Stellacyanin ligands

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... 

Another interesting blue protein is stellacyanin (FW = 20 000) from the Japanese lacquer tree Rhus vernicifera, in which, with respect to the other cupredoxins, glutamine replaces the methionine ligand.64 Stellacyanin also bears an overall positive charge (p/=9.9). It, therefore, gives a reversible Cu(II)/Cu(I) response at a glassy carbon electrode in aqueous solution (pH 7.6).61 The formal electrode potential of the Cu(II)/Cu(I) reduction (E01 = + 0.18 V vs. NHE) is the lowest among cupredoxins. 

Mavicyanin (Mj = 18,000) is obtained from green squash (Cucurbito pepo medullosa), where it occurs alongside ascorbate oxidase [64]. It has a peak at 600 nm (e 5000 M cm and reduction potential of 285 mV. Further studies on this and the mung bean and rice bran proteins [65, 66] would be of interest. All the above type 1 Cu proteins have an intense blue color and characteristic narrow hyperfine EPR spectrum for the Cu(II) state. Table 3 summarizes the properties of those most studied. There is some variation in reduction potential and position of the main visible absorbance peak. In the case of azurin, for example, the latter is shifted from 597 to 625 nm. Stellacyanin has no methionine and the identity of the fourth ligand is therefore different [75]. The possibility that this is the 0(amide) of Gln97 has been suggested [63b]. It now seems unlikely that the disulfide is involved in coordination. Stellacyanin has 107 amino acids, with carbohydrate attached at three points giving a 40% contribution to the M, of 20,000 [75]. 

Spectra, but, in general, leaves the copper site the most exposed of the four cupredoxins. The sequence of Cbp is quite similar to that of stella-cyanin. Stellacyanin is a plant protein, also of unknown function, having visible spectra characteristic of type I copper, but lacking the methionine ligand found in all other type I proteins. A disulfide bond has been suggested as a potential copper ligand in stellacyanin the Cbp has both a methionine and the disulfide, so that prior to the structure determina-... 

The type-1 blue copper proteins act as electron carriers azurin, plastocyanin, stellacyanin, umecyanin e.g. They are characterized by a rather strong LMCT (ligand to metal charge transfer) band near 600 nm and by small hyperline coupling constants A in EPR. Copper is bound to two imidazole groups of histidine and to two... 

Stellacyanin from Rhus vemicifera is less well studied. The polypeptide does not contain a methionine residue showing that the ligands of the type 1 site may vary. There is EXAFS evidence for a short Cu—S(Cys) bond,920 while UV, visible and near IR studies confirm similarities with the other blue proteins. It has been suggested that methionine is replaced as a ligand by an —S—S— group. This is based on resonance Raman921 and NMR studies.922... 

Assignments of the signals corresponding to copper-ligands in Cu(II) and Cu(I) azurin and stellacyanin recorded at 800 MHz... 

Spectroscopic studies of Co (II) derivatives of stellacyanin, plastocyanin, and azurin have established that the charge transfer interpretation is preferred (10, 11). Intense bands (c 2 X 103) that appear to be analogous to the 600-nm system of blue proteins are observed between 300 and 350 nm in the Co (II) derivatives. The shift in band position of about 16 kK [Cu(II) << Co (II)] accords well with expectation for an LMCT transition. The visible and near-infrared absorption, CD, and MCD spectra of Co (II) derivatives of stellacyanin, plastocyanin, and azurin have been interpreted (12) successfully in terms of the d-d transitions expected for distorted tetrahedral metal centers (Table I). Average ligand field parameters are the same for all three Co (II) proteins (Dq = 490, B = 730 cm"1), which strongly suggests that the donor atom... 

The derived ligand field parameters (/3 = 60°, Ds = 765, Dt = 444 cm"1) predict 2Ai to be 11,540 cm"1 above the 2B2 ground state for stellacyanin. An absorption band and CD maximum are observed near this... 

Strong evidence for cysteine sulfur coordination in stellacyanin has been obtained (18) in XPS experiments. Thioether coordination is ruled out in this case, as the protein does not possess any methionine (6). It is probable that the other ligands are similar, but not necessarily identical, to those of bean plastocyanin. 

Fig. 2. Proteins that bind Cu(I). (a) Saccharomyces cerevisiae metallothionein (Cupl, pdb code laqr). Cupl binds up to seven Cu(I) ions (medium gray spheres) using 10 cysteine sulfur atoms (light spheres) in a polythiolate cluster (Peterson et al., 1996). All bonds shorter than 2.8 A are shown as dotted lines, (b) Cucumis sativus stellacyanin (pdb code Ijer). Both Cu(l) and Cu(ll) are bound by a pseudo-trigonal planar arrangement of (His)2Cys residues with an axial Gin ligand (Hart et al., 1996). In other cupredoxins such as plastocyanin, a Met residue is the axial ligand (Adman, 1991).

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). 

From the assignment of the hyperhne shifted signals of Cu(II) plastocyanin, azurin, and stellacyanin (Bertini et al., 1999, 2000), information on the electron delocalization onto the metal ligands was gained hy calculating the contact and pseudocontact contrihutions to the hyperhne shifts. In any case, since the magnetic anisotropy of the Cu(ll) ion is low, the observed shifts can be approximated to the contact contribution, which can be used as an initial criterion to compare the electron spin density on the different nuclei and in the various proteins. 

As the axial ligand is weakly bound in BCP (Randall et al., 2000), the spin density delocalized on it is small. Indeed, in azurin the resonances of the axial methionine protons do not experience a significant hyperfine shift contribution. Electron delocalization onto a Hy of the axial Met has been detected in plastocyanin (signal F in Fig. 3B), suggesting some covalency for the Cu-S(Met) bond. The absence of spin density on the axial Gin ligand in stellacyanin has been attributed to the fact that the y-CH2 Gin geminal couple is four bonds away from the metal ion, whereas the equivalent protons in a bound Met residue (such as in plastocyanin) are only three bonds away (Bertini et al., 2000). 

When an axial Gin ligand is present (either natural as in stellacyanin or introduced by site-directed mutagenesis), the same orientation of the magnetic anisotropy tensor is maintained, and comparison of the average shift of the two P-CH2 Cys protons in different proteins may prove helpful (Diederix et aL, 2000 Fernandez et at., 1997 Salgado et al., 1996 Vila and Fernandez, 1996). 

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