Normal Copper Proteins

SOD produces an intense NJ — Cu(II) CT transition (Fig. 7, lower) at 373 nm (e 2700 M-1 cm-1)31a). This energy is consistent with equatorial binding of azide to a tetragonal copper site (Fig. 5). This relatively intense charge transfer transition raises the possibility of a UV resonance Raman spectroscopic investigation of competitive inhibitor311 (i.e., NJ) binding to this active site. 

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These systems are also described as normal copper proteins due to their conventional ESR features. In the oxidized state, their color is light blue (almost undetectable) due to weak d-d transitions of the single Cu ion. The coordination sphere around Cu, which has either square planar or distorted tetrahedral geometry, contains four ligands with N and/or 0 donor atoms [ 12, 22]. Representative examples of proteins with this active site structure (see Fig. 1) and their respective catalytic function include galactose oxidase (1) (oxidation of primary alcohols) [23,24], phenylalanine hydroxylase (hydroxy-lation of aromatic substrates) [25,26], dopamine- 6-hydroxylase (C-Hbond activation of benzylic substrates) [27] and CuZn superoxide dismutase (disproportionation of 02 superoxide anion) [28,29]. 

The copper proteins containing the type 2 active site are also known as normal copper proteins, because their spectroscopic features are similar to those of common Cu coordination compounds. The copper ion in these proteins is surrounded by four N and/or O donor atoms in either square-planar or distorted tetrahedral geometry [3, 4]. Examples of proteins with this active site include... 

Several copper enzymes will be discussed in detail in subsequent sections of this chapter. Information about major classes of copper enzymes, most of which will not be discussed, is collected in Table 5.1 as adapted from Chapter 14 of reference 49. Table 1 of reference 4 describes additional copper proteins such as the blue copper electron transfer proteins stellacyanin, amicyanin, auracyanin, rusticyanin, and so on. Nitrite reductase contains both normal and blue copper enzymes and facilitates the important biological reaction NO) — NO. Solomon s Chemical Reviews article4 contains extensive information on ligand field theory in relation to ground-state electronic properties of copper complexes and the application of... 

Contrary to popular belief, ceruloplasmin5, the principal copper-containing protein in plasma, ceruloplasmin, is not involved in copper transport. This is clearly underlined by the clinical observation that patients with aceruloplasminaemia (i.e. lacking ceruloplasmin in their blood) have perfectly normal copper metabolism and homeostasis. Copper is transported in plasma mostly by serum albumin with smaller amounts bound to low-molecular weight ligands like histidine. Likewise zinc is mostly transported in plasma bound to proteins (albumin and ot2-macroglobulin). 

Figure 1 also contains a drawing of the electronic structure of plastocy-anin (Penfield et al., 1981, 1985), oriented according to the first part of Fig. 1. The d-orbital plane appears to be normal to the peptide plane that is extended by the hydrogen-bonding pair the it orbital of the thiolate would then also be affected, poising the cluster for electron transfer and, interestingly, apparently in the direction utilized by the multi-copper protein ascorbate oxidase (see Section V,A).

The type-2 copper proteins, on the contrary, have a nearly square-planar copper environment, which is accessible to water molecules They show only weak absorption bands in the visible and normal A.. values in EPR. The main enzymatic activities are listed in Table 1. 

Figure 97 Blue copper protein (a) electronic spectrum (b) ESR spectrum (-------------- blue copper ------- normal copper) ...

A number of copper-containing proteins show spectral features like those of normal copper complexes, and therefore do not appear to contain blue copper centres. Amongst these are galactase oxidase and the amine oxidases. It is noteworthy that it appears unlikely that the copper is involved in the activation of dioxygen. 

Type II copper proteins, or low blue copper proteins, have less intense colours at normal concentrations, but even low blue copper sites have quite high absorption coefficients when compared with simple copper(II) coordination compounds. Bovine erthyrocyte superoxide dismutase (BSOD) is an example of a low blue copper(II) protein with Xmax 680 nm (e = dm3 300 mol-1 cm-1). 

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 ... 

Type II Cu(II), or low-blue copper, is less colored at common research concentrations. These systems have received less attention than Type I copper. However, even low-blue cupric copper can possess high molar absorbtivities when compared with simple coordination complexes of Cu(II). The Cu(II) sites in such proteins also yield Azz values normally greater than 140 G, i.e., more like that of low molecular weight square planar Cu(II) complexes (2, 8). The only available crystal structure of a copper protein is that of a low blue protein bovine erythrocyte superoxide dismtuase (9). The two copper atoms in this protein are each coordinated to four histidine nitrogens in an approximate square planar array. 

Figure 3. Blue copper proteins. A X-ray structure of poplar plastocyanin (21). B Absorption spectrum of plastocyanin and normal D 4 cCuCl42 (e scale expanded by 10). C X-band EPR spectrum of plastocyanin and Y>4 cCuCl42. ...

One of the major goals of studying active sites in copper proteins has therefore been to understand the spectroscopic features associated with the active site. This has led to a classification of three general types of copper protein active sites based on their unique spectral features Blue copper, normal copper and coupled binuclear copper. An additional class of copper proteins, the multi-copper oxidases, contains a combination of these three types of copper active sites. A reasonably firm understanding of the optical and EPR spectra of a number of copper proteins has now been achieved1,2K This article presents an overview of these electronic spectral features and their relationship to geometric and electronic structure. 

Before the unique spectral features of copper in proteins can be discussed, the geometric and electronic structures of normal copper must be considered. In an octahedral environment the cupric ion, which has nine d electrons, would possess a degenerate 2Eg electronic ground state (Fig. 1, left). A geometric distortion which removes this degeneracy would produce a more stable electronic structure, in accordance with the Jahn-Teller... 

The active sites of oxidized blue copper proteins are characterized by unique features relative to those of normal Cu(II) complexes. These features include an intense absorption... 

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