Azurin denitrificans

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

The blue, or type 1, copper proteins, azurin from Pseudomonas aeruginosa (Adman et ai, 1978 Adman and Jensen, 1981) and from Al-caligenes denitrificans (Norris et al., 1983, 1986) and poplar plastocyanin (Guss and Freeman, 1983 Guss et al., 1986), have been studied by X-ray diffraction. These involve a Cu(I)/Cu(II) redox system. Cu(I) d ) is... 

Shepard et al. (1990) reported the refined structure of reduced azurin from A. denitrificans. The reduced protein was formed at pH 6.0 in the native (oxidized) crystals using ascorbate. The refined moclel shows that the most significant structural differences occur at the copper. Its geometry remains much the same (already quite trigonal), and the distances from copper to the axial methionine and the carbonyl oxygen each increase by about 0.1 A. This is in contrast to results reported by... 

A study by Petrich et al. (1987) on the fluorescence lifetimes of excited tryptophans in azurin has proved exceptionally interesting, especially in light of the studies to be reviewed below on ascorbate oxidase. By comparing the lifetimes of tryptophan fluorescence of three azurins— Az-Pae (only one tryptophan, Trp-48), A.faecalis [Az-Afe (one tryptophan, Trp-118)] and A. denitrificans [Az-Ade (two tryptophans, Trp-48), and Trp-118)] in both holo and apo forms—the authors found that (1) there is virtually no fluorescence quenching in the apo forms (2) the decay of... 

The X-ray crystal structure of auracyanin B has been recently reported. It revealed a structure quite similar to that of azurin (Bond et al., 2001). The root mean square deviations between the positions of 89 Ca atoms of the auracyanin B and Alcaligenes denitrificans azurin have been estimated to be only 0.795 A. 

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 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). 

Bond Distances and Angles for the Cu Active Site of Azurin from Alcaligenes denitrificans ... 

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.)... 

Fig. 5. The a-carbon positions in the structures of Alcaligenes denitrificans azurin. The cross-hatched circle denotes the Cu atom. A disulfide bridge links Cys 3 and Cys 26. Two important insertions are observed as compared to plastocyanin. The flap region is shown on the right, and an extra loop is at the top of the molecule. (Reproduced with permission from Ref 8.)...

Groeneveld, C. M., Ouwerling, M. C., Erkelens, C. and Canters, G. W. (1988) HNuclear Magnetic Resonance Study of the Portonation Behaviour of the Histidine Residues and the Electron Selfexchange Reactions of Azurin from Alcaligenes denitrificans, J. Mol. Biol. 200, 189-199. 

Fig. 10. Visible spectra of the Cua amicyanin variant and the Cua domain of COX from B. subtilis. (A) The Cua amicyanin sample was in HEPES buffer at pH 7.0 whereas the Cua domain was in Tris buffer at pH 8.0. (B) The EPR spectra were obtained with the samples at 23 K, with glycereol added as a cryoprotectant. (C) The resonance Raman spectra of Cua sites on 488-nm ( 150-mW) excitation at 15 K. CCO fragments from P. denitrificans (2.0 mM in Cua), T. thermophilus (1.8 mAf in Cua), B. subtilis (1.5 mM in Cua), Cua construct in P. aeruginosa azurin ( 0.4 mM in Cua), and Cua construct in P. versutus amicyanin (1.7 mM in Cua).

CCPs from P. aeruginosa and P. denitrificans catalyze similar reactions, where the electron donors are either cytochrome C551 or azurin. Eq. (6.1) represents the P. aeruginosa CCP-catalyzed reaction. Ellfolk and Soininen purified P. aeruginosa CCP for the first time in 1970 [12]. P. aeruginosa CCP has been extensively used as a model for di-heme peroxidases. 

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