Easy Way to Figure Out Coenzymes in Pathays

New Approaches for Flavin Catalysis

Anna N. Khusnutdinova , ... Alexander F. Yakunin , in Methods in Enzymology, 2019

Abstract

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.

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Vitamins and Nutrition

Gerald Litwack PhD , in Human Biochemistry (Second Edition), 2022

Riboflavin (Vitamin B2)

The coenzyme forms of riboflavin are flavin mononucleotide (FMN) and flavin adenine dinucleotide (Fig. 20.4).

FMN is formed first by the action of riboflavin kinase on riboflavin. FMN is then converted to FAD by the action of FAD pyrophosphorylase with ATP. These reactions are shown in Fig. 20.5.

Many flavoproteins contain Mg2⁺ or other metals. Examples of such enzymes are succinate dehydrogenase and xanthine dehydrogenase catalyzing reactions shown in Fig. 20.6.

Succinate dehydrogenase has four domains: (1) located in the mitochondrial matrix containing the binding site for succinate and is the location of the reduction to fumarate and also where FAD is converted to FADH2 (histidine is the covalent link between the flavin and the peptide chain); (2) also located in the mitochondrial matrix; (3) located in the inner mitochondrial membrane where heme is located and where ubiquinone is reduced; and (4) also in the inner mitochondrial membrane adjacent to C containing heme and the reduction of ubiquinone. Succinate dehydrogenase constitutes Complex II of the mitochondrial respiratory transport chain.

Riboflavin is degraded when exposed to light, generating lumichrome as the product as shown in Fig. 20.7.

Riboflavin can become deficient in newborns treated with phototherapy for hyperbilirubinemia. In general, riboflavin deficiency causes aversion to light (photophobia), inflammation of the mouth, face, and tongue (glossitis), excessive oiliness of face and scalp (seborrhea), and angular stomatitis (fissures and inflammation of the lower lip). Recommended daily intakes for persons of various ages in milligram are as follows: babies (birth–6 months), 0.3 mg; infants (7–12 months), 0.4 mg; children (1–3 years), 0.5 mg; children (4–8 years), 0.6 mg; children (9–13 years), 0.9 mg; boys (14–18 years), 1.3 mg; girls (14–18 years), 1.0 mg; men (19 years plus), 1.3 mg; women (19 years plus), 1.1 mg; pregnant females, 1.4 mg; breastfeeding females, 1.6 mg (data from http://umm.edu/health/medical/altmed/supplement/vitamin-b2-riboflavin). If riboflavin is given as a supplement, the best absorption is between meals.

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Respiratory Chain and ATP Synthase

David G. Whitehouse , ... Anthony L. Moore , in Reference Module in Biomedical Sciences, 2019

Components of the Respiratory Chain

The conversion of the electromotive force (NADH and FADH oxidation) into a proton-motive force is carried out by three electron-driven proton-pumping respiratory chain complexes. These large transmembrane complexes contain numerous reduction–oxidation (redox) components that include flavins, iron–sulfur proteins, cytochromes, ubiquinone, and metal ions.

Flavins

Flavin mononucleotide (FMN) and flavin dinucleotide (FAD) are tightly bound (to their enzymes) cofactors that can accept (or donate) two electrons and two protons (to become fully reduced) or a single electron and proton to form the semiquinone intermediate.

Iron–Sulfur Proteins

Iron–sulfur (or nonheme iron proteins) contain iron atoms covalently bound to the apo-protein by cysteine sulfurs and to other iron atoms via acid labile sulfur bridges. In general, there are three types of iron–sulfur proteins namely a single iron atom coordinated to the sulphydryl groups of four cysteine residues of the protein, a protein containing two irons and two inorganic sulfides (2Fe–2S) and four irons and four inorganic sulfides (4Fe–4S). Similar to the single iron cluster, both are coordinated by four cysteine residues. All three types of iron–sulfur proteins can act as single electron carriers.

Cytochromes

Cytochromes are proteins containing a prosthetic group called a heme. The basis of the heme is a porphyrin, which consists of four pyrrole rings linked in a cyclic manner by methene bridges with a central coordinated iron atom that acts as a single electron carrier. There are at least four classes of cytochromes (cytochrome A–D) differentiated from each other by the ability to absorb light at different wavelengths (due to differences in the side chains on the porphyrin).

Ubiquinone

Ubiquinone or coenzyme Q is a hydrophobic quinone derivative that in mammalian mitochondria has a side chain of ten 5-carbon isoprene units (Q₁₀). Ubiquinone can be fully reduced to the quinol form by the acceptance of 2H⁺ and 2e⁻ (or the semiquinone derivative can be formed through the acceptance of a single proton and electron).

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Respiratory Chains

David G. Nicholls , Stuart J. Ferguson , in Bioenergetics (Fourth Edition), 2013

5.2.2.3 Flavins, quinones and quinols

Flavin mononucleotide (FMN) is a component of complex I, whereas flavin adenine dinucleotide (FAD) is present in complex II, ETF and α-glycerophosphate dehydrogenase. FAD is additionally present in a number of enzymes, including pyruvate and α-ketoglutarate dehydrogenases. The oxidised flavin moiety can gain an electron and a proton, forming an intermediate free radical (FMNH^• or FADH^•) detectable by EPR, followed by a second electron plus another proton forming the fully reduced state (FMNH₂ or FADH₂) (Figure 5.4b). The oxidised and fully reduced forms are difficult to detect optically, especially if other redox centres are present. The flavin is well adapted to undergo both a one-electron oxidation/reduction step (e.g., when one electron at a time is passed to Fe–S centres) and a two-electron reduction (e.g., accepting electrons from NADH in complex I).

The 50-carbon hydrocarbon side chain of ubiquinone renders UQ₁₀ highly hydrophobic (Figure 5.4). UQ undergoes an overall 2H⁺ + 2e⁻ reduction to form UQH₂ (ubiquinol), although in general the reaction will take place in two one-electron steps (Figure 5.4); the partially reduced free radical form UQ^•− (ubisemiquinone) plays a defined role in both the photosynthetic reaction centre (Chapter 6) and the cyt bc ₁ complex, where it is stabilised by binding sites in the proteins. Ubiquinone reduction and ubiquinol oxidation will always occur at catalytic sites provided by the membrane proteins for which they are substrates; the (de)protonation and oxidation/reduction steps are believed to always proceed as shown in Figure 5.4b.

The radical form can be detected by its ESR spectrum or a characteristic absorption band in the visible region, but ubiquinone and ubiquinol are more difficult to detect because in common with proteins, they absorb at approximately 280 nm, although the absorbance of the oxidised and reduced forms differs.

The simplest postulate for the role of UQ is as a mobile redox carrier linking complexes I and II (and the other flavin-linked dehydrogenases; Figure 5.1) with complex III, although the 'Q-cycle' of electron transfer in complex III involves a more sophisticated and integral role (Section 5.8). Although UQ₁₀ is the physiological mediator, its hydrophobic nature makes it difficult to handle, and ubiquinones with shorter side chains, and consequently greater water solubility, are usually employed in vitro. Some anaerobic bacterial respiratory chains employ menaquinone in place of UQ (Section 5.13.2), whereas in the chloroplast the corresponding redox carrier (Section 6.4.3) is plastoquinone (Figure 5.4).

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Cofactors

Rebecca L. Fagan , Bruce A. Palfey , in Comprehensive Natural Products II, 2010

7.03.3.3.1 Dihydroorotate dehydrogenase

DHODs are FMN-containing enzymes that convert dihydroorotate (DHO) to orotate (OA) in the only redox step in the de novo synthesis of pyrimidines ( Scheme 12 ). DHODs have been grouped into two classes based on sequence. ²⁴² Class 2 DHODs are membrane-bound monomers that are reoxidized by ubiquinone, coupling pyrimidine biosynthesis to the respiratory chain. ²⁴³ On the other hand, Class 1 DHODs are cytosolic proteins that have been further divided into two subclasses. Class 1A DHODs are homodimers that are reoxidized by fumarate. ²⁴⁴ Class 1B DHODs are α₂β₂ heterotetramers with an FMN-containing subunit very similar to Class 1A enzymes and a second subunit that contains an iron–sulfur cluster and FAD, allowing Class 1B DHODs to be reoxidized by NAD. ²⁴⁵

Both classes of DHODs are interesting drug targets. Currently, several Class 2 DHODs are being investigated as drug targets including the enzymes from humans, ²⁴⁶ Candida albicans (a pathogenic yeast species), ²⁴⁷ and Plasmodium falciparum (the malaria parasite). ^248–250 Human DHOD is targeted for the treatment of arthritis by the drug leflunomide, whose active metabolite is a potent inhibitor. ²⁵¹ Crystal structures have shown that Class 2 DHODs have two binding sites on opposite sides of the flavin, one for DHO oxidation and the other for quinone binding and electron transfer. ^{248,252–254} Inhibitors of Class 2 DHODs are directed at the so-called species-selective inhibitor site, which is near and possibly overlapping the quinone-binding site. ²⁵⁰ The sequence variability of this site has allowed species-specific inhibitors to be created. In Class 1A DHODs, both DHO and fumarate (the oxidizing substrate) bind to the same site, ^255,256 whose structure is very similar to the pyrimidine-binding site of Class 2 enzymes. Nonetheless, small molecules that bind specifically to Class 1A DHODs have been discovered. ^257,258 The underlying reason for the specificity of these compounds is not understood, especially since the pyrimidine-binding sites in both classes of enzymes appear to be nearly identical. However, these compounds demonstrate the possibility of creating inhibitors specific for Class 1A enzymes. Genetic studies have shown that DHOD is essential for the survival of Trypanosoma cruzi, ²⁵⁹ validating the idea that inhibitors specific for Class 1A DHODs could treat trypansomal diseases such as Chagas' disease and African sleeping sickness.

In the oxidation of DHO, the C5 pro-S hydrogen ²⁶⁰ is removed as a proton by an active site base, while the C6 hydrogen is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. Structures of product complexes of all DHODs show that C6 of OA is in van der Waals contact with N5 of the flavin and that C5 of OA is positioned correctly for proton abstraction by the active site base ( Figure 4 ), either serine in Class 2 enzymes or cysteine in Class 1 enzymes. ^{252,254,255,261,262}

The two C–H bonds of DHO could break at the same time in a concerted mechanism or they could break sequentially in stepwise mechanisms. This mechanistic question was addressed by determining kinetic isotope effects using DHO labeled at the 5- or 6-position or both. Isotope effects determined in the steady state for the Class 2 DHOD from bovine liver ²⁶³ and the Class 1B enzyme from Clostridium oroticum ²⁶⁴ were consistent with a concerted mechanism, while isotope effects for the Class 1A enzyme from Crithidia fasciculata ²⁶⁰ were consistent with a stepwise mechanism. Similar experiments to probe the order of bond breaking were conducted by directly observing the isotope effect on flavin reduction in anaerobic stopped-flow experiments. A concerted mechanism was found for the Class 1A enzyme from Lactococcus lactis, ²⁶⁵ while the isotope effects observed with the Class 2 enzymes from humans and E. coli are consistent with a stepwise mechanism or a concerted mechanism with tunneling. ²⁶⁶ The conclusions from the reductive half-reaction contradict those from steady-state kinetics, presumably due to the complexities of interpreting steady-state measurements. Alternatively, the mechanism of DHO oxidation could be species specific.

The pH dependence of kinetics of DHODs has been studied both by steady-state and transient methods. By watching flavin reduction directly in anaerobic stopped-flow experiments, a pK _a of ∼8.3 controlling reduction was observed for the Class 1A DHOD from L. lactis, ²⁶⁵ while in the Class 2 enzymes from humans and E. coli, ^266,267 the pK _a observed was ∼9. The pK _a observed for L. lactis DHOD was attributed to ionization of the active site base, Cys130. The pK _a observed in the Class 2 DHODs was a bit of a mystery. The structures of Class 2 DHODs show nothing in the active site that would lower the pK _a of the serine so dramatically ^252,254 from ∼13.6 for an unperturbed residue. ²⁶⁸ Kinetic studies suggest that this is a consequence of the stepwise mechanism of DHO oxidation, rather than a true thermodynamic pK _a. The deuterium isotope effect for label at the 5-position of DHO is lost when experiments are performed at pH values above the observed pK _a. Above pH ∼9, deprotonation becomes too fast to contribute to the rate of reduction, and the rate is determined only by hydride transfer. Conversely, the pH dependence of steady-state turnover rates by the Class 2 enzyme from bovine liver, ²⁶³ the Class 1A enzyme from yeast, ²⁶⁹ and the Class 1B enzyme from C. oroticum ²⁶⁴ all show a pK _a value ∼7, which was ascribed to the active site base needed to deprotonate C5 of DHO. The kinetic complexity of steady-state measurements could account for the observed differences in pH dependencies.

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Vitamin B2

D. Nohr , H.K. Biesalski , in Reference Module in Food Science, 2016

Riboflavin or Vitamin B2

The chemical name for riboflavin is 7,8-dimethyl-10-(1′-D-ribityl)isoalloxazine; riboflavin exists in an oxidized and a reduced form (Figure 1 ) and from which two coenzymes are formed, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD; Figure 2).

The ending 'flavin' refers to its yellowish color (latin 'flavus' = yellow).

Free as well as protein-bound riboflavin occurs in the diet, and milk in general is the best source. In cow's milk, the free form, with the higher bioavailability, is the major one (61% riboflavin, 26% FAD, 11% hydroxyethyl form and others) whereas the protein-bound and thus less bioavailable form predominates in other foods. In human breast milk, approximately one to two thirds of riboflavin occurs as FAD.

Riboflavin is very heat stable but it is extremely photosensitive. It is photo-degraded to lumiflavin (under alkaline conditions) or lumichrome (under acidic conditions), both of which are biologically inactive. Concentrations are significantly reduced in high-pressure low-temperature treated milk compared to raw milk. UV light excites riboflavin to a high degree of natural fluorescence that is used for its detection and determination in yogurt or non-fat dry milk.

Functions of Riboflavin

Riboflavin-dependent enzymes are called flavoproteins or flavoenzymes, because of their yellowish appearance. They catalyze hydroxylations, oxidative decarboxylations, dioxygenations and reduction of oxygen to hydrogen peroxide serving as electron carriers, mediators of electron transfer from pyridine nucleotides to cytochrome c or to other one-electron acceptors and as catalysts of electron transfer from a metabolite to molecular oxygen. The two flavoenzymes, FMN and FAD, play major roles in the metabolism of glucose, fatty acids, amino acids, purines, drugs and steroids, folic acid, pyridoxine, vitamin K, niacin and vitamin D. Riboflavin is used together with UV-light for crosslinking corneal collagen to treat Keratoconus.

The FAD-dependent enzyme, glutathione reductase, plays a major role in the antioxidant system by restoring reduced glutathione (GSH) from oxidized glutathione (GSSH). GSH is important in protecting lipids from peroxidation, in stabilizing the structure and function of red blood cells, it is the most important antioxidant in erythrocytes and in keeping lens-proteins in solution (thus preventing cataracts).

The formation of FMN and FAD is ATP dependent, it takes place mainly in the liver, kidney and heart. All enzymatic steps are under the control of thyroid hormones.

•: Flavokinase: Riboflavin + ATP → flavin mononucleotide + ADP
•: FAD pyrophosphorylase: FMN + ATP → flavin adenine dinucleotide + PP
•: FAD + apoenzyme/protein → covalently bound flavins

Sources of Riboflavin

Tables 1 and 2 summarize dietary sources of riboflavin and its content, especially in the milk of various species and dairy products.

Table 1. Riboflavin concentrations in food

Food	Concentration (μg 100 g⁻¹)
Brewers' yeast	3800
Pig's liver	3200
Ox liver	2900
Wheat germ	720
Almonds	620
Wheat bran	510
Soya bean, seed dry	460
Mushroom	436
Egg	408
Mackerel	360
Eel	320
Lentil, seed dry	262
Beef	260
Pork	230
Herring	220
Maize	200

Reproduced from Souci, S.W., Fachmann, W., Kraut, H., 2008. Food Composition and Nutrition Tables, seventh ed. Medpharm Scientific Publishers, Stuttgart.

Table 2. Riboflavin in milk, dairy products and cheese

Food	Concentration (μg 100 g⁻¹)
Dried whole milk	1400
Parmigiano	620
Camembert (45% fat in dry matter)	600
Blue cheese (50% fat in dry matter)	500
Condensed milk (min. 10% fat)	480
Limburger (40% fat in dry matter)	350
Quark/fresh cheese (from skim milk)	300
Cream cheese (min. 60% fat in dry matter)	230
Consumer milk (3,5% fat)	180
UHT milk	180
Yoghurt (min. 3,5% fat)	180
Skim milk	170
Buttermilk	160
Cream (min. 30% fat)	150
Sweet whey	150
Sterilized milk	140
Milk from
Sheep	230
Cow	180
Goat	150
Buffalo	100
Donkey	64
Human	38

Reproduced from Souci, S.W., Fachmann, W., Kraut, H., 2008. Food Composition and Nutrition Tables, seventh ed. Medpharm Scientific Publishers, Stuttgart.

Heat treatment has only negligible effects on riboflavin concentrations, whereas exposure of milk to sunlight results in a loss of 20–80% of riboflavin. Thus, storage in dark bottles, light-tight wax cartons or special polyethylenterephthalat (PET) bottles is recommended.

The photodegradation of riboflavin catalyses photochemical oxidation and a loss of ascorbic acid. Gamma radiation of 10 Gy destroys about 75% of riboflavin in liquid milk, while milk powder shows no losses even at higher doses.

Storage influences riboflavin concentrations as follows: condensed milk loses 28% or 33% of its initial riboflavin content when stored at 8–12 °C for 2 years or 10–15 °C for 4 years, respectively, ice-cream loses 5% when stored at −23 °C for 7 months. No losses were found in fresh milk stored at 4–8 °C for 24 h or in dried milk powder stored for 16 months.

In cheese, most losses (66–88%) of the original riboflavin content of the milk appear to occur during whey drainage, while ripening has almost no effects. However, in some cheese varieties, the concentration is higher in the outer layers due to microbial synthesis. High-pressure tests for thermal sterilization processes led to different results concerning the decay of the vitamin dependent from the matrix of the respective food tested.

Riboflavin Deficiency

Riboflavin is essential for humans, animals and some microorganisms, amongst humans seniors and adolescents seem to be at particular risk of deficiency; the recommended uptake is given in Table 3. In some cases, recommended uptake is related to energy intake and 0.6 mg riboflavin per 1000 kcal is considered adequate. Milk and milk products (without butter) can contribute about 30% of total riboflavin supply.

Table 3. Recommended daily uptake of riboflavin

Age	Riboflavin (mg day⁻¹)
Age	Male	Female
Sucklings <4 months	0.3
Sucklings 4–12 months	0.4
Children 1–4 years	0.7
Children 4–7 years	0.9
Children 7–10 years	1.1
Children 10–13 years	1.4	1.2
Children 13–15 years	1.6	1.3
Adults 15–25 years	1.5	1.2
Adults 25–51 years	1.4	1.2
Adults 51–65 years	1.3	1.2
Adults >65 years	1.2	1.2
Pregnant		1.5
Breast feeding		1.6

Reproduced from DGE (Deutsche Gesellschaft für Ernährung), 2015. Die Referenzwerte für die Nährstoffzufuhr. http://www.dge.de/wissenschaft/referenzwerte/riboflavin/ (last check January 2015).

The major portion of riboflavin is bound to proteins and these flavoproteins have to be hydrolyzed before absorption by specialized transporters in the upper gastrointestinal tract. The amount that can be stored depends on the availability of proteins providing binding sites. Although a limited uptake makes sense in preventing accumulation in tissues, it increases the body's dependence on dietary supply. Under normal conditions, riboflavin stores last for 2–6 weeks, but in cases of protein deficiency, they last significantly shorter.

Symptoms of a marginal deficiency are often non-specific: weakness, fatigue, mouth pain, glossitis, stomatitis, burning and itching of the eyes, personality changes. Signs of increased deficiency are cheilosis, angular stomatitis, seborrheic dermatitis at the mouth, nasolabial sulcus and ears (later extending to the trunk and extremities) desquamative dermatitis with itching in genital regions, opacity of the cornea, cataract and brain dysfunction. Major reasons for riboflavin deficiency are:

•: Insufficient dietary intake by seniors and adolescents (mainly girls)
•: Endocrine abnormalities, insufficient adrenal and thyroid hormones
•: Drugs (psychotropic, antidepressant, cancer therapeutics, antimalarial)
•: Alcohol intake interfering with digestion of food flavins and absorption
•: Agents that chelate- or form complexes with riboflavin or FMN affecting their bioavailability: copper, zinc, iron, caffeine, theophylline, saccharine, nicotinamide, ascorbic acid, tryptophane, urea.

As riboflavin (via FAD-dependent glutathione reductase) is involved in antioxidant mechanisms, riboflavin deficiency may considerably affect erythrocyte metabolism. However, several studies reported protective effects of a deficiency against malaria infections. A study in the USA showed that the uptake of yogurt, milk, cereals and also riboflavin was inversely correlated with homocysteine levels of plasma which in turn seem to be positively correlated with a higher risk of developing atherosclerosis.

Assessment of the riboflavin (mainly by HPLC methods) status uses the following parameters:

•: Erythrocyte glutathione reductase activity coefficient,
•: Excretion in urine (mg g⁻ ¹ creatinine to assess short term effects)
•: Riboflavin in erythrocytes (mg g⁻ ¹ hemoglobin)
•: Erythrocyte glutathione reductase activation (EGRAC) test

Concerning supplementation, no case of intoxication has been described. Thus, riboflavin is regarded as safe even at high doses. Supplements are usually given to reverse deficiency symptoms or to support high-risk groups:

Regular intake of drugs (e.g., antidepressants, oral contraceptive)

•: Malnutrition
•: Patients after trauma
•: Malabsorption
•: Chronic alcoholics

Hyperbilirubinaemia can be treated much quicker by phototherapy when 0.5 mg riboflavin kg⁻ ¹ bodyweight are given. Finally, persons with congenital methemoglobinaemia might benefit from 20 to 40 mg day⁻ ¹.

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Electron Paramagnetic Resonance Investigations of Biological Systems by Using Spin Labels, Spin Probes, and Intrinsic Metal Ions, Part A

Andrei V. Astashkin , in Methods in Enzymology, 2015

8.3.3 NOS: Calmodulin–Heme Domain Docking

The release of the FMN domain from the rest of the reductase domain is enabled in NOS by the Ca^{2 +}-dependent (except for iNOS) calmodulin (CaM) binding to the CaM-binding region of the linker connecting the FMN and oxygenase domains (Roman, Martasek, & Masters, 2002). It was suggested that the NOS-bound CaM itself can dock to the oxygenase domain and, in this way, facilitate the FMN domain docking (Smith, Underbakke, Kulp, Schief, & Marletta, 2013). This possibility was tested in a RIDME investigation of the oxyFMN construct of neuronal NOS (nNOS), using the spin-labeled CaM as spin A and the low-spin heme centers as spins B (Astashkin et al., 2014).

The experiments were performed using the conditions nearly identical to those in the previous work (mw K _a band; refocused stimulated ESE sequence; $T_{R}^{(1)} = T_{R}^{(2)} = 25 μ s$ ; T ⁽¹⁾ = 8 K, T ⁽²⁾ = 25 K). The RIDME effect from the spin-labeled CaM consisted of two contributions: the contribution from the docking complex (similar to Fig. 6c) in the form of a rapidly damping oscillation (about half a period), and the contribution from the undocked state (similar to Fig. 6b), where the position of CaM is distributed in broad limits (up to R _AB ~ 100 Å), which results in a monotonic decay of the ESE signal. The analysis of these RIDME effects took into account the presence of two heme centers in the oxygenase domain and used the X-ray structure of the oxygenase domain to provide the lower limits for the possible distances between CaM and the heme centers. The analysis allowed one to establish the CaM docking position, which is in good agreement with the computational docking model.

In addition, the analysis resulted in the statistical weights of the docked (~ 15%) and undocked (~ 85%) states. If the conformational distribution in the frozen solution represents a "snapshot" of the dynamic situation in liquid, then the equivalence of time and ensemble averagings requires that these statistical weights directly reflect the relative time intervals CaM spends in the docking complex (t _dock) and in diffusion-controlled open (undocked) conformations (t _open). Using for the absolute time reference the ET rate in nNOS oxyFMN construct of ~ 262 s^{− 1} (Feng et al., 2006), one can then estimate t _dock ~ 0.57 ms and t _open ~ 3.24 ms. This example shows the link between the conformational statistics in frozen solution and the dynamics in liquid solution.

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Flavin-Dependent Enzymes: Mechanisms, Structures and Applications

Helen S. Toogood , Nigel S. Scrutton , in The Enzymes, 2020

4.1.2 'Indirect' light-dependent FMN reduction

Indirect photochemical enzyme-bound FMN reduction occurs when electrons from a light-activated photosensitizer are transferred to the FMN via a chemical mediator (Fig. 5). For example, the reducing equivalents for the TsOYE-catalyzed reduction of ketoisophorone were supplied by a photosensitized Au-TiO₂ semiconductor, and mediated to the enzyme flavin via free flavin (Table 3; [63]). This study illustrated that in this case water was the sacrificial substrate, generating oxygen gas as the only by-product [63].

Photosensitive transition metal complexes based on Ru(II) or Ir(III) were tested for their ability to support alkene reduction by TOYE and variant PETNR_R324C (Table 3; [64]). This indirect route relied on methyl viologen as the mediator and the sacrificial substrate triethylamine. A variety of α,β-unsaturated aldehydes and ketones were successfully reduced using [Ru(bpz)₂(dClbpy)]Cl₂, with product yields and enantiopurity at least comparable to reactions with NADPH as the hydride source.

A novel approach to supplying reducing equivalents to TsOYE was the construction of a photoelectrochemical cell (PEC; [65]). This contained a protonated graphitic carbon nitride (p-g-C₃N₄) and carbon nanotube hybrid (CNT/p-g-C₃N₄) film cathode, and a FeOOH-deposited bismuth vanadate (FeOOH/BiVO₄) photoanode. In this system, photoexcited electrons from the anode traveled to the CNT/p-g-C₃N₄ hybrid film which subsequently electrocatalytically performed a two-electron reduction of the mediator. This in turn reduced the TsOYE-bound FMN, allowing the highly enantioselective conversion of ketoisophorone to (R)-levodione to proceed (83% ee). This is another example where water was the sacrificial substrate, and showed the potential to develop a PEC approach for preparative scale OYE-catalyzed alkene reduction [65].

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Flavin-Dependent Enzymes: Mechanisms, Structures and Applications

Caterina Martin , ... Andrea Mattevi , in The Enzymes, 2020

1 Introduction

Biocatalytic oxidations have attracted the interest of academic and industrial research since many decades [1]. The ability to carry out oxidations with high enantioselectivity together with the broad substrate scope and mild reaction conditions render enzymes attractive, environmentally sustainable catalysts [2]. Redox reactions are performed by peroxidases, oxidases, oxygenases, reductases and dehydrogenases. Such enzymes can be used to produce for example: alcohols, aldehydes, ketones and carboxylic acids that may be problematic to obtain in a single step via traditional chemical approaches. Oxidative biocatalysts employ various mechanisms that involve a variety of different electron acceptors, electron donors and/or redox cofactors. Common redox cofactors are metals (e.g., iron or copper), heme cofactors, nicotinamide cofactors (NADH and NADPH), and flavin cofactors (FAD and FMN). Typical electron acceptors/donors are small redox-active proteins (such as cytochrome C), nicotinamide coenzymes, cosubstrates, molecular oxygen and hydrogen peroxide [3].

An oxidase is defined as an oxidoreductase that utilizes molecular oxygen (O₂) as electron acceptor [4]. In contrast to oxygenases, reductases, and dehydrogenases, oxidases are of major interest because they do not depend on coenzymes (such as nicotinamide cofactors or quinones) that need to be regenerated, but rely only on molecular oxygen as electron acceptor. Their ability to work without any coenzyme regeneration make them cost-effective and less complicated, and therefore more suitable for industrial applications [5]. In contrast to other redox enzymes, such as dehydrogenases, oxidases are not very abundant in nature [6]. This is probably due to the fact that in most cases oxidases catalyze the reduction of dioxygen into hydrogen peroxide. This means that the electrons generated through the oxidation of an organic substrate are irreversibly lost and cannot be used in metabolic route. Moreover, the typical byproduct of the reaction, hydrogen peroxide, is a reactive and toxic molecule. In some cases, reduction of dioxygen can even lead to more damaging reactive oxygen species, such as superoxide. Only in a few oxidases, such as some reported NADH oxidases, reduction of dioxygen into harmless water is accomplished. Oxidases can be subdivided in two major groups based on the employed cofactor: copper- and flavin-containing oxidases [6].

In this chapter we focus on the flavin-containing oxidases. Many members of the flavoprotein oxidase family are regarded as valuable tools for biotechnological applications. Flavoprotein oxidases often are highly selective (for a particular substrate or exhibiting high enantio- or chemoselectivity), display a high activity, do not rely on any metals, and require no expensive coenzymes or cosubstrates. These features make them good candidates for various biotechnological applications, for synthesis of high value compounds [7] or biosensors [8].

1.1 Flavin cofactors

Flavin-containing oxidases contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as redox cofactor ( Fig. 1). In only a few cases, flavoprotein oxidases contain one or more additional cofactors [9]. FAD and FMN are synthetized starting from riboflavin (vitamin B₂). Phosphorylation of riboflavin by action of riboflavin kinase results in FMN. Adenylation of FMN yields FAD, the most common flavin cofactor in flavoenzymes [10].

The majority of flavin-containing enzymes, using either FAD or FMN, contain a tightly non-covalently bound flavin. In a relatively small number of flavoproteins, the flavin cofactor is covalently bound. In most cases, this involves a covalent tethering of the protein to the benzyl moiety of a FAD cofactor. For some covalent flavoproteins, the role of such covalent linkage has been shown to be essential to increase the redox potential of the flavin cofactor, to allow oxidations of compounds that are difficult to oxidize. In other cases, the covalent attachment may serve other purposes such as allowing enzymes to adopt a relatively open active site, increase the stability or protecting the protein from proteolysis [11,12].

The spectroscopic characteristics of riboflavin, FAD and FMN are comparable. This is mainly due to the shared isoalloxazine core structure [13]. The variability in the UV/Vis absorption spectra depend largely on the oxidation state of the flavin (quinone, semiquinone or hydroquinone) [13]. The flavin absorption spectra in the oxidized state in water exhibit four distinct peaks at around 445, 375, 265, and 220 nm, with extinction coefficients above 10⁴ M^{− 1} cm^{− 1} [14]. The absorbance spectra of 1- and 2-electron reduced flavins are totally different, with the fully reduced flavins exhibiting very little absorbance in the visible range. While the direct protein environment around a protein-bound flavin can affect UV/Vis absorbance features to a small extent, the fluorescence of flavin cofactors in proteins is typically influenced to a large extent. The neutral forms of oxidized flavins exhibit an intense yellow-green fluorescence at around 520 nm [14]. The polypeptide chain and especially certain amino acids (tryptophan and tyrosine) are potent quenchers of flavin fluorescence [13,15]. The effect of the protein on flavin fluorescence is often exploited for biochemical studies of flavoenzymes. For example, the ThermoFAD assay has been developed that allows an easy way of determining the apparent melting temperature (thermostability) of flavoproteins. It relies on detecting the difference in flavin fluorescence upon unfolding a (purified) flavoprotein using a temperature gradient, which is typically performed by using a real-time PCR machine [16]. The protein amount and assay time required for this assay are minimal and it can be used in a high-throughput fashion [16]. It is the ideal system to evaluate libraries of variants in thermostability studies. The assay is so sensitive that, if the flavoenzyme is well expressed, it can be performed using cell-free extracts [17].

The redox potentials for the two-electron reductions of FAD and FMN free in solution at pH 7.0 are respectively − 219 and − 205 mV [18,19]. In aqueous solution the flavin cofactor can be found in three different redox states: the oxidized redox state, the flavin semiquinone (one-electron reduced), or the fully reduced state (two-electron reduced) (Scheme 1).

The catalytic cycle of flavoenzymes typically comprises of two half-reactions. In the reductive half-reaction the oxidized flavin is reduced by its first substrate. In the second half-reaction, the reduced flavin is reoxidized. In the case of flavoprotein oxidases, molecular oxygen is able to act as efficient electron acceptor in the latter oxidative half-reaction (Scheme 2). The ability to swiftly react with molecular oxygen, to reduce it to hydrogen peroxide, sets flavoprotein oxidases apart from other flavoproteins [20]. The exact details on how molecular oxygen is converted into hydrogen peroxide, with the concomitant reoxidation of the flavin cofactor, are still not well understood. It is thought that the first step of the reaction involves the generation of a superoxide anion and flavin semiquinone [21]. In a second electron transfer process, hydrogen peroxide is generated along with fully oxidized flavin. The rate-limiting step is thought to be the first electron transfer (from the fully reduced flavin to O₂) [22].

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METABOLIC PATHWAYS | Metabolism of Minerals and Vitamins

M. Shin , ... T. Shin , in Encyclopedia of Food Microbiology (Second Edition), 2014

Riboflavin (Vitamin B₂)

Riboflavin is the direct precursors of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), the chemically versatile redox cofactors of a large number of flavoenzymes. In many microorganisms, riboflavin is biosynthetically produced or transported from the external environment due to the expression of specific transporters.

One molecule of GTP and two molecules of ribulose 5-phosphate are required as substrates for the biosynthesis of one riboflavin molecule. The first step in the biosynthesis of riboflavin is the conversion of GTP to 2,5-diamino-6-ribosylamino-4-pyrimidinone 5′-phosphate. The subsequent steps are different in bacteria, yeasts, and fungi, although they ultimately lead to the same intermediate, 5-amino-6-ribitylamino-2, 4-pyrimidinedione 5′-phosphate. To yield the intermediate, the ribosyl side chain of 2,5-diamino-6-ribosylamino-4-pyrimidinone 5′-phosphate is reduced and then deaminated in yeasts and fungi, whereas the deamination of the pyrimidine ring is followed by reduction of the side chain in E. coli. These reactions are catalyzed by bifunctional protein containing an N-terminal deaminase and a C-terminal reductase domain, encoded by the gene ribD or ribG from E. coli or B. subtilis, respectively. The final step in the biosynthesis of riboflavin is dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine by riboflavin synthetase to yield riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione, the latter is recycled and condensate with 3,4-dihydroxy-2-butanone 4-phosphate derived from ribulose 5′-phosphate to produce 6,7-dimethyl-8-ribityllumazine. The mechanistically unusual reaction involves the transfer of a four-carbon fragment between two identical substrate molecules.

The genes encoding the riboflavin biosynthetic enzymes of B. subtilis were found clustered in a single operon (rib operon). The products of the rib operon (RibG, RibB, RibA as GTP cyclohydrolase II, and RibH) form an enzyme complex with an unusual quaternary structure to catalyze the conversion of GTP and ribulose 5-phosphate to riboflavin. Riboflavin kinase catalyzes the conversion of riboflavin to FMN, and FAD synthetase converts FMN to FAD. In B. subtilis, ribC gene encodes a bifunctional flavokinase and FAD synthetase. In B. subtilis, FMN or FAD, but not riboflavin, acts as effector molecules controlling riboflavin biosynthesis.

Both FAD and FMN are covalently bound to a histidine residue of the apoflavoprotein polypeptide by an 8α-(N ³-histidyl)-riboflavin linkage.

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Easy Way to Figure Out Coenzymes in Pathays

New Approaches for Flavin Catalysis

Abstract

Vitamins and Nutrition

Riboflavin (Vitamin B2)

Respiratory Chain and ATP Synthase

Components of the Respiratory Chain

Flavins

Iron–Sulfur Proteins

Cytochromes

Ubiquinone

Respiratory Chains

5.2.2.3 Flavins, quinones and quinols

Cofactors

7.03.3.3.1 Dihydroorotate dehydrogenase

Vitamin B2

Riboflavin or Vitamin B2

Functions of Riboflavin

Sources of Riboflavin

Riboflavin Deficiency

Electron Paramagnetic Resonance Investigations of Biological Systems by Using Spin Labels, Spin Probes, and Intrinsic Metal Ions, Part A

8.3.3 NOS: Calmodulin–Heme Domain Docking

Flavin-Dependent Enzymes: Mechanisms, Structures and Applications

4.1.2 'Indirect' light-dependent FMN reduction

Flavin-Dependent Enzymes: Mechanisms, Structures and Applications

1 Introduction

1.1 Flavin cofactors

METABOLIC PATHWAYS | Metabolism of Minerals and Vitamins

Riboflavin (Vitamin B₂)

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