Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. ...Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a 2Fe-2S cluster on a scaffold protein; Hsp70 chaperone-mediated trafficking of the cluster and insertion into 2Fe-2S target apoproteins; and catalytic conversion of the 2Fe-2S into a 4Fe-4S cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein-mediated maturation of mitochondrial Fe S proteins and the importance for human disease.
Mechanistic concepts of iron-sulfur protein biogenesis in Biology Braymer, Joseph J.; Freibert, Sven A.; Rakwalska-Bange, Magdalena ...
Biochimica et biophysica acta. Molecular cell research,
January 2021, 2021-01-00, 20210101, Letnik:
1868, Številka:
1
Journal Article
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Iron-sulfur (Fe/S) proteins are present in virtually all living organisms and are involved in numerous cellular processes such as respiration, photosynthesis, metabolic reactions, nitrogen fixation, ...radical biochemistry, protein synthesis, antiviral defense, and genome maintenance. Their versatile functions may go back to the proposed role of their Fe/S cofactors in the origin of life as efficient catalysts and electron carriers. More than two decades ago, it was discovered that the in vivo synthesis of cellular Fe/S clusters and their integration into polypeptide chains requires assistance by complex proteinaceous machineries, despite the fact that Fe/S proteins can be assembled chemically in vitro. In prokaryotes, three Fe/S protein biogenesis systems are known; ISC, SUF, and the more specialized NIF. The former two systems have been transferred by endosymbiosis from bacteria to mitochondria and plastids, respectively, of eukaryotes. In their cytosol, eukaryotes use the CIA machinery for the biogenesis of cytosolic and nuclear Fe/S proteins. Despite the structural diversity of the protein constituents of these four machineries, general mechanistic concepts underlie the complex process of Fe/S protein biogenesis. This review provides a comprehensive and comparative overview of the various known biogenesis systems in Biology, and summarizes their common or diverging molecular mechanisms, thereby illustrating both the conservation and diverse adaptions of these four machineries during evolution and under different lifestyles. Knowledge of these fundamental biochemical pathways is not only of basic scientific interest, but is important for the understanding of human ‘Fe/S diseases’ and can be used in biotechnology.
•FeS proteins assist numerous cellular processes and many are essential for life.•FeS proteins are assembled by one or more out of four different machineries.•Similar mechanistic concepts underlie FeS protein biogenesis in all machineries.•Biogenesis involves synthesis, trafficking and apoprotein targeting of FeS clusters.•Minimal machineries reveal the basic biochemical requirements for biogenesis.
Synthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 ...scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging 2Fe-2S cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent 2Fe-2S cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced 2Fe-2S cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial 2Fe-2S cluster synthesis.
Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis ...occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.
Ferredoxins (FDXs) comprise a large family of iron–sulfur proteins that shuttle electrons from NADPH and FDX reductases into diverse biological processes. This review focuses on the structure, ...function and specificity of mitochondrial 2Fe‐2S FDXs that are related to bacterial FDXs due to their endosymbiotic inheritance. Their classical function in cytochrome P450‐dependent steroid transformations was identified around 1960, and is exemplified by mammalian FDX1 (aka adrenodoxin). Thirty years later the essential function in cellular Fe/S protein biogenesis was discovered for the yeast mitochondrial FDX Yah1 that is additionally crucial for the formation of haem a and ubiquinone CoQ6. In mammals, Fe/S protein biogenesis is exclusively performed by the FDX1 paralog FDX2, despite the high structural similarity of both proteins. Recently, additional and specific roles of human FDX1 in haem a and lipoyl cofactor biosyntheses were described. For lipoyl synthesis, FDX1 transfers electrons to the radical S‐adenosyl methionine‐dependent lipoyl synthase to kickstart its radical chain reaction. The high target specificity of the two mammalian FDXs is contained within small conserved sequence motifs, that upon swapping change the target selection of these electron donors.
Mitochondrial 2Fe‐2S ferredoxins are electron donors involved in numerous processes. While most eukaryotes contain only one ferredoxin, some species such as mammals including humans contain two isoforms, the FDX1 (aka adrenodoxin) and FDX2. They differ in their usually non‐overlapping substrate specificities.
Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) after sensing viral/bacterial RNA or DNA by toll-like receptor (TLR) 7 or TLR9, respectively. However, aberrant pDCs activation ...can cause adverse effects on the host and contributes to the pathogenesis of type I IFN-related autoimmune diseases. Here, we show that heparin interacts with the human pDCs-specific blood dendritic cell antigen 2 (BDCA-2) but not with related lectins such as DCIR or dectin-2. Importantly, BDCA-2-heparin interaction depends on heparin sulfation and receptor glycosylation and results in inhibition of TLR9-driven type I IFN production in primary human pDCs and the pDC-like cell line CAL-1. This inhibition is mediated by unfractionated and low-molecular-weight heparin, as well as endogenous heparin from plasma, suggesting that the local blood environment controls the production of IFN-α in pDCs. Additionally, we identified an activation-dependent soluble form of BDCA-2 (solBDCA-2) in human plasma that functions as heparin antagonist and thereby increases TLR9-driven IFN-α production in pDCs. Of importance, solBDCA-2 levels in the serum were increased in patients with scrub typhus (an acute infectious disease caused by
) compared to healthy control subjects and correlated with anti-dsDNA antibodies titers. In contrast, solBDCA-2 levels in plasma from patients with bullous pemphigoid or psoriasis were reduced. In summary, this work identifies a regulatory network consisting of heparin, membrane-bound and solBDCA-2 modulating TLR9-driven IFN-α production in pDCs. This insight into pDCs function and regulation may have implications for the treatment of pDCs-related autoimmune diseases.
Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex ...NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes 2Fe-2S clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381
to Cys138
and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138
rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological 2Fe-2S cluster assembly and clarifies frataxin's molecular role in this fundamental process.
Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of 2Fe-2S ferredoxins, FDX1 (aka ...adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.
The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet ...defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.
Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO2, but the molecular ...mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating FeFe-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H2). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P)+ and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P)+ binding affinity via reduction of a nearby iron–sulfur cluster, HydABC switches between the exergonic NAD(P)+ reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases.
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