Mechanism of Iron–Sulfur Cluster Assembly: In the Intimacy of Iron and Sulfur Encounter

Iron–sulfur (Fe–S) clusters are protein cofactors of a multitude of enzymes performing essential biological functions. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe–S clusters are assembled and a cysteine desulfurase that provides sulfur in the form of a persulfide. The sulfide ions are produced by reductive cleavage of the persulfide, which involves specific reductase systems. Several other components are required for Fe–S biosynthesis, including frataxin, a key protein of controversial function and accessory components for insertion of Fe–S clusters in client proteins. Fe–S cluster biosynthesis is thought to rely on concerted and carefully orchestrated processes. However, the elucidation of the mechanisms of their assembly has remained a challenging task due to the biochemical versatility of iron and sulfur and the relative instability of Fe–S clusters. Nonetheless, significant progresses have been achieved in the past years, using biochemical, spectroscopic and structural approaches with reconstituted system in vitro. In this paper, we review the most recent advances on the mechanism of assembly for the founding member of the Fe–S cluster family, the [2Fe2S] cluster that is the building block of all other Fe–S clusters. The aim is to provide a survey of the mechanisms of iron and sulfur insertion in the scaffold proteins by examining how these processes are coordinated, how sulfide is produced and how the dinuclear [2Fe2S] cluster is formed, keeping in mind the question of the physiological relevance of the reconstituted systems. We also cover the latest outcomes on the functional role of the controversial frataxin protein in Fe–S cluster biosynthesis.

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Iron–sulfur cluster biosynthesis

Biochemical Society Transactions ◽

10.1042/bst0361112 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1112-1119 ◽

Cited By ~ 112

Author(s):

Sibali Bandyopadhyay ◽

Kala Chandramouli ◽

Michael K. Johnson

Keyword(s):

Scaffold Proteins ◽

Cluster Assembly ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Eukaryotic Organisms ◽

Almost All ◽

Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

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Biogenesis and Transfer of Iron-Sulfur Clusters from Acidithiobacillus ferrooxidans

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.825.198 ◽

2013 ◽

Vol 825 ◽

pp. 198-201 ◽

Cited By ~ 1

Author(s):

Jian She Liu ◽

Lin Qian ◽

Chun Li Zheng

Keyword(s):

Acidithiobacillus Ferrooxidans ◽

Binding Protein ◽

Cluster Assembly ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

Iron Sulfur Clusters ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Iron Sulfur Proteins

Iron-sulfur (Fe-S) proteins are ubiquitous and participate in multiple essential functions of life. However, little is currently known about the mechanisms of iron-sulfur biosynthesis and transfer in acidophilic microorganisms. In this study, the IscS, IscU and IscA proteins from Acidithiobacillus ferrooxidans were successfully expressed in Escherichia coli and purified by affinity chromatography. The IscS was a cysteine desulfurase which catalyzes desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly. Purified IscU did not have an iron-sulfur cluster but could act as a scaffold protein to assemble the [2Fe-2S] cluster in vitro. The IscA was a [4Fe-4S] cluster binding protein, but it also acted as an iron binding protein. Further studies indicated that the iron sulfur clusters could be transferred from pre-assembled scaffold proteins to apo-form iron sulfur proteins, the reconstituted iron sulfur proteins could restore their physiological activities.

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N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization

Nature Communications ◽

10.1038/s41467-021-27122-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sven-A. Freibert ◽

Michal T. Boniecki ◽

Claudia Stümpfig ◽

Vinzent Schulz ◽

Nils Krapoth ◽

...

Keyword(s):

De Novo ◽

Cluster Formation ◽

Cluster Assembly ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

Mutant Proteins ◽

Iron Sulfur ◽

Initial Iron ◽

Oppositely Charged

AbstractSynthesis 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.

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The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011419 ◽

2020 ◽

Vol 295 (33) ◽

pp. 11891-11901 ◽

Cited By ~ 1

Author(s):

Brigitta Németh ◽

Henrik Land ◽

Ann Magnuson ◽

Anders Hofer ◽

Gustav Berggren

Keyword(s):

Gas Production ◽

Cluster Assembly ◽

Paramagnetic Resonance ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Spectroscopy Studies ◽

Electron Paramagnetic

[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly, i.e. in binding and delivering the precatalyst.

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Mechanism of activation of the human cysteine desulfurase complex by frataxin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909535116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19421-19430 ◽

Cited By ~ 15

Author(s):

Shachin Patra ◽

David P. Barondeau

Keyword(s):

Acyl Carrier Protein ◽

Cysteine Residue ◽

Cluster Assembly ◽

Cysteine Desulfurase ◽

Iron Sulfur ◽

Cluster Biosynthesis ◽

Low Activity ◽

New Strategies ◽

Allosteric Activator

The function of frataxin (FXN) has garnered great scientific interest since its depletion was linked to the incurable neurodegenerative disease Friedreich’s ataxia (FRDA). FXN has been shown to be necessary for iron-sulfur (Fe-S) cluster biosynthesis and proper mitochondrial function. The structural and functional core of the Fe-S cluster assembly complex is a low-activity pyridoxal 5′-phosphate (PLP)–dependent cysteine desulfurase enzyme that consists of catalytic (NFS1), LYRM protein (ISD11), and acyl carrier protein (ACP) subunits. Although previous studies show that FXN stimulates the activity of this assembly complex, the mechanism of FXN activation is poorly understood. Here, we develop a radiolabeling assay and use stopped-flow kinetics to establish that FXN is functionally linked to the mobile S-transfer loop cysteine of NFS1. Our results support key roles for this essential cysteine residue in substrate binding, as a general acid to advance the Cys-quinonoid PLP intermediate, as a nucleophile to form an NFS1 persulfide, and as a sulfur delivery agent to generate a persulfide species on the Fe-S scaffold protein ISCU2. FXN specifically accelerates each of these individual steps in the mechanism. Our resulting architectural switch model explains why the human Fe-S assembly system has low inherent activity and requires activation, the connection between the functional mobile S-transfer loop cysteine and FXN binding, and why the prokaryotic system does not require a similar FXN-based activation. Together, these results provide mechanistic insights into the allosteric-activator role of FXN and suggest new strategies to replace FXN function in the treatment of FRDA.

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