Human Nfu is an iron–sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe–2S] (GS)4 cluster complex, and extraction of the Nfu-bound iron–sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] cluster trafficking and delivery.

Iron-sulfur clusters (Fe-S) are one of the most ancient, ubiquitous and versatile classes of metal cofactors found in nature. Proteins that contain Fe-S clusters constitute one of the largest families of proteins, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, radical generation, and, more recently discovered, DNA repair. Research during the past two decades has shown that mitochondria are central to the biogenesis of Fe-S clusters in eukaryotic cells via a conserved cluster assembly machinery (ISC assembly machinery) that also controls the synthesis of Fe-S clusters of cytosolic and nuclear proteins. Several key steps for synthesis and trafficking have been determined for mitochondrial Fe-S clusters, as well as the cytosol (CIA - cytosolic iron-sulfur protein assembly), but detailed mechanisms of cluster biosynthesis, transport, and exchange are not well established. Genetic mutations and the instability of certain steps in the biosynthesis and maturation of mitochondrial, cytosolic and nuclear Fe-S cluster proteins affects overall cellular iron homeostasis and can lead to severe metabolic, systemic, neurological and hematological diseases, often resulting in fatality. In this review we briefly summarize the current molecular understanding of both mitochondrial ISC and CIA assembly machineries, and present a comprehensive overview of various associated inborn human disease states.

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS4) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe-S cluster transfer reactions. UV-vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS4) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS4) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe-S cluster proteins, thereby supporting a possible physiological role for such centers.

Iron-sulfur (Fe/S) cluster-containing proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an iron-sulfur scaffold protein, via an in vitro investigation into the structural and functional consequences. Analysis of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure composition. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an iron-sulfur cluster from physiologically relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change.

Complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb of the Hepatitis C Virus (HCV) Internal Ribosomal Entry Site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein are described additional studies focused on understanding the cleavage mechanism, as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determination, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all d-amino acid form of this metallopeptide, complex 2-Cu, is reported and compared to complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD of 76 ± 3 nM, and Michaelis-Menten parameters of kcat of 0.14 ± 0.01 min−1 and KM of 7.9 ± 1.2 µM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to the parent peptide, complex 1-Cu, with IC50 of 1.9 ± 0.4 µM and cytotoxicity exceeding 100 µM. RT-PCR experiments confirm a significant reduction in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three day dosing increments. This study shows the influence that the α-carbon stereocenter has for this the new class of compounds, while detailed mass spectrometry and computational analysis provide new insights into the mechanisms of recognition, binding, and reactivity.