Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity.

人从头铁硫（Fe-S）组装复合物由半胱氨酸脱硫酶NFS1，辅助蛋白ISD11，酰基载体蛋白ACP，支架蛋白ISCU和变构活化剂frataxin（FXN）组成。FXN结合NFS1-ISD11-ACP-ISCU复合物（SDAU），以激活脱硫酶活性和Fe-S团簇的生物合成。据报道，在没有FXN的情况下，NFS1-ISD11-ACP（SDA）复合物受到重组ISCU结合的抑制。最近的研究还报道了在酵母ISCU直向同源物Isu的Met141位置被Ile，Leu，Val或Cys取代，可以绕过FXN对Fe-S簇生物合成和细胞活力的需求。在这里，我们显示重组人ISCU结合锌（II）离子，如先前在大肠杆菌中证明的那样直向同源IscU。出人意料的是，纯化批次之间锌结合形式和贫锌形式之间的相对比例有所不同。重要的是，ISCU中锌的存在会影响SDAU脱硫酶的活性。实际上，与单独的SDA相比，从ISCU中除去锌（II）离子会导致活性适度但显着增加，并且FXN可以激活与SDA络合的ISCU的缺锌和锌结合形式。考虑到锌结合的ISCU对脱硫酶活性的抑制作用，我们表征了野生型ISCU和锌结合和缺锌条件下的M140I，M140L和M140V变体，并且在生化和生物物理方面未观察到显着差异类型和变体之间的特性。重要的是，在没有FXN的情况下，ISCU变体表现得像野生型，并且不刺激SDA复合物的脱硫酶活性。因此，本研究确定了锌结合的ISCU在调节人类Fe-S组装系统中的重要调节作用。在体外研究中，没有报道对人ISCU中Met140位置的突变没有“ FXN旁路”作用。此外，该研究在解释涉及重组ISCU的研究时也需要谨慎，要考虑到结合的锌（II）离子对SDAU复合物活性的影响。