Isolation of in vivo intermediates in iron sulfur cluster biogenesis
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Using A. vinelandii as a model organism, we have sought to better understand the mechanism of in vivo isc cluster assembly. In order test the scaffold hypothesis, we constructed strains that allowed for quick and rapid isolation of IscU. The purification of IscU with a bound [2Fe-2S] cluster strongly supports the model that IscU serves as the site of cluster synthesis in vivo. Additionally, using this same genetic system we isolated an IscU39DA variant with an oxygen stable bound [2Fe-2S] cluster. The IscU39DA scaffold came in tight Î±2Î²2 complex with IscS and was not separated by high salt, size exclusion, or reducing conditions. On the other hand, wild-type IscU also associated with IscS in a Î±2Î²2 complex, but readily dissociated upon increased salt concentration. The tight association of IscU39DA and IscS was found to occur regardless of the presence of a bound [Fe-S] cluster. We conclude that the IscU Asp-39 residue is essential for mediating the dissociation of IscU and IscS.
In addition to studying IscS and IscU, we were interested to further understand how the isc system is regulated in response to external factors. Previous work has demonstrated that IscR controls expression of the isc operon in Escherichia coli. When IscR is holo this protein represses isc expression, while in its apo-form it allows isc expression. In A. vinelandii we found that â iscR strains exhibit in a 5 â 7 fold elevation of isc expression. Additionally, â iscR strains reveal a small growth phenotype on plates, and a tendency to form spontaneous suppressor mutations allowing reversion to wild-type growth. Loss of apo-IscR function was found to cause a more severe effect on growth than the loss of holo-IscR function, suggesting IscR has cellular roles in addition to the regulation of the isc operon.
- Doctoral Dissertations