This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Metals can accumulate in cellular compartments during the normal aging process, through excessive dietary or environmental exposure and from genetic errors in proteins that regulate metal flux within cells. Mitochondrial iron overload is a common feature of several neurological and metabolic diseases. We have shown that in yeast with various errors in iron-sulfur cluster biosynthesis, the form of iron that accumulates differs. In particular, ferrihydrite of the type sequestered by frataxin in vitro is found only in yeast that express frataxin. We now propose to expand this work to compare mitochondrial iron in two human diseases, Friedreich's ataxia (FRDA) and sideroblastic anemia (SA) and to begin to address the chemistry of treatment strategies. The following questions will be addressed using XAS 1. How does frataxin's ferroxidase activity contribute to iron core assembly? Iron K-edge spectra will be collected for XANES and EXAFS analysis of iron cores assembled after amino acid substitutions to frataxin's ferroxidation site. 2. Does frataxin assemble an iron core in vivo that is structurally similar to that assembled in vivo? Frataxin assembled in vivo will be separated on a non-denaturing gel and K-edge spectra collected in situ. 3. Is mitochondrial iron that accumulates in yeast FRDA models and human FRDA the same? Mitochondria will be isolated from FRDA fibroblast cultures and K-edge spectra collected for XANES analysis. 4. Can selenium form a complex with iron in frataxin deficient mitochondria? Mitochondria will be isolated from frataxin-deficient yeast grown on selenium-supplemented media and spectra collected at the iron and selenium K-edges for EXAFS analysis. 5. Do iron deposits differ between SA and FRDA? Mitochondria will be isolated from an SA model and iron K-edge spectra collected for XANES analysis. This work will contribute to the development of better treatment strategies for these mitochondrial iron overload disorders.
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