The Fumarate Nitrate Reductase (FNR) transcription factor is the main anaerobic switch in Escherichia coli bacteria. In the absence of oxygen, the FNR protein exists as a dimer with a [4Fe-4S] cluster and binds to DNA to regulate the transcription of genes needed for anaerobic respiration. However, when oxygen is present, the [4Fe-4S] cluster degrades causing FNR to return to its inactive monomeric form. Examination of the crystal structure of Alivibrio fischeri FNR, a close variant to E. coli FNR, suggested that the dimerization and oxygen stability of FNR are dependent upon fine interactions between multiple amino acid residues. In our studies, site-directed mutagenesis was used to analyze the mutations highlighted in the crystal structure observations. The activity of all these mutants was investigated in an E. coli reporter strain with the narG-lacZ fusion by a β-galactosidase assay to determine the effect of the interactions described on FNR dimerization and oxygen stability. Activity analysis of mutants where the D130 and R140 residues were changed showed that the suggested salt bridge interaction between these positions may not be playing a role in FNR dimerization. Additionally, data showed that double alanine and leucine mutants at positions 130 and 140 had even higher activity than wild type FNR under anaerobic conditions suggesting that the presence of hydrophobic interactions may be stabilizing the FNR dimer. The crystal structure also proposed that a hydrogen bond between the histidine in the L28H mutant and the Q183 residue may form a conformation that prevents the entrance of oxygen, therefore inhibiting the degradation of the iron-sulfur cluster and resulting in a protein that is active under aerobic conditions. The activity of FNR under aerobic conditions was measured in mutants at the 183 position in an L28H mutant background to investigate this hypothesis.The initial results suggest that removing the proposed hydrogen bond does not affect the iron-sulfur cluster stability in the L28H mutant background. Further mutations that investigate these hypotheses will be discussed.

Investigating the effect of multiple interactions on the dimerization and oxygen stability of the fumarate nitrate reductase transcription factor in E. coli bacteria

2019 Spring National Meeting

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