Pseudoazurin

As illustrated in Figure 35, the same redox behaviour is exhibited by the positively charged blue protein pseudoazurin (FW=13 400 p/ = 8.9), a class of proteins devoted to reduction of NCV to NO in denitrifying bacteria. 

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. 

Amicyanin appears to be more homologous with the plastocyanin sequence than with the pseudoazurin sequence. 

The blue protein from A. faecalis strain S-6, which was isolated as a requirement for transferring electrons to a copper-containing nitrite reductase, has since been shown to have sequence homology with proteins arbitrarily designated pseudoazurin by Ambler and Tobari (1985), from Achromobacter cycloclastes and from Pseudomonas AMI. [Pseudomonas AMI also produces amicyanin, which is the recipient of electrons from methylamine dehydrogenase, (see below)]. In A. cycloclastes reduced pseudoazurin donates electrons to a copper nitrite reductase (Liu et ai, 1986), as it does in A. faecalis. Ambler and Tobari (1985)... 

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. 

The salient features of A. faecalis pseudoazurin are that (1) it has a Cu-Met bond length shorter than that of either plastocyanin or azurin (see Table III) (2) it has only one NH - S bond, as does plastocyanin and (3) its overall architecture resembles plastocyanin (see Fig. 4), with an extended carboxy terminus folded into two a helices [a preliminary sequence comparison suggested that the folding would resemble plastocyanin (Adman, 1985)]. It retains the exposed hydrophobic face found in azurin and plastocyanin. Just how it interacts with nitrite reductase is still a subject of investigation. It is intriguing that the carboxy-terminal portion folds up onto the face of the molecule where the unique portions of other blue proteins are the flap in azurin, and, as we see below in the multi-copper oxidase, entire domains. 

Fig. 4. (a) Copper site in pseudoazurin. (b) Ribbon drawing of the pseudoazurin backbone. (c and d) Schematic of pseudoazurin topology. 

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. 

A recently characterized single-domain copper protein, auracyanin (Trost et al., 1988), is a dimeric protein which has a visible spectrum more like that of the A. faecalis cupredoxin (pseudoazurin, subgroup II see Table II) than that of either azurin or plastocyanin, but, because of its cysteine content and rhombic EPR, it has been put in the other class in Table II. 

Amicyanin (Husain and Davidson, 1986 Groeneveld etal., 1988) spectroscopically resembles plastocyanin more than pseudoazurin and has about the same number of amino acids, so that its classiflcation has been changed from subgroup II to III (the plastocyanin group see Table II). However, its sequence is distinctly different than the plastocyanins, and the new function may indicate yet another class. 

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... 

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... 

Two types of dissimilatory nitrite reductases catalyze step b of Eq. 18-30. Some bacteria use a copper-containing enzyme, which contains a type 1 (blue) copper bound to a (3 barrel domain of one subunit and a type 2 copper at the catalytic center. The type 1 copper is thought to receive electrons from the small copper-containing carrier pseudoazurin (Chapter... 

Electron-transfer carrier plastocyanin, azurin, pseudoazurin... 

Bond Cu(SCPh3)L Azurin Populus plastocyanin Enteromorpha prolifera plastocyanin Pseudoazurin Cucumber basic blue copper protein"... 

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). 

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