Azurin sequences

Studies on the bacterial type 1 protein azurin have been extensive. Ten different azurin amino-acid sequences have been determined with 47 out of 129 residues (Mf 14,(XX)) conserved. Reduction potentials are in the range 280 339 mV at... 

The Ru(NH3)j+ moiety can be attached to histidine-83 on the azurin surface. It can then be oxidized to Ru(III) without altering the conformation of the protein. This ruthenated protein is mixed with Ru(bpy)3+ and laser irradiated. The sequence of events which occurs is shown in the scheme... 

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. 

The gene for this protein has also been isolated from A. faecalis and sequenced and expressed in Escherichia coli cells (Yamamoto et al., 1987). Like azurin, it contains an amino-terminal signal sequence, suggesting that it is secreted into the periplasm in A. faecalis, although not all of the blue protein is found in this fraction. Mutants of this protein are being made and characterized in T. Beppu s laboratory (Univ. of Tokyo) (personal communication, 1989). 

Cucumber basic blue protein (Cbp) is a protein without known function, also known as cusacyanin or plantacyanin. Its structure (Guss et al., 1988) completes the repertoire of cupredoxins with known structures. The topology of its folding is similar (Fig. 5) to those of plastocyanin and azurin, as might have been expected from sequence similarities and... 

The azurins are electron-transfer proteins in the respiratory chains of certain bacteria. They have been particularly well studied from Pseudomonas aeruginosa and other pseudomonads, and contain one type 1 copper bound to a single polypeptide chain of molecular weight about 16 000. Amino acid sequence data for a number of azurins show that about one third of residues are conserved. All contain three cysteine residues. Three are also sequence homologies with the plastocyanins. 

Many of the reactions of the plastocyanins and azurins with other redox proteins follow Marcus behaviour.946 These reactions all show a single mechanism of electron transfer, with no kinetic selectivity and no specific interactions between the proteins. The notable exception to this behaviour is cytochrome / (c552), where a specific interaction occurs,934 appropriate for its natural redox partner. Equation (48) represents a probable sequence of electron carriers, although it is difficult to extrapolate conclusions to the membrane-bound proteins. 

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). 

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 azurins have 128 or 129 amino acids (Mr —14,000) at least 10 have been sequenced (16). Of the nine sequences considered by Rydon and Lundgren, 47 residues are invariant, including His 46, Cys 112, His 117, and Met 121, which coordinate the Cu, and a further 32 are conservatively substituted (27). There is an additional interaction of... 

The structures of three Cu(II) azurins from A. denitrificans (8), P. aeruginosa (10, 11), and Pseudomonas denitrificans (50) have been determined to 1.8, 2.7, and 3.0 A resolution, respectively. In the case of P. aeruginosa there are four molecules in the asymmetric unit but only two in the case of A. denitrificans, which has yielded the most detailed information. Although these two azurins differ in their sequences at 49 positions, their three-dimensional structures are remarkably similar. The A. denitrificans Cu(I) structure has recently been determined, and relevant data are summarized in Table III (9). 

Fic, 4. The polypeptide chain folding of Alcaligenes denitrificans azurin. Solid circles are residues inserted for comparison with plastocyanin. Probable H bonds are shown by dotted lines. Strands of /3 structures are numbered according to their positions in the amino acid sequence the inserted flap 52-81 contains an a-helix section that is seen on the right-hand side in Fig. 5. The cross-hatched circle denotes the position of the Cu atom. (Reproduced with permission from Ref S.)... 

A final point concerns the variability in number of amino acids separating the residues coordinating the Cu active site, and the possible controlling influence this might have. Available information is summarized in Table VI. In the case of azurin and pseudoazurin, there is the additional influence of the peptide C=0 on coordination at the Cu. All of the blue copper proteins have similar features, with coordination of the copper to one His from the first part of the sequence, and three other residues from the latter part. At present there appears to be no... 

Figure 7-18. A sequence of in situ STM images of azurin adsorbed on Au(lll) in 50 mM NTLtAc (pH 4.6). Taken for increasing positive substrate potentials -0.05 (a), 0.0 (b), +0.05 (c) and +0.10 V (d). Potentials quoted are relative to the equilibrium redox potential of the centre. Note that the image contrast has decreased in images (c) and especially (d), which are above the equihbrium potential at the copper redox site (see the text). Reprinted from ref 57 with permission.

The central copper ion of the auracyanins is probably coordinated by two histidines, one cysteine, and a methionine residue. The auracyanins are unique among the small blue proteins in that they possess a methionine and a glutamine residue (see phytocyanins) which both could act as the fourth ligand coordinating the central copper ion. This copper center is surrounded by a hydro-phobic environment similar to that of the other small blue proteins [68]. Amino acid sequence comparisons place auracyanin in a phylogenetic tree at approximately equal distances from azurin and plastocyanin [68,92]. 

Azurins are single copper proteins that function as electron mediators in the energy conversion systems of many bacteria (15). While azurins isolated from distinct bacteria are highly homologous, subtle sequence differences do exist. 

---