Isoalloxazine ring

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

Figure 11-2. Oxidoreduction of isoalloxazine ring in fiavin nucieotides via a semi-quinone (free radicai) intermediate (center).

Riboflavin is also known as vitamin B2. It contains a complex isoalloxazine ring that humans are unable to synthesize. The complex ring is hooked onto a live-carbon sugar derivative, ribitol, closely related to the ribose that occurs in RNA. The RDA for adult males is 1.3 mg/day and for adult females 1.1 mg/day. Values decrease with increasing age but increase in pregnancy and lactation. Organ meats, milk, bread products, and fortified cereals are substantial sources of riboflavin. 

Riboflavin contains an isoalloxazine ring linked to the reduced sugar ribitol. The sugar unit in riboflavin is the non-cyclic ribitol, so that FAD and FMN differ somewhat from the nucleotides we encounter in nucleic acids. 

This group includes the coenzyme forms of water-soluble vitamin B2 or riboflavin. Synthesis occurs by initial cyclohydrolase action on the guanine ring of GTP and subsequent steps lead to the synthesis of the isoalloxazine ring structure (see structures below). 

Flavin redox states in a dual flavin enzyme. (Left) Single-electron reduction of the isoalloxazine ring generates the semiquinone radical, while reduction by two electrons generates the fully reduced species. (Right) Five possible oxidation levels of a dual flavin enzyme, where the FMN reduction potential is held at a more positive value relative U) FAD. The flavins can theoretically accept a maximum of four electrons obtained from two NADPH. However, in NADPH-cytochrome P450, reductase, full reduction of the flavins is not normally reached when NADPH serves as the reductant. 

Riboflavin (vitamin B2 6.18) consists of an isoalloxazine ring linked to an alcohol derived from ribose. The ribose side chain of riboflavin can be modified by the formation of a phosphoester (forming flavin mononucleotide, FMN, 6.19). FMN can be joined to adenine monophosphate to form flavin adenine dinucleotide (FAD, 6.20). FMN and FAD act as co-enzymes by accepting or donating two hydrogen atoms and thus are involved in redox reactions. Flavoprotein enzymes are involved in many metabolic pathways. Riboflavin is a yellow-green fluorescent compound and, in addition to its role as a vitamin, it is responsible for the colour of milk serum (Chapter 11). 

The three-dimensional structures, or part of it, are also known for Desulfovibrio vulgaris and Anacystis nidulans flavodoxins. These results, including those obtained on C.MP., were recently summarized by Adman . Hence, these results will be discussed only briefly. The x-ray structures show that the isoalloxazine ring is embedded in a hydrophobic pocket of the apoprotein, i.e. flanked by at least one aromatic amino acid residue. During the redox transitions, especially from the oxidized to the semiquinone state, small conformational changes occur and contacts with the isoalloxazine ring are formed or broken. These conformational transitions form probably a kinetic barrier so that the semiquinone state is trapped by the apoprotein and, therefore, rather stable towards oxidation by molecular oxygen. 

Since flavin semiquinones are tricyclic heteronuclear aromatic systems, the spin density due to the unpaired electron can be distributed at a number of sites on the isoalloxazine ring. Knowledge of the location and the extent of spin density at these locations on the ring system is important for the determination of sites that may participate... 

The results of these studies show that little or no spin density occurs in the pyrimidine ring, but rather the unpaired spin distribution is concentrated in the pyrazine and benzenoid portions of the isoalloxazine ring (Table 2). The site of highest spin density is the N(5) position with substantial densities also occuring at C(8) and at N(10). From ESR studies of 4a enriched flavins. Walker et al. 

No major differences in rate were observed with the ionic form (anion or neutral) of the flavin semiquinone. Comparison of the rates of reduction by a number of flavin analogs suggests that cytochrome interaction occurs through the N(5)-dimethylbenzene region of the isoalloxazine ring. 

Finally, the precise sites on the isoalloxazine ring which participate in semiquinone oxidation reactions in flavoenzymes will depend on whether the protein-bound semiquinone exhibits the same distribution of spin density as in the free system or in fact differs. Current ENDOR data suggests that the spin density in the 8-position of protein-bound flavin semiquinones is similar to that of the free systems. Further work is required to monitor spin distribution in the N(5) and... 

The catalytically functional portion of the coenzymes is the isoalloxazine ring, specifically N-5 and C-4a (see fig. 10.8 ), which is thought to be the immediate locus of catalytic function, although the entire chromophoric system extending over N-5, C-4a, C-lOa, N-l, and C-2 should be... 

Riboflavin is a combination of the isoalloxazine ring and ribitol. Conversion of the vitamin to FMN and FAD occurs via phosphorylation and adenylation, respectively, as indicated in Table 6.1. In the case of FAD or FMN, both the electrons and the hydrogen atoms are bound by the isoalloxazine ring structure of the riboflavin portion. The site of this reduction is pointed out in Table 6.1. FAD is often bound very tightly to the enzyme. For both the NAD+ and FAD-type coenzymes, the adenylate portion of the coenzyme is necessary for binding to the enzyme. Table 6.3 also lists some representative reactions in which FAD and FMN are cofactors. 

The molecule consists of a d-ribitol unit attached to an isoalloxazine ring (Figure 9-15). Anything more than a minor change in the molecule results in a loss of vitamin activity. Aqueous solutions of riboflavin are yellow with a yellowish-green fluorescence. The vitamin is a constituent of two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN is... 

Figure 16-8. Structure and labeling of the isoalloxazine ring and related flavins. Isoalloxazine (benzo[g]pteridine-2,4(3H,10H)-dione) R = R = H. FMN (Flavin mononucleotide) R = CH3 R = CH2- (CH0H)3-CH20-P0f-...

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