Ascorbate oxidase, characterization

Copper oxidases are widely distributed in nature, and enzymes from plants, microbes, and mammals have been characterized (104,105). The blue copper oxidases, which include laccases, ascorbate oxidases, and ceruloplasmin, are of particular interest in alkaloid transformations. The principle differences in specificity of these copper oxidases are due to the protein structures as well as to the distribution and environment of copper(II) ions within the enzymes (106). While an in vivo role in metabolism of alkaloids has not been established for these enzymes, copper oxidases have been used in vitro for various alkaloid transformations. 

Type III copper is characterized by antiferromagnetic coupling of a pair of copper atoms and strong absorbance at 330 nm. A single type III pair is found in hemocyanin, in which it is involved in O2 transport, and in tyrosinase, in which an oxygen is inserted into substrate. A pair of copper atoms is also found in the multi-copper ascorbate oxidase, but it is coupled to the type II copper in a trinuclear arrangement. 

Messerschmidt, A., Steigemann, W., Huber, R., Lang, G., and Kroneck, P. M. H. (1992b). X-ray crystallographic characterization of type 2 depleted ascorbate oxidase from zucchini. Eur. J. Biochem. 209, 597-602. 

Arabidopsis thaliana has three different genes encoding ascorbate oxidases. They display 50-70% sequence identity with one another and only 20-25% identity with the proteins of the LC family. They all have a Met as the axial ligand for blue copper, whereas most plant laccases have Leu or He (see Fig. 9). A multicopper blue oxidase has also been characterized from fungus Acremonium sp. HI-25 and identified as AO because of its... 

The reaction of nitric oxide with laccase (76) and ascorbate oxidase (147) has been studied as well. Nitric oxide fully reduces fungal and tree laccase when it is added to the oxidized enzyme under anaerobic conditions. In addition the binding of one NO molecule to laccase can be detected. This is characterized by a new EPR signal and has been described as coordinated with the type-2 copper (76). Only the reduction of the type-1 copper has been observed when NO has been added to ascorbate oxidase under anaerobic conditions. 

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... 

The first successful observation and characterization of the ascorbate free radical was carried out with ESR (14,15). A 1.7-G ESR doublet was reported and it was correctly concluded that the observed spectrum represented the anionic form (A ) of the radical. These measurements (14,15) showed that the enzyme-generated radical (horseradish peroxidase-hydrogen peroxide-ascorbate) was present as a free radical and decayed by second-order kinetics (see Figure 2). Recent experiments (16,17) have shown that ascorbate oxidase and dopamine-monooxygenase also generate unbound ascorbate radicals. 

This chapter summarizes some recent developments in the purification of ascorbate oxidase, the number of copper atoms per active molecule, and the stoichiometry of the different copper sites with reference to the classification introduced by Malkin and Malmstrom (5). Furthermore, physical properties of the metal centers are discussed in relation to other simple copper proteins that have been characterized in recent years. Finally, kinetic investigations of ascorbate oxidase reduction are presented as studied by anaerobic stopped-fiow and rapid-freeze techniques. 

Faced with the problem of elucidating the individual roles of the diflFerent copper centers in the blue oxidases, the researcher has naturally focused in recent years on the laccases (9). Being easier to isolate, better characterized, and containing fewer copper atoms than cemloplasmin or ascorbate oxidase, the laccases from the Japanese lacquer tree Rhus vernicifera and the fungus Polyporus versicolor have been the subject of several transient kinetic studies in the millisecond range, that is, studies using stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy (9,49,50). 

Cyt c and azurin are structurally and in other respects very well characterized, and in-situ STM can be referred to many other structural, spectral, and kinetic data. No three-dimensional structure of laccase is available, but the structure of the closely related enzyme ascorbate oxidase is available with high resolution and srq ports a view of facile ET through die protein, involving all the copper atoms. 

Laccase, ascorbate oxidase, and ceruloplasmin are the classical members of the multicopper oxidase family also known as blue oxidases. Recently, a small number of bacterial members of this family have been characterized, including CueO from E. coli a spore-coat laccase (CotA) from Bacillus suhtilis and phenoxazinone synthase from Streptomyces antibioticus The catalyzed reaction of these enzymes except for phenoxazinone synthase is given in Equation (11). A comprehensive overview of the broad and active research on blue copper oxidases is presented in Messerschmidt. Recent results have been included in a review on the reduction of dioxygen by copper-containing enzymes. The nature and number of the different copper sites in blue oxidases has been described in the sections about the type-1 copper site and the trinuclear copper cluster. 

The blue multicopper oxidases constitute a heterogeneous family of enzymes from different sources (7). In addition to the well characterized members of this family, ascorbate oxidase (45,46), laccase (47,48), and ceruloplasmin (49,50), all from higher organisms, two other proteins have attracted much recent interest FetSp, which is involved in iron uptake in yeast (51), and CueO, which is required for copper homeostasis in Escherichia coli (52). The characteristic reactivity of these enzymes is the one-electron oxidation of four substrate equivalents coupled to the four-electron reduction of dioxygen to water (1). These processes occur at a catalytic unit constituted by four copper atoms classified according to their spectroscopic properties in... 

Mosery O, KaneUis AK. Ascorbate oxidase of Cucumis melo L. var. reticulatus purification, characterization and antibody production. J Exp Bot 1994 45 717-724. 

A number of enzymes have been characterized that catalyze reactions involving DHA. In addition, other aspects of DHA biochemistry can be deduced from metabolic studies of ascorbic acid. Experiments demonstrating the biological oxidation of AA and reduction of DHA were first made in 1928 (10) and during the next decade several groups studied these reactions. By 1941 Crook (62) was able to separate the ascorbic acid oxidase and DHA reductase activities and to show that glutathione was used in the reductase reaction. 

The best characterized of the enzymes involving DHA is ascorbic acid oxidase (L-ascorbate O2 oxidoreductase, EC 1.10.3.3). This plant enzyme catalyzes the reaction of AA and oxygen to give DHA and 1 mol of water (63). 

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. 

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. 

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