Flavin coenzymes, complex

Pantothenic acid, sometimes called vitamin B3, is a vitamin that makes up one part of a complex coenzyme called coenzyme A (CoA) (Figure 18.23). Pantothenic acid is also a constituent of acyl carrier proteins. Coenzyme A consists of 3, 5 -adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage. Phosphopantetheine in turn consists of three parts /3-mercaptoethylamine linked to /3-alanine, which makes an amide bond with a branched-chain dihydroxy acid. As was the case for the nicotinamide and flavin coenzymes, the adenine nucleotide moiety of CoA acts as a recognition site, increasing the affinity and specificity of CoA binding to its enzymes. 

The other classes of flavoproteins in table 10.2 interact with molecular oxygen either as the electron-acceptor substrates in redox reactions catalyzed by oxidases or as the substrate sources of oxygen atoms for oxygenases. Molecular oxygen also serves as an electron acceptor and source of oxygen for metalloflavoproteins and dioxygenases, which are not listed in the table. These enzymes catalyze more complex reactions, involving catalytic redox components, such as metal ions and metal-sulfur clusters in addition to flavin coenzymes. 

Mieloszyk and colleagues [226] determined that flavins which form charge-transfer complexes with proteins exist in both the ground and excited electronic states. In the flavin-riboflavin-binding protein the trytophan is considered to be the donor. Other such complexes of flavin coenzymes and apoenzymes are known [227-229]. 

Several coenzymes are involved in the biosynthesis of their own precursors. Thus, thiamine is the cofactor of the enzyme that converts 1-deoxy-D-xylulose 5-phosphate (43) (the precursor of thiamine pyrophosphate, pyridoxal 5 -phosphate and of iso-prenoids via the nomnevalonate pathway) into 2 C-methyl-D-erythritol 4-phosphate (90, Fig. 11). Similarly, two enzymes required for the biosynthesis of GTP, which is the precursor of tetrahydrofolate, require tetrahydrofolate derivatives as cofactors (Fig. 3). When a given coenzyme is involved in its own biosynthesis, we are faced with a hen and egg problem, namely how the biosynthesis could have evolved in the absence of the cmcially required final product. The answers to that question must remain speculative. The final product may have been formed via an alternative biosynthetic pathway that has been abandoned in later phases of evolution or that may persist in certain organisms but remains to be discovered. Alternatively, the coenzyme under study may have been accessible by a prebiotic sequence of spontaneous reactions. An interesting example in this respect is the biosynthesis of flavin coenzymes, in which several reaction steps can proceed without enzyme catalysis despite their mechanistic complexity. 

A final distinction from nicotinamides is that the flavin coenzymes generally form tight non-dissociable non-covalent complexes with the apoenzyme. Nicotinamides are released at the end of each catalytic cycle and so are consumed as substrate as part of the redox stoichiometry. Because flavins are tightly bound to the apoprotein (/irD= 10 -10 " M) the coenzyme must be oxidised/reduced at the end each turnover before the enzyme complex again becomes catalytically active. Differential binding of flavin and dihydroflavin is responsible for the wide range of redox potentials for flavoproteins so that oxidation or reduction can be thermodynamically favourable. For example, D-amino acid oxidase binds FAD with a dissociation constant of 10 M but FADHj with one of 10 M which changes the reduction potential from —200 for the FAD/FADHj couple free in solution to 0 mV when bound to the enzyme. 

Complex I The hrst complex, NADH-CoQ oxidoreductase, catalyzes the first steps of electron transport, namely the transfer of electrons from NADH to coenzyme Q (CoQ). This complex is an integral part of the inner mitochondrial membrane and includes, among other suhunits, several proteins that contain an iron-sulfur cluster and the flavoprotein that oxidizes NADH. (The total number of subunits is more than 20. This complex is a subject of active research, which has proven to he a challenging task because of its complexity. It is particularly difficult to generalize about the nature of the iron—sulfur clusters because they vary from species to species.) The flavoprotein has a flavin coenzyme, called flavin mononucleotide, or FMN, which differs from FAD in not having an adenine nucleotide (Figure 20.4). 

Dithiol-disulphide redox roles in primitive systems would have favoured the development of dicysteinyl peptides, restraining two thiols in close proximity. Thioredoxin type molecules would have evolved from these small prototype dithiol peptides. Similar centres would also have developed as parts of more complex enzymes. The binding of a flavin coenzyme near a dithiol centre could eventually have produced the combined disulphide-flavoprotein centre with its special redox properties. 

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. 

Riboflavin, also called vitamin B2, is stmcturally composed of an isoafloxazine ring with a ribityl side chain at the nitrogen at position 10. This vitamin functions metabol-icafly as the essoitial component of two flavin coenzymes, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), complexed with proteins, which act as intmnediaries in transfers of electrons in biological oxidation-reduction reactions. Both FAD and FMN function as coenzymes for flavoproteins of flavoenzymes. Flavoproteins are essoitial for the metabolism of carbohydrates, amino acids, and lipids and for pyridoxine and folate conversion to their respective coenzyme forms. 

As its name implies, this complex transfers a pair of electrons from NADH to coenzyme Q a small, hydrophobic, yellow compound. Another common name for this enzyme complex is NADH dehydrogenase. The complex (with an estimated mass of 850 kD) involves more than 30 polypeptide chains, one molecule of flavin mononucleotide (FMN), and as many as seven Fe-S clusters, together containing a total of 20 to 26 iron atoms (Table 21.2). By virtue of its dependence on FMN, NADH-UQ reductase is a jlavoprotein. 

We next focus on the use of fixed-site cofactors and coenzymes. We note that much of this coenzyme chemistry is now linked to very local two-electron chemistry (H, CH3", CH3CO-, -NH2,0 transfer) in enzymes. Additionally, one-electron changes of coenzymes, quinones, flavins and metal ions especially in membranes are used very much in very fast intermediates of twice the one-electron switches over considerable electron transfer distances. At certain points, the chains of catalysis revert to a two-electron reaction (see Figure 5.2), and the whole complex linkage of diffusion and carriers is part of energy transduction (see also proton transfer and Williams in Further Reading). There is a variety of additional coenzymes which are fixed and which we believe came later in evolution, and there are the very important metal ion cofactors which are separately considered below. 

All of the complexes in the respiratory chain are made up of numerous polypeptides and contain a series of different protein bound redox coenzymes (see pp. 104, 106). These include flavins (FMN or FAD in complexes I and II), iron-sulfur clusters (in I, II, and III), and heme groups (in II, III, and IV). Of the more than 80 polypeptides in the respiratory chain, only 13 are coded by the mitochondrial genome (see p. 210). The remainder are encoded by nuclear genes, and have to be imported into the mitochondria after being synthesized in the cytoplasm (see... 

Fig. 1. Energy metabolism in the normal myocardium (ATP adenosine-5 -triphosphate, ADP adenosine-5 -diphosphate, P phosphate, PDH pyruvate dehydrogenase complex, acetyl-CoA acetyl-coenzyme A, NADH and NAD" nicotinamide adenine dinucleotide (reduced and oxidized), FADH2 and FAD flavin adenine dinucleotide (reduced and oxidized).

The catalytic effect of metal ions such as Mg2+ and Zn2+ on the reduction of carbonyl compounds has extensively been studied in connection with the involvement of metal ions in the oxidation-reduction reactions of nicotinamide coenzymes [144-149]. Acceleration effects of Mg2+ on hydride transfer from NADH model compounds to carbonyl compounds have been shown to be ascribed to the catalysis on the initial electron transfer process, which is the rate-determining step of the overall hydride transfer reactions [16,87,149]. The Mg2+ ion has also been shown to accelerate electron transfer from cis-dialkylcobalt(III) complexes to p-ben-zoquinone derivatives [150,151]. In this context, a remarkable catalytic effect of Mg2+ was also found on photoinduced electron transfer reactions from various electron donors to flavin analogs in 1984 [152], The Mg2+ (or Zn2+) ion forms complexes with a flavin analog la and 5-deazaflavins 2a-c with a 1 1 stoichiometry in dry MeCN at 298 K [153] ... 

Whereas many coenzymes form noncovalent complexes with their respective apoenzymes, various flavoenzymes are characterized by covalently bound FMN (25) or FAD (Fig. 3). Covalent linkage involves the position 8a methyl group or the benzenoid carbon atom 6 of the flavin and a cysteine or histidine residue of the protein. The covalent CN or CS bond can be formed by autoxidation of the noncovalent apoen-zyme/coenzyme precursor complex as shown in detail for nicotine oxidase (59). 

The redox states of the flavin cofactor in a purified flavoenzyme can be conveniently studied by optical spectroscopy (see also Elavoprotein Protocols article). Oxidized (yellow) flavin has characteristic absorption maxima around 375 and 450 nm (Fig. lb and Ic). The anionic (red) and neutral (blue) semiquinone show typical absorption maxima around 370 nm and 580 nm, respectively (Fig. lb and Ic). During two-electron reduction to the (anionic) hydroquinone state, the flavin turns pale, and the absorption at 450 nm almost completely disappears (Fig. lb and Ic). The optical properties of the flavin can be influenced through the binding of ligands (substrates, coenzymes, inhibitors) or the interaction with certain amino acid residues. In many cases, these interactions result in so-called charge-transfer complexes that give the protein a peculiar color. 

The deprotonation and addition of a base to thiazolium salts are combined to produce an acyl carbanion equivalent (an active aldehyde) [363, 364], which is known to play an essential role in catalysis of the thiamine diphosphate (ThDP) coenzyme [365, 366]. The active aldehyde in ThDP dependent enzymes has the ability to mediate an efScient electron transfer to various physiological electron acceptors, such as lipoamide in pyruvate dehydrogenase multienzyme complex [367], flavin adenine dinucleotide (FAD) in pyruvate oxidase [368] and Fc4S4 cluster in pyruvate ferredoxin oxidoreductase [369]. 

Flavin mononucleotide was first isolated from the yellow enzyme in yeast by Warburg and Christian in 1932 (4). The yellow enzyme was spht into the protein and the yellow prosthetic group (coenzyme) 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 [6184-17-4] with a water solubility of more than 200 rimes that of riboflavin. It has wide appHcation in multivitamin and B-complex solutions, where it does not require the solubilizers needed for riboflavin. 

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. 

---