Trinuclear copper sites

Hakulinen N, Kiiskinen LL, Kruus K, Saloheimo M, Paananen A, Koivula A, Rouvinen J. 2002. Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nature Struct Biol 9 601-605. 

The probable binding site for the reducing substrate is close to the Type I copper. Two channels can be identified providing access from the solvent to the trinuclear copper site which is the likely binding site for dioxygen. 

Type IV. Trinuclear copper site having an isosceles triangle shape two copper ions are strongly magnetically coupled. 

Type 2 Type 3(2) Type 3(3) Trinuclear copper site... 

C. Electron Transfer within the Trinuclear Copper Site Summary... 

The trinuclear copper site (see Fig. 6) has eight histidine ligands symmetrically supplied by domain 1 and domain 3 and two oxygen ligands. Seven histidines are ligated by their NE2 atoms to the copper ions, whereas His62 is ligated to CU3 K3 by its NDl atom. In the... 

Fig. 6. Stereo drawing of the trinuclear copper site. The displayed bond distances are for subunit A.

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. 

About 40% (938 residues) of the amino-acid sequence of factor V has been established (103). It shows similarities to the amino-acid sequences of human ceruloplasmin and human factor VIII. The partial sequence contains a region that is similar to domains 5 and 6 in ceruloplasmin and A3 in factor VIII. However, it does not have the canonical ligands for type-1 copper or the trinuclear copper site. On the other hand, cysteine residues are present at the homologous position of ceruloplasmin. They form a disulfide bridge in this structure. 

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 pronounced threefold repeat in the six-domain molecule ceruloplasmin contradicts evolution by a duplication of an ascorbate oxidaselike molecule and suggests evolution from the tandem two-domain molecule by triplication, possibly before acquisition of the trinuclear copper site. This might have occurred later, between the N- and C-terminal domains. 

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... 

From these data, it follows that the dioxygen binds to the trinuclear copper site. This species may store three electrons and transfer them to the bound dioxygen followed by a final one-electron transfer. 

Fig. 10. Averaged FOx2d-FC p2d difference electron density map plus atomic model around the trinuclear copper site. Contour levels -18.0, solid line 18.0, dashed line. Magnitudes of the hole are less than -35.0.

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. 

Electron exchange within the trinuclear copper site is expected to be very fast due to the short distances between the copper atoms (from 3.7 to 5.2 A in the reduced form), as is ET to the bound dioxygen. This fast electron exchange is necessary to include CU4 into the redox processes. CU4 is at a greater distance from the type-1 copper CUl than from CU2 and CU3. 

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. 

Multicopper oxidases containing a type-1 copper center and a trinuclear copper site 527... 

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... 

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