Copper sites in protein

Based on spectroscopic properties, mainly electron paramagnetic resonance (EPR), the active sites of copper proteins have been classified into three groups, types I, II, and III. This nomenclature was originally applied to blue oxidases to distinguish the four copper ions contained in these proteins. The original classification has been extended to the copper sites of other proteins. The recent increase in structural information on the copper sites in proteins has, however, revealed greater diversity in the type of copper site. For instance, the type III and type II sites in ascorbate oxidase are in close proximity, forming a trinuclear site, in which all three copper ions are essential for the reactivity. Some proteins, once believed to contain a copper site with normal spectroscopic properties, and thus referred as type II, have been shown to contain copper coordinated by an unusual side chain. Therefore, in this review, new nomenclature is used to classify the copper sites more precisely with respect to their structural features and spectroscopic properties. The definitions are as follows ... 

A considerable number of crystal structures of type I copper sites in proteins are now available, so there may be no particular advantage in the synthetic model approach to prove the coordination structure of type I. Yet, inorganic chemists still have an opportunity to utilize the spectroscopic and structural bases established by model studies to understand the precise electronic structure of type I copper. One should keep in mind that the generally accepted interpretation derived from spectroscopic and theoretical studies on the proteins (47-49) has not been definitely proved experimentally. A systematic comparison of a series of copper(II) thiolate complexes having an unusual distorted coordination structure is required for a conclusive description of the electronic structure of the type I copper. The synthetic approach is ultimately the most adequate way to clarify how the ligand donors and geometry affect the electronic property and function of type I copper as an electron transfer center. 

Canters GW, Gilardi G (1993) Engineering type 1 copper sites in proteins. EEBS Lett 325 39 18... 

Li H, Webb SP, Ivanic J, Jensen JH (2004) Determinants of the relative reduction potentials of type-1 copper sites in proteins. J Am Chem Soc 126(25) 8010-8019... 

Lancaster KM, Sproules S, Palmer JH, Richards JH, Gray HB (2010) Outer-sphere effects on reduction potentials of copper sites in proteins the curious case of high potential type 2 C112D/M121E Pseudomonas aeruginosa azurin. J Am Chem Soc 132 14590-14595... 

Copper sites in proteins have traditionally been classified into three classes blue type 1 sites (present in the blue copper proteins), normal type 2 sites (tetragonal mononuclear copper sites), and type 3 (spin-coupled pairs of copper ions). The type 1 sites have been further classified as axial or rhombic depending on their EPR (and other spectroscopic) characteristics." Plastocyanin (axial) and nitrite reductase (rhombic) are typical examples of type 1 proteins. Proteins with properties intermediate between those of type 1 and type 2 sites have been termed type 1.5." ... 

For biomolecular S = 1/2 systems subject to central hyperfine interaction the intermediate-field situation (B S S I) is not likely to occur unless the micro-wave frequency is lowered to L-band values. When v = 1 GHz, the resonance field for g = 2 is at B = 357 gauss. Some Cu(II) sites in proteins have Az 200 gauss, and this would certainly define L-band EPR as a situation in which the electronic Zeeman interaction is comparable in strength to that of the copper hyperfine interaction. No relevant literature appears to be available on the subject. An early measurement of the Cun(H20)6 reference system (cf. Figure 3.4) in L-band, and its simulation using the axial form of Equation 5.18 indicated that for this system... 

Norris, G. E., Anderson, B. F., and Baker, E. N. (1986). Blue copper proteins. The copper site in azurin from Alcaligenes denitr cans.J. Am. Chem. Soc. 108, 2784-2785. 

Fig. 5. (a) Copper site in cucumber basic blue protein (Cbp). (b) Ribbon drawing of the Cbp backbone, (c and d) Schematic of Cbp topology. 

One of the questions surrounding the mechanism of tyrosinase concerns the initial site of attack. As a control, LFMD simulations of a model for the sTy active site, Meim6 (Fig. 28), give identical behavior for each Cu center consistent with its symmetry. In contrast, the LFMD simulations clearly distinguish the two copper sites in the sTy enzyme which must result from the protein environment (Fig. 29). 

Type 2 Copper Sites. - Mononuclear proteins with Cu(II) in a nearly... 

Some progress has been made in the characterization of the redox centres. The presence of a potential type 1 blue copper site in subunit I is in accord with EXAFS data that have demonstrated Cu—S interactions, in particular the possibility of two sulfur atoms bound to CuA.1309 It seems possible therefore that CuA is a type 1 copper, typical of copper electron-transfer proteins. The nature of CuB is less certain ESR parameters indicate that CuB is similar to type 3 copper, which occurs pairwise in copper oxidases as the 02-binding site. 

For the most part, adequate copper is received in diet and widespread human deficiencies do not occur, but deficiencies may arise because of antagonists. The metals Cd, Hg, Ag and Zn interfere with copper metabolism, probably by competing for copper-binding sites in proteins. Ascorbic acid depresses intestinal absorption of copper56 (in contrast to iron). Some proteins in the diet adversely affect utilization of copper. The sulfide ion is a well known inhibitor of copper absorption, since it forms copper(II) sulfide which is insoluble.56... 

Copper(II) sites in proteins can be classified into three types based on their spectral properties. The blue (Type I) copper proteins are characterised by a visible absorption... 

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... 

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... 

Several copper-containing NiRs have been identified, but the most extensive structural and mechanistic studies have focused on the enzyme from Achromobacter cycloclastes (17-25). A 2.3-A resolution X-ray crystal structure for this NiR in its oxidized form at pH 5.2 has been reported (17), and a representation of the active site is shown in Figure 1. Each monomer in the trimeric protein contains two copper ions, one of which (Cu-1) is ligated to a cysteine, a methionine, and two histidine residues in a geometry similar to that of type 1 copper centers in proteins such as plastocyanin (26). The second type 2 copper ion in NiR (Cu-2) is only 12.5-A distant from the first and is bound to three histidine imidazoles (two from one monomer, the third from an associated subunit) and a fourth small ligand in an unusual tetrahedral arrangement. The... 

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. 

When the coupled binuclear copper site in the protein becomes structurally characterized by x-ray crystallography, single-crystal spectroscopy should provide a more detailed picture of its electronic structure and bonding, as has been achieved for the Blue Copper site described in the previous section. 

Included in this latter set is a double mutant protein, a T1D/T2D protein that lacks both the type 1 and the type 2 copper atoms. Only the type 3 binuclear cluster contributes to the nonprotein absorbance in this protein, demonstrating that the shoulder at 330 nm is due to this cluster. This cluster also contributes a broad absorbance centered at 720 nm as the spectrum of this double mutant demonstrates. The absorbance of the wild-type protein at 608 nm is clearly due to the type 1 Cu(II) since it is seen only in protein forms that possess this site. The spin Hamiltonian and absorbance values for the copper sites in FetSp are summarized in Table I. Additional properties of these copper site-depleted Fet3p mutant proteins are discussed below. 

Lerch, K., and German, U. A. (1988). Evolutionary relationships among copper proteins containing coupled binuclear copper sites. In Oxidases and Related Redox Systems (K. T. S., Mason, H. S., and Morrison, M., Eds.), pp. 331-348. A. R. Liss, New York. 

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