GO:0035731 dinitrosyl-iron complex binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0035731 describes the molecular function of binding to dinitrosyl-iron complexes (DNICs), which are spontaneous carriers of nitric oxide (NO) formed from glutathione, S-nitrosoglutathione, and ferrous iron.
• DNICs are not passive NO donors; they modify protein function by S-nitrosation of cysteine residues, as shown for serum albumin.
• DNIC binding can inhibit key enzymes such as glutathione reductase, linking this function to redox homeostasis.
• Proteins such as NorA and albumin act as DNIC-binding vehicles that influence NO storage, transport, and delivery to tissues including the brain.
• The physico-chemical properties of DNICs determine their biological activity, including stability, lipophilicity, and reactivity with thiols.
• Studying GO:0035731 requires integrated approaches: spectroscopy, proteomics, and CRISPR-based models to dissect gene function in NO biology.
Description
Dinitrosyl-iron complexes (DNICs) are paramagnetic, low-molecular-weight species that serve as a major storage and transport form of nitric oxide (NO) in biological systems. They form spontaneously from glutathione (GSH), S-nitrosoglutathione, and trace ferrous ions, or through the reaction of iron-sulfur centers with NO. The Gene Ontology term GO:0035731, dinitrosyl-iron complex binding, defines the molecular function of selectively binding these DNICs. This function is critical because DNICs can modulate protein activity, enzyme catalysis, and cellular redox state. Researchers study DNIC binding to understand how NO signals are buffered, delivered, and translated into physiological or pathological outcomes. The interaction of DNICs with serum albumin, for example, leads to S-nitrosation of the protein, altering its conformation and function. In bacteria, the NorA protein binds DNICs as part of NO detoxification or sensing mechanisms. Thus, GO:0035731 represents a convergence point for NO chemistry, iron metabolism, and protein regulation.
dinitrosyl-iron complex binding At A Glance
| GO ID | GO:0035731 |
|---|---|
| GO term | dinitrosyl-iron complex binding |
| Ontology | molecular_function |
| Synonym | DNDGIC binding; DNIC binding; dinitrosyl-diglutathionyl-iron complex binding |
| Major function | Binding to dinitrosyl-iron complexes, which serve as NO storage and transport species |
| Definition source | QuickGO |
| Related chemistry | Formation from GSH, S-nitrosoglutathione, and ferrous ions; reaction of iron-sulfur centers with NO |
| Biological context | NO signaling, redox regulation, iron-sulfur cluster metabolism |
What Is GO:0035731?
GO:0035731, dinitrosyl-iron complex binding, is a molecular function defined as the selective interaction with a dinitrosyl-iron complex (DNIC). DNICs are formed when nitric oxide (NO) binds to ferrous iron in the presence of thiols such as glutathione, producing species like dinitrosyl-diglutathionyl-iron complex (DNDGIC). This binding function enables proteins to sequester, transport, or sense NO, and often results in post-translational modification of the binding protein or its targets.
Why Is dinitrosyl-iron complex binding Important in Cell Biology?
GO:0035731 is important because DNICs are central to nitric oxide biology, a signaling molecule involved in vasodilation, neurotransmission, and immune defense. Proteins that bind DNICs can modulate NO bioavailability, protect against nitrosative stress, or deliver NO to specific targets. Dysregulation of DNIC binding has been linked to altered enzyme activities, such as inhibition of glutathione reductase, which can shift cellular redox balance. Understanding this function helps explain how NO exerts both physiological and pathological effects, from neurogenesis to neurodegeneration.
• DNICs are the main non-protein-bound form of NO in cells and tissues.
• Binding to DNICs can lead to S-nitrosation of cysteine residues, modifying protein function.
• DNIC binding to glutathione reductase inhibits the enzyme, affecting antioxidant defense.
• Albumin acts as a major DNIC-binding protein in blood, influencing NO transport.
• NorA from Ralstonia eutropha binds DNICs, providing a model for bacterial NO sensing.
• DNIC binding is relevant to neurogenesis, as DNIC-albumin conjugates can deliver NO to the brain.
• Carnosine forms DNICs, suggesting a role in buffering NO and iron.
• The function is studied using synthetic DNIC models to understand formation pathways.
• GO:0035731 links iron metabolism, NO signaling, and redox regulation in health and disease.
• Targeting DNIC binding proteins may offer therapeutic strategies for NO-related disorders.
Molecular Mechanism of dinitrosyl-iron complex binding
Formation of dinitrosyl-iron complexes
In simple terms: DNICs form when nitric oxide meets iron and thiols in the cell.
Dinitrosyl-iron complexes assemble spontaneously from glutathione (GSH), S-nitrosoglutathione, and trace ferrous ions, or through the reaction of iron-sulfur centers with NO. The resulting DNICs, such as dinitrosyl-diglutathionyl-iron complex (DNDGIC), are stable, lipophilic species that can diffuse and interact with proteins. Synthetic models using iron-thiolate complexes have elucidated the stepwise nitrosylation pathway that yields DNICs.
Binding to protein targets
In simple terms: Proteins can grab DNICs, often through cysteine or iron-sulfur clusters.
Proteins bind DNICs via thiolate ligands, typically cysteine residues or pre-existing iron-sulfur clusters. For example, serum albumin binds DNICs and undergoes S-nitrosation, altering its conformation. NorA, a di-iron protein from Ralstonia eutropha, binds NO to form a DNIC, which is part of its NO-binding function. The binding affinity and specificity depend on the protein environment and the DNIC's thiol ligands.
Consequences of DNIC binding
In simple terms: When a protein binds a DNIC, it can change the protein's activity or release NO later.
DNIC binding can inhibit enzyme activity, as seen with glutathione reductase, where the dinitrosyl-iron-dithiolate complex blocks catalysis. In other cases, binding serves as a reservoir for NO, allowing its delivery to distant sites; albumin-DNIC conjugates can transport NO to the brain and activate hippocampal neurogenesis. The binding event may also transfer NO to other thiols, propagating S-nitrosation.
Physico-chemical determinants of binding
In simple terms: The chemical nature of the DNIC decides how it binds and what it does.
The biological activity of DNICs is determined by their physico-chemical properties, including charge, lipophilicity, and stability. These properties influence whether DNICs bind to proteins, diffuse through membranes, or release NO. For instance, carnosine-bound DNICs exhibit distinct stability and reactivity compared to glutathione-bound DNICs. Understanding these determinants is key to predicting the functional outcomes of GO:0035731.
Key Genes Involved in GO:0035731 dinitrosyl-iron complex binding
The following genes and proteins are experimentally linked to dinitrosyl-iron complex binding or its downstream effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ALB | Binds DNICs and undergoes S-nitrosation | Major NO carrier in blood; studied for conformation changes |
| GSR | Glutathione reductase; inhibited by DNIC binding | Links DNIC binding to redox homeostasis |
| NorA | Di-iron protein that binds NO to form DNIC | Bacterial model for NO sensing and detoxification |
| CARN | Carnosine dipeptide; forms DNICs | Physiologically active dipeptide that buffers NO and iron |
| GSSG | Oxidized glutathione; involved in DNIC formation | Redox couple affecting DNIC stability |
| GSNO | S-nitrosoglutathione; precursor for DNIC formation | NO donor that reacts with iron to form DNICs |
| HBB | Hemoglobin; potential DNIC interaction | NO transport in blood; not directly cited but related |
| HBA1 | Hemoglobin subunit; potential DNIC interaction | NO transport in blood; not directly cited but related |
| TXN | Thioredoxin; may interact with DNICs | Redox regulation; not directly cited but related |
| NFE2L2 | Nrf2; regulates antioxidant response | May be affected by DNIC-mediated redox changes; not directly cited |
| HMOX1 | Heme oxygenase-1; iron metabolism | Indirectly linked to iron availability for DNICs; not directly cited |
| FTL | Ferritin light chain; iron storage | Affects ferrous iron availability for DNIC formation; not directly cited |
| FTH1 | Ferritin heavy chain; iron storage | Affects ferrous iron availability for DNIC formation; not directly cited |
| SLC7A11 | Cystine/glutamate transporter; affects GSH synthesis | Modulates glutathione levels for DNIC formation; not directly cited |
| GCLC | Glutamate-cysteine ligase; GSH synthesis | Influences GSH pool for DNIC formation; not directly cited |
| GCLM | Glutamate-cysteine ligase modifier | Influences GSH pool for DNIC formation; not directly cited |
| NOS1 | Neuronal nitric oxide synthase | Produces NO for DNIC formation; not directly cited |
| NOS2 | Inducible nitric oxide synthase | Produces NO for DNIC formation; not directly cited |
| NOS3 | Endothelial nitric oxide synthase | Produces NO for DNIC formation; not directly cited |
How Is dinitrosyl-iron complex binding Regulated?
The formation and binding of dinitrosyl-iron complexes are regulated by the availability of nitric oxide, ferrous iron, and reduced thiols such as glutathione. S-nitrosoglutathione (GSNO) serves as a major NO donor and precursor for DNIC assembly. Cellular redox state, particularly the GSH/GSSG ratio, influences DNIC stability and reactivity. In bacteria, NorA expression is controlled by NO-sensing regulators, ensuring DNIC binding is tuned to NO levels. In mammals, albumin acts as a sink and carrier, and its DNIC-binding capacity may be modulated by oxidative modifications. Additionally, carnosine can compete for DNIC binding, affecting NO bioavailability.
dinitrosyl-iron complex binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ALB | Cardiovascular disease; NO transport | Albumin knockout or point-mutant cell lines; DNIC binding assays |
| GSR | Oxidative stress; redox imbalance | GSR knockout cells; DNIC inhibition studies |
| NorA | Bacterial NO resistance | NorA knockout in R. eutropha; NO sensitivity assays |
| CARN | Neuroprotection; NO buffering | Carnosine-treated neuronal cultures; DNIC formation assays |
| NOS2 | Inflammation; NO overproduction | iNOS knockout macrophages; DNIC detection |
Neurodegeneration and neurogenesis
DNIC-albumin conjugates can deliver NO to the brain and activate hippocampal neurogenesis, suggesting a role in neural plasticity. Conversely, excessive DNIC formation under nitrosative stress may contribute to neurodegeneration by modifying proteins and inhibiting antioxidant enzymes like glutathione reductase. The balance between these effects is critical for neuronal health.
Cardiovascular disease
Albumin is the most abundant DNIC-binding protein in plasma, and its S-nitrosation affects NO transport and vascular tone. Dysregulation of DNIC binding may impair NO bioavailability, contributing to endothelial dysfunction and hypertension. The physico-chemical properties of DNICs determine their vasoactive potential.
Infectious disease and bacterial NO sensing
NorA from Ralstonia eutropha binds NO to form a DNIC, representing a bacterial strategy to sense or detoxify NO during infection. Understanding DNIC binding in pathogens may reveal targets for antimicrobial therapy, especially for bacteria that resist nitrosative stress.
From dinitrosyl-iron complex binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does ALB bind DNICs and affect NO transport? | ALB knockout or point-mutation cell lines; DNIC binding assays |
| How does GSR inhibition by DNICs affect redox balance? | GSR knockout cells; DNIC treatment; glutathione measurements |
| What is the role of NorA in bacterial NO sensing? | NorA knockout in Ralstonia eutropha; NO exposure |
| Can carnosine modulate DNIC bioavailability? | Carnosine overexpression or knockout in neuronal cells |
| Does DNIC binding to albumin promote neurogenesis? | Albumin-DNIC conjugate treatment in hippocampal cultures |
| What are the structural determinants of DNIC binding? | Point mutations in cysteine residues of target proteins; spectroscopy |
How to Study the dinitrosyl-iron complex binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| EPR spectroscopy | DNIC paramagnetic signal | Detection and quantification of DNICs |
| UV-visible spectroscopy | Absorbance changes upon DNIC binding | Binding kinetics and stoichiometry |
| Biotin-switch assay | S-nitrosation of proteins | Identifying cysteine modifications by DNICs |
| Glutathione reductase activity assay | Enzyme inhibition by DNICs | Redox enzyme functional studies |
| Pull-down with tagged DNICs | DNIC-binding proteins | Proteomic discovery of new binders |
| CRISPR knockout | Gene function in DNIC binding | Causal testing of candidate genes |
| CRISPR point mutation | Specific residue requirement | Mapping binding sites in target proteins |
| Mass spectrometry | Protein identification and modification sites | Proteomic profiling of DNIC interactome |
Spectroscopic detection of DNICs
Electron paramagnetic resonance (EPR) spectroscopy is the gold standard for detecting DNICs, which exhibit a characteristic g=2.03 signal. UV-visible spectroscopy can monitor DNIC formation and binding through absorbance changes. These methods are used to quantify DNIC binding to proteins like albumin.
Proteomic identification of DNIC-binding proteins
Biotinylated or tagged DNICs can be used as probes to pull down binding proteins from cell lysates, followed by mass spectrometry. This approach identifies novel DNIC-binding proteins and maps binding sites. S-nitrosation sites can be detected by the biotin-switch technique.
Functional assays for enzyme inhibition
Glutathione reductase activity assays measure the inhibitory effect of DNICs on the enzyme. Similar assays can be adapted for other redox enzymes. These functional readouts link DNIC binding to cellular consequences.
CRISPR-based genetic models
Knockout, point-mutation, and knock-in cell lines for genes like ALB, GSR, and NorA enable causal testing of DNIC binding in cellular and animal models. CRISPR screens can identify modifiers of DNIC sensitivity.
How CRISPR Can Be Used to Study GO:0035731 dinitrosyl-iron complex binding
Knockout
CRISPR knockout of genes such as ALB, GSR, or NorA allows researchers to test whether loss of the protein affects DNIC binding, NO transport, or redox homeostasis. For example, GSR knockout cells show altered sensitivity to DNIC-mediated inhibition.
Point Mutation
Introducing point mutations in cysteine residues of candidate DNIC-binding proteins (e.g., ALB) can determine which residues are required for binding and S-nitrosation. This approach has been used to map DNIC binding sites in albumin.
Knock-in
Knock-in of tagged versions of DNIC-binding proteins (e.g., FLAG-tagged NorA) enables affinity purification and localization studies. Tagged knock-ins can also be used to monitor DNIC binding in live cells.
Overexpression
Overexpression of DNIC-binding proteins or their mutants can amplify NO storage or delivery. For instance, overexpression of albumin-DNIC conjugates enhances NO delivery to the brain and promotes neurogenesis.
How EDITGENE Supports dinitrosyl-iron complex binding Research
Researchers studying dinitrosyl-iron complex binding-related genes often need to determine whether a candidate gene is causally involved in DNIC binding, NO transport, or downstream redox effects. EDITGENE provides custom CRISPR cell models and screening services to accelerate this functional validation.
Contact EDITGENE today to design your custom CRISPR model for dinitrosyl-iron complex binding research.
Frequently Asked Questions About dinitrosyl-iron complex binding
What is dinitrosyl-iron complex binding?
It is the molecular function defined by GO:0035731, where a protein binds to dinitrosyl-iron complexes (DNICs), which are NO storage and transport species formed from glutathione, S-nitrosoglutathione, and ferrous iron.
What genes are involved in dinitrosyl-iron complex binding?
Key genes include ALB (albumin), GSR (glutathione reductase), NorA (bacterial NO-binding protein), and CARN (carnosine), among others.
How are dinitrosyl-iron complexes formed?
DNICs form spontaneously from glutathione, S-nitrosoglutathione, and trace ferrous ions, or by reaction of iron-sulfur centers with nitric oxide.
What is the role of albumin in DNIC binding?
Albumin binds DNICs and undergoes S-nitrosation, which alters its conformation and allows it to transport NO in the blood.
Can DNICs inhibit enzymes?
Yes, DNICs inhibit glutathione reductase by binding to its active site, affecting cellular redox balance.
How is DNIC binding detected in the lab?
Electron paramagnetic resonance (EPR) spectroscopy and UV-visible spectroscopy are commonly used to detect DNICs and their binding to proteins.
What diseases are linked to DNIC binding?
DNIC binding is linked to neurodegeneration, cardiovascular disease, and bacterial infections through its role in NO signaling and redox regulation.
What is the bacterial NorA protein?
NorA is a di-iron protein from Ralstonia eutropha that binds NO to form a DNIC, serving as a model for bacterial NO sensing.
How can CRISPR help study DNIC binding?
CRISPR knockout, point mutation, knock-in, and overexpression models allow causal testing of genes involved in DNIC binding and NO biology.
What is the difference between DNIC and DNDGIC?
DNDGIC (dinitrosyl-diglutathionyl-iron complex) is a specific type of DNIC where the iron is coordinated by two glutathione molecules; it is a synonym for DNIC binding in GO:0035731.
Conclusion
GO:0035731, dinitrosyl-iron complex binding, represents a critical molecular function at the intersection of nitric oxide signaling, iron metabolism, and redox regulation. Proteins that bind DNICs, such as albumin, glutathione reductase, and NorA, modulate NO bioavailability and cellular responses to nitrosative stress. Understanding this function has implications for neurodegeneration, cardiovascular disease, and infectious disease. CRISPR-based models and advanced spectroscopic methods are essential tools for dissecting the mechanisms and therapeutic potential of DNIC binding.
References
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