Zinc(II) binding on human wild-type ISCU and Met140 variants modulates NFS1 desulfurase activity

Abstract

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.

Mechanism of frataxin “bypass” in human iron–sulfur cluster biosynthesis with implications for Friedreich’s ataxia

“…Our results, along with two recent studies (31,32), indicate that native ISCU2 and the ISCU2 M140I variant have similar biochemical and stability properties. First, we found that the M140I substitution does not affect the protein's secondary structure (Fig.…”

“…Initial radiolabeling studies suggest that Isu1 Sup increases the accumulation of persulfide species on Nfs1 similar to the addition of FXN (12,27). In contrast, two subsequent studies using standard reconstituted assays of partial reactions failed to identify a mechanism by which the human ISCU2 M140I (equivalent to Isu1 Sup ) variant could functionally replace FXN in iron-sulfur cluster biosynthesis (31,32).…”

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism

“…[10][11][12] Zn 2+ ion has been found to completely inhibit the SDAU complex in vitro, although its activity is restored by addition of FXN. 13 Recently, crystal structures of SDA/SDAU/SDAU-Zn 2+ complexes without the key component FXN have been published, 5,14 which attributed the zinc inhibition to the sequestration of key NFS1 catalytic residue Cys381, but could not serve as template to understand the molecular roles of FXN activator. To this end, we pursued structure determination of the SDAUF complex, coupled with FXN binding studies, to decipher the FXN-mediated activation mechanism.…”

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

“…Moreover, the supporting SAXS and chemical crosslinking studies were not tested for the presence of the open form or appropriately evaluated for their ability to distinguish the closed and ready architectures (22,40,41). This model also fails to explain (i) the inherently low cysteine However, when Zn 2+ was removed by EDTA, the activity enhancement was modest and to a lower extent compared to the activating effect of FXN (43). Again, this model also neither considers the open architecture, which has been demonstrated to be the dominant SDAec species in solution, nor explains the inherently low cysteine desulfurase activity of the SDAec complex (5,18,24).…”