Флавин аденин динуклеотиді: тотығу-тотықсыздану коферменті

Redox active coenzyme

In biochemistry, flavin adenine dinucleotide (FAD) is a redox active coenzyme associated with various proteins, which is involved with several enzymatic reactions in metabolism. A flavoprotein is a protein that contains a flavin group, which may be in the form of FAD or flavin mononucleotide (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex, α ketoglutarate dehydrogenase, and a component of the pyruvate dehydrogenase complex. FAD can exist in four redox states, which are the flavin N(5) oxide, quinone, semiquinone, and hydroquinone. FAD is converted between these states by accepting or donating electrons. FAD, in its fully oxidized form, or quinone form, accepts two electrons and two protons to become FADH2 (hydroquinone form). The semiquinone (FADH·) can be formed by either reduction of FAD or oxidation of FADH2 by accepting or donating one electron and one proton, respectively. Some proteins, however, generate and maintain a superoxidized form of the flavin cofactor, the flavin N(5) oxide.

Flavoproteins were first discovered in 1879 by separating components of cow's milk. They were initially called lactochrome due to their milky origin and yellow pigment. Warburg and Christian then found FAD to be a cofactor of D amino acid oxidase through similar experiments in 1938. Warburg's work with linking nicotinamide to hydride transfers and the discovery of flavins paved the way for many scientists in the 40s and 50s to discover copious amounts of redox biochemistry and link them together in pathways such as the citric acid cycle and ATP synthesis.

Flavin adenine dinucleotide consists of two portions: the adenine nucleotide (adenosine monophosphate) and the flavin mononucleotide (FMN) bridged together through their phosphate groups. Adenine is bound to a cyclic ribose at the 1' carbon, while phosphate is bound to the ribose at the 5' carbon to form the adenine nucledotide. Riboflavin is formed by a carbon nitrogen (C N) bond between the isoalloxazine and the ribitol. The phosphate group is then bound to the terminal ribose carbon, forming a FMN. Because the bond between the isoalloxazine and the ribitol is not considered to be a glycosidic bond, the flavin mononucleotide is not truly a nucleotide. This makes the dinucleotide name misleading; however, the flavin mononucleotide group is still very close to a nucleotide in its structure and chemical properties. FAD can be reduced to FADH2 through the addition of 2 H+ and 2 e−. FADH2 can also be oxidized by the loss of 1 H+ and 1 e− to form FADH. The FAD form can be recreated through the further loss of 1 H+ and 1 e−. FAD formation can also occur through the reduction and dehydration of flavin N(5) oxide. Based on the oxidation state, flavins take specific colors when in aqueous solution. flavin N(5) oxide (superoxidized) is yellow orange, FAD (fully oxidized) is yellow, FADH (half reduced) is either blue or red based on the pH, and the fully reduced form is colorless. Changing the form can have a large impact on other chemical properties. For example, FAD, the fully oxidized form is subject to nucleophilic attack, the fully reduced form, FADH2 has high polarizability, while the half reduced form is unstable in aqueous solution. FAD is an aromatic ring system, whereas FADH2 is not. This means that FADH2 is significantly higher in energy, without the stabilization through resonance that the aromatic structure provides. FADH2 is an energy carrying molecule, because, once oxidized it regains aromaticity and releases the energy represented by this stabilization. The spectroscopic properties of FAD and its variants allows for reaction monitoring by use of UV VIS absorption and fluorescence spectroscopies. Each form of FAD has distinct absorbance spectra, making for easy observation of changes in oxidation state. Flavins in general have fluorescent activity when unbound (proteins bound to flavin nucleic acid derivatives are called flavoproteins). This property can be utilized when examining protein binding, observing loss of fluorescent activity when put into the bound state. Therefore, humans must obtain riboflavin, also known as vitamin B2, from dietary sources. This wide variety of ionization and modification of the flavin moiety can be attributed to the isoalloxazine ring system and the ability of flavoproteins to drastically perturb the kinetic parameters of flavins upon binding, including flavin adenine dinucleotide (FAD). The number of flavin dependent protein encoded genes in the genome (the flavoproteome) is species dependent and can range from 0.1% 3.5%, with humans having 90 flavoprotein encoded genes. FAD is the more complex and abundant form of flavin and is reported to bind to 75% of the total flavoproteome Cellular concentrations of free or non covalently bound flavins in a variety of cultured mammalian cell lines were reported for FAD (2.2 17.0 amol/cell) and FMN (0.46 3.4 amol/cell). FAD has a more positive reduction potential than NAD+ and is a very strong oxidizing agent. The cell utilizes this in many energetically difficult oxidation reactions such as dehydrogenation of a C C bond to an alkene. FAD dependent proteins function in a large variety of metabolic pathways including electron transport, DNA repair, nucleotide biosynthesis, beta oxidation of fatty acids, amino acid catabolism, as well as synthesis of other cofactors such as CoA, CoQ and heme groups. One well known reaction is part of the citric acid cycle (also known as the TCA or Krebs cycle); succinate dehydrogenase (complex II in the electron transport chain) requires covalently bound FAD to catalyze the oxidation of succinate to fumarate by coupling it with the reduction of ubiquinone to ubiquinol. by oxidative phosphorylation. Some redox flavoproteins non covalently bind to FAD like Acetyl CoA dehydrogenases which are involved in beta oxidation of fatty acids and catabolism of amino acids like leucine (isovaleryl CoA dehydrogenase), isoleucine, (short/branched chain acyl CoA dehydrogenase), valine (isobutyryl CoA dehydrogenase), and lysine (glutaryl CoA dehydrogenase). Additional examples of FAD dependent enzymes that regulate metabolism are glycerol 3 phosphate dehydrogenase (triglyceride synthesis) and xanthine oxidase involved in purine nucleotide catabolism. Noncatalytic functions that FAD can play in flavoproteins include as structural roles, or involved in blue sensitive light photoreceptors that regulate biological clocks and development, generation of light in bioluminescent bacteria. 90 flavoproteins are encoded in the human genome; about 84% require FAD, and around 16% require FMN, whereas 5 proteins require both to be present.

Glucose oxidase (GOX) catalyzes the oxidation of β D glucose to D glucono δ lactone with the simultaneous reduction of enzyme bound flavin. GOX exists as a homodimer, with each subunit binding one FAD molecule. Crystal structures show that FAD binds in a deep pocket of the enzyme near the dimer interface. Studies showed that upon replacement of FAD with 8 hydroxy 5 carba 5 deaza FAD, the stereochemistry of the reaction was determined by reacting with the re face of the flavin. During turnover, the neutral and anionic semiquinones are observed which indicates a radical mechanism. The P450 systems that are located in the mitochondria are dependent on two electron transfer proteins: An FAD containing adrenodoxin reductase (AR) and a small iron sulfur group containing protein named adrenodoxin. FAD is embedded in the FAD binding domain of AR. The FAD of AR is reduced to FADH2 by transfer of two electrons from NADPH that binds in the NADP binding domain of AR. The structure of this enzyme is highly conserved to maintain precisely the alignment of electron donor NADPH and acceptor FAD for efficient electron transfer. The structures of the reductase of the microsomal versus reductase of the mitochondrial P450 systems are completely different and show no homology. This $150 to 500 million market is not only for medical applications, but is also used as a supplement to animal food in the agricultural industry and as a food colorant. Already, scientists have determined the two structures FAD usually assumes once bound: either an extended or a butterfly conformation, in which the molecule essentially folds in half, resulting in the stacking of the adenine and isoalloxazine rings. FAD imitators that are able to bind in a similar manner but do not permit protein function could be useful mechanisms of inhibiting bacterial infection.

Optogenetics allows control of biological events in a non invasive manner. The field has advanced in recent years with a number of new tools, including those to trigger light sensitivity, such as the Blue Light Utilizing FAD domains (BLUF). BLUFs encode a 100 to 140 amino acid sequence that was derived from photoreceptors in plants and bacteria. Scientists have taken advantage of this by using them to monitor disease progression or treatment effectiveness or aid in diagnosis. For instance, native fluorescence of a FAD and NADH is varied in normal tissue and oral submucous fibrosis, which is an early sign of invasive oral cancer. Doctors therefore have been employing fluorescence to assist in diagnosis and monitor treatment as opposed to the standard biopsy.