Ascorbate oxidase reoxidation

Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

The reoxidation studies on laccase and ascorbate oxidase are listed in Table IX. The reoxidation of the type-1 copper and of the trinuclear copper site occurs at a rate of 5 x 10 M" sec" both for tree laccase 134) and for ascorbate oxidase 135). During reoxidation with H2O2, an 02 " intermediate is formed in several minutes, which is documented for tree laccase by changes in the CD spectrum 136) and for ascorbate oxidase in the formation of an absorption band at 350 nm... 

In studies on the anaerobic reduction of tree laccase by hydroquinone and ascorbate (49), the existence of a plateau phase at low substrate concentration was reported for the reaction of the type 1 copper. This observation was explained in terms of an intramolecular reoxidation by the type 3 copper pair. A similar plateau phase is a dominant feature of the reduction of both chromophores of ascorbate oxidase by reductate (Figure 7). However, the plateau phase is only observed in the presence of "contaminating dioxygen rigorous removal of these dioxygen traces removes the plateau phase at all wavelengths. The reaction of reduced ascorbate oxidase with dioxygen is very rapid, k = 5 X 10 at pH... 

Finally, it is of interest to compare intramolecular ET in hCp with the corresponding processes in laccase and ascorbate oxidase. As in hCp, pulse radiolytically produced RSSR radicals are also the primary reaction products in tree laccase (73), and the reduction equivalents are further transferred to the TFCu ) center in an intramolecular process. The rate of Tl(Cu ) reoxidation by intramolecular ET to T2/T3 takes place unimolecularly with a rate constant of 2s at room temperature, similar to that observed in hCp (2.9 s ), which is hardly surprising, since the stmctural arrangements of the T1-T2/3 sites in these two proteins are quite similar (the driving forces also are comparable). The situation in ascorbate oxidase, however, was found to be more complex (Section... 

Asard et al. (1992) showed that sealed plasma membrane vesicles (isolated from Phaseolus vulgaris) containing a particular / -type cytochrome with a midpoint reduction potential between +120 and +160 mV were able to transfer electrons to external impermeable electron acceptors, such as ferricyanide and cytochrome c, when loaded with ascorbate. The cytochrome b was reduced by internal ascorbate but not by NADH. Generation of AFR in the external medium using a mixture of ascorbate and ascorbate oxidase resulted in the reoxidation of the 6-cytochrome, suggesting that AFR may be the natural acceptor to cytochrome 6-mediated transmembrane electron transport (Horemans et al., 1994). 

D-glucose and the three-enzyme system GOx, mutarotase and invertase for sucrose estimation. A common format was adopted to facihtate design and operation, in this case immobilization method, the fact that all enzymes used were oxidases and that a common detection principle, reoxidation of H2O2 generated product, was chosen (except for ascorbic acid which was estimated directly). Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilization in the sensors. Interferences related to electrochemi-caUy active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate membrane diffusional barrier or by enzymatic inactivation with ascorbate oxidase. Enzyme sensors demonstrated expected response with respect to their substrates, on analyte average concentration of 5 mM. 

The Wacker process, of course, gives highly selective oxidation of olefins to aldehydes or ketones (42) the function of the 02 is to reoxidize the catalyst, and again any formation of a dioxygen complex is incidental, although such a species could be involved in the reoxidation step. Reoxidation of Cu(I) to Cu(II)/Cu(III) by 02 appears to be involved in certain Cu-containing oxidase systems, for example, ascorbic-acid oxidase (43, 44). 

With the exception of a study carried out with a partially characterized multicopper oxidase isolated from tea leaves (85), there has been very little detailed work concerned with the steady state kinetic behavior of laccases. Early work on the transient kinetics indicated, however, that (1) enzyme bound Cu + was reduced by substrate and reoxidized by O2, and (2) substrate was oxidized in one-electron steps to give an intermediate free radical in the case of the two electron donating substrates such as quinol and ascorbic acid. The evidence obtained suggested that free radicals decayed via a non-enzymatic disproportionation reaction rather than by a further reduction of the enzyme (86—88). In the case of substrates such as ferrocyanide only one electron can be donated to the enzyme from each substrate molecule. It was clear then that the enzjmie was acting to couple the one-electron oxidation of substrate to the four-electron reduction of oxygen via redox cycles involving Cu. 

Ferric iron must be reduced to Fe for absorption by the mucosal cells in the proximal duodenum, the primary site of absorption. The most effective enhancer of iron absorption is ascorbic acid, especially with food of low iron bioavailability where a 10-fold, or greater, enhancement has been observed (8). Inhibitors of iron absorption include tea, soy protein, bran, and other fibers. Gastric and intestinal juices assist in solubilizing and releasing bound Fe in food. Absorbed Fe is reoxidized to Fe by ferroxidase(s) before it is bound by apotransferrin or apoferritin (storage). Ferroxidase activity has been demonstrated with xanthine oxidase and cemloplasmin [11-13]. 

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