Iron-sulfur clusters (Fe-S) are essential protein cofactors for most life forms and function in processes such as electron transfer, enzyme catalysis, and initiating radical chemistry. In humans, disruption of the ISC Fe-S cluster assembly pathway results in a variety of diseases including Friedrich's Ataxia and IscU myopathy. Previously, the bacterial protein CyaY, which is not part of the ISC operon, was shown to inhibit Fe-S cluster biosynthesis. IscX is located in the ISC operon, but its function remains elusive. Mutational analysis of the cysteine desulfurase (IscS) has shown that the binding surface of CyaY and IscX may overlap, suggesting that they compete for the same binding site on IscS. Here we performed binding experiments and Fe-S cluster assembly assays to determine the function of IscX and investigate the potential for binding competition on IscS. Native IscX along with constructed IscX mutants were purified, and a maleimide-thiol labeling strategy was used to incorporate Texas Red fluorophore. Fluorescence anisotropy experiments revealed that IscX exhibits cooperative behavior and binds to IscS and IscS-IscU with 0.130 and 0.09 �M affinities, respectively. Finally, IscX exhibited spectral properties consistent with a role as an iron donor for Fe-S cluster biosynthesis and competed for binding with CyaY.

Elucidating the role of IscX in bacterial Fe-S cluster assembly

247th National Meeting (2014)

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