Hemocyanin copper site

The enzyme requires two copper ions per subunit for full expression of activity (18), but, unlike tyrosinase and hemocyanin, there is an absence of magnetic coupling between the two Cu(II) sites and both appear to be separate, isolated mononuclear copper sites (17). The process of dioxygen binding and activation appears to involve interaction of the doxygen molecule with only one copper ion, and it is also found that a proton is requir for the hydroxylation of substrate (19). 

In addition to the fascinating and complex properties of the subunit architecture of hemocyanins [described, for example, by Preaux and Gielens (1984) and Herskovits (1988)], the nature of the copper site has... 

Fig. 9. (a) Copper sites in hemocyanin. (b) Ribbon drawing of the hemocyanin backbone. 

This protein contains a coupled binuclear copper site that appears to be very similar to that found in hemocyanin (Section 62.1.12.3.8).1399 Tyrosinase catalyzes the hydroxylation of monophenols, and also behaves as an oxidase in the oxidation of orfho-diphenols. The deoxy protein [copper(I)] binds dioxygen to give oxytyrosinase, which is a Cu11 peroxide species with antiferromagnetic coupling between the two Cu11 centres. The oxybinuclear site is diamagnetic to the most sensitive detectors. 

Figure 7. Structural aspects of purple hemocyanin. Coppers in the circles marked with dots illustrate different coordination geometry from those in the black circles, and shaded parts indicate the slightly deformed active site of oxyhemocyanin.

Figure 17. Comparison of the reactivity and magnetism ofdeoxy and met hemocyanin and the laccase type 3 copper site in the T2D derivative.

Figure 18. Comparison of half-met hemocyanin with the half-met type 3 (in T2D) laccase copper sites. A EPR spectra and binding constants of exogenous azide binding. B Spectroscopically effective structural models for exogenous ligand binding to the half-met derivatives and their relation to differences in dioxygen reactivity.

A series of hemocyanin and tyrosinase active site derivatives (Fig. 23) can be prepared61"66), allowing systematic variation of the binuclear copper active site and chemical perturbation for spectral studies. In the simplest derivative, met-apo, one copper has been removed and the remaining copper oxidized to the spectroscopically accessible Cu(II). Next in complexity is a mixed-valent binuclear copper site. The Cu(II), in this half-met derivative, exhibits open-shell d9 spectroscopic features and the Cu(I), though spectroscopically inaccessible, can still be studied by comparison to the met-apo derivative. Two derivatives have formally binuclear cupric sites met, which is EPR-non-detect-able, and dimer, which exhibits an intense broad EPR signal. Spectroscopic study of these derivatives has led to the present picture of the coupled binuclear copper protein active site shown at the bottom of Fig. 23. 

Spectral studies of the native enzyme are difficult to interpret due to simultaneous contributions from all three types of copper. The EPR spectrum (Fig. 38) of native laccase98) shows features attributable to the type 2 (g = 2.237, gx = 2.053, A = 206 x 10-4 cm-1) and type 1 (gz = 2.300, gy = 2.055, g = 2.030, Az = 43 x 10-4 cm-1) coppers. The type 3 site is EPR-non-detectable and diamagnetic (-2 J >550 cm-1)56,57). This site should be compared to the met derivative of hemocyanin and tyrosinase only in the sense that it appears to contain two anti-ferromagnetically coupled copper(II) s lacking an EPR signal. A characteristic Blue band of the type 1 copper is seen in the optical spectrum at 614 nm (c = 5700 M-1 cm-1). The shoulder observed at 330 nm (e 2800 M-1 cm-1) was originally associated with the type 3 copper site, as it reduces with two electrons at... 

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. 

Active sites containing two copper ions that are antiferromagneti-cally coupled in the oxidized state are often referred to as type 3 copper sites (129). It has recently become evident that these centers cannot all be considered alike and that in the blue copper oxidases the type 3 sites are in fact part of a tricopper cluster these will be considered in Section VA. The proteins containing dinuclear type 3 copper sites comprise hemocyanin and a number of oxygenase enzymes, of which the best known are tyrosinase and dopamine j8-hydroxylase. 

The structure of the ascorbate oxidase tricopper site is illustrated in Fig. 45. The three copper atoms form an almost equilateral triangle of sides ca. 3.7 A. The Cul and Cu2 atoms are bridged by OH" or O " and make up the EPR-silent type 3 pair each copper atom is coordinated to three histidine residues and the Cu—N(His) distances are all comparable and unexceptional. In contrast to the hemocyanin active site (Section IVD), the copper ions have approximately tetrahedral coordination geometry and are not in identical environments. The third copper ion is coordinated to two histidine residues and to either hydroxide or water. There is no evidence for a fJ-s-OH or fiyO donor at the center of the cluster (and the Cu—Cu distances are too long to support such a bridge). 

Figure 5) and the copper-copper distance is 3.6 A. The geometry of the type-3 copper site in tyrosinase is very similar to that of hemocyanin due to the similar spectroscopic features and has now been derived from the X-ray structure of a bacterial tyrosinase. ... 

The crystal structures of tyrosinase from Streptomyces castaneoghbisporus HUT 6202 and catechol oxidase from the sweet potato Ipomoea batata have been determined. They confirm that the coordination of the type-3 copper site in tyrosinase and catechol oxidase is very similar to that found in hemocyanin. This had been deduced before from the similarity of spectroscopic properties and a comparison of many tyrosinase and hemocyanin primary structures. On the basis of the biological source of the proteins seven different domain organizations could be identified. Plant catechol oxidases of different organisms have a sequence identity of about 40-60%. The sequence identity between catechol oxidases and mulluscan hemocyanins is about 35% over almost the whole length of the sequences. In contrast, the sequence identity between plant catechol oxidases and other type-3 copper proteins from any nonplant source is limited to the two copperbinding regions. 

Remarkably, reduced T2D Lc does not react with atmospheric 02. The behavior of the dinuclear T3 center thus contrasts starkly with that displayed by other reduced dinuclear copper sites of enzymes such as hemocyanin and tyrosinase or synthetic model dinuclear complexes (see Chapter 8.15). " These promptly perform a two-electron reduction of O2 to the peroxide level (reversibly, in the case of hemocyanin). 

X-ray analysis of both copper(I) (e.g. 9) and copper(II) complexes, independent synthesis, and ammonolysis to the free ligands 35 and 36, confirmed the hydroxylation pathway.42 02-binding and hydroxylation in complex 10 were shown to be sensitive to electronic effects of the para-substituent (X = OMe, Me, C02Me, N02>. Tyrosinase, which contains a dinuclear copper active site strongly resembling the hemocyanine active site, binds O2 reversibly and activates O2 for arene hydroxylation (stoichiometry Cu O2 = 2 These are key features observed in the dinuclear copper complexes shown in... 

Spectroscopic and biochemical studies reveal that the active site of tyrosinase is very similar to the dinuclear copper site in hemocyanins (Hcs) (13), which are dioxygen carriers in the hemolymph of mollusks and arthropods. 

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