| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Iron-sulfur (Fe-S) proteins are present in almost all living organisms and exhibit diverse functions, which include electron transport, redox and non-redox catalysis, and sensing for regulatory processes. They contain clusters of iron and sulfur atoms with variable complexity, such as [2Fe-2S], [3Fe-4S], and [4Fe-3S] clusters. We have recently found two independent systems, called ISC and SUF, essential for the biosynthesis of Fe-S clusters, although still very little is known about the mechanistic details. In this study we have undertaken genetic, biochemical and structural approaches to elucidate the complex mechanism by which the Fe-S clusters are assembled.
1. We have isolated and analyzed 14 psuedorevertants generated from a mutant strain lacking HscA and HscB in the ISC machinery, which revealed various suppressor mutations located in IscU. We also found that the IscU homolog from the thermophilic bacterium Aquifex aeolicus exceptionally carries the intermediate Fe-S cluster in the stable form that was further stabilized by introduction of several mutations.
2. Purification and biochemical characterization of the components of the SUF machinery have revealed dimeric form of SufD and the SufC_2SufD_2 oligomer as well as the SufB_1SufC_2 SufD_1 complex, suggesting that dynamic alteration of the oligomeric status is involved in the reaction. Essential residues for the SufD function have also been identified by mutagenic studies.
3. We have determined the crystal structures of E.coli yfhJ (IscX), SufA, SufC, and SufD. The structure of SufA revealed the active-site architecture comprising of four cysteine residues. We have also identified a salt bridge that appears to trigger the conformational change of SufC. The structure of SufD revealed an unprecedented β-helix dimer. The docking model between SufC and SufD suggests the role of SufC to alter the oligomeric status of SufD.
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