Flavin-containing nucleotides

Conversion of dihydroorotate to orotate is catalyzed by dihydroorotate dehydrogenase, a metalloflavoprotein that contains nonheme-iron atoms and flavin adenine nucleotides (FMN and FAD). In this reaction, the electrons are probably transported via iron atoms and flavin nucleotides that are reoxidized by NAD+. 

Low et al. (2004) have proposed a model to explain thioacetamide-induced hepatotox-icity and cirrhosis in rat livers. The pathways of thioacetamide-induced liver fibrosis were found to be initiated by thioacetamide S-oxide derived from the biotransformation of thioacetamide by the microsomal flavin-adenine nucleotide containing monooxygenase and cytochrome P450 systems and involve oxidative stress and depletion of succinyl-CoA, thus affecting heme and iron metabolism. Karabay et al. (2005) observed such hepatic damage in rats with elevation of total nitrite level in livers and decrease in arginase activity. The authors have reported that nitrosative stress was essentially the critical factor in thioacetamide-induced hepatic failure in rats. 

Both NADPH cytochrome P450 reductase (P450 reductase) and NAD(P)H-quinone oxidoreductase (NQO) are flavin adenine nucleotide-containing enzymes that catalyze the reduction of quinones and quinone-like structures. However, P450 reductase is a microsomal enzyme that catalyzes a one-electron reduction to yield semiquinone radieals that can redox cycle to produce superoxide anion radicals, whereas NQO is a cytosolic enzyme that catalyzes a two-electron reduction to yield hydroquinones (Matsunaga et al., 2006). P450 reductase also can catalyze the one-electron reduction of nitroaromatics to the nitro anion radical, which can redox cycle ... 

An NADH diaphorase has been purified from human erythrocytes. The enzyme activity can be determined in a system containing 2.6 dichlorophenol-indo-phenol, Tris HCl buffer (pH 7.55), EDTA, and the enzyme. The reduction of 2.6 dichlorophenol-indo-phenol is followed spectrophotometrically. The purified enzyme contains one mole of a flavin adenine nucleotide (probably FAD) per 195,000 g of protein. The enzyme functions with either NADPH or NADH as hydrogen donor, but the affinity for NADH is almost 10 times greater than that for NADPH. 

Flavin adenine dinucleotide (FAD) has been electropolymerized using cyclic voltammetry. Cyclic voltammograms of poly (FAD) modified electrode were demonstrated dramatic anodic current increasing when the electrolyte solution contained NADH compare with the absence of pyridine nucleotide. 

Several classes of vitamins are related to, or are precursors of, coenzymes that contain adenine nucleotides as part of their structure. These coenzymes include the flavin dinucleotides, the pyridine dinucleotides, and coenzyme A. The adenine nucleotide portion of these coenzymes does not participate actively in the reactions of these coenzymes rather, it enables the proper enzymes to recognize the coenzyme. Specifically, the adenine nucleotide greatly increases both the affinity and the speeifieity of the coenzyme for its site on the enzyme, owing to its numerous sites for hydrogen bonding, and also the hydrophobic and ionic bonding possibilities it brings to the coenzyme structure. 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

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. 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

Muscle tissue contains two glycerol-3-phosphate dehydrogenases a cytosolic enzyme, which uses NADH, and a flavin-nucleotide-dependent mitochondrial enzyme. What is the metabolic significance of these two enzymes ... 

The oxidizing agents here are related to FAD. We said little about FADH2 as a reducing agent earlier in this chapter because it is rather similar to NADH which we have discussed in detail. FAD is another dinucleotide and it contains an AMP unit linked through the 5 position by a pyrophosphate group to another nucleotide. The difference is that the other nucleotide is flavin mononucleotide. Here is the complete structure. 

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