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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG015709-08
Application #
6644742
Study Section
Geriatrics and Rehabilitation Medicine (GRM)
Program Officer
Kohanski, Ronald A
Project Start
1997-09-30
Project End
2005-08-31
Budget Start
2003-09-01
Budget End
2004-08-31
Support Year
8
Fiscal Year
2003
Total Cost
$250,250
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K et al. (2016) Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN. J Biol Chem 291:10378-98
Gakh, Oleksandr; Ranatunga, Wasantha; Smith 4th, Douglas Y et al. (2016) Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery. J Biol Chem 291:21296-21321
Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina et al. (2016) The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS. J Biol Chem 291:11887-98
Jobling, Rebekah K; Assoum, Mirna; Gakh, Oleksandr et al. (2015) PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia. Brain 138:1505-17
Isaya, Grazia (2014) Mitochondrial iron-sulfur cluster dysfunction in neurodegenerative disease. Front Pharmacol 5:29
Oglesbee, Devin; Kroll, Charles; Gakh, Oleksandr et al. (2013) High-throughput immunoassay for the biochemical diagnosis of Friedreich ataxia in dried blood spots and whole blood. Clin Chem 59:1461-9
Li, Hongqiao; Gakh, Oleksandr; Smith 4th, Douglas Y et al. (2013) Missense mutations linked to friedreich ataxia have different but synergistic effects on mitochondrial frataxin isoforms. J Biol Chem 288:4116-27
Vaubel, Rachael A; Isaya, Grazia (2013) Iron-sulfur cluster synthesis, iron homeostasis and oxidative stress in Friedreich ataxia. Mol Cell Neurosci 55:50-61
Söderberg, Christopher A G; Rajan, Sreekanth; Shkumatov, Alexander V et al. (2013) The molecular basis of iron-induced oligomerization of frataxin and the role of the ferroxidation reaction in oligomerization. J Biol Chem 288:8156-67

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