Moore, Michael John (2017-06). An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool. Doctoral Dissertation. Thesis uri icon


  • Mitochondria contain a low-molecular-mass (LMM) pool of weakly bound iron complexes, called the labile iron pool (LIP). Although its composition and biological function remain largely uncharacterized, the LIP has been implicated in cellular iron metabolism and disease pathogenesis. In this dissertation, results obtained from Mossbauer, EPR, and UV-Vis studies were integrated with LC-ICP-MS data to investigate the chemical nature of the mitochondrial LIP. LMM Fe, Mn, Cu, Zn, and Co complexes were detected in yeast and mammalian mitochondria. Such complexes were reproducibly observed and hypothesized to metalate mitochondrial apo-metalloproteins. The approximate mass of each complex was estimated along with its mitochondrial concentration. The predominant LMM Fe species detected in mitochondria had a mass of ~580 Da (called Fe580) and was present in both yeast and mammalian mitochondria. Increasing the Fe concentration in the medium increased the intensity of Fe580. Interestingly, the mitochondrial concentration of Fe580 was ~100 uM, which was consistent with previous estimates of the mitochondrial LIP. Thus, Fe580 was hypothesized as the cytosolic iron species that is imported into mitochondria to form the LIP. Treatment with metal chelators demonstrated that Fe580 was labile. A yeast strain lacking mitochondrial Fe importers, Mrs3/4, was characterized using biophysical and bioanalytical methods (??). Respiring Fe-deficient ?? cells exhibited a "slow-growth" phenotype that was ameliorated under Fe-sufficient conditions. ?? cells accumulated more Fe than wild-type (WT) cells, even under Fe-sufficient conditions, indicating Fe dysregulation. ?? cells accumulated NHHS FeII and FeIII in the cytosol and vacuoles, respectively. Fe-deficient ?? mitochondria accumulated FeIII oxyhydroxide nanoparticles and were devoid of central doublet ([Fe4S4] 2+ and LS FeII hemes), whereas Fe-sufficient ?? mitochondria contained comparable levels of central doublet but less NHHS FeII relative to WT. This suggested the mitochondrial NHHS FeII pool was reduced in Fe-sufficient ?? cells. Fe580 was absent in Fe-deficient ?? mitochondria but present under Fe-sufficient conditions. Two candidate masses {664.46 and 665.46 amu} and {685.39 and 686.39 amu} were determined for Fe580. Viewed comprehensively, this study provides strong evidence that the mitochondrial NHHS FeII pool is composed predominantly of Fe580 and that the size of this pool controls cellular Fe homeostasis.

publication date

  • August 2017