Ascorbate oxidase redox potentials

Visible MCD spectra of plastocyanin, azurin, Rhus vernicifera laccase, ascorbate oxidase and ceruloplasmin are similar on a per copper basis, but show differences from those of stellacyanin and fungal laccase. This is of interest in view of the absence of methionine from the coordination sphere of copper in stellacyanin, and the very high redox potential of fungal laccase.925... 

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

The driving force, calculated from the difference in the redox potentials ( + 344 mV for the type-1 copper in ascorbate oxidase (see Table VII) +295 mV for the couple ascorbate/ascorbate-free radical (176)) is 49 mV. In the proposed modeled encounter complex (74), there is a short distance of about 7 A between the two redox centers (distance GUI—01 ASC = 6.8 A distance GUI—02 ASG = 7.5 A) and an effective parallel arrangement of the rings, with good overlap of the TT-electron density systems facilitating a rapid electron transfer (see Fig. 15). 

Anaerobic Reduction of Ascorbate Oxidase—Number of Redox Equivalents and Midpoint Potentials of the Copper Sites... 

Blue (Type 1) copper proteins are found widely in nature. Typical examples are plasiocyaiiin (MW 10,500) and ascorbale oxidase (MW 150,000) which contain one and eight Cu atoms per protein, respectively. The former serves as a component of the electron transfer chain in plant photosynthesis while the latter is an enzyme involved in the oxidation of ascorbic acid. The oxidized form is characterized by intense blue color due to electronic absorption near 600 nm. In addition, blue copper proteins exhibit unusual properties such as extremely small hyperfine splitting constants (0.003 0.009 cm" ) in ESR spectra and rather high redox potential (4-0.2 0.8 V) compared to the Cu(ll)/Cu(I) couple in aqueous solution. 

The reaction scheme used in the first commercial electrochemical test strip from MediSense (now Abbott Diabetes Care) is shown later. Electron transfer rates between the reduced form of glucose oxidase and ferricinium ion derivatives are very rapid compared with the unwanted side reaction with oxygen (Cass et al. 1984 Forrow et al. 2002). The Abbott Diabetes Care Precision QID strip includes the l,l -dimethyl-3-(2-amino-l-hydroxyethyl) ferrocene mediator, which has the desirable characteristics of high solubility in water, fast electron-shuttling (bimolecular rate constant of 4.3 X 10 M s ), stability, and pH independence of the redox potential (Heller and Feldman 2008). Electrochemical oxidation of the ferrocene derivative is performed at 0.6 V. Oxidation of interferences, such as ascorbic acid and acetaminophen present in blood, are corrected for by measuring the current at a second electrode on the strip that does not contain glucose oxidase. 

Blue multicopper oxidases (BMCOs) such as laccase, ceruloplasmin, bilirubin oxidase (BOx), and ascorbate oxidase (AOx) have been extensively investigated as cathodic biocatalysts for DET-based biodevices [44]. BMCOs have a catalytic center consisting of four coppers a type 1 (Tl) Cu site, which accepts electrons from the substrate and from the electrode surface, and a type 2/type 3 (T2/T3) cluster, where O2 is reduced directly to water. High redox potential laccases and BOx, with redox potential up to 780 and 670 mV versus normal hydrogen electrode (NHE), respectively [44,45], can be used to create efficient biocathodes with current densities up to a few mA cm . In 2012, Shleev and coworkers used the DET ability of these enzymes to create several completely DET-based BFCs [42]. The enzymes have also been used in different MET-based approaches [46,47] specifically, Heller and coworkers... 

Ascorbate Oxidase. The enzyme catalyzes the oxidation of ascorbate to dihydroascorbate and water by O2 and contains three types of copper complexes mononuclear Cu (I) and Cu II) and binuclear [Cu ]2 (HI). The redox potential of the primary electron acceptor of Cu l) Eo = 500 mV, which is quite sufficient for the one electron oxidation of ascorbate (Eo = 310 mV) but is not enough for the one-electron reduction of O2 (Eo = -330 mV). The most probable mechanism of reaction involves two consecutive one-electron reduction of the copper complexes followed by the two-electron reduction of O2 in the binuclear [Cu+(III)]2 complex accompanied by synchronuos proton transfer. 

Many hypotheses have been advanced for these differences in oxidative susceptibility, including the oxidation potential of milks, and the action of xanthine oxidase and lactoperoxidase, which is controversial. However, there are no substrates for these enzymes in milk. The action of various metallo proteins in milk may be confused as enzymes. These metallo proteins act as powerful lipid oxidation catalysts in the presence of oxygen and redox systems involving ascorbic acid. 

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