This proposal is relevant to the antioxidant defenses of mitochondria and their role in degenerative diseases and aging. Oxidative phosphorylation (OXPHOS) is an essential source of energy for the cell, but also represents the main source of reactive oxygen species (ROS) such as O2 and H2O2. In addition, OXPHOS depends on a constant supply of iron, required for heme and iron-sulfur cluster biosynthesis. Uncomplexed iron can catalyze the decomposition of H2O2 to OH. that attacks mitochondrial DNA, proteins, and membranes, causing a decline in mitochondrial function that can culminate in cell death. Known antioxidant defenses include agents that catalytically remove or scavenge ROS. How mitochondria handle """"""""free"""""""" iron, however, is unknown. The mitochondrial protein frataxin is a candidate to fulfil this role. Frataxin deficiency leads to mitochondrial iron accumulation and hypersensitivity to oxidative stress in yeast, and is responsible for Friedreich ataxia in humans. We have shown that recombinant yeast frataxin (Yfh1p) assembles into regular spherical multimers that sequester iron in a soluble form and prevent oxidative damage of other biomolecules, such as deoxyribose and lipids. We have also detected frataxin multimers in vivo, and have demonstrated that yeast and human frataxin are functional homologues. These findings indicate that the role of frataxin is to enable iron utilization within mitochondria in a manner that minimizes iron-induced oxidative damage. The goal of this proposal is to define the mechanism of action of frataxin using Yfh1p as a model.
The specific aims are (1) to identify the protein functional domains required for assembly, iron storage, and iron release; (2) to characterize the path of Yfh1p assembly and (3) the role of iron-catalyzed radical reactions in this process; (4) to define the reactions involved in iron deposition in frataxin, and how the protein minimizes oxidative damage of other biomolecules; (5) to obtain the three-dimensional structure of Yfhlp multimers; to develop (6) assays to identify biomolecules involved in iron release from frataxin and (7) purification procedures to characterize frataxin in vivo. This work has a potential to elucidate a previously unknown antioxidant system, and to advance our understanding of the role played by mitochondria in degeneration and aging.
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