Blue copper proteins crystal structures

Interestingly, in all blue copper protein crystal structures so far determined it is the of the imidazole that coordinates to the Cu. Since a proton attached at the is more acidic (pKg values 5.0-7.5) than the covalently bound H atom at N, this observation is of considerable interest and will be returned to. 

Guss, J. M., et al. (1988). Phase determination by multiple-wavelength x-ray diffraction crystal structure of a basic blue copper protein from cucumbers. Science 241, 806-811. 

The EPR spectrum of the blue copper protein plastocyanin (Figure 3C) has gu > g > 2.00, and thus the copper site must have a dx2 y2 ground state. First, we are interested in determining the orientation of the dx2 y2 orbital relative to the distorted tetrahedral geometry observed in the protein crystal structure. Single crystal EPR spectroscopy allowed us to obtain this orientation and located the unique (i.e., z) direction in this distorted site (29). Plastocyanin crystallizes in an orthorhombic space group with four symmetry related molecules in the unit cell. The orientation of the plastocyanin copper sites in the unit cell are shown in... 

Having summarized the types of transitions which are likely to contribute to the absorption spectrum of tetragonal copper (II), it is important to consider methods which probe these transitions and maximize the amount of information which can be ascertained. Linear dichroism13) provides the specific orientation of the electric dipole transition moment (Eq. 3) with respect to the copper site. This method is quite valuable in assigning an absorption spectrum (as discussed for Blue Copper proteins in Sect. Ill) but requires a structurally defined site oriented in a single crystal. A number of other physical... 

Crystal structures of three phytocyanins are currently available. Two are for plantacyanins, from cucumber (also known as cucumber basic protein) (Guss et al., 1988, 1996) and from spinach (Einsle et al., 2000), and one is for the recombinant BCB domain of cucumber stella-cyanin (Hart et al., 1996). The three proteins display folding topology identical to one another, suggesting that phytocyanins fold into a uniform structure, which can be designated as a phytocyanin fold. As a historical note, the crystallization of the cucumber basic protein and its preliminary crystallographic data were reported in 1977, before any structure of a blue copper protein was available (Colman et al., 1977). However, the structure was solved in 1988 only by application of the then newly... 

The subunits are arranged in the crystals as homotetramers with D2 symmetry. The structure of a subunit is shown schematically in Fig. 1 (87). Each subunit of 552 amino acid residues has a globular shape with dimensions of 49 x 53 x 65 A and is built up of three domains arranged sequentially on the polypeptide chain, tightly associated in space. The folding of all three domains is of a similar jS-barrel type. It is distantly related to the small blue copper proteins, for example, plastocyanin or azurin. Domain 1 is made up of two four-stranded jS-sheets (Fig. lb), which form a jS-sandwich structure. Domain 2 consists of a six-stranded and a five-stranded jS-sheet. Finally, domain 3 is built up of two five-stranded jS-sheets that form the jS-barrel structure and a four-stranded j8-sheet that is an extension at the N-terminal part of this domain. A topology diagram of ascorbate oxidase for all three domains and of the related structures of plastocyanin and azurin is shown in Fig. 2. Ascorbate oxidase contains seven helices. Domain 2 has a short a-helix (aj) between strands A2 and B2. Domain 3 exhibits five short a-helices that are located between strands D3 and E3 (a ), 13 and J3 (a ), and M3 and N3 (a ) as well as at the C terminus (ag and a ). Helix 2 connects domain 2 and domain 3. 

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). 

Three-dimensional structures. The TPQ-con-taining amine oxidase from E. coU is a dimer of 727-residue subunits with one molecule of TPQ at position 402 in each subunit. 7458 Methylamine dehydrogenase is also a large dimeric protein of two large 46.7-kDa subunits and two small 15.5-kDa subunits. Each large subunit contains a TTQ cofactor Reduced TTQ is reoxidized by the 12.5-kDa blue copper protein amicyanin. Crystal structures have been determined for complexes of methylamine dehydrogenase with amicyanin and of these two proteins with a third protein, a small bacterial cytochrome... 

The first crystal structure information on a blue copper protein, for poplar plastocyanin in the Cu(II) state, was published in 1978 (2, 3). Since then, the Cu(I) state and related apo and Hg(II) substituted forms (5, 6), the green algal plastocyanin from Enteromorpha prolifera [Cu(II)] (7), azurin from Alcaligenes denitrificans [Cu(II) and Cu(D] (8, 9), azurin from Pseudomonas aeruginosa [Cu(II)] (10, 11), as well as pseudoazurin from Alcaligenes faecalis S-6 (12), and the cucumber basic protein, both in the Cu(II) state, have been published (13), making this one of the best-documented class of proteins. In addition, information as to three-dimensional structure in solution has been obtained from two-dimensional NMR studies on French bean and Scenedesmus obliquus plastocyanins (14,15). This review is concerned in the main with the active site chemistry. Other recent reviews are listed (16-20). 

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] ...

Single-crystal structural data have provided valuable information about blue copper proteins containing Type 1 Cu centres. Figure 28.10a shows a representation of the folded protein chain of spinach plastocyanin. The Cu(II) centre lies within a pocket in the chain, bound by a Cys, a Met and two His residues (Figure 28.10b) the S(Met) atom is significantly further away from the Cu(II) centre than is S(Cys). Figure 28.10c shows the backbone of the protein chain in azurin isolated from the bacterium Pseudomonas putida. The coordination environment of the Cu(II) centre resembles that in plastocyanin with Cu—S(Met) > Cu—S(Cys), but in addition, an O atom from an adjacent Gly residue is involved in a weak coordinate interaction (Figure 28.10d). Structural... 

Single-crystal structural data have provided valuable information about blue copper proteins containing Type 1 Cu centres. Figure 29.11a shows a representation of the... 

It is known, from the crystal structures of some natural proteins, such as plastocyanin [1] and azurin [2], that the so-called blue-copper proteins. Type 1, have one copper(II) ion, with two Cu-S and two Cu-N coordinate bonds, in their active sites. 

The crystal structures of blue copper proteins show that the T1 site is in a trigonally distorted tetrahedral environment (Fig. la) [34—40]. The trigonal plane has a very short ( 2.1 A) Cu-S(Cys) and two typical ( 2.0 A) Cu-N His bonds. An additional axial thioether ligand is usually present with a long Cu-S(Met) bond ( 2.8 A), although the Met residue is replaced by a non-ligating one, such as Lys or Phe, in some tree and fungal laccases [41-44]. 

Table 1 Comparison of the Optimized Geometry of Cu(imidazole)3(SCH3)(S(CH3)2)+ and the Crystal Structures of Oxidized Blue Copper Protein.s...

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