein of nitrogenase, and a limited number of substitutions at this position resulted in inactive nitrogenase. It was also found that the counterpart of β-95-Cys, α-88-Cys, which also acts as a cysteinyl ligand to the P cluster, is replaceable without a complete loss of activity. In order to study the structure-function relationship of the protein environment in this region with respect to the P-cluster, subtle changes were introduced at β-95-Cys in <i>Azotobacter vinelandii</I>nitrogenase through site-directed mutagenesis and gene replacement method. Some crude extracts from the mutants with substitutions at β-Cys contain typical FeMo cofactor EPR signal. The β-95<sup>Asp</sup> MoFe protein also has significant nitrogenase activity, but lower, suggesting that β-Cys is not absolutely required for both FeMo cofactor insertion and nitrogenase activity.
In order to characterize its catalytic features, the β-95<sup>Asp</sup> MoFe protein was purified from mutant strain DJ1096. It has significantly reduced H⁺ reduction, C₂H₂-reduction and N₂-reduction activity. It was found that a higher percentage of electron flux goes to H⁺ compared to the wild type MoFe protein. It was also found that reductant independent ATP hydrolysis occurs during H⁺ reduction, suggesting that the altered MoFe protein has an increased affinity for Fe protein-ADP complex. Surprisingly, CO has a significant enhancement effect on H⁺ reduction at low electron flux, but not at high electron flux, and highly couples the electron transfer to ATP hydrolysis. These results indicate that the binding of CO to the MoFe protein may either decrease the affinity of Fe-ADP complex for the β-95<sup>Asp</sup> MoFe protein or facilitate electron acceptance by the P cluster, thus improving the electron transfer to substrate. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/46509 |
| Date | 26 August 1998 |
| Creators | Xie, Haibing |
| Contributors | Biochemistry, Newton, William E., Larson, Timothy J., Chen, Jiann-Shin |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | Final-1.pdf |
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