ISCU(M108I) and ISCU(D39V) differ from wild type ISCU in their failure to form cysteine desulfurase complexes containing both frataxin and ferredoxin

Kai Cai, Ronnie O. Frederick, Marco Tonelli, and John L. Markley; Biochemistry, Just Accepted Manuscript DOI: 10.1021/acs.biochem.7b01234

Whereas iron-sulfur (Fe-S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human cysteine desulfurase complex (SDA), exhibits a requirement for frataxin (FXN), assembly on variant ISCU(M108I) has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that ISCU(M108I) populates only the structured state as does another variant ISCU(D39V). We have compared the properties ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the cysteine desulfurase step of Fe-S cluster assembly and in the overall Fe-S cluster assembly reaction. In the cysteine desulfurase step with DTT as the reductant, FXN was found to stimulate cluster assembly with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with wild-type or ISCU(M108I). In overall Fe-S cluster assembly, frataxin was found to stimulate cluster assembly with both the wild-type and structured variants when the reductant was DTT; however, with the physiological reductant, reduced ferredoxin 2 (rdFDX2), frataxin stimulated the reaction with wild-type ISCU but not with the fully-structured variants. Through NMR titration experiments, we discovered that, wild-type ISCU, frataxin, and rdFDX2 all bind to SDA. However, when ISCU was replaced by the fully-structured variant ISCU(M108I), the addition of rdFDX2 to the SDA-ISCU(M108I)-FXN complex led to the release of FXN. Thus, the displacement of FXN by rdFDX2 explains the failure of FXN to stimulate Fe-S cluster assembly on ISCU(M108I).

 ISCU(M108I) and ISCU(D39V) differ from wild type ISCU in their failure to form cysteine desulfurase complexes containing both frataxin and ferredoxin