The Flavin Coenzymes FAD and Riboflavin Phosphate

The majority of flavoproteins have FAD as the prosthetic group rather than riboflavin phosphate. Some have both flavin coenzymes, and some have other prosthetic groups. 

In solution, the flavin semiquinone radical is highly unstable, undergoing rapid equilibration to a mixture of the oxidized and fully reduced flavins. It is stabilized by protein binding in enzymes. 

The neutral flavin radical has an absorption maximum at 580 nm and hence a blue color it is sometimes referred to as the blue radical. It can undergo either protonation atN-1 to yield a cation radical or deprotonation atN-5 to yield an anion radical, if the enzyme has appropriate proton donating or withdrawing amino acid residues at the catalytic site. Both protonation and deprotonation result in the same spectral shift to give an absorption maximum at 470 nm and hence a red color. Both the blue and red radicals are seen as intermediates in enzyme reactions, suggesting that some enzymes form the neutral radical, whereas others form one of the charged radicals. 

Dihydroflavin can be oxidized by reaction with a substrate, NAD(P)+ or cytochromes in a variety of dehydrogenases, or can react with molecular oxygen in oxygenases and mixed-function oxidases. 

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Why are there four major hydrogen transfer coenzymes, NAD+, NADP+, FAD, and riboflavin phosphate (FMN), instead of just one Part of the answer is that the reduced pyridine nucleotides NADPH and NADH are more powerful reducing agents than are reduced flavins (Table 6-7). Conversely, flavin coenzymes are more powerful oxidizing agents than are... 

Hyperthyroidism is not associated with elevated tissue concentrations of flavin coenzymes, despite increased activity of flavokinase. Again, this demonstrates the importance of the enzyme binding of flavin coenzymes and the rapid hydrolysis of unbound FAD and riboflavin phosphate in the regulation of tissue concentrations of the vitamin. 

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. 

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.

FMN, also known as riboflavin phosphate, is a flavin containing electron carrier in the cell (Figure 14.7). It participates in oxidation/ reduction reactions and, like FAD, differs from the nicotinamide coenzymes (NAD+ and NADP ) in being able to accept electrons either singly or in pairs (Figure 14.8). NAD+ and NADP+ can only accept electrons in pairs. 

So what does riboflavin do As such riboflavin does nothing. Like thiamine, riboflavin must undergo metabolic change to become effective as a coenzyme. It fact, it undergoes two reactions. The first converts riboflavin to riboflavin-5-phosphate (commonly known as flavin adenine mononucleotide, FMN), about which we will say no more, and the second converts it to flavin adenine dinucleotide, FAD. The flavins are a class of redox agents of very general importance in biochemistry. FAD is the oxidized form and FADH2 is the reduced form. ... 

Control over tissue concentrations of riboflavin coenzymes seems to be largely by control of the activity of flavokinase, and the synthesis and catabolism of flavin-dependent enzymes. Almost all the vitamin in tissues is enzyme bound, and free riboflavin phosphate and FAD are rapidly hydrolyzed to riboflavin. If this is not rephosphorylated, it rapidly diffuses out of tissues and is excreted. 

In higher mammals, riboflavin is absorbed readily from the intestines and distributed to all tis.sues. It is the precursor in the biosynthesis of the cocnzyme.s flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The metabolic functions of this vitamin involve these Iwocoenzymes. which participate in numerous vital oxidation-reduction proces.ses. FMN (riboflavin 5 -phosphate) is produced from the vitamin and ATP by flavokinasc catalysis. This step con be inhibited by phcnothiazincs and the tricyclic antidepressants. FAD originates from an FMN and ATP reaction that involves reversible dinucicotide formation catalyzed by flavin nucleotide pyrophosphorylase. The.se coenzymes function in combination with several enzymes as coenzyme-en-zyme complexes, often characterized as, flavoproteins. 

Fig. 19.10. Reduction of FAD. FAD accepts two electrons as two hydrogen atoms and is reduced. The reduced coenzyme is denoted in this text as FAD(2H) because it often accepts a total of two electrons one at a time, never going to the fully reduced form, FADH2. FMN (flavin mononucleotide) consists of riboflavin with one phosphate group attached.

Ann O Rexia has been malnourished for some time, and has developed subclinical deficiencies of many vitamins, including riboflavin. The coenzymes FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) are synthesized from the vitamin riboflavin. Riboflavin is actively transported into cells, where the enzyme flavokinase adds a phosphate to form FMN. FAD synthetase then adds AMP to form FAD. FAD is the major coenzyme in tissues and is generally found tightly bound to proteins, with about 10% being covalently bound. Its turnover in the body is very slow, and people can live for long periods on low intakes without displaying any signs of a riboflavin deficiency. 

Flavin coenzymes are very susceptible to enzymatic or chemical hydrolysis. FAD is hydrolysed in acidic solutions to FMN. In acidic solutions, the phosphate group of FMN migrates from the C-5 position to C -4, C -3 and C -2 positions and subsequent hydrolysis of phosphates yields free riboflavin. 

Subsequently, the functions of the vitamin were better established and requirements for the vitamin were set. Riboflavin is an Integral part of two coenzymes, flavin-5 -phosphate (FMN) and flavin adenine dinucleotide (FAD), which function in oxidation/reductlon reactions. Indeed, riboflavin is an enzyme cofactor which is necessary in metabolic processes in which oxidation of glucose or fatty acid is used for production of adenosine triphosphate (ATP) as well as in reactions in which oxidation of amino acids is accomplished. The minimum requirement for riboflavin has been established as that amount which actually prevents the signs of deficiency. A range of intakes varying from 0.55 to 0.75 mg/day of riboflavin has been established as the minimum amount which is required to prevent appearance of deficiency signs. 

As shown in Figure 7.1, riboflavin consists of a tricyclic dimethyl-isoalloxazine ring conjugated to the sugar alcohol ribitol. The metabolically active coenzymes are riboflavin 5 -phosphate and flavin adenine dinucleotide (FAD). In some enzymes the prosthetic group is riboflavin, bound covalently at the catalytic site. 

It is still unknown how the pyrimidine intermediate 5 is dephosphorylated (reaction VI). However, it is well established that the dephosphorylation product 6 is condensed with 3,4-dihydroxy-2-butanone 4-phosphate (8) by the catalytic action of lumazine synthase (reaction VIII). The carbohydrate substrate 8 is in turn obtained from ribulose phosphate (7) by a complex reaction sequence that is catalyzed by a single enzyme, 3,4-dihydroxy-2-butanone 4-phosphate synthase (reaction VII). As mentioned above, the lumazine 9 is converted to riboflavin (10) by the catalytic action of riboflavin synthase (reaction IX). Ultimately, riboflavin is converted to the coenzymes, riboflavin 5 -phosphate (flavin mononucleotide (FMN), 11) and flavin adenine dinucleotide (FAD, 12) by the catalytic action of riboflavin kinase (reaction X) and FAD synthase (reaction XI). These reaction steps are required in all organisms, irrespective of their acquisition of riboflavin from nutritional sources or by endogenous biosynthesis. 

Riboflavin is an important constituent of the flavoproteins.The prosthetic group of these compound proteins contains riboflavin in the form of the phosphate (flavin mononucleotide, FMN) or in a more complex form as flavin adenine dinucleotide (FAD). There are several flavoproteins that function in the animal body they are all concerned with chemical reactions involving the transport of hydrogen. Further details of the importance of flavoproteins in carbohydrate and amino acid metabolism are discussed in Chapter 9. Flavin adenine dinucleotide plays a role in the oxidative phosphorylation system (see Fig. 9.2 on p. 196) and forms the prosthetic group of the enzyme succinic dehydrogenase, which converts succinic acid to fumaric acid in the citric acid cycle. It is also the coenzyme for acyl-CoA dehydrogenase. 

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