Azurin importance

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

His35 to ligated His46 may be important in an electron transfer role or in His35 exercising some conformational control of the active site. The reduction potential of P. aeruginosa azurin increases from 300 mV (pH 8) to 360 mV (pH 5), which is believed to be related to His35 protonation. A pK of 6.6 is observed for this process, or alternatively pK s for azurin in the oxidized (6.1) and reduced (7.2) forms can be obtained [56]. 

Last but not least, as this review has attempted to illustrate, there are many other type 1 blue Cu proteins which deserve attention. Azurin has already been fairly extensively studied. As more structural information becomes available, other proteins will no doubt be further investigated, and add to an understanding of this area. The recent ascorbate oxidase structure is extremely important in the further understanding of multi-Cu oxidases. 

More subtle factors that might affect k will be the sites structures, their relative orientation and the nature of the intervening medium. That these are important is obvious if one examines the data for the two copper proteins plastocyanin and azurin. Despite very similar separation of the redox sites and the driving force (Table 5.12), the electron transfer rate constant within plastocyanin is very much the lesser (it may be zero). See Prob. 16. In striking contrast, small oxidants are able to attach to surface patches on plastocyanin which are more favorably disposed with respect to electron transfer to and from the Cu, which is about 14 A distant. It can be assessed that internal electron transfer rate constants are =30s for Co(phen)3+, >5 x 10 s for Ru(NH3)jimid and 3.0 x 10 s for Ru(bpy)3 , Refs. 119 and 129. In the last case the excited state Ru(bpy)3 is believed to bind about 10-12 A from the Cu center. Electron transfer occurs both from this remote site as well as by attack of Ru(bpy)j+ adjacent to the Cu site. At high protein concentration, electron transfer occurs solely through the remote pathway. 

Copper ions in the (I) and (II) oxidation states are biologically important. Basically, three different types of copper centre are shown. Blue of type I copper occurs in the blue electron carrying proteins such as stellacyanin, plastocyanin and azurin. There is also non-blue or type II copper and a type III copper centre that is non-detectable by EPR, apparently containing a pair of contiguous copper atoms. Tyrosinase is a type III protein that is not detected EPR because of antiferromagnetism of a pair of copper in it [137],... 

The blue copper proteins azurin, plastocyanin, stellacyanin, and umecyanin incorporate Cu bound to a combination of N/thiolate/thioether ligands. An important feature of these metalloenzymes is the facile copper(II)/(I) couple that these species exhibit, which is linked to the highly strained, asymmetric coordination geometry at the metal center. The synthesis of model complexes for these so-called Type 1 copper proteins has been reviewed. ... 

The group of small plant proteins, azurin, stellacyanin, and plasto-cyanin, appear to be electron transfer proteins. They are listed because they share a type of copper site with the intensely blue representatives of the first class like laccase and ascorbate oxidase. The evidence that they participate in plant electron transfer chains remains circumstantial. Azurins, for example, purify along with well-known respiratory chain proteins like cytochrome C. A good deal of evidence exists, however, that plastocyanin is important in the photoreduction of NADP see below). 

Covalent attachment has also been exploited for protein incorporation of non-native redox active cofactors. A photosensitive rhodium complex has been covalently attached to a cysteine near the heme of cytochrome c (67). The heme of these cytochrome c bioconjugates was photoreducible, which makes it possible for these artificial proteins to be potentially useful in electronic devices. The covalent anchoring, via a disulfide bond, of a redox active ferrocene cofactor has been demonstrated in the protein azurin (68). Not only did conjugation to the protein provide the cofactor with increased water stability and solubility, but it also provided, by means of mutagenesis, a means of tuning the reduction potential of the cofactor. The protein-aided transition of organometallic species into aqueous solution via increased solubility, stability and tuning are important benefits to the construction of artificial metalloproteins. 

Moreover, other effects are as important as the ligands. The dielectric properties of the protein matrix are very different from those of water. It has often been argued that it behaves as a medium with a low dielectric constant (around 4 compared to 80 in water) [47,123,124]. Figure 11 shows that this gives rise to a very prominent change in the reduction potential of a blue-copper site [45]. It increases by 0.8-1 V as the site is moved from water solution to the centre of a protein with a radius of 1.5 nm (like plastocyanin) or 3.0 nm (like an azurin tet-ramer). It can also be seen that it is not necessary to move the site to the centre of the protein to get a full effect. Already at the surface of the protein, 80% of the maximum effect is seen, and when the site is 0.5 nm from the surface (as is typi-... 

The luminescence properties of polynucleic acids and proteins have been reviewed in detail previously. However recent time-resolved studies have important implications for existing concepts of the excited states of biopolymers. The readily observable luminescence from proteins at room temperature has made these systems particularly attractive to study. The fluorescence properties of the arrxnatic amino acid zwitter-ions which determine the emission from proteins are summarised in Table 11. The wide range of emission maxima observed in proteins (ranging from 308 nm in azurin to 342 nm in lysozyme) has been exfdained by many authors to reflect the different environments of the tryptophan residues in the proteins However, it is now... 

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.)...

Summarizing the results of studies of intramolecular ET in azurins, it is important to stress that in all probability, the induced ET between the disulfide radical and the blue copper(II) center is not part of any physiological function, and the role of the disulfide is stmctural, solely. Still, azurin has turned out to be a very useful model system for examination of different parameters controlling LRET rates in proteins. The impact of specific stmctural changes introduced by single-site mutations has been studied in order to obtain a better understanding... 

Perhaps the three most important redox systems in bioinorganic chemistry are (I) high spin, tetrahedral Fe(II)/Fe(III) in rubredoxin, ferredoxin, etc. (2) low spin, octahedral Fe(II)/Fe(IlI) in the cytochromes and (3) pseudotetrahedral Cu(I)/Cu(II) in the blue copper proteins, such as slellacyanin, plastocyanin, and azurin. Gray94 has pointed out that these redox centers are ideally adapted for electron exchange in that no change in spin state occurs. Thus there is little or no movement of the ligands—the Franck-Condon activation barriers will be small. 

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