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.

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.

Four new coordination polymers [Cd(L)(OAc)]·H₂O (1), [Cd(L)(µ₂-OH)]·H₂O (2), [Cd₂(L)₂(µ₃-SO₄)(H₂O)₂]·5H₂O (3) and [Cd(L)₂]·H₂O (4) with different structures and topologies were obtained by solvo/hydrothermal reactions of varied Cd(II) salts with 3,5-di(1H-imidazol-1-yl)benzoic acid (HL) and 1,3-bis(4-pyridyl)propane (bpp). The structures of 1–4 were characterized by single-crystal X-ray diffraction analysis and the results showed that no bpp ligands were incorporated in the resulting complexes, and more interestingly, complexes 1–3 were obtained by reactions of metal to ligand ratio of 1 : 1, however, when the reactions were carried out with a metal to ligand ratio of 1 : 2, the resulting complexes had the same structure, namely [Cd(L)₂]·H₂O (4). In contrast to the 2D network with 6³-hcb topology of complex 1 with acetate as terminal ligand, complex 2 is a 3D (4,6)-connected net with µ₂-hydroxyl bridging group. Complex 3 is an unprecedented 3D self-penetrating (3,4,5)-connected net with each sulfate as a µ₃-bridge to link three Cd(II) atoms, and complex 4 is a 3D self-penetrating (3,5)-connected net formed by two alternate chiral helical chains linking 2-fold interpenetrating 2D (4,8²) nets. The results showed that the L⁻ ligand has varied coordination modes and the counteranions have a great impact on the structure of the complexes.

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min⁻¹ at 25 °C, henceforth reported in units of min^–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer; however, while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA₃₈₆), NBD with a partial linker (HscA₃₈₉), and NBD with the full linker (HscA₃₉₅). We found that the rate of ATP hydrolysis of HscA₃₉₅ (0.45 min^–1) is nearly 15-fold higher than that of HscA₃₈₆ (0.035 min^–1), although their apparent affinities for ATP are equivalent. HscA₃₉₅, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence and basal thermostability that were higher than those of HscA₃₈₆. Furthermore, HscA₃₉₅ displayed narrower ¹H^N line widths in its two-dimensional ¹H–¹⁵N TROSY-HSQC spectrum in comparison to HscA₃₈₆, indicating that the peptide in the cis configuration binds to and stabilizes the NBD. The addition to HscA₃₈₆ of a synthetic peptide with a sequence identical to that of the interdomain linker (L³⁸⁷LLDVIPLS³⁹⁵) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.