Azurin function

It is interesting to speculate why nitrite reductase has its type I coppers in domains 1, whereas in hCP the mononuclear copper binding sites are retained in the domains 2,4, and 6 where they are comparatively buried in the protein. One possible reason can be related to the difference in functions of the two proteins. NR has to interact with a relatively large pseudo-azurin macromolecule in order for electron transfer to take place,... 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

Since the rate constants of bimolecular diffusion-limited reactions in isotropic solution are proportional to T/ these data testify to the fact that the kt values are linearly dependent on the diffusion coefficient D in water, irrespective of whether the fluorophores are present on the surface of the macromolecule (human serum albumin, cobra neurotoxins, proteins A and B of the neurotoxic complex of venom) or are localized within the protein matrix (ribonuclease C2, azurin, L-asparaginase).1 36 1 The linear dependence of the functions l/Q and l/xF on x/t] indicates that the mobility of protein structures is correlated with the motions of solvent molecules, and this correlation results in similar mechanisms of quenching for both surface and interior sites of the macromolecule. 

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... 

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... 

Fig. 5.40. (A) H NMR spectra at 298 K of oxidized spinach plastocyanin at 800 MHz (adapted from [117]). (B) Far downfield region of the H NMR spectra of oxidized (i) P. aeruginosa azurin, (ii) spinach plastocyanin and (iii) cucumber stellacyanin containing signals not observable in direct detection (adapted from [198]). The positions and line widths of the signals were obtained using saturation transfer experiments by plotting the intensity of the respective exchange connectivities with the reduced species as a function of the decoupler irradiation frequency.

The azurin structural gene has been cloned and expressed in large amounts in E. coli (Karlson et al., 1989). The copper site in azurin is distorted-planar with two additional weakly interacting groups in axial positions. Site directed mutagenesis has been used to exchange His-46 for Met, Cys-112 for His and Met-121 for all other amino acids, in order to study the relationship between structure and function and to determine the prerequisites for the blue copper site. The Met-121 mutant proteins were characterised by their absorption and ESR spectra (Karlson et al., 1991). At low pH, all mutants exhibit the characteristics of the blue (Type I) copper protein, indicating... 

In this chapter, we overview first some recent examples of interfacial electrochemical ET of composite metalloproteins where molecular mechanistic detail has in some way been achieved. We discuss next some theoretical issues regarding in situ STM of large molecules, where resonance or environmentally activated tunnel channels are opened by the redox metal centre. This is followed by an overview of some recent achievements in the area of in situ STM/AFM of the single-metal proteins cytochrome c and azurin on polycrystalline and single-crystal platinum and gold surfaces. Such an integrated approach offers new perspectives for experimental and theoretical characterization of metalloproteins at solid surfaces in contact with the natural aqueous medium for metalloprotein function. 

The function of the electron-mediating proteins which contain a single redox active site (e.g., rubredoxin, azurins, flavodoxins, plasto-cyanins) is mainly related to the first aspect. Still, the pronounced specificity encountered in their function in biological energy conversion processes indicates that their redox center, often a transition metal ion, is embedded in an evolutionarily optimized polypeptide envelope. The... 

We have used a range of different physical and chemical approaches in the effort to better understand how the different blue copper proteins function. With the relatively simpler, electron-mediating proteins like azurin, the ultraviolet chromophores were shown to be informative in terms of copper-protein interactions. These proteins are also a useful system for detailed examination of the electron transfer pathways to and from their single copper site. 

Structure-function roles have been suggested for unique tryptophan residues in other copper proteins as well (44,45, 46). Moreover, the single tryptophan that is quenched by including the copper atom in azurin is apparently not in contact with the indole ring, as evidenced by metal replacement and phosphorescence results (45, 46). 

Surprisingly, its biological redox partners remain largely unknown. It has been implicated in anaerobic nitrite respiration and it has been shown that azurin can donate electrons to nitrite reductase, a function that is proposed to be carried out by another cupredoxin, pseudoazurin (see Section IV, E). On the other hand, azurin is not an inducible protein and denitrifying bacteria express azurin constitutively under aerobic conditions. 

Figure 4 Cd PAC data—example, (a) Experimentally determined perturbation function that contains the information on the local structure and dynamics at the PAC probe site (data points with error bars and fit (fiiU line)).(b) Fourier transform of the experimental data (red) and of the fit (blue). This dataset was recorded for the cadnumn-substituted blue copper protein azurin. (After Figure 6 in )...

Substitution of amino acid residues at the metal center by site-directed mutagenesis can alter the geometry and properties of the active site, thus allowing elucidation of the minimal requirements for the proper functioning of the metalloprotein. As an example. Figure 21 and Table 5 show the results of RR scattering from a series of P. aeruginosa azurins in which the active site Met 121 has... 

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