Pseudoazurin structure

Figure 35 (a) X-Ray structure of pseudoazurin (from Alcaligenes faecalisj. (b) Cyclic... 

The crystal structure of the pseudoazurin from Alcaligenes faecalis S-6 sometimes referred to as the blue protein (also as cupredoxin), has been reported to 2.0 A [74]. The protein folds in /3-sandwich which is described as being similar to plastocyanin and azurin. 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

The structure of pseudoazurin from A. faecalis strain S-6 was determined in two laboratories (in part, because it crystallizes so readily excellent crystals are formed in a matter of hours ) (Petratos et al., 1987, 1988a Adman et al, 1989). The crystals, space group P6j, have the interesting property that they are intensely blue when viewed along the sixfold axis, but are nearly colorless when viewed normal to this axis. This is assumed to be due to the fact that the plane of the Cu-Sy-C/3 atoms is perpendicular to the sixfold axis, consistent with the fact that the copper— thiolate bond is responsible for the blue color of the protein. 

Unfortunately, bond lengths have not been reported for the copper center of Cbp. Its spectrum (like that of plastocyanin) is much more like that of the A. faecalis pseudoazurin than azurin. Since little variability of the Cu-Sy bond has been seen in the three structures described above, and since the major difference between pseudoazurin and plastocyanin (or azurin) is the length of the Cu-Met S8 bond, this would suggest that the Cu-Met bond is short in this protein, as well. Its EPR is also rhombic, again, like that of pseudoazurin. 

P,2, S transition, ratio r, 35 368 Prussian Blue, 46 41 -like phases, 43 245-249 magnetic ordering, 43 246-247 magnetic properties, 43 248 relation to Berlin green, 8 120-121 structure studies on, 8 119-120 Prussiates, M6ssbauer spectrum of, 6 464-466 Pseudoazurins, 36 382 x-ray crystal structures, 36 389-390 Pseudocatalase, 40 379... 

The first class is cupredoxins—single-domain blue copper proteins composed of only one BCB domain. These proteins include plastocyanin, azurin, pseudoazurin, amicyanin, auracyanins, rusticyanin, halocyanin, and sulfocyanin (see Section IV). Plantacyanin of the phytocyanin family (Section V), subunit II of the cytochrome c oxidase, and the recently characterized nitrosocyanin also fall into this class. The last two are single BCB domain polypeptides closely related structurally to cupredoxins, but harboring, respectively, a binuclear copper site known as CuA and a novel type of copper-binding site called red (see Sections IX and X). 

At this writing, the three-dimensional sttuctures of eight different naturally occurring type 1 copper proteins are known. These include the cupredoxins plastocyanin at 1.33 A resolution (pdb code 1 PTC), azurin at 1.8 A (pdb code 2AZA), pseudoazurin at 1.55 A (pdb code IPAZ), amicyanin at 1.3 A (pdb code lAAC), auracyanin at 1.55 A (pdb code IQHQ), rusticyanin at 1.9 A (pdb code IRCY), and the phytocyanins cucumber basic protein at 1.8 A (pdb code2CBP), and stellacyanin at 1.6 A (pdb code IJER) Atomic coordinates for these and all other single-domain type 1 copper proteins are available from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) and can be accessed online at www.rcsb.org/pdb/. 

Pseudoazurin (123-124 residues) closely resembles plastocyanin in many respects. It has a minimal /3-barrel with short connecting loops. Strand 5 contains fewer residues than the equivalent strand in plastocyanin, however, as it has a regular, extended /3-structure without the extra kinks and loops of plastocyanin. The major difference is the presence of an extra 25 residues at the C-terminal end of the polypeptide chain these form two a-helices which pack on the surface of the /3-barrel in a location adjacent to but structurally distinct from the flap in azurin. 

The structure of amicyanin (99-106 residues) also more closely resembles that of plastocyanin than azurin as shown by both sequence comparisons and the crystal-stracture data. It has short connecting loops and no flap . Strand 5 is shorter and more regular than that in plastocyanin. Amicyanin, however, has an extension at the N-terminal end of the polypeptide chain (rather than the C-terminus as in pseudoazurin) the extra 20 amino acids form a ninth /3-strand which extends the /3-barrel by hydrogen bonding to the exposed edge of one of the two /3-sheets (i.e. to strand 6 in Figure 3). 

A fundamental premise of the entatic state concept is that the metal simply fits into a site that is preformed by the protein and determined by the many interactions which stabilize the protein structure. This idea has been tested by crystallographic studies of apo-forms of azurin, plastocyanin, pseudoazurin and amicyanin. 

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). 

Even if the calculations were performed on a simple model, the results presented in Figure 8 nicely reflect the structure-electronic spectroscopy relationship between the various types of copper-cysteinate proteins. The copper coordination geometry of axial type 1 proteins is close to trigonal, and their spectroscopic characteristics are reflected by the results obtained for (p > 80°. Rhombic type 1 proteins like pseudoazurin and cucumber basic protein, on the other hand, have (p angles between 70° and 80°. As can be seen from Figure 8, even at such a small... 

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). 

In 1985 the existence of two new classes of bacterial blue proteins, the pseudoazurins and amicyanins, was demonstrated (28). Sequence information is available for pseudoazurin from Pseudomonas AMI (28), Achromobacter cycloclastes (29), as well as A. faecalis, for which the structure has been determined (12). The name pseudoazurin rather than cupredoxin is used here. The sequence for amicyanin, also present in Pseudomonas AMI, has been reported, and that for amicyanin from Thiobacillus versutus is being determined. Preliminary X-ray crystallographic information has been reported for amicyanin from T. versutus (30). 

From X-ray crystal structures the AT of the imidazole rings of the two histidine residues are coordinated to the Cu in plastocyanin (3, 4, 7), azurin (8), pseudoazurin (12), and CBP il3). However, in studies on Co(II)-substituted stellacyanin (71), it has been demonstrated that both histidines bind the metal via the N atom. Similar differences have been observed in the case of binuclear Fe proteins for example. Thus in ribonucleotide reductase the of histidine is coordinated, whereas in hemerythrin it is the N atom which is involved (85). In carbonic anhydrase the two coordinated imidazoles have and N atoms respectively bonded to the same Zn (85). The differences are most likely attributable to steric factors involving the polypeptide. 

Estrada, E. and Uriarte, E. (2005) Eolding degrees of azurins and pseudoazurins. Implications for structure and function. Comp, Biol, Chem., 29, 345-353. 

The sequence homologies of plastocyanin, pseudoazurin, amicyanin, and halocyanin are reflected in the similarities of their tertiary structures and in the crystal structures of plastocyanin [22], pseudoazurin [76, 77] and amicyanin [20,78], The peptide chains of plastocyanin and pseudoazurin consist of eight, and that of amicyanin nine, /1-sheets which are connected by loops. The protein consists of two sandwiched planes, formed by strands 2,8,7, and 4 on the one side and strands 2,1,3, and 6 on the other. Strand 2 belongs to both planes, and strands 0 and 5 are not part of the sandwich [20,71,78]. Strand 0 is the N-ter-minal strand in amicyanin which is formally an elongation of the peptide chain as compared to plastocyanin and pseudoazurin (Fig. 13). 

The physiological electron donors for NIR are either the cupredoxins pseudoazurin or azurin depending on the organism. Pseudoazurin, for example, reduces the type-1 copper and subsequently the electron is transferred to the type-2 copper site, which is also the binding site for nitrite. Nitrite is bound to the type-2 copper site as demonstrated by electron nucleus double resonance studies on NIR from Achromohacter xylosoxidans and in a crystal structure of the complex between nitrite and NIR from A. cycloclastes The crystal structure of the complex shows that nitrite binds asymmetrically with the oxygens toward the copper. 

The metal-binding site structures of pseudoazurin and auracyanin are similar to that of other cupredoxins, with one cysteine and two histidines forming a trigonal plane and a weak axial methionine to complete the distorted tetrahedral geometry. The Cu-S (Met) distance (2.76 A) in pseudoazurin is shorter than those of blue copper centers in plastocyanin and azurin, and is still longer than those in plantacyanin and NiR (see Table 4). 

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