Trinuclear copper structure

ESR spectroscopy and cryomagnetic susceptibility measurements suggest that the active site of pMMO has a trinuclear copper structure [256]. The mechanism of the methane monooxygenation by pMMO is proposed as shown in Scheme 14. 

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. 

In 1996 Stack and co-workers reported an unusual 3 1 (copper 02 stoichiometry) reaction between a mononuclear copper(I) complex of a A-permethylated (lR,2R)-cyclohexanediamine ligand with dioxygen. The end product of this reaction, stable at only low temperatures (X-ray structure at —40 °C) is a discrete, mixed-valence trinuclear copper cluster (1), with two Cu11 and a Cu111 center (Cu-Cu 2.641 and 2.704 A).27 Its spectroscopic and magnetic behavior were also investigated in detail. The relevance of this synthetic complex to the reduction of 02 at the trinuclear active sites of multicopper oxidases4-8 was discussed. Once formed, it exhibits moderate thermal stability, decomposed by a non-first-order process in about 3h at —10 °C. In the presence of trace water, the major isolated product was the bis(/i-hydroxo)dicopper(II) dimer (2). 

Figure 1. (a) A schematic representation of the overall organization of the molecule of human ceruloplasmin. Domains 2,4, and 6 contain mononuclear copper centers, while the trinuclear copper cluster is located at the interface of domains 1 and 6. (b) An a-carbon ribbon diagram of the human ceruloplasmin molecule viewed along the pseudo threefold axis highlighting the triplication of the structure. Domains 1, 3, and 5 are depicted by striped motifs, whereas domains 2, 4, and 6 are dark shaded. The copper... 

In the elucidation of the X-ray structure of hCP by the method of isomorphous replacement, gold and mercury heavy atom derivatives were utilized. In the case of the mercury derivative, p-chloromercury-benzoate, the heavy atom bound to the free sulphydryl residue, C221, but for the gold cyanide derivative the gold atom was found to bind in the vicinity of the trinuclear copper cluster. A realistic explanation of this... 

Figure 7. Traces of the a-carbon polypeptide backbone of domains 1 and 6 in the hCP structure. Domain 1 is shown (shaded) on the left hand side of the diagram this domain contributes four histidine residues (not shown) to the trinuclear cluster copper atoms are depicted as black spheres. Domain 6 is on the right hand side of the figure and also contributes four histidine residues to the cluster. The portion of the polypeptide chain colored black is that which is missing in the truncated enzyme. This polypeptide, residues 991 to 1046 inclusive, includes two histidine residues bound to the trinuclear copper center and three residues bound to the mononuclear copper in domain 6 these residues are depicted in black. The absence of the C-terminal polypeptide would also remove over 50% of the interdomain hydrogen-bond and iron-pair interactions observed in the intact enzyme.

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. 

Figure 22. Comparison of oxygen intermediates. A Electronic absorption spectra of the peroxy-intermediate in laccase versus oxyhemocyanin and oxytyrosinase. B Proposed structural differences between peroxide binding in oxyhemocyanin and oxytyrosinase relative to the end-on bound hydroperoxide intermediate at the trinuclear copper cluster in laccase.

Cole et al. (97) studied the electronic structure of the laccase trinuclear copper active site by the use of absorption, circular dichroism, and low-temperature magnetic circular dichroism spectroscopies. The assigned ligand field transition energies indicated that all three coppers have tetragonal geometries and that the two type-3 coppers are inequivalent. 

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. 

Thereafter, crystals were brought back to the aerobic 25% MPD solution, buffered with 50 mAf sodium phosphate, pH 5.5. This procedure is based on Avigliano et al. s (157) method of preparing T2D ascorbate oxidase in solution and was modified by Merli et al. (159) for use with ascorbate oxidase crystals. The 2.5-A-resolution X-ray structure analysis by difference-Fourier techniques and crystallographic refinement shows that about 1.3 copper ions per ascorbate oxidase monomer are removed. The copper is lost from all three copper sites of the trinuclear copper species, whereby the EPR-active type-2 copper is the most depleted (see Fig. 10). Type-1 copper is not affected. The EPR spectra from polycrystalline samples of the respective native and T2D ascorbate oxidase were recorded. The native spectrum exhibits the type-1 and type-2 EPR signals in a ratio of about 1 1, as expected from the crystal structure. The T2D spectrum reveals the characteristic resonances of the type-1 copper center, also observed for T2D ascorbate oxidase in frozen solution, and the complete disappearance of the spectroscopic type-2 copper. This observation indicates preferential formation of a Cu-depleted form with the holes equally distributed over all three copper sites. Each of these Cu-depleted species may represent an anti-ferromagnetically coupled copper pair that is EPR-silent and that could explain the disappearance of the type-2 EPR signal. 

There are many structural studies of copper coordination compounds with azide ligands, mainly of mononuclear and binuclear copper complexes but a few also of trinuclear copper complexes. A comprehensive... 

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