Azurin metalloprotein

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... 

Direct electron transfer has also been achieved with many metalloproteins such as cytochrome C, horseradish peroxidase, microperoxidase (MP-11), myoglobin, hemoglobin, catalase, azurin, and so on, immobilized on different CNT-modified electrodes [45, 61, 144—153]. 

The mechanism of the regulation of electron transfer in metalloproteins has been investigated 61) and two relevant examples have been discussed in the first one the molecular mechanism controlling the electron transfer reactions is restricted to the immediate chemical environment of the metal center (azurin), while in the second one it involves a conformational transition of the whole quaternary structure of the enzyme. The power of the kinetic approach in detecting significant intermediates was emphasized 6t>. The Cu metal complex site of azurin has a distorted tetrahedral... 

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. 

IN SITU AND AFM OF SINGLE-METAL METALLOPROTEINS HORSE HEART CYTOCHROME C AND PSEUDOMONAS AERUGINOSA AZURIN... 

Stellacyanin, the plastocyanins, and the azurins are the most widely studied copper-containing metalloproteins of the next active-site class, the Blue Copper sites. These proteins, which generally appear to be involved in redox chemistry, have quite unique spectral features32,33). The potential for complementary interaction between inorganic spectroscopy and protein crystallography is well demonstrated by the roles that they have played in generating fairly detailed geometric and electronic structural pictures of the Blue Copper metal centers. 

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. ... 

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... 

Metalloproteins, where the active site includes one or more metals, represent a very different class of proteins than those discussed above. The particular kinds of metalloproteins discussed here are those where the metal is redox active and represents a functional and not structural component of the system. Many mechanistic studies of metalloproteins have been carried out using radiation chemistry in the past 50 years. Two different ways of using radiation chemistry to query mechanisms will be illustrated here. The first, as described in the earliest of these studies using blue copper proteins such as azurin, involves using pulse radiolysis to change an oxidation state and thus... 

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. 

Recently DFM, TFM and other methionine analogues were utilized to explore the contribution that methionine makes to the reduction-oxidation potential of the P. aeruginosa metalloprotein azurin [26, 45], Azurin is a copper metalloprotein that is involved in... 

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] ...

A model of the structure of the blue single copper centre metalloprotein Pseudomonas aeruginosa azurin, is shown in Fig. 7-16. 

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