Cucumber basic blue protein copper site

Fig. 5. (a) Copper site in cucumber basic blue protein (Cbp). (b) Ribbon drawing of the Cbp backbone, (c and d) Schematic of Cbp topology. 

The cucumber basic blue protein is made up of a single polypeptide chain of 96 amino acids containing a single copper ion with a molecular weight of 10000 (Adman, 1985). The amino acid sequence (Bergman et al., 1977) is very similar to those of stellacyanin and umecyanin. The copper in cucumber blue protein displays the characteristic spectroscopic properties of a typical blue copper site (Table 5-5). 

The crystal structure of cucumber basic blue protein has now been refined to 3.0 A resolution (Adman, 1985). The protein consists of eight strands, only five of which form a P-sandwich and the protein has less P-sheet character than plastocyanin or azurin. The ligands to copper are provided by the side chains of His-39, Cys-79, His-84 and Met-89. The copper site has the N2SS coordination seen in plastocyanin. The imidazole rings of the His-39 and His-94 residues are exposed to the solvent providing a likely entry site for electon transfer to the copper centre. 

The type I copper sites function as electron transfer centers in the blue copper proteins and in multicopper enzymes, particularly oxidases (33). They are characterized by their intense blue color, their unusually small A values, and their very positive redox potentials (Table II). X-ray crystal structures of several blue copper proteins have been determined, notably plastocyanin (34), azurin (35), cucumber basic blue protein (36), and pseudoazurin (37). The active site structures show marked similarities but also distinct differences (Fig. 8). 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

Solvent is usually excluded from the blue copper site, which is buried 6 A inside the protein, having only the His ligand from the copperbinding loop exposed to the surface. The phytocyanins, stellacyanin and plantacyanin (cucumber basic protein), are exceptions, in which both His ligands are solvent exposed and the copper ion is only 3 A beneath the protein surface. This situation makes the copper center in this family of blue copper proteins more accessible to low-molecular-weight solutes (see Section V). 

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

The first crystal structure information on a blue copper protein, for poplar plastocyanin in the Cu(II) state, was published in 1978 (2, 3). Since then, the Cu(I) state and related apo and Hg(II) substituted forms (5, 6), the green algal plastocyanin from Enteromorpha prolifera [Cu(II)] (7), azurin from Alcaligenes denitrificans [Cu(II) and Cu(D] (8, 9), azurin from Pseudomonas aeruginosa [Cu(II)] (10, 11), as well as pseudoazurin from Alcaligenes faecalis S-6 (12), and the cucumber basic protein, both in the Cu(II) state, have been published (13), making this one of the best-documented class of proteins. In addition, information as to three-dimensional structure in solution has been obtained from two-dimensional NMR studies on French bean and Scenedesmus obliquus plastocyanins (14,15). This review is concerned in the main with the active site chemistry. Other recent reviews are listed (16-20). 

The classic blue copper sites in plastocyanin and azurin exhibit essentially identical EPR spectra, with approximately axial (gj >= gy) EPR signals. This argues that the long 3.0 Cu-0 A carbonyl oxygen makes little contribution to the electronic structure of azurin, consistent with other spectroscopy and the fact that the relatively compact 0 2p orbitals would be expected to have little contribution to bonding at this distance. On the basis of EPR, the perturbed blue copper sites can be divided into 2 classes (1) those which exhibit a rhombic EPR signal (i.e. A gi = g — g, > 0.01, as in cucumber basic protein, nitrite reductase and stellacyanin, Figure 9)159,160 2) those which are perturbed, but still... 

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