Ascorbate oxidase trinuclear copper site

The average copper-copper distance in the trinuclear copper site of ascorbate oxidase is 3.74 A (o- = 0.08 A) and the individual distances do not deviate by more than 0.16 A from this mean value. The average copper-copper distance in hemocyanin is 3.54 A 100). The copper-copper distances are too long for copper-copper bonds but magnetic interactions are possible. 

The similarity matrix calculated in Messerschmidt and Huber (202) indicates clearly the six-domain structure of ceruloplasmin and three-domain structures for laccase and ascorbate oxidase. The internal triplication within the ceruloplasmin amino-acid sequence is reflected by values of about 60% difference. Comparison of both the N-terminal domains and the C-terminal domains of the blue oxidases indicates, respectively, a relationship that is closer and relevant values for percent difference that are significantly lower than those for other comparisons. This might reflect the requirements for the trinuclear copper site. The lowest values of about 70 to 73% difference are observed for both N-terminal and C-terminal domains of laccase and ascorbate oxidase, showing that the two oxidases are more closely related to ceruloplasmin than either of them. 

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... 

Fig. 11. Schematic drawing of the reduced form of ascorbate oxidase around the trinuclear copper site. The included copper-copper distances are the mean values between both subunits.

In the case of laccase and ascorbate oxidase, the observed ET rates for the reduction of the type-3 coppers (see Table VIII) are lower than the observed turnover number. This can be explained only by the possibility that the enzymes are in a resting form under the experimental conditions. A considerable reorganization energy seems to be necessary to get to the reduced state of the type-3 coppers (release of the bridging OH" and movement of the copper GU2 and GU3). From these data it cannot be decided what the rate-limiting step is in the catalytic cycle, either this intramolecular ET or the reaction of the dioxygen at the trinuclear copper site. 

Laccase contains four copper atoms and catalyzes the four-electron reduction of dioxygen to water. X-Ray absorption edge spectroscopy has been used to determine the oxidation states of copper in Rhus vernicifera laccase, following the reaction of the reduced enzyme with dioxygen (202). This study included the incorporation of mercury(II) in the Type 1 copper site (see Section IV,B). The results demonstrate that the Type 2/Type 3 trinuclear copper site, as found in ascorbate oxidase (103), represents the minimal active site required for the multielectron reduction of dioxygen. 

Besides the blue type-1 copper sites, the blue oxidases contain a trinuclear copper center, which is located between the N- and C-terminal domains. It can be described spectroscopically as a coupled type-2/type-3 copper center. The atomic structure of the trinuclear copper site for oxidized ascorbate oxidase as derived from X-ray crystallography is displayed in Figure 6. The trinuclear cluster has eight histidine ligands symmetrically supplied from... 

Laccase has been isolated from lacquer trees (e.g. Rhus vernifera) and from various fungi. The crystal structure of laccase obtained from the fungus Trametes versicolor was reported in 2002 and confirms the presence of a trinuclear copper site containing Type 2 and Type 3 copper atoms, and a monocopper (Type 1) site. The structure of the trinuclear copper site is similar to that in ascorbate oxidase (Fig. 29.14). However, the Type 1 copper atom in laccase is 3-coordinate (trigonal planar and bound by one Cys and two His residues) and lacks the axial ligand... 

With the structure of ascorbate oxidase in hand, a new structurally based alignment of the sequences of ascorbate oxidase, laccase, and ceruloplasmin has been performed (Messerschmidt and Huber, 1990). In brief, while gene triplication for ceruloplasmin is still revelant, its sequence can be further subdivided into two domains per unit of triplicated sequence, or six domains in total. Each of these sequences bears some resemblance to each of the three domains of ascorbate oxidase, as does each of the two domains in laccase. The coppers of the trinuclear site of ceruloplasmin then are predicted to be bound between domains 1 and 6, with a type I site also lying in both domains 6 and 4 (see Huber, 1990). The relative orientation of each of these domains is not predicted by this alignment, but it turns out that the structure of nitrite reductase may shed some light on this (see Section V,C). 

Usually, these metalloproteins contain both type 2 and type 3 copper centers, together forming a triangular-shaped trinuclear active site, such as found in laccase (polyphenol oxidase) [38-41] and ascorbate oxidase (3) [42]. Recent evidence for a related arrangement has been reported for the enzyme particulate methane monooxygenase as well [43], but in this case the Cu Cu distance of the type 2 subunit (2.6 A) appears to be unusually short and the third Cu ion is located far from the dinuclear 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. 

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 ... 

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 low-temperature MCD and absorption titration studies (Figure 10) have determined that azide binds to both the type 2 and type 3 centers with similar binding constants. A series of chemical perturbations and stoichiometry studies have shown that these effects are associated with the same azide. This demonstrates that one N3 bridges between the type 2 and type 3 centers in laccase. These and other results from MCD spectroscopy first defined the presence of a trinuclear copper cluster active site in biology (89). At higher azide concentration, a second azide binds to the trinuclear site in laccase. Messerschmidt et al. have determined from X-ray crystallography that a trinuclear copper cluster site is also present in ascorbate oxidase (87, 92) and have obtained a crystal structure for a two-azide-bound derivative (87). It appears that some differences exist between the two-azide-bound laccase and ascorbate oxidase derivatives, and it will be important to spectroscopically correlate between these sites. 

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