Riboflavin and the Flavin Coenzymes

The ribityl moiety is not linked to the isoalloxazine ring by a glycosidic linkage, and it is not strictly correct to caU FAD a dinucleotide. Nevertheless, this trivial name is accepted, as indeed is the even less correct term flavin mononucleotide for riboflavin phosphate. 

Riboflavin phosphate and FAD may be either covalently or noncovalenfly bound at the catalytic sites of enzymes. Even in those enzymes in which the binding is not covalent, the flavin is tightly bound in many cases, the flavin has a role in maintaining or determining the conformation of the enzyme protein. In some cases, the flavin is incorporated into the nascent polypeptide chain, while it is stUl attached to the ribosome. However, in others a flavin-free apoenzyme is synthesized and accumulates in riboflavin deficiency (Section 7.5.2). 

Covalent binding of the flavin coenzymes is normally through the 8-a-methyl group. 8-Hydroxymethyl-riboflavin is formed by microsomal mixed-function oxidases (Section 7.2.5), but it is not known whether or not this is a precursor of covalently bound flavin coenzymes. A variety of amino acid residues may be involved in covalent binding of flavin coenzymes to enzymes, including the following  

flavin 8-a-carbon linkage to imidazole N-3 of a histidine residue (e.g., in succinate, sarcosine, and dimethylglycine dehydrogenases in mammals and bacterial 6-hydroxynicotine oxidase)  

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Figure 2. 14 Riboflavin and the flavin coenzymes, riboflavin monophosphate and flavin adenine dinucleotide.

The separate determination of riboflavin and the flavin coenzymes FMN and FAD has been described in dairy products (26,43,48) and liquid tonics (40) (Table 4). 

Figure45-10. Riboflavin and the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

Figure 15-7 The flavin coenzymes flavin adenine dinucleotide (FAD) and riboflavin 5 -phosphate (FMN). Dotted lines enclose the region that is altered upon reduction.

The attention of biochemists was first attracted to flavins as a result of their color and fluorescence. The study of spectral properties of flavins (Fig. 15-8) has been of importance in understanding these coenzymes. The biochemical role of the flavin coenzymes was first recognized through studies of the "old yellow enzyme"144 145 which was shown by Theorell to contain riboflavin 5 -phosphate. By 1938, FAD was recognized as the coenzyme of a different yellow protein, D-amino acid oxidase of kidney tissue. Like the pyridine nucleotides, the new flavin coenzymes were reduced by dithionite to nearly colorless dihydro forms (Figs. 15-7 and 15-8) revealing the chemical basis for their function as hydrogen carriers. 

Flavin coenzymes are usually bound tightly to proteins and cycle between reduced and oxidized states while attached to the same protein molecule. In a free unbound coenzyme the redox potential is determined by the structures of the oxidized and reduced forms of the couple. Both riboflavin and the pyridine nucleotides contain aromatic ring systems that are stabilized by resonance. Part of this resonance stabilization is lost upon reduction. The value of E° depends in part upon the varying amounts of resonance in the oxidized and reduced forms. The structures of the coenzymes have apparently evolved to provide values of E° appropriate for their biological functions. 

Dietary deficiency is relatively widespread, yet is apparently never fatal there is not even a clearly characteristic riboflavin deficiency disease. In addition to intestinal bacterial synthesis of the vitamin, there is very efficient conservation and reutilization of riboflavin in tissues. Flavin coenzymes are tightly enzyme bound, in some cases covalently, and control of tissue flavins is largely at the level of synthesis and catabolism of flavin-dependent enzymes. 

Figure 7.1. Riboflavin, the flavin coenzymes and covalently bound flavins in proteins. Relative molecular masses (Mr) riboflavin, 376.4 riboflavin phosphate, 456.6 and FAD, 785.6.

Although the ribitol moiety is not involved in the redox function of the flavin coenzymes, both the stereochemistry and nature of the sugar alcohol are important. Although some riboflavin analogs have partial vitamin action. 

Riboflavin deficiency is relatively common, yet there is no clear deficiency disease and the condition never seems to be fatal. This presumably reflects the high degree of conservation of riboflavin in tissues (Section 7.2.3). There is only a relatively small difference between the concentration of flavins at which tissues are saturated and the lowest levels in prolonged depletion of experimental animals. In deficiency, most of the flavin coenzymes released by the catabolism of enzymes are reutilized. 

Riboflavin, the flavin coenzymes and covalently bound flavins... 

FIGURE 5.3 The flavin coenzymes FAD and FMN. Whereas FMN consists simply of riboflavin monophosphate, FAD has an AMP unit joined to riboflavin monophosphate. Note that in contrast to NAD+, flavins can be half-reduced to the stable radical FADH or fully reduced to the dihydroflavin shown. 

FIGURE 15.4 The structures of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Even in organisms that rely on the nicotinamide coenzymes (NADH and NADPH) for many of their oxidation-reduction cycles, the flavin coenzymes fill essential roles. Flavins are stronger oxidizing agents than NAD and NADP. They can be reduced by both one-electron and two-electron pathways and can be reoxidized easily by molecular oxygen. Enzymes that use flavins to carry out their reactions—flavoenzymes—are involved in many kinds of oxidation-reduction reactions. 

Vitamin B2 Food contains three B2 vitamers, riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, which are the predominant vitamers in foods and are usually bound to proteins. Their analysis usually takes place after extraction with dilute mineral acids with or without enzymatic hydrolysis of the coenzymes (which is necessary to convert all forms to riboflavin and to quantify them as total riboflavin). The extracts may be purified using SPE with Cig cartridges. All the operations performed prior to analysis need to be done under subdued lighting to avoid decomposition of riboflavin upon exposure to light. RP chromatography with Cig columns is used along with fluorescence detection (excitation, 440 nm emission, 520 nm). 

Figure 2.15 Oxidation and reduction of the flavin coenzymes. The reaction may proceed as either a single two-electron reaction or as two single-electron steps by way of intermediate formation of the riboflavin semiquinone radical.

A member of the water-soluble B complex of vitamins. It is a constituent of the flavin coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which participate in biological reduction-oxidation reactions. Riboflavin is usually obtained in the diet from plant sources. Deficiency of the vitamin (ariboflavinosis) can result in a rough scaly skin and oral, anal and vaginal lesions. 

As mentioned earlier, nearly all tissues require riboflavin. The free vitamin is trapped as one of its phosphorylated coenzyme forms, which then become specifically associated (and in a few cases covalently linked) to the protein chains of catalytic flavoenzymes. If not already covalently linked, the flavin coenzyme can often be liberated by extremes of pH or by other nonphysiological maneuvers. In a few biological locations, such as the mature red cell, flavoenzymes such as glutathione reductase (NADPH oxidized glutathione oxidoreductase EC 1.6.4.2) may exist partly in their apoenzyme form, i.e., without the flavin coenzyme and therefore without enzyme activity. An increased supply of riboflavin will permit the depleted coenzyme (in this case FAD) to be synthesized so that enzyme activity can be restored. 

The fused system, pyrimido[4,5-h]pyrazine (219), is more commonly known as pteridine, a ring system which is present in such biologically important molecules as alloxazine, riboflavin, leucopterin, xanthopterin, and the coenzyme, flavin-adenine dinucleotide. 

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