Azurin three-dimensional structure

Fig. 5.8. Three-dimensional structure of P. aeruginosa azurin. Coordinates from ref. [80] and Brookhaven Data Bank. Graphic representation by Molscript [66], The copper atom is indicated by the large sphere at the top, the disulphide group by the two smaller, lightly...

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). 

Figure 2.12 Overview of three-dimensional structures and in situ STM images of metalloproteins representative of the three ET protein classes characterized by single-crystal PFV and in situ STM to single-molecule resolution, (a) Blue copper protein P. aeruginosa azurin (PDB 4AZU) [94] ...

The Ru-protein rates in Fig. 1-2 are scattered around the Ru-azurin P = 1.1. A. exponential distance decay. Rates at a single distance can differ by as much as a factor of 10 and D/A distances that differ by as much as 5 A can produce virtually identical rates. Clearly, the absence of a simple exponential distance dependence in the Ru-protein rate data is a reflection of the heterogeneity of the coupling medium. The efficiency of the coupling between redox centers is determined by the three-dimensional structure of the intervening polypeptide. 

Figure 1 (Chapter 1). Three-dimensional structure of Pseudomonas aeruginosa azurin (16). In addition to the protein backbone, the side chains of three copper ligating residues, His46, HisllT, and Cysll2 are shown near the top together with the disulfide bridge (bottom) and Trp48 (center). Coordinates were taken from the Protein Data Bank (PDB), code 4AZU.

Cyt c and azurin are structurally and in other respects very well characterized, and in-situ STM can be referred to many other structural, spectral, and kinetic data. No three-dimensional structure of laccase is available, but the structure of the closely related enzyme ascorbate oxidase is available with high resolution and srq ports a view of facile ET through die protein, involving all the copper atoms. 

Figure 1. Three-dimensional structure of the polypeptide backbone o/Pseudomo-nas aeruginosa azurin, with some amino add residues of particular interest included. Coordinates were obtained from reference 21.

The emission maximum of tryptophan in proteins can range fiom 308 mn for a residue buried in a completely apolar enviroiunent (as in the single tryptophan variant of azurin (37)) to 350 nm for a fully solvent-exposed residue (38-40). Due to their apolar character, tryptophan residues are usually either fully or partially buried in the three-dimensional structures of proteins and are expected to have emission maxima from 320 to 340 nm. Unfolding of a protein, and concomitant increase in the solvent exposure of tiyptophan residues will invariably lead to a red-shift in the fluorescence of a tryptophan-containing protein. 

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/. 

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