The Fumarate Nitrate Reductase (FNR) regulator protein is a transcription factor that is found in E. coli. It contains a 4Fe-4S cluster, which is ligated by 4 cysteines in the protein (Cys 20, Cys23, Cys29 and Cys 122). The FNR transcription factor is a dimer and active when the 4Fe-4S cluster is present. Introduction of oxygen degrades the iron sulfur cluster causing it to become a monomer, thus inactivating the protein. Previous research has shown that altering the amino acid at position 28, which is adjacent to a ligating cysteine, can lead to a Fe-S cluster that is stable in the presence of O2. In this research, we examine how altering the amino acid at position 22, which adjacent to a different ligating cysteine, can affect the stability of the cluster to oxygen. Site directed mutagenesis performed on the FNR gene supplied on a pET11a plasmid. The in vivo activity of the FNR protein was evaluated under aerobic growth conditions by transforming the mutated plasmids into a lacZ reporter strain that contains the FNR dependent narG promoter. β -Galacosidase assays were used to find the activity of the mutated protein. To date, the FNR mutations that have been made (D22A, D22H, D22G, and D22W) do not show significant in vivo aerobic activity when compared to the cluster stabilizing mutations at position 28. Further studies will examine more amino acid substitutions at this position.

Analysis of amino acid substitutions at position 22 in the FNR transcription factor: The effect on oxygen stability of the 4Fe-4S cluster

251st National Meeting (2016)

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