| cytochrome-b5 reductase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
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| Identifiers | |||||||||
| EC no. | 1.6.2.2 | ||||||||
| CAS no. | 9032-25-1 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / QuickGO | ||||||||
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Cytochrome-b5 reductase is a NADH-dependent enzyme that converts ferricytochrome from a Fe3+ form to a Fe2+ form. [1] It contains FAD and catalyzes the reaction:
In its b5-reducing capacity, this enzyme is involved in desaturation and elongation of fatty acids, cholesterol biosynthesis, and drug metabolism. This enzyme can also reduce methemoglobin to normal hemoglobin, gaining it the inaccurate synonym methemoglobin reductase. Isoforms expressed in erythrocytes (CYB5R1, CYB5R3) perform this function in vivo. Ferricyanide is another substrate in vitro.[ citation needed ]
Cytochrome b5 reductase (c5br) is a NADH-dependent enzyme known as a flavoprotein that results in the chemical reduction to two different isoforms, a soluble form and a membrane-bound form. [2] This enzyme is involved in the transfer of reducing equivalents from NADH due to the FAD electron acceptor in cytochrome b5, located in complex III of the electron transport chain, which results in the two isoforms due to alternative splicing. The overall reduction reaction from cytochrome b5 reductase aids in the control of iron in red blood cells, which dictates the amount of oxygen cells carry. [3]
Cytochromes are redox proteins that are essential for energy transfer in the electron transport chain with the help of an enzyme such as a reductase. Cytochromes are categorized into three classes (a,b, and c) in accordance with the type of heme that is present in the core and their light-absorption spectra. [4]
The specialized protein cytochrome b5 is a class B cytochrome with a high and low potential heme b attached to the central iron on the protein. [5] The cytochrome b class is especially unique because it is attached to proteins within the inner mitochondrial membrane instead of on the outer portion, and this particular class has high sequence variation. With the ability to express approximately 1080 base pairs, cytochrome b proteins are commonly studied to analyze mitochondrial DNA and determine phylogenetic relationships across evolution. [6]
Cytochrome reductase enzymes are therefore an essential component of the electron transport chain that carry out the function of cytochrome proteins and activate their reactions. Cytochrome b5 reductase successfully catalyzes the electron transfer of reducing equivalents to then activate cytochrome b for it to carry out its role in organisms. [7]
The cytochrome b5 reductase enzyme contains a typical oxidoreductase structure with a diaphorase binding domain complex for NADH and a FAD-binding domain. A three-stranded linker domain is present, as well as water-mediated hydrogen bonds, in order to biochemically connect the complex. [9] Once cytochrome b5 reductase catalyzes electron transfer, the resulting reduced form of cytochrome b5 reduces the oxidized ferric ion of hemoglobin from Fe 3+ to Fe 2+. [10]
Mechanism: NADH + H+ + 2 ferricytochrome b5 -> NAD+ + 2 ferrocytochrome b5
The features of cytochrome b5 reductase enzymes allow the successful reduction of molecules of cytochrome b5 to be used for various functions across the electron transport chain and metabolism. In metabolism, c5br is active in lipid conversion involving the elongation and desaturation of fatty acids and cholesterol biosynthesis. [11]
Specialized isoforms of the enzyme c5br are heavily functional in the blood to help deliver oxygen to the body’s tissue by conformationally changing methemoglobin to hemoglobin. Other isoforms are useful in chemical reactions throughout the body, with assistance in the breakdown of various substances. [12]
In living organisms, because methemoglobin (MetHb) is unable to bind oxygen, it must be reduced to hemoglobin (Hb) through the action of the soluble isoform of cytochrome b5 reductase. Overall, the mechanics of this reaction include electron transfer through oxidation steps, which can be accomplished through a couple of different mechanisms involving the reduction of pyridine nucleotides. [13] One mechanism, which is also the most naturally present pathway, involves electron transfer catalyzed by cytochrome b5 reductase through the oxidation of NADH to NAD+. The electron donor, NADH, that supports this reaction is a product of glucose oxidation from glycolysis. The reduction reaction converts the oxidized methemoglobin to the reduced hemoglobin form, that now has an affinity for oxygen. [14] Another mechanism involves the conversion of the reduced pyridine nucleotide triphosphopyridine nucleotide (TPNH) to methylene blue, which is induced by the electron transfer in the oxidation of NADPH to NADP+ by NAPHD methemoglobin reductase. [13] TPNH is the most favorable pyridine nucleotide to reduce methemoglobin; however, other cofactors can be used, including leukomethylene blue. [15] Additional reduced nucleotide pyridines can also catalyze the oxidation of hemoglobin to methemoglobin. For example, in the reverse reaction of NAPHD methemoglobin reductase, methylene blue can be used to catalyze the oxidation of hemoglobin to methemoglobin. Other enzymes, including diaphorase enzymes, can convert reduced diphosphopyridine nucleotide (DPNH) from glyceraldehyde-3-phosphate in order to replenish methemoglobin levels in the cell. [16]
The membrane-bound isoform of cytochrome b5 reductase is found in all cell types and is not limited to red blood cells. It is typically found embedded in the membranes of various cellular compartments, with a domain inserted into the lipid bilayer on the outer leaflet of the endoplasmic reticulum. [12] This specific isoform is made up of approximately 300 amino acid residues with an N-terminal tail of 24 residues that anchors the protein to the membrane. [7] There is a subsequent soluble domain that is part of this isoform that attaches to the cytosol. Due to its structure and location, the membrane-bound isoform of c5br is essential for biological functions within organisms. [7] By possessing a membrane binding domain and a water-soluble domain, this isoform is able to carry out chemical and redox reactions for the electron transport chain and is functional in the formation of fatty acids, the formation of cholesterol, and the breakdown of molecules and drugs. [11]
The soluble isoform of cytochrome b5 reductase is found only present in red blood cells. On erythrocytes, red blood cells, the c5br enzyme is responsible for the recycling and conversion of methemoglobin to hemoglobin. [7] Methemoglobin is an oxidized form of hemoglobin attached to a ferric-state iron (Fe3+), which can therefore not carry and deliver oxygen to tissues. [17] The formation of methemoglobin occurs when electrons are not returned to the iron of a normal state hemoglobin, which is not preferred for a functioning organism. Methemoglobin is not favorable for a functional organism since oxygen needs to constantly be transferred; therefore, the soluble isoform of c5br is essential to keep levels of methemoglobin low in humans. [18]
Mutations in cytochrome b5 reductase can lead to many disorders, including autosomal recessive congenital methemoglobinemia. There are over 65 mutations of the enzyme that can lead to various types of the disorder. [12] Some include:
Most cases of methemoglobinemia are treatable and not chronic. The most common and successful treatment used to treat patients with high levels of methemoglobinemia is the antidote methylene blue. Methylene blue is already recognized as a product of the reversible reaction fueled by NAPHD methemoglobin reductase, catalyzed by leukcomethylene, to reduce methemoglobin to hemoglobin. Therefore, when methemoglobin levels are high in a patient, additional methylene blue can be introduced to be reduced to leukcomethylene to now catalyze the reduction of excess methemoglobin to hemoglobin. [25] While the addition of methylene blue to treat cases of methemoglobinemia has been scientifically tested and proven, there are some side effects to note and monitor with high dosages of the antidote. Minor side effects include green or blue discoloration of urine; however, significant side effects include worsening of the present methemoglobinemia. Because methylene blue is an oxidizing agent itself, when it is not effectively reduced, NADPH will not be properly restored in the cell for electron transfer, resulting in increased levels of non reduced methemoglobin to support methemoglobinemia in patients. [25] Additional studies have found that the use of methylene blue during pregnancy is associated with a high risk of small intestinal atresia, which can be fatal to the fetus. [26]
It is recommended that treatment with methylene blue requires two doses before being deemed ineffective. If symptoms of methemoglobinemia are still present after the second dosage, alternative treatments, including ascorbic acid, exchange transfusion, and hyperbaric oxygen therapy, can be considered. However, no additional antidote has been tested and confirmed to the extent of methylene blue, and in most cases, additional antidotes are generally ineffective. It is also noted that high doses of ascorbic acid are associated with increased urinary excretion of oxalate and renal failure. [25] [27]
Cytochrome b5 reductase is a prevalent topic in research and clinical tests to understand the additional functions of the enzyme in other metabolic pathways in the body. Mice and flies are common model organisms used to test for the relationship of cytochrome b5 reductase with the overall health of living organisms.
A recent study from 2023 used mice as a model to test the extended effects of c5br on oxygen supply in the presence of additional oxidative stress, such as from sickle cell disease or ischemic stroke. The results showed that c5br not only increases oxygen supply and transport in a wild-type organism but also regulates the erythropoietin response to ischemic stroke. These findings have made cytochrome b5 reductase a target for future research on managing stroke risk and providing selective advantage for those with genetic disorders such as sickle cell disease. [28]
Another study with mice as well as flies tested the physiological role of cytochrome b5 reductase on lipid metabolism, health, and aging. By activating the cb5r-expressing gene in both model organisms, it was observed that lifespan and lipid metabolism were positively affected. In the model flies, the drug tetrahydroindenoindole was used to activate cytochrome b5 reductase activity, and observations concluded that the increased function of cb5r extended the fly's lifespan. The increased expression of cytochrome b5 reductase in mice resulted in high levels of long-chain polyunsaturated fatty acids, improved mitochondrial function, and a decrease in oxidative damage, indicating improved metabolic pathways with high levels of cb5r activity. These results indicate that cytochrome b5 reductase is a new target for new research and development of lipid metabolism and health in living organisms. [29]
The citric acid cycle—also known as the Krebs cycle, Szent–Györgyi–Krebs cycle or the TCA cycle (tricarboxylic acid cycle)—is a series of biochemical reactions to release the energy stored in nutrients through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The chemical energy released is available under the form of ATP. The Krebs cycle is used by organisms that respire (as opposed to organisms that ferment) to generate energy, either by anaerobic respiration or aerobic respiration. In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous other reactions. Its central importance to many biochemical pathways suggests that it was one of the earliest components of metabolism. Even though it is branded as a "cycle", it is not necessary for metabolites to follow only one specific route; at least three alternative segments of the citric acid cycle have been recognized.
Oxidative phosphorylation or electron transport-linked phosphorylation or terminal oxidation is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing chemical energy in order to produce adenosine triphosphate (ATP). In eukaryotes, this takes place inside mitochondria. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is so pervasive because it releases more energy than alternative fermentation processes such as anaerobic glycolysis.
A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as forward reactions, and in some cases this has physiological significance: for example, alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde in animals, but in yeast it catalyzes the production of ethanol from acetaldehyde.
An electron transport chain (ETC) is a series of protein complexes and other molecules that transfer electrons from electron donors to electron acceptors via redox reactions (both reduction and oxidation occurring simultaneously) and couples this electron transfer with the transfer of protons (H+ ions) across a membrane. Many of the enzymes in the electron transport chain are embedded within the membrane.
Heme, or haem, is a ring-shaped iron-containing molecular component of hemoglobin, which is necessary to bind oxygen in the bloodstream. It is composed of four pyrrole rings with 2 vinyl and 2 propionic acid side chains. Heme is biosynthesized in both the bone marrow and the liver.
Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. As a medication, it is mainly used to treat methemoglobinemia by chemically reducing the ferric iron in hemoglobin to ferrous iron. Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despite oxygen therapy. It has previously been used for treating cyanide poisoning and urinary tract infections, but this use is no longer recommended.
Methemoglobinemia, or methaemoglobinaemia, is a condition of elevated methemoglobin in the blood. Symptoms may include headache, dizziness, shortness of breath, nausea, poor muscle coordination, and blue-colored skin (cyanosis). Complications may include seizures and heart arrhythmias.
Cytochromes P450 are a superfamily of enzymes containing heme as a cofactor that mostly, but not exclusively, function as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various compounds, as well as for hormone synthesis and breakdown, steroid hormone synthesis, drug metabolism, and the biosynthesis of defensive compounds, fatty acids, and hormones. CYP450 enzymes convert xenobiotics into hydrophilic derivatives, which are more readily excreted. In almost all of the transformations that they catalyze, P450's affect hydroxylation.
Blue baby syndrome can refer to conditions that cause cyanosis, or blueness of the skin, in babies as a result of low oxygen levels in the blood. This term has traditionally been applied to cyanosis as a result of:.
Methemoglobin (British: methaemoglobin, shortened MetHb) (pronounced "met-hemoglobin") is a hemoglobin in the form of metalloprotein, in which the iron in the heme group is in the Fe3+ (ferric) state, not the Fe2+ (ferrous) of normal hemoglobin. Sometimes, it is also referred to as ferrihemoglobin. Methemoglobin cannot bind oxygen, which means it cannot carry oxygen to tissues. It is bluish chocolate-brown in color. In human blood a trace amount of methemoglobin is normally produced spontaneously, but when present in excess the blood becomes abnormally dark bluish brown. The NADH-dependent enzyme methemoglobin reductase (a type of diaphorase) is responsible for converting methemoglobin back to hemoglobin.
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP+ or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NADPH as a reducing agent ('hydrogen source'). NADPH is the reduced form, whereas NADP+ is the oxidized form. NADP+ is used by all forms of cellular life.
Any enzyme system that includes cytochrome P450 protein or domain can be called a P450-containing system.
Nitrite reductase refers to any of several classes of enzymes that catalyze the reduction of nitrite. There are two classes of NIR's. A multi haem enzyme reduces NO2− to a variety of products. Copper containing enzymes carry out a single electron transfer to produce nitric oxide.
Microbial metabolism is the means by which a microbe obtains the energy and nutrients it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe's ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.
Trimethylamine N-oxide reductase is a microbial enzyme that can reduce trimethylamine N-oxide (TMAO) into trimethylamine (TMA), as part of the electron transport chain. The enzyme has been purified from E. coli and the photosynthetic bacteria Roseobacter denitrificans.
In enzymology, a camphor 5-monooxygenase (EC 1.14.15.1) is an enzyme that catalyzes the chemical reaction
Nitric oxide dioxygenase (EC 1.14.12.17) is an enzyme that catalyzes the conversion of nitric oxide (NO) to nitrate (NO−
3) . The net reaction for the reaction catalyzed by nitric oxide dioxygenase is shown below:
NADH-cytochrome b5 reductase 3 is an enzyme that in humans is encoded by the CYB5R3 gene.
The phytoglobin-nitric oxide cycle is a metabolic pathway induced in plants under hypoxic conditions which involves nitric oxide (NO) and phytoglobin (Pgb). It provides an alternative type of respiration to mitochondrial electron transport under the conditions of limited oxygen supply. Phytoglobin in hypoxic plants acts as part of a soluble terminal nitric oxide dioxygenase system, yielding nitrate ion from the reaction of oxygenated phytoglobin with NO. Class 1 phytoglobins are induced in plants under hypoxia, bind oxygen very tightly at nanomolar concentrations, and can effectively scavenge NO at oxygen levels far below the saturation of cytochrome c oxidase. In the course of the reaction, phytoglobin is oxidized to metphytoglobin which has to be reduced for continuous operation of the cycle. Nitrate is reduced to nitrite by nitrate reductase, while NO is mainly formed due to anaerobic reduction of nitrite which may take place in mitochondria by complex III and complex IV in the absence of oxygen, in the side reaction of nitrate reductase, or by electron transport proteins on the plasma membrane. The overall reaction sequence of the cycle consumes NADH and can contribute to the maintenance of ATP level in highly hypoxic conditions.
Hemoglobin M disease is a rare form of hemoglobinopathy, characterized by the presence of hemoglobin M (HbM) and elevated methemoglobin (metHb) level in blood. HbM is an altered form of hemoglobin (Hb) due to point mutation occurring in globin-encoding genes, mostly involving tyrosine substitution for proximal (F8) or distal (E7) histidine residues. HbM variants are inherited as autosomal dominant disorders and have altered oxygen affinity. The pathophysiology of hemoglobin M disease involves heme iron autoxidation promoted by heme pocket structural alteration.
