GO:0051537 2 iron, 2 sulfur cluster binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0051537 defines the molecular function of binding a 2Fe-2S cluster, a cofactor composed of two iron atoms bridged by two inorganic sulfur atoms.
• 2Fe-2S clusters are ancient, versatile cofactors that enable electron transfer, substrate binding, and structural stabilization in proteins across all domains of life [1, 5].
• Mitochondrial ferredoxin-2 (FDX2) binds the core iron-sulfur cluster assembly complex in a two-stage manner, highlighting the dynamic nature of 2Fe-2S cluster delivery.
• The SARS-CoV-2 helicase contains a 2Fe-2S cluster within its zinc-binding domain that modulates RNA binding and unwinding activities.
• AlphaFold2-based predictions have dramatically expanded the known metalloproteome by identifying thousands of potential 2Fe-2S and zinc-binding sites.
• Dysregulation of 2Fe-2S cluster proteins such as CISD2 is linked to ferroptosis and cancer, making these proteins attractive therapeutic targets.
Description
Iron-sulfur (Fe-S) clusters are among the most ancient and versatile protein cofactors, and the 2 iron, 2 sulfur (2Fe-2S) cluster is a particularly widespread variant. The Gene Ontology term GO:0051537, 2 iron, 2 sulfur cluster binding, describes the molecular function of selectively binding a 2Fe-2S cluster, which consists of two iron atoms bridged by two inorganic sulfur atoms. This binding event is critical for the structural integrity and catalytic activity of numerous proteins involved in electron transport, enzymatic catalysis, and gene regulation [1, 5]. Researchers study 2Fe-2S cluster binding because it underpins fundamental biological processes, from mitochondrial respiration to DNA repair and viral replication [1, 6]. Defects in Fe-S cluster biogenesis or binding are associated with a growing list of human diseases, including neurodegenerative disorders, cancer, and metabolic syndromes. The recent application of deep learning tools like AlphaFold2 has predicted thousands of previously unrecognized Fe-S cluster-binding sites, expanding the known metalloproteome and opening new avenues for functional genomics. Understanding the precise molecular interactions that govern 2Fe-2S cluster binding is essential for deciphering how cells manage iron homeostasis, respond to oxidative stress, and execute essential metabolic pathways [1, 5]. This article provides a comprehensive overview of the GO:0051537 term, its mechanistic basis, associated genes, disease relevance, and the experimental strategies used to study it.
2 iron, 2 sulfur cluster binding At A Glance
| GO ID | GO:0051537 |
|---|---|
| GO term | 2 iron, 2 sulfur cluster binding |
| Ontology | molecular_function |
| Synonym | 2Fe-2S cluster binding; diiron disulfide cluster binding; iron-sulfur cluster 2Fe-2S binding |
| Major function | Binding to a 2Fe-2S cluster for electron transfer, catalysis, or structural roles |
| Cluster composition | Two iron atoms and two inorganic sulfur atoms acting as bridging ligands |
| Common ligands | Cysteine thiolates, occasionally histidine or other residues |
| Representative proteins | Ferredoxins, Rieske proteins, iron-sulfur assembly proteins |
| Disease relevance | Cancer, neurodegeneration, mitochondrial disorders, viral replication |
What Is GO:0051537?
GO:0051537 (2 iron, 2 sulfur cluster binding) is a molecular function term that describes the binding to a 2Fe-2S cluster. This cluster is a prosthetic group composed of two iron atoms and two inorganic sulfur atoms, where the sulfur atoms act as bridging ligands between the irons. Proteins that possess this function can coordinate the cluster through cysteine or histidine residues, enabling electron transfer, substrate activation, or structural stabilization [1, 7].
Why Is 2 iron, 2 sulfur cluster binding Important in Cell Biology?
The 2Fe-2S cluster binding function is essential for life because it enables proteins to perform electron transfer, sense iron and oxygen levels, and catalyze diverse chemical reactions [1, 5]. Disruption of this function leads to mitochondrial dysfunction, impaired DNA repair, and increased susceptibility to oxidative stress, underscoring its importance in human health and disease.
• 2Fe-2S clusters are critical for mitochondrial electron transport and cellular respiration.
• They serve as cofactors in enzymes involved in DNA replication and repair, influencing genome stability.
• The SARS-CoV-2 helicase requires a 2Fe-2S cluster for optimal RNA unwinding, linking this function to viral pathogenesis.
• Mutations in Fe-S cluster assembly proteins cause human diseases such as Friedreich's ataxia and ISCU myopathy.
• 2Fe-2S cluster binding is involved in iron homeostasis and the regulation of ferroptosis.
• Predictive proteomics has revealed thousands of potential 2Fe-2S proteins, many with unknown functions.
• Histidine-ligated 2Fe-2S peptides expand the structural diversity of cluster coordination.
• Small molecules targeting 2Fe-2S clusters show promise as anticancer and antiviral agents.
• The 2Fe-2S cluster in CISD2 regulates autophagy and cell survival pathways.
• Understanding 2Fe-2S binding aids in engineering metalloproteins for biotechnology.
Molecular Mechanism of 2 iron, 2 sulfur cluster binding
Cluster Coordination and Ligand Environment
In simple terms: The cluster is held in place by surrounding amino acids, usually cysteines.
The 2Fe-2S cluster is typically coordinated by four cysteine thiolate ligands, although histidine and other residues can substitute in some proteins [1, 7]. The cluster adopts a rhombic structure with two iron atoms bridged by two inorganic sulfurs. The protein environment fine-tunes the redox potential of the cluster, enabling it to function in electron transfer chains or as a catalytic center. Recent studies on histidine-ligated Fe-S peptides demonstrate that alternative ligation can modulate cluster stability and reactivity.
Two-Stage Binding of Ferredoxin-2 to the Core Assembly Complex
In simple terms: Ferredoxin-2 docks onto the assembly machinery in two steps to deliver the cluster.
Mitochondrial ferredoxin-2 (FDX2) binds to the core iron-sulfur cluster assembly complex in a two-stage process. First, FDX2 forms an initial encounter complex, followed by a tighter binding stage that facilitates cluster transfer. This dynamic interaction ensures efficient assembly of 2Fe-2S clusters and their delivery to target apoproteins. The study by Steinhilper et al. (2024) provides structural insights into this mechanism, highlighting the role of electrostatic interactions and conformational changes.
Electron Transfer and Redox Properties
In simple terms: The cluster can shuttle electrons because iron atoms change their oxidation state.
2Fe-2S clusters can undergo reversible one-electron oxidation-reduction between the [2Fe-2S]2+ and [2Fe-2S]+ states. This property is exploited by ferredoxins and Rieske proteins in electron transport chains [1, 5]. The redox potential is influenced by the protein environment, including hydrogen bonding and solvent exposure. In mitochondrial ferredoxin-2, the cluster participates in electron transfer to cytochrome P450 enzymes and other acceptors.
Regulation by Cluster Availability and Oxidative Stress
In simple terms: Cells adjust cluster binding based on iron levels and oxidative stress.
The binding of 2Fe-2S clusters is regulated by iron availability, the activity of the ISC (iron-sulfur cluster) assembly machinery, and cellular redox status [1, 8]. Oxidative stress can damage the cluster, leading to protein dysfunction and triggering degradation or repair pathways. The CISD2 protein, which contains a 2Fe-2S cluster, is involved in ferroptosis regulation through ferritinophagy and the p62-Keap1-NRF2 pathway. This illustrates how cluster binding is integrated with stress-responsive signaling.
Viral and Bacterial Exploitation of 2Fe-2S Clusters
In simple terms: Some viruses and bacteria use these clusters for their own replication.
The SARS-CoV-2 helicase contains a 2Fe-2S cluster within its zinc-binding domain that modulates RNA binding and unwinding activities. This highlights how pathogens hijack host iron-sulfur cluster machinery for replication. Similarly, nitrogenase enzymes in bacteria use complex Fe-S clusters, including 2Fe-2S units, for dinitrogen activation. These examples underscore the broad biological significance of 2Fe-2S cluster binding.
Key Genes Involved in GO:0051537 2 iron, 2 sulfur cluster binding
The following genes encode proteins that bind or assemble 2Fe-2S clusters, and they are frequently studied to understand the function and regulation of GO:0051537.
| Gene | Major Role | Research Relevance |
|---|---|---|
| FDX2 | Mitochondrial ferredoxin-2; delivers electrons and clusters | Two-stage binding to ISC assembly complex |
| ISCU | Scaffold protein for Fe-S cluster assembly | Mutations cause myopathy; central to cluster biogenesis |
| CISD2 | 2Fe-2S cluster protein in ER/mitochondria | Regulates ferroptosis and autophagy |
| NFS1 | Cysteine desulfurase; provides sulfur for clusters | Essential for Fe-S cluster synthesis |
| FXN | Frataxin; involved in iron-sulfur cluster assembly | Deficiency causes Friedreich's ataxia |
| LYRM4 | Accessory protein for Fe-S assembly | Required for FDX2 function |
| BOLA3 | Involved in Fe-S cluster maturation | Mutations linked to mitochondrial disease |
| GLRX5 | Glutaredoxin; transfers clusters to target proteins | Facilitates 2Fe-2S cluster delivery |
| HSCB | Co-chaperone for Fe-S cluster assembly | Assists in cluster transfer |
| GRPEL1 | Mitochondrial chaperone | Supports Fe-S cluster assembly |
| NUBPL | Assembly factor for respiratory complex I | Contains 2Fe-2S cluster; mutations cause disease |
| SDHB | Succinate dehydrogenase subunit B | Contains 2Fe-2S clusters; cancer relevance |
| Rieske proteins (e.g., UQCRFS1) | Electron transfer in cytochrome bc1 | 2Fe-2S cluster binding for respiration |
| NDUFS1 | Complex I subunit | Contains 2Fe-2S clusters; mitochondrial function |
| SARS-CoV-2 helicase (nsp13) | Viral RNA unwinding | 2Fe-2S cluster modulates activity |
| Nitrogenase (nif genes) | Dinitrogen activation | Mo-Fe-S cluster with 2Fe-2S units |
| CIAO1 | Cytosolic Fe-S assembly | Required for cluster maturation |
| MMS19 | Cytosolic Fe-S protein assembly | Facilitates cluster transfer |
How Is 2 iron, 2 sulfur cluster binding Regulated?
The binding of 2Fe-2S clusters is regulated at multiple levels. Iron availability controls the expression of genes involved in cluster assembly through the IRP/IRE system. The ISC assembly machinery is regulated by the availability of sulfur from cysteine desulfurase NFS1 and electrons from FDX2. Oxidative stress can damage clusters, leading to protein degradation or activation of stress responses such as the integrated stress response. Additionally, the CISD2 protein is regulated by the p62-Keap1-NRF2 pathway, linking cluster binding to ferroptosis and autophagy.
2 iron, 2 sulfur cluster binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CISD2 | Cancer, ferroptosis, Wolfram syndrome 2 | CISD2 knockout cancer cell lines; ferroptosis induction assays |
| FDX2 | Mitochondrial myopathy, neurodegeneration | FDX2 knockout iPSC-derived neurons; mitochondrial function assays |
| ISCU | Myopathy with lactic acidosis | ISCU mutant knock-in mice; muscle biopsies |
| SARS-CoV-2 nsp13 | COVID-19 viral replication | nsp13 point mutants in viral replicon systems |
| SDHB | Paraganglioma, pheochromocytoma | SDHB knockout cell lines; metabolic profiling |
Cancer and Ferroptosis
2Fe-2S cluster proteins such as CISD2 play a role in cancer cell survival and ferroptosis. Inhibition of CISD2 promotes ferroptosis through ferritinophagy-mediated ferritin turnover and regulation of the p62-Keap1-NRF2 pathway. This suggests that targeting 2Fe-2S cluster binding could be a therapeutic strategy for cancers resistant to conventional treatments.
Neurodegeneration and Mitochondrial Disorders
Mutations in genes encoding Fe-S cluster assembly proteins, such as FDX2 and ISCU, cause mitochondrial myopathies and neurodegenerative diseases. Defective 2Fe-2S cluster binding leads to impaired mitochondrial respiration and increased oxidative stress, contributing to neuronal death. Friedreich's ataxia, caused by frataxin deficiency, is a classic example of a disease linked to Fe-S cluster dysfunction.
Viral Infections
The SARS-CoV-2 helicase requires a 2Fe-2S cluster for efficient RNA unwinding, making it a potential antiviral target. Disruption of cluster binding could inhibit viral replication. This highlights the broader relevance of 2Fe-2S clusters in infectious diseases.
Metabolic and Iron Homeostasis Disorders
2Fe-2S clusters are essential for iron-sulfur cluster biogenesis and iron homeostasis. Dysregulation can lead to iron overload or deficiency, affecting multiple organs [1, 8]. The CISD2 protein is also involved in longevity and metabolic regulation, linking cluster binding to aging and metabolic syndromes.
From 2 iron, 2 sulfur cluster binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of FDX2 impair 2Fe-2S cluster delivery? | FDX2 knockout cell line (e.g., HEK293) |
| How does a point mutation in the cluster-binding motif affect protein function? | Point-mutation knock-in of cysteine-to-serine in target gene |
| Can a tagged version of CISD2 reveal its interactome? | Knock-in of FLAG-HA tag at endogenous CISD2 locus |
| What is the effect of CISD2 overexpression on ferroptosis? | CISD2 overexpression stable cell line |
| Does the SARS-CoV-2 helicase require its 2Fe-2S cluster for RNA unwinding? | Point mutation of cluster-coordinating residues in nsp13 |
| Can AlphaFold2-predicted 2Fe-2S proteins be validated? | Knockout of predicted genes followed by proteomics |
How to Study the 2 iron, 2 sulfur cluster binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| EPR spectroscopy | Paramagnetic states of Fe-S clusters | Detection of 2Fe-2S clusters in purified proteins |
| Mössbauer spectroscopy | Iron oxidation and spin states | Characterization of cluster type |
| AlphaFold2 prediction | Potential metal-binding sites | Proteome-wide discovery of 2Fe-2S proteins |
| ICP-MS | Iron and sulfur content | Quantification of cluster stoichiometry |
| Helicase unwinding assay | RNA unwinding activity | Testing SARS-CoV-2 nsp13 function |
| Ferroptosis assay | Cell death due to lipid peroxidation | Evaluating CISD2 inhibition |
| CRISPR knockout screen | Gene essentiality and pathway discovery | Identifying regulators of 2Fe-2S cluster binding |
| Co-immunoprecipitation | Protein-protein interactions | Studying FDX2 binding to ISC complex |
Structural and Spectroscopic Characterization
Electron paramagnetic resonance (EPR), circular dichroism (CD), and Mössbauer spectroscopy are used to confirm 2Fe-2S cluster binding and determine redox states [1, 4]. X-ray crystallography and cryo-EM provide atomic-level details of cluster coordination.
Proteomic and Predictive Approaches
AlphaFold2-based predictions have identified thousands of potential 2Fe-2S cluster-binding sites across proteomes. Mass spectrometry-based proteomics can validate these predictions by detecting iron-sulfur cluster-containing peptides.
Functional Assays for Cluster Binding
Enzymatic activity assays, such as succinate dehydrogenase or helicase unwinding assays, measure the functional impact of 2Fe-2S cluster binding. Iron content can be quantified using colorimetric or inductively coupled plasma mass spectrometry (ICP-MS).
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes required for 2Fe-2S cluster binding and function. For example, screens for ferroptosis regulators have highlighted CISD2. These screens are powerful for discovering novel components of the Fe-S cluster machinery.
How CRISPR Can Be Used to Study GO:0051537 2 iron, 2 sulfur cluster binding
Knockout
CRISPR knockout of genes encoding 2Fe-2S cluster-binding proteins, such as FDX2 or CISD2, allows researchers to assess loss-of-function phenotypes. For example, FDX2 knockout cells exhibit impaired mitochondrial respiration and reduced cluster assembly. CISD2 knockout promotes ferroptosis, providing insights into its role in cell survival.
Point Mutation
Introducing point mutations in cluster-coordinating residues (e.g., cysteine to serine) via CRISPR base editing or HDR can dissect the contribution of individual ligands to 2Fe-2S binding. Such mutations in the SARS-CoV-2 helicase abolish RNA unwinding activity.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) at endogenous loci enables proteomic and imaging studies of 2Fe-2S proteins. Tagged CISD2 can be used to study its interactome and subcellular localization.
Overexpression
CRISPR activation (CRISPRa) or cDNA overexpression can elevate levels of 2Fe-2S cluster-binding proteins to study gain-of-function effects. Overexpression of CISD2 protects cells from ferroptosis, highlighting its cytoprotective role.
How EDITGENE Supports 2 iron, 2 sulfur cluster binding Research
Researchers studying 2 iron, 2 sulfur cluster binding-related genes often need to determine whether a candidate gene is causally involved in cluster assembly, electron transfer, or disease pathogenesis. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research, from gene knockout to precise point mutations and library screening.
Contact EDITGENE today to design your custom CRISPR model for 2 iron, 2 sulfur cluster binding research.
Frequently Asked Questions About 2 iron, 2 sulfur cluster binding
What is GO:0051537?
GO:0051537 is a Gene Ontology molecular function term that describes the binding to a 2 iron, 2 sulfur (2Fe-2S) cluster, a cofactor composed of two iron atoms bridged by two inorganic sulfur atoms.
What genes are involved in 2 iron, 2 sulfur cluster binding?
Key genes include FDX2, ISCU, CISD2, NFS1, FXN, and others encoding ferredoxins and Fe-S assembly proteins [1, 8].
What is the function of 2Fe-2S clusters?
2Fe-2S clusters facilitate electron transfer, substrate binding, and structural stabilization in proteins involved in respiration, DNA repair, and metabolism [1, 5].
How are 2Fe-2S clusters assembled?
They are assembled by the ISC machinery, involving cysteine desulfurase NFS1, scaffold ISCU, and ferredoxin FDX2, which delivers electrons and clusters.
What diseases are associated with 2Fe-2S cluster binding defects?
Diseases include mitochondrial myopathies, neurodegeneration (e.g., Friedreich's ataxia), cancer, and viral infections like COVID-19 [1, 6, 8].
How can I study 2Fe-2S cluster binding in the lab?
Methods include EPR, Mössbauer spectroscopy, AlphaFold2 prediction, CRISPR knockout, and functional assays such as helicase unwinding [1, 5, 6].
What is the role of CISD2 in ferroptosis?
CISD2 contains a 2Fe-2S cluster and regulates ferroptosis through ferritinophagy and the p62-Keap1-NRF2 pathway; its inhibition promotes ferroptosis.
Does SARS-CoV-2 use 2Fe-2S clusters?
Yes, the SARS-CoV-2 helicase nsp13 contains a 2Fe-2S cluster that modulates its RNA binding and unwinding activities.
Can AlphaFold2 predict 2Fe-2S binding sites?
Yes, AlphaFold2 has been used to predict Fe-S cluster and zinc-binding sites across proteomes, dramatically expanding the known metalloproteome.
What CRISPR models are available for studying 2Fe-2S cluster genes?
EDITGENE offers knockout, point mutation, knock-in, tagged knock-in, and overexpression models for genes like FDX2, CISD2, and ISCU, as well as custom library screening.
Conclusion
GO:0051537 (2 iron, 2 sulfur cluster binding) represents a fundamental molecular function that underpins diverse biological processes, from mitochondrial respiration to viral replication. The integration of structural biology, predictive proteomics, and CRISPR-based genetics has greatly expanded our understanding of 2Fe-2S cluster proteins and their roles in health and disease [1, 5, 6]. Continued research into this term promises to reveal new therapeutic targets for cancer, neurodegeneration, and infectious diseases. EDITGENE provides end-to-end CRISPR solutions to study 2Fe-2S cluster-binding genes, enabling researchers to move from candidate gene to functional validation with speed and precision.
References
- 1. Steinhilper R et al.. 2024. Two-stage binding of mitochondrial ferredoxin-2 to the core iron-sulfur cluster assembly complex.. Nat Commun 15(1):10559 PMID: 39632806
- 3. McSkimming A et al.. 2021. Dinitrogen binding and activation at a molybdenum-iron-sulfur cluster.. Nat Chem 13(7):666-670 PMID: 34045715
- 4. Gong W et al.. 2024. Insight into the photodynamic mechanism and protein binding of a nitrosyl iron-sulfur [Fe(2)S(2)(NO)(4)](2-) cluster.. Spectrochim Acta A Mol Biomol Spectrosc 320:124603 PMID: 38878720
- 5. Wehrspan ZJ et al.. 2022. Identification of Iron-Sulfur (Fe-S) Cluster and Zinc (Zn) Binding Sites Within Proteomes Predicted by DeepMind's AlphaFold2 Program Dramatically Expands the Metalloproteome.. J Mol Biol 434(2):167377 PMID: 34838520
- 6. Maio N et al.. 2023. An iron-sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities.. Proc Natl Acad Sci U S A 120(33):e2303860120 PMID: 37552760
- 7. Valer L et al.. 2022. Histidine Ligated Iron-Sulfur Peptides.. Chembiochem 23(14):e202200202 PMID: 35674331
- 8. Li Y et al.. 2022. Inhibition of CISD2 promotes ferroptosis through ferritinophagy-mediated ferritin turnover and regulation of p62-Keap1-NRF2 pathway.. Cell Mol Biol Lett 27(1):81 PMID: 36180832
