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 和变构激活剂 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 簇生物合成。据报道，在没有 FXN 的情况下，NFS1-ISD11-ACP (SDA) 复合物会被重组 ISCU 的结合所抑制。最近的研究还报道了酵母 ISCU 直系同源物 Isu 上 Met141 位置替换为 Ile、Leu、Val 或 Cys，可以绕过 FXN 对 Fe-S 簇生物合成和细胞活力的要求。在这里，我们证明重组人 ISCU 结合锌 (II) 离子，正如之前用大肠杆菌直系同源物 IscU 所证明的那样。令人惊讶的是，锌结合型和贫锌型之间的相对比例在纯化批次之间有所不同。重要的是，ISCU 中锌的存在会影响 SDAU 脱硫酶活性。事实上，与单独使用 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 复合的 ISCU 的锌耗尽形式和锌结合形式。考虑到锌结合 ISCU 对脱硫酶活性的抑制，我们在锌结合和缺锌条件下对野生型 ISCU 和 M140I、M140L 和 M140V 变体进行了表征，没有观察到生化和生物物理方面的显着差异。野生型和变异体之间的特性。重要的是，在没有 FXN 的情况下，ISCU 变体的行为与野生型相似，并且不会刺激 SDA 复合物的脱硫酶活性。 因此，这项研究确定了锌结合 ISCU 在体外调节人 Fe-S 组装系统中的重要调节作用，并报告对人 ISCU 中 Met140 位置的突变没有“FXN 旁路”效应。此外，本研究还呼吁在解释涉及重组 ISCU 的研究时谨慎考虑结合的锌 (II) 离子对 SDAU 复合物活性的影响。