Coupled oxidation ascorbate reaction

In the presence of ascorbate and oxygen, oxyMb and other heme proteins undergo a series of reactions that resemble the catalytic cycle of HO, albeit with less efficiency 278-281). Although the spectroscopic similarities of Mb and corresponding derivatives of HO are remarkable 264, 267, 272), the mechanism of the coupled oxidation reaction... 

Other reactions of hemoglobin also permit a free radical interpretation, notably the coupled oxidation with ascorbic acid by molecular 02 which yields choleglobin, but further discussion requires a full kinetic analysis. Even though the denaturation reactions described above have not been examined kinetically it is worth emphasizing that their chief features can be explained by the formation of 02- as in the mechanism advanced for the autoxidation. The liberation of an activated O2 molecule is no longer required—02 is the active oxygen. 

Antioxidant capacities of common individual curcuminoids were determined in vitro by phosphomolybdenum and linoleic acid peroxidation methods. Antioxidant capacities expressed as ascorbic acid equivalents (pmol/g) were 3099 for curcumin, 2833 for demethoxycurcumin, and 2677 for bisdemethoxycurcumin at concentrations of 50 ppm. The same order of antioxidant activity (curcumin > demethoxycurcumin > bisdemethoxycurcumin) was observed when compared with BHT (buty-lated hydroxyl toluene) in linoleic peroxidation tests. The antioxidant activity of curcumin in the presence of ethyl linoleate was demonstrated and six reaction products were identified and structurally characterized. The mechanism proposed for this activity consisted of an oxidative coupling reaction at the 3 position of the curcumin with the lipid and a subsequent intramolecular Diels-Alder reaction. ... 

In the normal prolyl 4-hydroxylase reaction (Fig. 4a), one molecule of a-ketoglutarate and one of 02 bind to the enzyme. The a-ketoglutarate is oxidatively decarboxylated to form C02 and succinate. The remaining oxygen atom is then used to hydroxylate an appropriate Pro residue in procollagen. No ascorbate is needed in this reaction. However, prolyl 4-hydroxylase also catalyzes an oxidative decarboxylation of a-ketoglutarate that is not coupled to proline hydroxylation—and this is the reaction that requires ascorbate (Fig. 4b). During this reaction, the heme Fe2+ becomes oxidized, and the oxidized form of the enzyme is inactive—unable to hydroxylate proline. The ascorbate consumed in the reaction presumably functions to reduce the heme iron and restore enzyme activity. 

FIGURE 4 The reactions catalyzed by prolyl 4-hydroxylase, (a) The normal reaction, coupled to proline hydroxylation, which does not require ascorbate. The fate of the two oxygen atoms from 02 is shown in red. (b) The uncoupled reaction, in which a-ketoglutarate is oxidatively decarboxylated without hydroxylation of proline. Ascorbate is consumed stoichiometrically in this process as it is converted to dehydroascorbate. 

In the discussion of the biochemistry of copper in Section 62.1.8 it was noted that three types of copper exist in copper enzymes. These are type 1 ( blue copper centres) type 2 ( normal copper centres) and type 3 (which occur as coupled pairs). All three classes are present in the blue copper oxidases laccase, ascorbate oxidase and ceruloplasmin. Laccase contains four copper ions per molecule, and the other two contain eight copper ions per molecule. In all cases oxidation of substrate is linked to the four-electron reduction of dioxygen to water. Unlike cytochrome oxidase, these are water-soluble enzymes, and so are convenient systems for studying the problems of multielectron redox reactions. The type 3 pair of copper centres constitutes the 02-reducing sites in these enzymes, and provides a two-electron pathway to peroxide, bypassing the formation of superoxide. Laccase also contains one type 1 and one type 2 centre. While ascorbate oxidase contains eight copper ions per molecule, so far ESR and analysis data have led to the identification of type 1 (two), type 2 (two) and type 3 (four) copper centres. 

Spectrophotometric techniques combined with flow injection analysis (FIA) and on-line preconcentration can meet the required detection limits for natural Fe concentrations in aquatic systems (Table 7.2) by also using very specific and sensitive ligands, such as ferrozine [3-(2-bipyridyl)-5,6-bis(4-phenylsulfonic acid)-l,2,4-triazine], that selectively bind Fe(II). Determining Fe(II) as well as the total Fe after on-line reduction of Fe(III) to Fe(II) with ascorbic acid allows a kind of speciation.37 A drawback is that the selective complexing agents can shift the iron redox speciation in the sample. For example, several researchers have reported a tendency for ferrozine to reduce Fe(III) to Fe(II) under certain conditions.76 Most ferrozine methods involve sample acidification, which may also promote reduction of Fe(III) in the sample. Fe(II) is a transient species in most seawater environments and is rapidly oxidized to Fe(III) therefore, unacidified samples are required in order to maintain redox integrity.8 An alternative is to couple FIA with a chemiluminescence reaction.77-78... 

Follow-up reaction of the product of an electrochemical reaction, usually producing a species that is not electroactive at potentials where the redox process of the electroactive couple occurs. The oxidation of ascorbic acid, followed by hydration of the product is one representative example for this type of reactions [i, ii]... 

The active sites of D/iM and PHM contain two catalytically essential copper ions. EPR spectra of the oxidized forms of these proteins are typical for Type 2 copper with N, O donor ligands. " The copper centers are not coupled and are at least 5 A apart.Note the stereochemistry of hydroxylation in equations (4) and (5) both reactions proceed with retention. 0 tracer experiments for the enzymes provided direct evidence for the incorporation of molecular oxygen into the substrate, and both exhibit large deuterium kinetic isotope effects. In both cases, ascorbate is the physiological reductant. These biochemical similarities are confirmed by sequence comparisons, which pinpoint a conserved catalytic domain. The copper ligands of both the oxidized or reduced states of D/3M and PHM are conserved the H-X-L (L = His or Met) motif is a common copperbinding sequence. 

The mnlticopper oxidases couple the one-electron or two-electron oxidation of their substrates to the four-electron rednction of dioxygen to water (36). The reaction with substrate can proceed via an onter-sphere or an inner-sphere mechanism, and as a resnlt, the snbstrate specificity varies substantially among the enzymes. The best-characterized enzymes are laccase, ascorbate oxidase, and cernloplasmin. Radical phenol and amine species formed by laccase and ascorbate oxidase... 

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