Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity

Abstract

The human de novo iron-sulfur (Fe-S) assembly complex consists of the cysteine desulfurase NFS1, accessory protein ISD11, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN has been shown to bind the NFS1-ISD11-ISCU complex (SDU), to activate the desulfurase activity and thus Fe-S cluster biosynthesis. Conversely, in the absence of FXN, the NFS1-ISD11 (SD) complex was reported to be inhibited by the binding of recombinant ISCU. Here, we show that recombinant ISCU binds zinc(II) ion, and that the presence of zinc in as-isolated ISCU has impacts on the SDU desulfurase activity as measured by sulfide production. Indeed, the removal of this zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SD alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SD. Recent yeast studies have reported a substitution on the yeast ISCU orthologue Isu, at position Met141 (Met140 in human numbering of precursor protein) to Ile, Leu, Val, or Cys that could bypass the requirement of FXN for Fe-S cluster assembly and cell viability. Using recombinant human proteins, we report no significant differences in the biochemical and biophysical properties observed between wild-type and variants M140I, M140 L, and M140 V of ISCU. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SD complex. This study therefore identifies an important regulatory role for ISCU-bound zinc in modulation of the human Fe-S assembly system in vitro but no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU.ABBREVIATIONSACPacyl carrier transfer proteinBLIbiolayer interferometryBSAbovine serum albuminCDcircular dichroismDMPDNN-dimethyl-p-phenylenediamineDSFdifferential scanning fluorimetryDTTdithiothreitol; EDTA, ethylenediaminetetracetic acidFe-Siron sulfurFRDAFriedreich’s ataxiaFXNfrataxinHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidIPTGisopropyl β-D-1-thiogalactopyranosidePLPpyridoxal 5′-phosphateSDprotein complex composed of NFS 1 and ISD11SDUprotein complex composed of NFS 1, ISD11ISCUSDUF, protein complex composed of NFS 1, ISD11, ISCU, and frataxinTCAtrichloroacetic acidTCEPtris(2-carboxyethyl) phosphineTristris(hydroxymethyl)aminomethane

Molecular Mechanism of ISC Iron–Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry

“…Zn 2+ also inhibited [2Fe-2S] cluster formation on IscU (Figure 3), consistent with previous studies that Zn 2+ stabilizes IscU by binding to the same cysteine residues that are used to ligate [2Fe-2S] cluster intermediates. 28,32,[55][56][57][58] Zn 2+ binding stabilizes uncomplexed IscU 58 and, in our experiments, decreases the propensity of the IscU subunits to dimerize (Figure S3). Reductants also decrease IscU dimerization (Figures S3, S4A), indicating that cysteine residues are being oxidized to form an intermolecular disulfide bond.…”

“…60 Moreover, native MS results indicate apo-IscU binds tighter than the Znbound form to IscS (Figure 2B). Regardless, Zn 2+ can be removed from Zn-IscU with the chelating agent DTPA (Figure 1B) 58,61 or by reactions that include both L-cysteine and a stoichiometric amount of IscS (Figure 3C), which might remove the Zn 2+ either by generating a persulfide species on IscU that decreases the Zn 2+ binding affinity or by precipitating the Zn 2+ with generated sulfide. Overall, the Zn 2+ stabilizes and protects IscU from oxidation and remains bound until the IscS-dependent turnover of L-cysteine promotes the loss of Zn 2+ and initiates the Fe-S cluster assembly reaction.…”

A comprehensive landscape of the zinc-regulated human proteome

“…ZnCPT also provided systematic insights into modes of dynamic zinc regulation over iron-binding proteins, for example the mitochondrial de novo iron-sulfur cluster (ISC) assembly machinery [40][41][42][43][44] . Zinc has been implicated as an important cofactor for the ISC assembly machinery, but its physiological role remained obscure 40,[43][44][45][46][47][48][49][50] . Zinc can be coordinated by ISCU residues Cys95, Asp71 and His137 and NFS1 Cys381 (Figure 5E) 40,44 .…”