Blue copper proteins characterization

Copper proteins have been classified according to their spectroscopic properties (Malkin and Malmstrbm, 1970 Fee, 1975) as type I, II, or III. Type I blue copper proteins are characterized by an extraordinarily intense absorption near 600 nm and by unusually small hyperfine coupling constants for the paramagnetic [oxidized Cu(II)] form of the protein. 

The significant experimental effort put into characterizing the blue copper proteins has made them attractive targets for computation (161-163). These computational studies have provided... 

The type-1 blue copper proteins act as electron carriers azurin, plastocyanin, stellacyanin, umecyanin e.g. They are characterized by a rather strong LMCT (ligand to metal charge transfer) band near 600 nm and by small hyperline coupling constants A in EPR. Copper is bound to two imidazole groups of histidine and to two... 

The first class is cupredoxins—single-domain blue copper proteins composed of only one BCB domain. These proteins include plastocyanin, azurin, pseudoazurin, amicyanin, auracyanins, rusticyanin, halocyanin, and sulfocyanin (see Section IV). Plantacyanin of the phytocyanin family (Section V), subunit II of the cytochrome c oxidase, and the recently characterized nitrosocyanin also fall into this class. The last two are single BCB domain polypeptides closely related structurally to cupredoxins, but harboring, respectively, a binuclear copper site known as CuA and a novel type of copper-binding site called red (see Sections IX and X). 

Blue copper proteins have been purified and biochemically characterized from Archaea, Bacteria, and Eukarya. Such ubiquitous distribution suggests an important ancient role. A survey of sequence databases reveals genes encoding blue copper proteins that display characteristics often quite different from those of well-studied canonical (traditional) blue copper proteins. For example, there are modular proteins where the domains that bind type 1 copper are fused with structurally distinct and evolutionarily unrelated sequence motifs (Figure 1). While these additional domains do not usually contribute directly to the ftmction of a blue copper protein, they do so indirectly by facilitating protein translocation to a specific cellular compartment. Together, these blue copper proteins can be combined into a large superfamily which can be subdivided into three classes as described below. 

The active sites of oxidized blue copper proteins are characterized by unique features relative to those of normal Cu(II) complexes. These features include an intense absorption... 

The type I copper sites function as electron transfer centers in the blue copper proteins and in multicopper enzymes, particularly oxidases (33). They are characterized by their intense blue color, their unusually small A values, and their very positive redox potentials (Table II). X-ray crystal structures of several blue copper proteins have been determined, notably plastocyanin (34), azurin (35), cucumber basic blue protein (36), and pseudoazurin (37). The active site structures show marked similarities but also distinct differences (Fig. 8). 

Some of the first protein systems where pulse radiolysis was used to help determine mechanism were those of blue copper proteins. These are proteins that are blue in solution and contain what are known as type (I) and type (2) copper centers. Two of the most well-known and well-characterized examples of these are azurin and cytochrome c. It was the studies of these systems that opened up the field of long-distance electron transfer in proteins and, by using the protein structure as a framework for electron transfer through space and through bonds, allowed for the development of a broad theoretical basis and many fascinating experiments on long-range electron transfer. Here, I will limit the discussion to electron transfer studies in azurin as illuminated by pulse radiolysis studies. ... 

Cobley JG, Haddock BA (1975). The respiratory chain of Thiobacillus ferrooxidans The reduction of cytochromes by Fe and the prehminary characterization of rasticyanirr, a novel blue copper protein. FENS Letters 60 29-33... 

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] ...

Cobley JG (1984) Oxidation of nitrite and formate in Nitrobacter membrane preparations evidence that both reactions are catalyzed by the same enzyme. In Strohl WR, Tuovinen OH (eds) Microbial chemoautotrophy. Ohio State University Press, Columbus, pp 169-183 Cobley JG, Haddock BA (1975) The respiratory chain of Thiobacillus ferrooxidans reduction of cytochrome by Fe2+ and preliminary characterization of rusticyanin, a novel blue copper protein. FEBS Eett 60 29-33... 

The structural studies on amicyanin from P. versutus show that the protein consists of nine B-strands that form into two antiparallel /3-sheets, giving the molecule an overall structural motif known as a /3-sandwich (see Fig. 4). This overall topology is very similar to that of other structurally characterized cupredoxins, with the homology to plastocyanin being greatest (87). The only notable difference between amicyanin and most other cupredoxin structures is the presence of a 21-residue N-tereminal extension that forms an extra /8-strand in the structure. The recently published structural studies (95, 96) on the type 1 blue copper protein rusticyanin show that it, too, possesses an... 

Blue (Type 1) copper proteins are found widely in nature. Typical examples are plasiocyaiiin (MW 10,500) and ascorbale oxidase (MW 150,000) which contain one and eight Cu atoms per protein, respectively. The former serves as a component of the electron transfer chain in plant photosynthesis while the latter is an enzyme involved in the oxidation of ascorbic acid. The oxidized form is characterized by intense blue color due to electronic absorption near 600 nm. In addition, blue copper proteins exhibit unusual properties such as extremely small hyperfine splitting constants (0.003 0.009 cm" ) in ESR spectra and rather high redox potential (4-0.2 0.8 V) compared to the Cu(ll)/Cu(I) couple in aqueous solution. 

Ubbink M, Worrall JA, Canters GW et al (2002) Paramagnetic resonance of biological metal centers. Annu Rev Biophys Biomol Struct 31 393-422 119. Hansen DF, Led JJ (2006) Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR. Proc Natl Acad Sci USA 103 1738-1743 Donaldson LW, Skrynnikov NR, Choy WY et al (2001) Stmctural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy. J Am Chem Soc 123 9843-9847... 

Type 2 Cu2+. This form of Cu is present in all the blue multi-copper oxidases. It is characterized by lacking sufficient optical absorption to be observed above that of the other Cu-chromophores in these molecules. Consequently it is sometimes referred to as the colorless Cu. Further, its EPR spectrum is similar to those exhibited by most small Cu2+ complexes. However, its presence is essential to the functioning of the multi-copper oxidases, and it has very unique chemical properties which distinguish it from Cu + bound to the non-blue Cu proteins. The T5q)e 2 designation should therefore be reserved for classification of the types of Cu2+ sites observed in the blue multi-copper oxidases, and it should not be used to classify the binding sites of non-blue copper proteins which have distinctly different chemical behavior. Thus, for example, any purported analogies between... 

Blue copper proteins transfer electrons between various biological systems, e.g., between the two photosystems in photosynthesis (plastocyanin). They are characterized by a number of unusual properties, viz., a bright blue color, an unusually high reduction potential, and distinctive... 

LMCT bands characterize the visible spectrum of this and other thioether complexes of Cu(II). Resonance Raman spctroscopy on [Cu(14S4)] and copper(II) complexes of other crown thioethers extended the parallel with the blue copper proteins, and assigned the Cu-S stretch to vibrations around 250 cm [179]. 

In other systems, W-band EPR and X-band Fe-ENDOR have been used to characterize a new stable diiron centre found in the E. coli RNR mutant R2-Y122H. The spectrum was fully resolved at 94 GHz and evidence was given to support the view that the centre was a Fe Fe " centre with a strongly coupled radical. Pulsed EPR at 95 GHz has been used to characterize the azurin mutant M121H, a blue copper protein, in a single-crystal study which allowed the complete g tensor to be determined relative to the molecular axes. ... 

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