Protein Azurin

Studies of ferredoxin [152] and a photosynthetic reaction center [151] have analyzed further the protein s dielectric response to electron transfer, and the protein s role in reducing the reorganization free energy so as to accelerate electron transfer [152], Different force fields were compared, including a polarizable and a non-polarizable force field [151]. One very recent study considered the effect of point mutations on the redox potential of the protein azurin [56]. Structural relaxation along the simulated reaction pathway was analyzed in detail. Similar to the Cyt c study above, several slow relaxation channels were found, which limited the ability to obtain very precise free energy estimates. Only semiquantitative values were... 

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... 

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 reduction of the Cu(II) protein, azurin, with excess dithionite, 8204 was monitored at 625 nm (first-order loss of azurin) at pH 9.2 and 25 °C with the following results... 

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... 

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. ... 

Figure 15 Structures of type 1 copper protein, Azurin (a) and Type 2 copper protein, Copper, zinc superoxidase dismutase (b). (Reprinted with permission from Ref. 23. 2001 the American Chemical Society)...

Metalloproteins that have been overexpressed may need to be reconstituted with their native metal(s). For example, overexpression of the blue copper protein azurin results in a mixture of apo- and Zn forms. When a gene of an uncharacterized metalloprotein is expressed, care must be taken to assure that the identity of the native metal is determined through studies of native protein. Overexpression of heme proteins typically requires reconstitution with hemin. In the case of c-type heme, the heme must be attached covalently to the polypeptide with the assistance of cellular machinery. Wide success in expressing a range of c-heme containing proteins has been achieved by expression of both the structural cytochrome gene and the apparatus for protein maturation. ... 

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 )...

Brill, A. S. Conformational distribution and vibronic coupling in the blue copper-containing protein azurin, in Tunneling in biological systems (eds. Chance, B. et al.) p. 561, New York, Academic Press 1979... 

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). 

Figure 3 Isostructural substitution of cysteine and methionine with unnatural analogs in the metal binding site of the electron transfer protein azurin. (Adapted from Reference 36, p. 15608, copyright 2006 with permission from The American Chemical Society.)...

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. 

Direct electrochemistry has also been used (72-78) to couple the electrode reactions to enzymes for which the redox proteins act as cofactors. In the studies, the chemically reduced or oxidized enzyme was turned over through the use of a protein and its electrode reaction as the source or sink of electrons. In the first report (72, 73) of such application, the electrochemical reduction of horse heart cjd,ochrome c was coupled to the reduction of dioxygen in the presence of Pseudomonas aeruginosa nitrite reductase/cytochrome oxidase via the redox proteins, azurin and cytochrome C551. The system corresponded to an oxygen electrode in which the four-electron reduction of dioxygen was achieved relatively fast at pH 7. 

Chi, Q.J., Zhang, J.D., Andersen, J.E.T., and Ulstrup, J. (2001) Ordered assembly and controlled electron transfer of the blue copper protein azurin at gold (111) single-crystal substrates. Journal of Physical Chemistry B, 105,... 

Interfadal Electrochemical Organization and Electron Transfer of the Blue Copper Protein Azurin - a Nanoscale Bioelectrochemical Paradigm... 

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