Blue oxidases

Blue copper electron transfer proteins, 6,712-717 Blue copper oxidases, 6,699 Blue copper proteins, 2, 557 6, 649 Blue electron transfer proteins, 6,649,652 spectroscopy, 6, 651 Blue oxidases copper, 6,654,655 Blueprint process, 6,124 Blue proteins model studies, 6,653 Boleite... 

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

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. 

Several site-directed mutations were carried out with a new member of the blue oxidases , the Fet3p protein from Saccharomyces cerevisiae which is involved in iron trafficking. The protein oxidizes Fe2+ to Fe3+ as first step in the iron uptake chain. The mutations were targeted to block this ferroxidase activity and thus to identify the residues involved in iron binding. The respective mutants showed no perturbed type 1 sites.91... 

Ceruloplasmin is involved in copper storage and transport as well as in iron mobilisation and oxidation. Among the blue oxidases it is unique since it contains, in addition to the usual motif of a type 1 combined with the trinuclear cluster, two other type 1 coppers. Electron transfer occurs, however, only between five of the six copper ions since one of the type 1 centres is not catalytically relevant due to its too high redox potential. The redox potentials of the centres were determined and possible electron transfer pathways among the copper sites were discussed.101... 

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... 

Table 74 Multiple-type Copper Enzymes in Blue Oxidases...

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. 

The copper centres in the multicopper blue oxidases have been classified into three groups. This classification may be extended to include other copper proteins. 

This is the copper centre responsible for the deep blue colour of the blue oxidases and the blue electron-transfer proteins. Type 1 copper centres have an intense absorption band near 600 nm,... 

This centre, present in the multicopper blue oxidases, is similar to the typical Cu11 sites of tetragonal geometry found in simple coordination complexes of copper. It is ESR-active and has a normal d-d spectrum, which is difficult to measure in the presence of type 1 Cu due to its intense absorptions. The copper in the non-blue oxidases is often regarded as type 2 copper, although this was not implied by the original definition. 

These are listed in Table 22, which includes details of the electron-transfer proteins and the blue oxidases. 

The type 3 centre is present in the blue oxidases, and in hemocyanin, tyrosine and dopamine /3-hydroxylase. Cytochrome oxidase, like laccase, contains four metal centres including an antifer-romagnetically linked Fe- -Cu pair. It appears that there are considerable similarities between the type 3 site and the cytochrome a3- -CuB site in cytochrome oxidase. 

While this section is concerned with structural aspects of the type 3 site, there will be some overlap with the discussion of the blue oxidases, notably laccase, presented in Section 62.1.12.6. 

The paramagnetic copper present in the non-blue oxidases such as galactose oxidase and amine oxidases, and also in the blue oxidases, has d-d and ESR spectra typical of coordination complexes of copper(II). 

This blue oxidase, present in the plasma of vertebrates, appears to be multifunctional.905,975 It accounts for some 95% of the circulating copper in a normal mammal, and its concentration fluctuates considerably in diseased states. It appears that ceruloplasmin has a major role in copper transport (as discussed in Section 62.1.11). In addition it has oxidase activity towards three groups of substrates, although its physiological role is not known with certainty. 

The several functions of ceruloplasmin cannot be explained at present. It seems reasonable that this diversity is related to the activity of the copper centres. The general pattern of oxidase activity is probably similar to that of the other blue oxidases, with a type 3 binuclear site serving to bind and reduce dioxygen, with electrons transferred from the type 1 site. The type 2 copper may represent a substrate-binding site. 

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. 

The enzyme catalyses the oxidation of L-ascorbic acid, 4, to dehydroascorbic, 5. The blue protein belongs to the group of blue oxidases together with laccase and ceruloplasmin. These are multicopper enzymes catalysing the electron reduction of molecular oxygen to water with concomitant one electron oxidation of the substrate. 

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... 

The elaborate mechanism by which blue oxidases react with dioxygen to produce water was tackled by studying the possible role of H202. We have observed the formation of a stable and high affinity complex between tree laccase and H202. Moreover, the finding that the oxidation of the reduced enzyme with H202 follows a pattern which is different from that operative in the reduction of the oxidized enzyme may have important implications for the mechanism of action of laccase. 

Product of SOD and non-blue oxidases (galactose and amine oxidases)... 

Messerschmidt, A., and Huber, R. (1990). The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin. Modeling and structural relationships. Eur. J. Biochem. 187, 341-352. 

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). 

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