Assembly of the [4Fe–4S] cluster of NFU1 requires the coordinated donation of two [2Fe–2S] clusters from the scaffold proteins, ISCU2 and ISCA1

Abstract

NFU1, a late acting iron–sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease Multiple Mitochondrial Dysfunctions Syndrome (MMDS1). In this work, using genetic and biochemical approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2 (FDX2). By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We observed that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochemical functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase (LIAS), which supports the Fe-S dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (∝-KGDH) and the glycine cleavage (GC) complex.

Allele-specific mitochondrial stress induced by Multiple Mitochondrial Dysfunctions Syndrome 1 pathogenic mutations modeled in Caenorhabditis elegans

“…Most of the nfu-1 mutations resulted in impaired delivery of lipoic acid to PDH and KGDH (Fig 1D and 1E) as has also been observed in MMDS1 samples [16,19], patient-derived fibroblasts [11,15,17,19,21,28,40], and NFU1 mutant rodents [41], plants [42], and yeast [8]. A single mutant, Arg140Gln appears unaffected in its ability to lipoylate PDH and KDGH.…”

“…This information, paired with the data herein about diffences between alleles, suggests that the amino acid changes are likely the causes of dysfunction rather than reduced NFU-1 protein abundance. Second, recent work has demonstrated that the C-terminus of NFU1, including residues surrounding the ISC-binding motif is important for interactions with both other members of the ISC biogenesis machinery and NFU1 targets [9,11]. We cannot exclude the possibility that the modeled nfu-1 mutants also disrupt those types of interactions and likewise alter ISC handling.…”

“…NFU1 binds 4Fe-4S ISCs and delivers them to targets such as lipoic acid synthase (LIAS), aconitase, and succinate dehydrogenase [5][6][7][8][9][10][11]. NFU1 also plays a role in delivery of ISCs to components of complex I of the electron transport chain (ETC), yet the details of this delivery remain to be clarified [12,13].…”

Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation

“…It has been proposed, on the basis of a combination of in vitro and in vivo studies [ 23 , 28 ], that the BOLA3–GLRX5 complex transfers its [2Fe–2S] 2+ cluster to protein partners, such as NFU1 [ 26 ], which are involved in the final steps of the maturation of mitochondrial [4Fe–4S] cluster-binding target proteins [ 28 , 58 ]. On this respect, it is also relevant to investigate how the Cys59Tyr mutation affects the Fe–S cluster-binding properties of the hetero-complex, as Cys59 is a ligand of the [2Fe–2S] 2+ cluster in the hetero-complex [ 22 ].…”

Iron–Sulfur Cluster Biogenesis as a Critical Target in Cancer

“…Like ISCA1/2, the mitochondrial [4Fe-4S] cluster protein, NFU1, helps assemble and facilitate intramitochondrial [4Fe-4S] trafficking. The [4Fe-4S] 2+ ISCA1/2 heterodimer can directly transfer the cluster to NFU1 ( Figure 3 A) [ 66 ]. Holo-ISCA1 and holo-ISCU can also bind directly to NFU1 to donate a [2Fe-2S] cluster for [4Fe-4S] cluster formation in an FDX2 dependent manner ( Figure 3 B) [ 67 ].…”