Monodehydroascorbate radical

By reaction with ascorbate to yield the monodehydroascorbate radical, which in turn can either be reduced to ascorbate or can undergo dis-mutation to yield dehydroascorbate and ascorbate (Section 13.4.7.1). In vitro, the formation of the tocopheroxyl radical can be demonstrated by the appearance of its characteristic absorbance peak, which normally has a decay time of 3 msec in the presence of ascorbate, the tocopheroxyl peak has a decay time of 10 /rsec, and its disappearance is accompanied by the appearance of the monodehydroascorbate peak. There is an integral membrane oxidoreductase that uses ascorbate as the preferred electron donor, linked either directly to reduction of tocopheroxyl radical or via an electron transport chain involving ubiquinone (see no. 4 below May, 1999). 

As shown in Figure 13.3, oxidation of ascorbic acid, for example, by the reduction of superoxide to hydrogen peroxide or Fe + to Fe +, and similar reduction of other transition metal ions, proceeds by a one-electron process, forming the monodehydroascorbate radical. The radical rapidly disproportionates into ascorbate and dehydroascorbate. Most tissues also have both nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione-dependent monodehydroascorbate reductases, which reduce the radical back to ascorbate. Ascorbate is thus an effective quencher of singlet oxygen and other radicals. 

The immediate product of the oxidative reaction, the monodehydroascorbate radical (Eq. [1]), is a fairly reactive and unstable species which, in the presence of a suitable reductase system, is reduced back to ascorbate. Monodehydroascorbate reductases have been identified and purified in a few cases (Ushimara et al., 1997 Dalton et al., 1992 Shigeoka et al., 1987 Borraccino et al., 1986 Hossain et al., 1984) and cDNA sequences have been published for the pea (Murthy and Zilinskas, 1994) and cucumber enzymes (Sano et al., 1995). A bacterial expression system is also available for the cucumber enzyme (Sano et al., 1995). In the absence of a suitably efficient reductase system, the monodehydroascorbate radicals disproportionate to dehydroascorbate and ascorbate in this case, ascorbate is regenerated using a glutathione-dependent dehydroascorbate reductase enzyme (Foyer and Mullineaux, 1998 and references therein). Under non-... 

Nakano, Y., and Asada, K., 1987, Purification of ascorbate peroxidase in spinach chloroplasts its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical, Plant Cell Physiol. 28 131nl40. 

The ready oxidation of ascorbic acid will catalyze chemical changes in a number of other substances. Thus, unsaturated fatty acids in lecithins and tissues are catalytically oxidized in the presence of ascorbic acid to a substance producing color with thiobarbiturate (B21). The product of the ascorbic acid-catalyzed oxidation is malonaldehyde, which can also inhibit L-gulonolactone oxidase, the enzyme forming ascorbic acid (Cl). It has been suggested that this enzyme inhibition may occur in vivo in animals deficient in vitamin E, a compound believed to have antioxidant actions which would prevent the ascorbic acid-catalyzed lipid oxidation from giving rise to malonaldehyde. It is quite probable that the active intermediate in the formation of malonaldehyde is the monodehydroascorbate radical which initiates the lipid oxidation. 

Grace S, Pace R and Wydrzynski T (1995) Formation and decay of monodehydroascorbate radicals in illuminated thylakoids as determined by ERR spectroscopy. Biochim Biophys Acta 1229 155-165... 

Mishra NP, Mishra RK and Singhal GS (1993) Involvement of active oxygen species in photoinhibition of Photosystem II protection of photosynthetic efficiency and inhibition of lipid peroxidation by superoxide dismutase and catalase. J Photo-chem Photobiol B Biol 19 19-24 Miyake C and Asada K (1992) Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiol 33 541-553 Miyake C and Asada K (1994) Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiol 35 539-549 Miyake C, Schreiber U, Hermann H, Sano S and Asada K (1996) Monodehydroascorbate radical reductase-dependent photoreduction of oxygen in chloroplast thylakoids. Plant Cell Physiol 37 s284... 

Kobayashi, K., Harada, Y., and Hayashi, K., 1991, Kinetic behavior of the monodehydroascorbate radical studied by pulse radiolysis. Biochemistry 30 8310-8315. 

It was noted above (section 7.2.3-3) that vitamin C can act at the surface of cells or lipoproteins to reduce the tocopheroxyl radical back to tocopherol, forming the stable monodehydroascorbate radical. Vitamin C can also react with superoxide and hydroxyl radicals ... 

Figure 7.18 Metabolism of the monodehydroascorbate radical to non-radical products.

In the oxidation of ascorbate by Oj catalysed by ascorbate oxidase, the formation of the monodehydroascorbate free radical was demonstrated by EPR spectroscopy in a flow cell. A steady state was usually reached within 50 ms. The production of the free radical was also followed by the reduction of Fe(in)-cytochrome c. Thus the oxidation of ascorbate occurs in a one-electron step The formation of the monodehydroascorbate free radical was also measured directly by spectrophotometry at 360 nm, where the free radical shows an absorption maximum... 

The formation of the monodehydroascorbate free radical by dopamine P-mono-oxygenase was similarly demonstrated by the acceleration of the reduction of Fe(III)-cytochrome c and by direct spectrophotometry at 360 nm The action of the laccase from the mushroom Agaricus hisporum on catechol yielded also the semiquinone as the result of a one-electron transfer... 

Two ascorbate radicals can react with each other in a disproportionation reaction to give ascorbate plus dehydroascorbate. However, most cells can reduce the radicals more directly. In many plants this is accomplished by NADH + H+ using a flavoprotein monodehydroascorbate reductase.0 Animal cells may also utilize NADH or may reduce dehydroascorbate with reduced glutathione.CC/ff Plant cells also contain a very active blue copper ascorbate oxidase (Chapter 16, Section D,5), which catalyzes the opposite reaction, formation of dehydroascorbate. A heme ascorbate oxidase has been purified from a fungus. 11 1 Action of these enzymes initiates an oxidative degradation of ascorbate, perhaps through the pathway of Fig. 20-2. 

Ascorbate can act as a radical-trapping antioxidant, reacting with superoxide and a proton to yield hydrogen peroxide, or with the hydroxy radical to yield water. In each case, the product is monodehydroascorbate. 

The antioxidant activity of ascorbate is variable. From consideration of the chemistry involved, it would be expected that, overall, 2 moles of peroxyl radical would be trapped per mole of ascorbate, because of the reaction of 2 moles of monodehydroascorbate to regenerate ascorbate and yield dehydroascorbate (see Figure 13.3). However, as the concentration of ascorbate increases, so the molar ratio decreases, and it is only at very low concentrations of ascorbate that it tends toward the theoretical 2 1. 

As well as its antioxidant role, ascorbate can be a source of hydroxyl and superoxide radicals. At high concentrations, it can reduce molecular oxygen to superoxide, being oxidized to monodehydroascorbate. At lower concentrations of ascorbate, both Fe + and Cu + ions are reduced by ascorbate, yielding monodehydroascorbate. Fe + and Cu+ are readily reoxidized by reaction with hydrogen peroxide to yield hydroxide ions and hydroxyl radicals. Cu+ also reacts with molecular oxygen to yield superoxide. 

Reduction of the Vitamin E Radical by Ascorbate Asdiscussed in Section 4.3.1, one of the major roles of vitamin E is as a radical-trapping antioxidant in membranes and lipoproteins. a-Tocopherol reacts with lipid peroxides forming the a-tocopheroxyl radical, which reacts with ascorbate in the aqueous phase, regenerating a-tocopherol, and forming monodehydroascorbate. Vitamin C may have a vitamin E-sparing antioxidant action, coupling lipophilic and hydrophilic reactions. 

The oxidation of ascorbic acid in certain reactions has given evidence of an intermediate with the properties of a free radical which could be formed by one-electron oxidation. Thus, the rate-limiting step of ascorbic acid oxidation by Fe + and H2O2 was this one-electron oxidation (G12). Such a radical has now been identified in hydrogen peroxide-ascorbic acid solutions at pH 4.8 by electron paramagnetic resonance spectroscopy. The free radical, commonly referred to as monodehydroascorbic acid, decayed in about 15 minutes at this acid pH. It was also formed during the enzymatic oxidation of ascorbic acid by peroxidase (Yl). The existence of the monodehydroascorbic acid radical makes possible very... 

Other effects of ascorbic acid are probably also dependent upon the monodehydroascorbic free radical. One is the depolymerization of hyaluronic acid, first shown by Robertson et al. (R14). This is particularly likely, since subsequent studies have shown that the depolymerization was not prevented by catalase, as should have occurred if peroxide were the active component. There is no reason to believe that this hyaluroni-dase-like action is physiological. There is every reason to believe, however, that the monodehydroascorbic acid radical is formed in vivo. It is unlikely that all of the reactions in the body of such a reactive molecule are catalyzed by enzymes. 

Staudinger s interpretation of this involves the central problem of such hydroxylation reactions the generation of free radicals from O2 as the possible hydroxylating agents (K4). The hypothesis is that the reaction of TPNH with O2 in Ae mitochondrial 11-P-hydroxylase generates such free radicals, and so does the oxidation of ascorbic acid by the DPNH-oxidase-cytochrome be system (Eq. 15). The monodehydroascorbic acid formed in the latter system is already well documented. 

Pulse radiolysis was also used to elucidate the mechanism of catalytic action of monodehydroascorbate reductase, an enzyme containing FAD and using Nicotinaminde atjenine dinucleotide (NADH) as reductant. The substrate is dehydroascorbate radical produced by pulse radiolysis (130). The authors show that this radical reacts with the protein to give the FADH radical and that the... 

Fig. 4. The mechanism-dependent inactivation of AsA peroxidase by HA, HU, and phenols, and their protection by AsA. DHA dehydroascorbate MDA monodehydroascorbate ROH HU, HA, AP and CR R0 radical of ROH.

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