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Abstract
The objectives of this work were threefold. To synthesize and characterize thioferrate, a linear inorganic anionic polymer of iron and sulfide with a (FeS2)1 repeating unit, and investigate thioferrate-mediated assembly of protein-bound Fe-S clusters. To determine the nature of the Fe2+ donor and the role of the Isc Fdx in bacterial Fe-S cluster assembly on the IscU scaffold protein. To investigate the ability of 5-, 6-, and 7-membered cyclic thiosulfinates to inhibit Fe2+ acquisition and [2Fe-2S]2+ cluster assembly by IscU. The approach involved using a combination of spectroscopic techniques to characterize thioferrate and protein-bound Fe2+ and Fe-S centers. The results confirm that thioferrate can be used to reconstitute [2Fe-2S]2+, linear [3Fe-4S]1+ and [4Fe-4S]2+ clusters in Fe-S proteins under anaerobic conditions. The presence of a protein-bound linear [3Fe-4S]1+ clusters as major or minor components of all thioferrate reconstitutions suggests a mechanism involving transfer of a linear [3Fe-4S]1+ fragment of thioferrate to the apo Fe-S protein, that can be used for in situ formation of [2Fe-2S]2+ and [4Fe-4S]2+ clusters. Spectroscopic and analytical studies demonstrated IscX binds one Fe2+ ion that is transferred rapidly to IscU, indicating IscX is the elusive Fe2+ donor for [2Fe-2S]2+ cluster assembly on IscU. Parallel UV-visible absorption/EPR studies showed reduced Fdx is oxidized in the presence of IscS and L-Cys and in a single turnover of IscS in the presence of stoichiometric IscU, Fe2+-bound IscX, and L-Cys. However, no evidence for formation a cysteine persulfide radical anion intermediate on IscS, that would generate a trisulfide radical ion on IscU was observed. The results indicate IscS-mediated persulfide rather than S• ̶ transfer to IscU that is reduced by electrons from cysteinate-bound Fe2+ and reduced Fdx to yield a cysteinate-bound [Fe-S]1+ intermediate. Finally, 5- to 7-membered cyclic thiosulfinate have been shown to be potent inhibitors of binding Fe2+ and [2Fe-2S]2+ cluster assembly on IscU. The unique properties of this new class of thiol cross-linkers offers a new approach to investigate the mechanism of [2Fe-2S]2+ cluster assembly on IscU and has the potential to function as a new type of anticancer therapy by inhibiting the growth of rapidly dividing cancer cells.