Flavin

Flavins can serve as either one-electron or two-electron transfer reagents, acting at the interface between the cytochromes and NADH. The vitamin, riboflavin, is converted by the body to the catalytically active coenzyme forms, FMN or FAD. Flavoproteins are involved in oxidation reactions with substrates which include the pyridine nucleotides, a-amino acids and a-hydroxy acids, and compounds containing saturated carbon-carbon bonds convertible into olefins. When oxidation occurs, the isoalloxazine ring of FMN (Fig. 8) or FAD becomes reduced. 

The mechanism of action of the flavin coenzymes has been quite refractory of solution, but recently some advances in our knowledge have come about. The state of knowledge has been reviewed by Bruice (1976b), and the reader is directed there for areas which cannot be covered here. 

Owing to the wide range of reactions in which flavin coenzymes are involved, a unitary mechanism of action appears unlikely. It seems clear that in enzymes where the fully reduced form is reacting with a one-electron acceptor (such as NADH-cytochrome b-reductase, NADPH-cytochrome c-reductase, and ferridoxin NADP reductase), the mechanism involves flavin semiquinones as intermediates. These are free-radical intermediates and can be detected by electron spin resonance (ESR). Flavin semiquinones can be stabilized because of multiple resonance forms, the odd electron being shared throughout the entire isoalloxazine ring. The free-radical intermediate may be further oxidized or reduced, one electron at a time. A number of enzjmies requir- 

Vastano et al. [8] have reported a method, based on solid phase extraction, with ion pair high performance liquid chromatography using fluorescence detection for the determination of flavins in seawater. Concentrations in the pm range could be determined. 

However, it must be stressed that not everyone is in agreement with such a scheme. For example, flavoproteins are thermodynamically capable of transferring electrons directly to O2 to form O2 , and not all functional oxidase preparations contain substantial amounts of FAD. Indeed, in one series of experiments the ratio of cytochrome b to FAD decreased from about 1 1 to 19 1 as the oxidase became progressively more purified. It may be, howev- 

FIGURE 11.7 Two-electron reduction of flavin. The attachment to the larger part of the FAD or FMN molecules is at the lower methyl group. 

Riboflavin (7,8-dimethyl- 10-ribityl-isoalloxazi-ne), known as vitamin B2 (cf. 6.3.2), is the building block of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Both act as prosthetic groups for electron transfer reactions in a number of enzymes. 

Due to the much wider redox potential of the flavin enzymes, riboflavin is involved in the transfer of either one or two electrons. This is different from nicotinamides which participate in double electron transfer only. Values between +0.19V (stronger oxidizing effect than NAD ) and -0.49 V (stronger reducing effect than NADH) have been reported. 

Like glucose oxidase, many flavin enzymes transfer the electrons to molecular oxygen, forming H2O2 and flavoquinone. The following intermediary products appear in this reaction  

---

Coemymes effecting transfer of hydrogen. These include the pyridine nucleotides, nicotinamide-adenine dinucleolide and nicotinamide-adenine dinucleolide phosphate the flavin nucleotides such as flavin-adenine dinucleotide and lipoic acid. 

FAD Flavin-adenine dinucleotide, fahl ore, CujSbSj. Tetrahedrite. 

The reduction of flavin in FAD is accompanied by loss of the characteristic yellow colour. The reduction-oxidation of flavo-proteins can thus be followed spectro-photomelrically. 

FMN See flavin mononucleotide, foamed plastics See cellular plastics. 

Figure 11.39 summarizes the reactions taking place in this amperometric sensor. FAD is the oxidized form of flavin adenine nucleotide (the active site of the enzyme glucose oxidase), and FAD1T2 is the active site s reduced form. Note that O2 serves as a mediator, carrying electrons to the electrode. Other mediators, such as Fe(CN)6 , can be used in place of O2. 

He/minthosporium (15). The mode of action is considered to be inhibition of the enzyme NADPH-cytochrome C reductase, which results in the generation of free radicals and/or peroxide derivatives of flavin which oxidize adjacent unsaturated fatty acids to dismpt membrane integrity (16) (see Enzyme inhibitors). 

Bacterial concentrations have also been determined by using the enzyme-catalyzed chemiluminescent reaction of reduced flavin mononucleotide (FMN) with oxygen and aldehydes. The detection limit was reported to be 10 ceUs of E. coli, which contains 7 x 10 g of FMN per ceU (303). 

An important advance with regard to light stabiUty was made with a group of yellow coumarin dyes with heterocycHc systems attached to the coumarin nucleus (4), eg, a greenish yellow cationic dye that is sold under the name Maxilon Brilliant Flavine 10 GFF [12221 -86-2] (Blue Wool 4), designated Cl Basic Yellow 40, available from several manufacturers. 

Paul Karrer chemistry research iato constitution of carotenoids, flavins, and vitamins A and B2... 

The chemistry of flavins, including several synthetic methods for the preparation of A/-D-ribityl-3,4-xyhdine (11) is reviewed in Reference 36. 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

Polarography is appHed in the presence of other vitamins, eg, in multivitamin tablets, without separation. The polarography of flavins is reviewed in Reference 71. 

In contrast to the nicotinamide nucleotide dehydrogenases, the prosthetic groups FMN and FAD are firmly associated with the proteins, and the flavin groups are usually only separated from the apoen2yme (protein) by acid treatment in water. However, in several covalently bound flavoproteins, the enzyme and flavin coen2ymes are covalently affixed. In these cases, the flavin groups are isolated after the proteolytic digestion of the flavoproteins. 

Riboflavin-5 -Phosphate. Riboflavin-5 -phosphate [146-17-8] (vitamin B2 phosphate, flavin mononucleotide, FMN, cytoflav), C2yH22N402P,... 

Flavin mononucleotide was first isolated from the yellow en2yme in yeast by Warburg and Christian in 1932 (4). The yellow en2yme was spHt into the protein and the yellow prosthetic group (coen2yme) by dialysis under acidic conditions. Flavin mononucleotide was isolated as its crystalline calcium salt and shown to be riboflavin-5Lphosphate its stmeture was confirmed by chemical synthesis by Kuhn and Rudy (94). It is commercially available as the monosodium salt dihydrate [6184-17 /, with a water solubiUty of more than 200 times that of riboflavin. It has wide appHcation in multivitamin and B-complex solutions, where it does not require the solubili2ers needed for riboflavin. 

Riboflavin-5 -Adenosine Diphosphate. Riboflavin-5 -adenosine diphosphate [146-14-5] (flavin—adenine dinucleotide, FAD), C27H33N9O15P2 (2), mol wt 785.56, was first isolated in 1938 from the D-amino acid oxidase as its prosthetic group (95), where it was postulated to be... 

Covalently Bound Flavins. The FAD prosthetic group in mammalian succinate dehydrogenase was found to be covalently affixed to protein at the 8 a-position through the linkage of 3-position of histidine (102,103). Since then, several covalently bound riboflavins (104,105) have been found successively from the en2ymes Hsted in Table 3. The biosynthetic mechanism, however, has not been clarified. 

Deazariboflavin. In 5-dea2ariboflavin (24), the N-5 of riboflavin is replaced by CH it serves as cofactor for several flavin-cataly2ed reactions (109). It was first synthesi2ed in 1970 (110) improved synthetic processes were reported later (111). 

Anhydrotetracycline oxygenase from Streptomjces aureofaciens which cataly2es the conversion of anhydrotetracycline to dehydrotetracycline, has been isolated and characterized as a flavin-dependent oxygenase (83). It consists of two subunits of mol wt = 57, 500 based on SDS/polyacrylamide—gel electrophoresis. The cosynthetic factor 1 of Streptomjces aureofaciens involved in the reduction of 5a,lla-dehydrochlortetracycline to chlortetracycline, has been identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin. This work was aided by comparison of spectral data with that of an authentic sample obtained from the hydrolysis of coenzyme F-420 (84). 

Hydrolases represent a significant class of therapeutic enzymes [Enzyme Commission (EC) 3.1—3.11] (14) (Table 1). Another group of enzymes with pharmacological uses has budt-ia cofactors, eg, in the form of pyridoxal phosphate, flavin nucleotides, or zinc (15). The synthases, and other multisubstrate enzymes that require high energy phosphates, are seldom available for use as dmgs because the required co-substrates are either absent from the extracellular space or are present ia prohibitively low coaceatratioas. 

---