Biophysical and Bioanalytical Probes to Study Iron Trafficking in Yeast, Archaea, and Mammals

Abstract

Iron (Fe) is essential for most forms of life due to its unique redox and substrate binding properties. It is also dangerous since it can generate reactive oxygen species (ROS). In Saccharomyces cerevisiae, 95 known Fe Fe-containing proteins are found in different cellular compartments. Though the physiological role of each molecular Fe species is understood, how they interact on the system or cellular level is not well characterized. To narrow the knowledge gap, we have developed a semi-quantitative model of the entire iron content of a yeast cell using Mössbauer (MB) spectroscopy. By comparing with experimental data, the model suggests that the concentration of cytosolic labile iron pool (LFeP) is 10-25µM. MB spectra of the isolated nuclei agreed with the model, and they suggested the presence of LFeP in this organelle.

Pyrococcus furiosus is a hyperthermophilic archaeon. Fe plays a critical role in the cell’s metabolism which shifts depending on the presence or absence of elemental sulfur (S⁰) in the growth medium. MB spectra of P. furiosus were dominated by magnetically ordered thioferrate-like (TFL) iron or [Fe₄S₄]²⁺ clusters depending on the availability of S⁰. IssA protein localizes with TFL particles and is overexpressed in the presence of S⁰. MB spectroscopy in combination with growth experiments using the ΔIssA strain suggested that IssA is not required for the production of TFL iron and that IssA perhaps helps in detoxifying harmful sulfides from the cell.

Hereditary Hemochromatosis (HH) is an iron-overload disease in which a toxic form of Fe called Non-Transferrin-Bound Iron (NTBI) is formed in the blood that causes multiple organ damage. Analysis of low (<10 kDa) and high-mass fractions of blood plasma from HH mice via LC-ICP-MS in combination with MB spectroscopy suggests that NTBI is an aggregated form of Feᴵᴵᴵ present in the high mass fraction of blood plasma and this material can be chelated by deferoxamine (DFO). This tentative conclusion is contrary to the popular view that NTBI is Feᴵᴵᴵ-citrate.

Taken together, the studies presented in this dissertation demonstrate that MB spectroscopy in combination with LC-ICP-MS can be used to effectively study Fe trafficking across different organisms.