Blue copper oxidases, ascorbate oxidase

Multicopper Oxidases (Blue Copper Oxidases) Ascorbate Oxidase, Ceruloplasmin, and Laccase. The multicopper oxidases (MCOs) are important enzymes, which are found in many plants (lignin formation), fungi (lignin degradation and detoxification), bacteria, as well as humans (ferroxidase activity) (13). MCOs catalyze the four-electron reduction of O2 to two waters with the electrons coming firom one-electron oxidation of four substrate molecules. The latter are organic reductants for ascorbate oxidase (AO) (32) and laccase (Lc) (130), and a metal ion (ferrous ion) for ceruloplasmin (Cp) (33) (Scheme 9). 

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... 

Ceruloplasmin is a member of the family of blue copper oxidases which also contains laccase and ascorbate oxidase. The relationship... 

Laccases (p-diphenol O2 oxidoreductase EC 1.10.3.2) catalyze the oxidation of p-diphenols with the concurrent reduction of dioxygen to water. However, the actual substrate specificities of laccases are often quite broad and vary with the source of the enzyme [116,117]. Laccases are members of the blue copper oxidase enzyme family. Members of this family have four cupric (Cu +) ions where each of the known magnetic species (type 1, type 2, and type 3) is associated with a single polypeptide chain. In the blue copper oxidases the Cu + domain is highly conserved and, for some time, the crystallographic structure of ascorbate oxidase, another member of this class of enzymes, has provided a good model for the structure of the laccase active site [124,125]. The crystal structure of the Type-2 Cu depleted laccase from Coprinus cinereus at 2.2. A resolution has also been elucidated [126]. 

The blue copper oxidases are similar to cytochrome oxidase in their ability to catalyze reduction of Oj to HjO. Catalysis is centered upon the protein-bound copper ions that can be differentiated into three classes according to their physical, chemical, and functional properties. They are designated Types 1, 2, and 3 copper . In the blue copper proteins (tree and fungal laccases, ceruloplasmin, ascorbate oxidase) these three classes of copper appear in varying amounts the laccases contain the minimum amounts of each (one each of Types 1 and 2 and two Type 3 coppers). 

Copper oxidases Blue oxidases (multicopper oxidases) Laccase Ascorbate oxidase Ceruloplasmin... 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

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. 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

Each of these proteins is blue and appears to have a minimum of four copper atoms per molecule one type 1, one type 11, and two type III. Laccase is not known to be multimeric, nor is ceruloplasmin, but ascorbate oxidase is apparently a dimer. 

So-called blue multinuclear copper oxidase enzymes, such as laccase and ascorbate oxidase, catalyze the stepwise oxidation of organic substrates (most likely in successive one-electron steps) in tandem with the four-electron reduction of O2 to water, i.e. no oxygen atom(s) from O2 are incorporated into the substrate (Eq. 4) [15]. Catechol oxidase, containing a type 3 center, mediates a two-electron substrate oxidation (o-diphenols to o-chinones), and turnover of two substrate molecules is coupled to the reduction of O2 to water [34,35]. The non-blue copper oxidases, e.g. galactose oxidase and amine oxidases [27,56-59], perform similar oxidation catalysis at a mononuclear type 2 Cu site, but H2O2 is produced from O2 instead of H2O, in a two-electron reduction. 

The blue oxidases contain these three types of copper together The stoichiometry is straightforward with laccase which contains one type-1 and one type-2 copper, and one type-3 dimeric copper site . One would expect two laccase-like sites in ascorbate oxidase and in ceruloplasmin, but the presence of respectively 3 and 1 and 1 and 3 type-1 and type-2 copper atoms has been deduced. Ceruloplasmin shows oxidase activities towards different substrates, like Fe (ferroxidase) and aromatic amines. It plays, moreover, an active role in the transport of copper With the proper precautions against the action of proteinases it can be isolated as a single polypeptide chain... 

In addition to its previously mentioned role in copper transport, ceruloplasmin is an amine oxidase, a superoxide dismutase, and a ferrooxidase able to catalyze the oxidation of Fe2+ to Fe3+. Ceruloplasmin contains three consecutive homologous 350-residue sequences which may have originated from an ancestral copper oxidase gene. Like ascorbate oxidase, this blue protein contains copper of the three different types. Blood clotting factors V and VIII (Fig. 12-17), and the iron uptake protein Fet3 (Section A,l) are also closely related. 

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. 

Copper has an essential role in a number of enzymes, notably those involved in the catalysis of electron transfer and in the transport of dioxygen and the catalysis of its reactions. The latter topic is discussed in Section 62.1.12. Hemocyanin, the copper-containing dioxygen carrier, is considered in Section 62.1.12.3.8, while the important role of copper in oxidases is exemplified in cytochrome oxidase, the terminal member of the mitochondrial electron-transfer chain (62.1.12.4), the multicopper blue oxidases such as laccase, ascorbate oxidase and ceruloplasmin (62.1.12.6) and the non-blue oxidases (62.12.7). Copper is also involved in the Cu/Zn-superoxide dismutases (62.1.12.8.1) and a number of hydroxylases, such as tyrosinase (62.1.12.11.2) and dopamine-jS-hydroxylase (62.1.12.11.3). Tyrosinase and hemocyanin have similar binuclear copper centres. 

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. 

Some proteins contain more than one copper site, and are therefore among the most complicated and least understood of all. The active site known as type 4 is usually composed of a type 2 and a type 3 active site, together forming a trinuclear cluster. In some cases, such proteins also contain at least one type 1 site and are in this case termed multicopper oxidases, or blue oxidases [3], Representatives of this class are laccase (polyphenol oxidase) [7-9], ascorbate oxidase (Figure 5.Id) [10], and ceruloplasmin [11], which catalyze a range of organic oxidation reactions. 

Copper proteins are involved in a variety of biological functions, including electron transport, copper storage and many oxidase activities. A variety of reviews on this topic are available (Sykes, 1985 Chapman, 1991). Several copper proteins are easily identified by their beautiful blue colour and have been labelled blue copper proteins. The blue copper proteins can be divided into two classes, the oxidases (laccase, ascorbate oxidase, ceruloplasmin) and the electron carriers (plastocyanin, stellacyanin, umecyanin, etc.). 

Based on spectroscopic properties, mainly electron paramagnetic resonance (EPR), the active sites of copper proteins have been classified into three groups, types I, II, and III. This nomenclature was originally applied to blue oxidases to distinguish the four copper ions contained in these proteins. The original classification has been extended to the copper sites of other proteins. The recent increase in structural information on the copper sites in proteins has, however, revealed greater diversity in the type of copper site. For instance, the type III and type II sites in ascorbate oxidase are in close proximity, forming a trinuclear site, in which all three copper ions are essential for the reactivity. Some proteins, once believed to contain a copper site with normal spectroscopic properties, and thus referred as type II, have been shown to contain copper coordinated by an unusual side chain. Therefore, in this review, new nomenclature is used to classify the copper sites more precisely with respect to their structural features and spectroscopic properties. The definitions are as follows ... 

The multicopper oxidases (laccase, ascorbate oxidase, and ceruloplasmin) catalyze a four-electron reduction of dioxygen to water (285-287). Consistent with the four-electron stoichiometry, the enzymes contain four copper ions. One of the copper ions is type I, causing an intensely blue color of the proteins, thus the enzymes of this family are referred to as blue oxidases. They also contain a monomeric copper site that exhibits normal spectroscopic features, whereas the other two copper... 

Figure 8. Proposed electron transfer pathway in blue copper proteins. The plastocyanin wave function contours have been superimposed on the blue copper (type 1) site in ascorbate oxidase (40). The contour shows the substantial electron delocalization onto the cysteine Spir orbital that activates electron transfer to the trinuclear copper cluster at 12.5 A from the blue copper site. This low-energy, intense Cys Sp - Cu charge-transfer transition provides an effective hole superexchange mechanism for rapid long-range electron transfer between these sites (2, 3, 28).

The second class consists of multidomain blue copper proteins composed of exclusively two or more BCB domains and includes nitrite reductase (Section IV, E), multicopper blue oxidases such as laccase, ascorbate oxidase, ceruloplasmin, and hephaestin (Section VII), and some sequences found in extreme halophilic archaea (see Section V, E). 

---