Collaborative Research:The ferric uptake regulator (Fur) regulates intracellular iron homeostasis via reversible binding of a [2Fe-2S] cluster in Escherichia coli

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This NSF award will support a collaborative project of Dr. Huangen Ding (Louisiana State University) and Dr. Codrina V. Ewbank-Popescu (University of St. Thomas) for research conducted with graduate and undergraduate students. Research activities ranging from molecular biology to spectroscopy seek to understand the molecular mechanism by which the protein Fur regulates iron content inside bacteria. Iron is an essential element for all living organisms. However, iron overload can cause dangerous oxidative damage to cells under aerobic conditions. Thus, tight regulation of the iron content is essential for cells to grow. In bacteria, the Ferric Uptake Regulator (Fur) is a global iron regulator. When iron content is too high, Fur becomes a transcription repressor to block formation of the iron transporters and prevent accumulation of excess iron in cells. However, the mechanism by which Fur senses the intracellular iron content remains largely elusive. This project is based on preliminary studies showing that in the bacterium E coli, Fur can bind a cluster of Iron bound to Sulfur when the intracellular iron content is high. The goal of the project is to determine how Fur may undergo the binding of this [2Fe-2S]-cluster in response to changes of the intracellular iron concentration and in turn regulate the expression of the genes related to iron metabolism in bacteria. This project will provide greater understanding of how bacteria, including infective bacteria in our tissues, can pull iron essential for growth from inside animal and plant cells, to be used instead for bacterial growth. This will inform our knowledge of the interactions of bacteria with larger creatures in the ecosystem, agriculture, and health. The project will also provide opportunities for undergraduate and graduate students to participate in vigorous and exciting interdisciplinary research activities.The ferric iron regulator (Fur) is a highly conserved global iron regulator in bacteria. The current hypothesis states that when the intracellular “free” iron content is elevated, Fur binds ferrous iron to form an “iron-bound” Fur and regulates the intracellular iron homeostasis by controlling the expression of specific genes. However, the “iron-bound” Fur has never been isolated from any bacteria. Preliminary studies revealed that the Escherichia coli Fur can bind a [2Fe-2S] cluster via the conserved cysteine residues in the E. coli mutant cells in which the intracellular iron content is elevated. The goal of this project is to test a hypothesis that Fur regulates the intracellular iron homeostasis via reversible binding of a [2Fe-2S] cluster. Aim 1 is to determine the redox properties and stability of the [2Fe-2S] cluster in Fur by combining biochemical and spectroscopic (including UV-Visible absorption spectroscopy, electron paramagnetic resonance (EPR) and Mössbauer spectroscopy) approaches. Aim 2 is to investigate the physiological function of the [2Fe-2S] cluster-bound Fur by analyzing its specific DNA binding activity in vitro and the gene expression profile regulated by the [2Fe-2S] cluster-bound Fur in E. coli cells. Aim 3 is to explore the conserved nature of the [2Fe-2S] cluster binding in Fur proteins from other bacteria. Because Fur is highly conserved among bacteria, success of the project is expected to illustrate a novel mechanism by which Fur regulates the intracellular iron homeostasis in E. coli and other bacteria via an iron-sulfur cluster.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.