The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome inhibitor-resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small-molecule compound elesclomol. Genome-wide CRISPR/Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and NMR-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies elucidate a fundamental mechanism by which cells adapt to proteotoxic stress and suggests strategies to mitigate proteasome inhibitor-resistance.

IscU from Escherichia coli, the scaffold protein for iron-sulfur cluster biosynthesis and delivery, populates a complex energy landscape. IscU exists as two slowly interconverting species: one (S) is largely structured with all four peptidyl–prolyl bonds trans; the other (D) is partly disordered but contains an ordered domain that stabilizes two cis peptidyl–prolyl peptide bonds. At pH 8.0, the S-state is maximally populated at 25 °C, but its population decreases at higher or lower temperatures or at lower pH. The D-state binds preferentially to the cysteine desulfurase (IscS), which generates and transfers sulfur to IscU cysteine residues to form persulfides. The S-state is stabilized by Fe–S cluster binding and interacts preferentially with the DnaJ-type co-chaperone (HscB), which targets the holo-IscU:HscB complex to the DnaK-type chaperone (HscA) in its ATP-bound from. HscA is involved in delivery of Fe–S clusters to acceptor proteins by a mechanism dependent on ATP hydrolysis. Upon conversion of ATP to ADP, HscA binds the D-state of IscU ensuring release of the cluster and HscB. These findings have led to a more complete model for cluster biosynthesis and delivery.

Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each ferredoxin became oxidized in the presence of the cysteine desulfurase complex when l-cysteine was added, leading to its conversion to l-alanine and the generation of sulfide. In an in vitro reaction, the reduced form of each ferredoxin was found to support Fe–S cluster assembly on ISCU; the rate of cluster assembly was faster with FDX2 than with FDX1. Taken together, these results show that both FDX1 and FDX2 can function in Fe–S cluster assembly in vitro.

SUMMARY Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N-and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermolecular disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-containing form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by molecular weight estimates from SAXS and size-exclusion chromatography. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. The activity of the holo-NFU1 preparation was verified by demonstrating its ability to activate apo-aconitase.

Frataxin (FXN) is involved in mitochondrial iron-sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron-sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe2+ but not Fe3+. While FXN (with or without bound Fe2+) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the cysteine desulfurase complex ([NFS1]2:[ISD11]2:[Acp]2), abbreviated as (NIA)2, where “N” represents the cysteine desulfurase (NFS1), “I” represents the accessory protein (ISD11), and “A” represents acyl carrier protein (Acp). FXN binds (NIA)2 weakly in the absence of ISCU but more strongly in its presence. Fe2+-FXN binds to the (NIA)2-ISCU2 complex without release of iron. However, upon the addition of both L-cysteine and a reductant (either reduced FDX2 or DTT), Fe2+ is released from FXN as consistent with Fe2+-FXN being the proximal source of iron for Fe-S cluster assembly.