Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe–S Cluster Biosynthesis

Abstract

Iron–sulfur (Fe–S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe–S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe–S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe–S cluster assembly process and establish that the initiation of Fe–S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

A comprehensive landscape of the zinc-regulated human proteome

“…Binding of ISC assembly activator Frataxin (FXN), displaces the NFS1 loop carying Cys381 and reorients zinc coordination to ISCU residues Cys95, Asp71 and Cys138 42,43,51,52 . This possibly primes the complex for a catalytic cycle 42,43,53,54 . Importantly, iron, but not zinc binding allowed FeS formation by an in vitro ISC assembly complex 53 .…”

Native mass spectrometric studies of IscSU reveal a concerted, sulfur-initiated mechanism of iron–sulfur cluster assembly

“…The Cys residue of IscU to which S 0 is transferred from IscS is not known. Evidence for the involvement of the residues equivalent to Cys37 and Cys106 in the human homologue ISCU is available 53,88,89 Inspection of the structure of IscS/IscU suggests that Cys37 is the most likely residue for initial transfer, prior to arrival at Cys106.…”

Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2

“…4; Table 3). As controls we used 57 Fe-loaded ISCU2 (Fe-U) 19 (Fig. 4a), (NIAX)2 plus added 57 Fe (lacking ISCU2; Fig.…”

“…Mitochondrial Fe/S protein biogenesis begins with the de novo assembly of a [2Fe-2S] cluster from Fe 2+ and Cys-derived persulfide (SSH) on the scaffold protein ISCU2 14 . Assembly requires the multi-protein 'core ISC complex' consisting of ISCU2, the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, FXN, and the ferredoxin FDX2 [15][16][17][18][19][20] (Fig. 1a).…”

“…1a). This residue is located on a flexible loop (Cys loop) that moves towards the Fe 2+ binding site of ISCU2 (denoted as U; 19 ), thereby forming the (Fe-N S IAU)2 complex ( S indicates persulfidation of the preceding ISC component). The (NIAU)2 complex was shown to have increased affinity for FXN (X) 16,[27][28][29] .…”