GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004128 describes the enzymatic activity that reduces two Fe(III) centers of cytochrome b5 to Fe(II) using NADH or NADPH as the electron donor.
• The reaction is a classic two-electron transfer from a reduced pyridine nucleotide to a b-type cytochrome, producing NAD(P)+ and a proton.
• This activity is central to fatty acid desaturation, sterol biosynthesis, and xenobiotic metabolism because cytochrome b5 is a key electron carrier for these pathways.
• Steady-state kinetic studies of FMN-containing NADH(NADPH)-cytochrome c reductase from yeast have provided mechanistic insight into how the enzyme handles NAD(P)H and cytochrome b5.
• Modulation of this activity by plant extracts such as Adhatoda vesica has been observed in mouse xenobiotic metabolism and antioxidant studies, linking it to redox homeostasis.
• CRISPR-based knockout, point-mutation, and knock-in models are powerful tools to dissect the physiological roles of cytochrome-b5 reductase activity in cells and organisms.
Description
Cytochrome-b5 reductase activity, acting on NAD(P)H (GO:0004128) is a molecular function that catalyzes the reduction of cytochrome b5 using either NADH or NADPH as the electron donor. This activity is essential for maintaining the redox state of cytochrome b5, a small heme-containing protein that participates in a wide range of oxidative reactions, including fatty acid desaturation and drug metabolism. Researchers study this activity to understand how cells handle electron transfer between pyridine nucleotides and b-type cytochromes, and how perturbations in this process contribute to metabolic and oxidative stress-related diseases [1,2]. The enzyme's ability to use both NADH and NADPH makes it a versatile node in cellular redox networks, and its kinetic properties have been characterized in model organisms such as yeast. In this article, we explore the definition, mechanism, key genes, disease relevance, and research methods for GO:0004128, with a focus on how CRISPR-based models can accelerate discovery.
cytochrome-b5 reductase activity, acting on NAD(P)H At A Glance
| GO ID | GO:0004128 |
|---|---|
| GO term | cytochrome-b5 reductase activity, acting on NAD(P)H |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Catalyzes the reduction of cytochrome b5 using NADH or NADPH as electron donor |
| Reaction | 2 Fe(III)-[cytochrome b5] + NAD(P)H = 2 Fe(II)-[cytochrome b5] + NAD(P)+ + H+ |
| Cofactor | FMN (flavin mononucleotide) in related enzymes |
| Subcellular location | Typically associated with membranes, including endoplasmic reticulum and outer mitochondrial membrane (inferred from cytochrome b5 reductase family) |
| Pathways | Fatty acid desaturation, sterol biosynthesis, xenobiotic metabolism [1,2] |
What Is GO:0004128?
GO:0004128 is defined as the catalysis of the reaction: 2 Fe(III)-[cytochrome b5] + NAD(P)H = 2 Fe(II)-[cytochrome b5] + NAD(P)+ + H+. In other words, it is the enzyme activity that transfers electrons from either NADH or NADPH to two molecules of oxidized cytochrome b5, reducing the heme iron from Fe(III) to Fe(II) and releasing a proton. This activity is a type of oxidoreductase and is classified under molecular_function in the Gene Ontology.
Why Is cytochrome-b5 reductase activity, acting on NAD(P)H Important in Cell Biology?
Cytochrome-b5 reductase activity, acting on NAD(P)H is important because it supplies reducing equivalents to cytochrome b5, which in turn supports essential metabolic pathways such as fatty acid desaturation and drug detoxification. Dysregulation of this activity can lead to altered lipid metabolism and increased oxidative stress, both of which are implicated in human diseases. Understanding its mechanism and regulation is therefore critical for developing therapeutic strategies targeting metabolic disorders and cancer.
• Provides electrons for fatty acid desaturation, affecting membrane fluidity and lipid signaling.
• Supports sterol biosynthesis, including cholesterol production.
• Participates in xenobiotic metabolism by supplying electrons to cytochrome P450 systems via cytochrome b5.
• Modulates antioxidant status and lipid peroxidation in vivo, as shown by plant extract studies in mice.
• Its kinetic properties can be studied using steady-state methods, offering insights into electron transfer efficiency.
• Potential target for modulating redox balance in cancer and metabolic diseases.
• Enables cross-talk between NADH and NADPH pools, linking cellular energy status to biosynthetic pathways.
• Can be regulated by dietary and environmental factors, as demonstrated by Adhatoda vesica extract.
• Its activity can be measured using cytochrome c as an artificial electron acceptor in vitro.
• CRISPR models allow precise dissection of its physiological roles in development and disease.
What Happens During cytochrome-b5 reductase activity, acting on NAD(P)H?
Substrate Binding and Electron Transfer
In simple terms: The enzyme grabs electrons from NADH or NADPH and hands them to cytochrome b5.
The reaction begins with the binding of NAD(P)H to the enzyme's active site, where the flavin cofactor (FMN) accepts two electrons. This reduces the flavin, which then transfers electrons to two molecules of oxidized cytochrome b5, converting Fe(III) to Fe(II). The overall reaction produces NAD(P)+ and a proton.
Catalytic Cycle and Steady-State Kinetics
In simple terms: The enzyme works in a cycle, and its speed can be measured in the lab.
Steady-state kinetic studies of FMN-containing NADH(NADPH)-cytochrome c reductase from yeast have revealed how the enzyme processes NAD(P)H and cytochrome b5. These studies show that the enzyme follows a ping-pong or sequential mechanism depending on conditions, and that the reaction rate depends on substrate concentrations.
Role in Cellular Redox Homeostasis
In simple terms: This activity helps keep the cell's redox balance in check.
By reducing cytochrome b5, the enzyme maintains the pool of reduced cytochrome b5 needed for various redox reactions. This indirectly influences antioxidant status and lipid peroxidation, as observed in mouse studies with Adhatoda vesica leaf extract.
Integration with Metabolic Pathways
In simple terms: The electrons from this reaction feed into fat and cholesterol production.
Reduced cytochrome b5 is required for fatty acid desaturation and sterol biosynthesis. Therefore, cytochrome-b5 reductase activity directly impacts lipid metabolism and membrane composition.
Key Genes Involved in GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H
The following genes and proteins are directly or indirectly involved in cytochrome-b5 reductase activity, acting on NAD(P)H, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| CYB5R1 | Cytochrome b5 reductase 1 | Main enzyme catalyzing the reaction in many tissues |
| CYB5R2 | Cytochrome b5 reductase 2 | Isoform with tissue-specific expression |
| CYB5R3 | Cytochrome b5 reductase 3 | Well-studied isoform, involved in methemoglobin reduction |
| CYB5R4 | Cytochrome b5 reductase 4 | Contains cytochrome b5 domain, may modulate activity |
| CYB5A | Cytochrome b5 type A | Electron acceptor substrate for the reductase |
| CYB5B | Cytochrome b5 type B | Outer mitochondrial membrane isoform |
| NADH | Electron donor | Cofactor providing reducing equivalents |
| NADPH | Electron donor | Alternative electron donor |
| FMN | Flavin cofactor | Prosthetic group in the enzyme active site |
| FAD | Flavin cofactor | May be used by some isoforms |
| MTRR | Methionine synthase reductase | Related enzyme using similar chemistry |
| NPR1 | Nitrate reductase | Plant enzyme with similar activity |
| CBR1 | Carbonyl reductase 1 | NADPH-dependent reductase, potential cross-talk |
| POR | Cytochrome P450 oxidoreductase | Shares electron transfer roles with cytochrome b5 |
| SCD | Stearoyl-CoA desaturase | Downstream enzyme requiring reduced cytochrome b5 |
| HMGCR | HMG-CoA reductase | Sterol biosynthesis pathway component |
| CYP450 | Cytochrome P450 family | Receives electrons from cytochrome b5 in some reactions |
How Is cytochrome-b5 reductase activity, acting on NAD(P)H Regulated?
The activity of cytochrome-b5 reductase can be regulated at multiple levels. Its expression may be influenced by dietary factors, as shown by the modulatory effect of Adhatoda vesica leaf extract on xenobiotic metabolism enzymes in mice. Additionally, the availability of NADH and NADPH, which are central to cellular energy metabolism, directly affects the reaction rate. Steady-state kinetic parameters such as Km and Vmax for NADH and NADPH have been determined for the yeast enzyme, indicating that substrate affinity can vary. Post-translational modifications and membrane association may also regulate activity, though specific mechanisms require further study.
cytochrome-b5 reductase activity, acting on NAD(P)H and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CYB5R3 | Methemoglobinemia, lipid metabolism disorders | Knockout mouse, point-mutation cell lines |
| CYB5A | Fatty acid desaturation defects | Knock-in of mutant cytochrome b5 |
| SCD | Obesity, insulin resistance | Overexpression in adipocytes |
| CYP450 | Drug metabolism variability | CRISPR knockout in hepatocytes |
| NPR1 | Plant nitrogen metabolism | Knockout in Arabidopsis |
Metabolic Disorders and Lipid Metabolism
Cytochrome-b5 reductase activity is essential for fatty acid desaturation and cholesterol synthesis. Deficiencies in this activity could lead to altered lipid profiles and metabolic disorders. Studies on xenobiotic metabolism in mice have shown that modulation of this activity affects antioxidant status and lipid peroxidation, suggesting a role in oxidative stress-related diseases.
Cancer and Redox Balance
Altered redox homeostasis is a hallmark of cancer. By influencing the reduction of cytochrome b5, this activity may impact drug metabolism and reactive oxygen species levels. However, direct evidence linking GO:0004128 to cancer requires further investigation.
Xenobiotic Metabolism and Detoxification
Cytochrome b5 is a known electron donor for certain cytochrome P450 reactions involved in drug and toxin metabolism. Therefore, changes in cytochrome-b5 reductase activity could affect the body's ability to detoxify xenobiotics, as suggested by mouse studies with plant extracts.
From cytochrome-b5 reductase activity, acting on NAD(P)H-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of CYB5R3 knockout on lipid metabolism? | CYB5R3 knockout cell line or mouse |
| How does a point mutation in the active site affect catalytic efficiency? | Point-mutation knock-in cell line |
| Can tagged CYB5R3 be used to study localization? | Tagged knock-in (e.g., GFP) cell line |
| What happens when CYB5R3 is overexpressed? | Overexpression cell line or transgenic mouse |
| Which genes interact with CYB5R3 in redox pathways? | CRISPR library screening |
| How does CYB5R3 activity change under oxidative stress? | Reporter cell line with redox sensor |
How to Study the cytochrome-b5 reductase activity, acting on NAD(P)H Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric assay | Enzyme activity via cytochrome c reduction | Kinetic characterization |
| CRISPR knockout screen | Gene essentiality and modifiers | Identify regulators of redox pathways |
| RNA-seq | Transcriptional changes | Expression profiling of CYB5R genes |
| Proteomics | Protein abundance and modifications | Post-translational regulation |
| Lipidomics | Fatty acid composition | Assess desaturation activity |
| Metabolomics | NAD(P)H/NAD(P)+ ratios | Redox state measurement |
| Imaging | Subcellular localization | Tagged knock-in cell lines |
Enzymatic Activity Assays
Cytochrome-b5 reductase activity can be measured spectrophotometrically by monitoring the reduction of cytochrome c or cytochrome b5 at specific wavelengths. Steady-state kinetic assays using NADH or NADPH as substrates provide Km and Vmax values.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout or activation screens can identify genes that modulate cytochrome-b5 reductase activity or its downstream effects. These screens are powerful for uncovering novel regulators and pathways.
Transcriptomics and Proteomics
RNA-seq and proteomics can reveal changes in expression of CYB5R genes and related metabolic enzymes under different conditions. Such studies help link genotype to phenotype.
Metabolic Flux Analysis
Isotope tracing and lipidomics can measure the impact of cytochrome-b5 reductase activity on fatty acid desaturation and sterol synthesis.
How CRISPR Can Be Used to Study GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H
Knockout
CRISPR knockout of CYB5R genes can abolish cytochrome-b5 reductase activity, allowing researchers to study its role in lipid metabolism, drug detoxification, and oxidative stress. Knockout cell lines and animal models are valuable for phenotypic analysis.
Point Mutation
Introducing specific point mutations in the active site of CYB5R can help dissect catalytic residues and cofactor binding. Such models are useful for structure-function studies.
Knock-in
Knock-in of tagged versions (e.g., GFP, FLAG) of CYB5R allows real-time tracking of protein localization and interaction. This is particularly useful for understanding membrane association and trafficking.
Overexpression
Overexpression of CYB5R can elevate cytochrome-b5 reductase activity, enabling studies on gain-of-function effects in metabolic pathways and redox balance.
How EDITGENE Supports cytochrome-b5 reductase activity, acting on NAD(P)H Research
Researchers studying cytochrome-b5 reductase activity, acting on NAD(P)H-related genes often need to determine whether a candidate gene is causally involved in a specific metabolic or redox phenotype. EDITGENE provides a comprehensive suite of CRISPR services to accelerate this discovery process.
Contact EDITGENE today to design your custom CRISPR model for cytochrome-b5 reductase activity, acting on NAD(P)H research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| POR Knockout HEK293 Cell Line | EDJ-KQ3088 | Human | 5447 | Details Get a Quote |
| CYB5R3 Knockout HEK293 Cell Line | EDJ-KQ4440 | Human | 1727 | Details Get a Quote |
| CYB5R4 Knockout HEK293 Cell Line | EDJ-KQ10958 | Human | 51167 | Details Get a Quote |
| CYB5R1 Knockout HEK293 Cell Line | EDJ-KQ11196 | Human | 51706 | Details Get a Quote |
| CYB5R2 Knockout HEK293 Cell Line | EDJ-KQ11197 | Human | 51700 | Details Get a Quote |
| CYB5RL Knockout HEK293 Cell Line | EDJ-KQ13067 | Human | 606495 | Details Get a Quote |
| NQO1 Knockout HEK293 Cell Line | EDJ-KQ14488 | Human | 1728 | Details Get a Quote |
| CYB5R3 Knockout A-549 Cell Line | EDJ-KQ26989 | Human | 1727 | Details Get a Quote |
| CYB5R3 Knockout HCT 116 Cell Line | EDJ-KQ26990 | Human | 1727 | Details Get a Quote |
| CYB5R3 Knockout HeLa Cell Line | EDJ-KQ26991 | Human | 1727 | Details Get a Quote |
| NQO1 Knockout A-549 Cell Line | EDJ-KQ44745 | Human | 1728 | Details Get a Quote |
| NQO1 Knockout HeLa Cell Line | EDJ-KQ44747 | Human | 1728 | Details Get a Quote |
| POR Knockout A-549 Cell Line | EDJ-KQ25763 | Human | 5447 | Details Get a Quote |
| POR Knockout HCT 116 Cell Line | EDJ-KQ25764 | Human | 5447 | Details Get a Quote |
| POR Knockout HeLa Cell Line | EDJ-KQ25765 | Human | 5447 | Details Get a Quote |
Displaying Records 1 To 15 Of 29 Records
Frequently Asked Questions About cytochrome-b5 reductase activity, acting on NAD(P)H
What is cytochrome-b5 reductase activity, acting on NAD(P)H?
It is an enzymatic activity (GO:0004128) that catalyzes the reduction of cytochrome b5 using NADH or NADPH as the electron donor, producing reduced cytochrome b5 and NAD(P)+.
What genes are involved in cytochrome-b5 reductase activity, acting on NAD(P)H?
Key genes include CYB5R1, CYB5R2, CYB5R3, and CYB5R4, which encode different isoforms of cytochrome b5 reductase.
What is the reaction catalyzed by GO:0004128?
The reaction is: 2 Fe(III)-[cytochrome b5] + NAD(P)H = 2 Fe(II)-[cytochrome b5] + NAD(P)+ + H+.
Which cofactors are required for cytochrome-b5 reductase activity?
The enzyme typically uses FMN (flavin mononucleotide) as a prosthetic group to transfer electrons from NAD(P)H to cytochrome b5.
How is cytochrome-b5 reductase activity measured in the lab?
It can be measured spectrophotometrically by monitoring the reduction of cytochrome c or cytochrome b5, often using steady-state kinetic assays.
What diseases are associated with cytochrome-b5 reductase deficiency?
Deficiency in cytochrome b5 reductase (especially CYB5R3) can cause methemoglobinemia and may affect lipid metabolism, though direct links to other diseases require further study [1,2].
Can CRISPR be used to study cytochrome-b5 reductase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are powerful tools to dissect the function of CYB5R genes and their role in metabolism.
What is the role of cytochrome b5 in the reaction?
Cytochrome b5 is the electron acceptor; it becomes reduced (Fe(II)) and then participates in downstream reactions such as fatty acid desaturation.
How does Adhatoda vesica affect cytochrome-b5 reductase activity?
Studies in mice showed that Adhatoda vesica leaf extract modulates xenobiotic metabolism enzymes and antioxidant status, which may involve changes in cytochrome-b5 reductase activity.
What are the research methods for studying GO:0004128?
Common methods include enzymatic assays, CRISPR screens, RNA-seq, proteomics, lipidomics, and metabolomics to assess activity and downstream effects [1,2].
Conclusion
Cytochrome-b5 reductase activity, acting on NAD(P)H (GO:0004128) is a fundamental molecular function that links pyridine nucleotide redox state to cytochrome b5-dependent processes such as fatty acid desaturation and xenobiotic metabolism. Despite its importance, many aspects of its regulation and disease relevance remain to be fully elucidated. CRISPR-based models offer unprecedented opportunities to dissect its physiological roles and identify therapeutic targets [1,2]. By combining precise gene editing with advanced omics and screening technologies, researchers can accelerate discoveries in metabolic and oxidative stress-related diseases.
References
- 1. Johnson MS et al.. 1986. Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast.. Arch Biochem Biophys 245(1):271-81 PMID: 3080958
- 2. Singh RP et al.. 2000. Modulatory influence of Adhatoda vesica (Justicia adhatoda) leaf extract on the enzymes of xenobiotic metabolism, antioxidant status and lipid peroxidation in mice.. Mol Cell Biochem 213(1-2):99-109 PMID: 11129964
