Iron Storage by Ferritin and Microbial Growth

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Iron is essential for almost all forms of life. It is a cofactor for cell sustaining reactions such as mediating single electron transfer reactions of the electron transport chain or the formation of DNA building blocks by ribonucleotide reductases. The unique reactivity of metals such as iron necessitates control of uptake and intracellular handling to avoid toxic side effects. Understanding the cellular mechanisms of iron homeostasis in microbes is the central direction of my research and the focus of this research program is the role of ferritins. Ferritins are iron storage proteins constructed of 12 or 24 subunits that assemble into a hollow sphere. Ferrous iron is oxidized to ferric ions by oxygen or hydrogen peroxide at ferroxidase sites before storage inside the sphere as an iron oxide mineral. By removing ferrous iron and consuming dioxygen or hydrogen peroxide, ferritins have an antioxidant role. Less is known about the mechanism of iron release from ferritin or how iron storage is regulated. I hypothesize that the mineral core of ferritin regulates the concentration of readily exchangeable cellular iron and that iron release is dependent on the reduction potential of the cell. Ferritin is proposed to be used by bloom-forming pennate diatoms to store excess iron for use in subsequent cell divisions thereby out competing centric diatoms that do not possess ferritin. Our hypothesis is that this ferritin has distinct functional features to supply iron to the photosynthetic machinery of the diatom. Escherichia coli has 3 different types of ferritin with potential distinct roles in iron storage and detoxification and is a model system to investigate ferritin function. Specific aims are: 1. To identify putative amino acid residues directly involved in Fe storage and release by ferritins by site-directed mutagenesis. Crystal structures, kinetics of the ferroxidase reaction, iron mineralization, and iron release of recombinant wild-type and variants will assayed. 2. To show that a ferritin stores iron in cell, metal content analysis by ICP-MS will be done with native ferritin isolated from cells grown under varying iron availability and oxidative stress. 3. Iron release from ferritins will be examined by incubating iron loaded samples in the presence of physiological levels of ferrous iron chelating metabolites (eg. histidine) and reductants such as flavins and NAPH. A screen will be undertaken to search for proteins involved in iron release. The results of this work will contribute to understanding of diatom blooms in the open ocean and the provide a foundation to understanding how pathogens cope with iron restriction imposed by host plants and animals.