Azurin copper site

Antholine, W.E., Hanna, P.M., and McMillan, D.R. 1993. Low frequency EPR of Pseudomonas aeruginosa azurin analysis of ligand superhyperfine structure from a type 1 copper site. Biophysical Journal 64 267-272. 

As an example on the relationship between proton relaxivity, electron relaxation and coordination environment, we report the case of azurin and its mutants. The relaxivity of wild type azurin is very low (Fig. 6) due to a solvent-protected copper site, the closest water being found at a distance of more than 5 A from the copper ion. The fit, performed with the Florence NMRD program, able to take into account the presence of hyperfine coupling with the metal nucleus (Ay = 62 x 0 cm , see Section II.B) indicates Tie values of 8 X 10 s. Although the metal site in azurin is relatively inaccessible, several mutations of the copper ligands open it up to the solvent. The H NMRD profiles indicate the presence of water coordination for the... 

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. 2. Copper site in azurin. In this and subsequent figures the following conventions have been used, (a) The copper site is generally an enlargement of (b). The copper site is a dotted sphere, the ligand residues are represented by bonds connecting atoms in the side chain, and, where possible, the atoms bonded to the copper atom are identified by atom type. Ribbons represent portions of the backbone structure near the copper. In (b) of each figure, the main-chain polypeptide is represented by a ribbon fit to the main-chain coordinates, and the amino and carboxy termini are indicated by N and C, respectively, (c) A schematic version drawn from (b). Solid arrows represent main-chain regions participating in the /3 sheet roughly above the plane of the paper, while dotted or light arrows are the...

Several metals bind at the vacant copper site of apoazurin, including Ni11 and Co11,928 and, less usually, gold.929 There were some differences between the H NMR spectra of the copper and gold azurins, notably in the methionine S—Me region. Au1 prefers linear coordination. 

The crystal structure of cucumber basic blue protein has now been refined to 3.0 A resolution (Adman, 1985). The protein consists of eight strands, only five of which form a P-sandwich and the protein has less P-sheet character than plastocyanin or azurin. The ligands to copper are provided by the side chains of His-39, Cys-79, His-84 and Met-89. The copper site has the N2SS coordination seen in plastocyanin. The imidazole rings of the His-39 and His-94 residues are exposed to the solvent providing a likely entry site for electon transfer to the copper centre. 

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 additional effects in the aromatic region of the difference spectrum (250-300 nm) are probably caused by aromatic transitions which are influenced by the redox state of the copper. The shoulder at 270 nm, which occurs in all three proteins, could result from an increase in tyrosine absorption. In this context, it is interesting to recall that Tyr 108 (azurin numbering), which is relatively close to the proposed copper ligands Cys 112 and Met 121, is completely invariant both in azurin and plastocyanin and may therefore be an obligatory constituent of the copper site. 

We have used a range of different physical and chemical approaches in the effort to better understand how the different blue copper proteins function. With the relatively simpler, electron-mediating proteins like azurin, the ultraviolet chromophores were shown to be informative in terms of copper-protein interactions. These proteins are also a useful system for detailed examination of the electron transfer pathways to and from their single copper site. 

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. 

In order to understand the charge transfer features of the Blue Copper site, the variable-temperature optical absorption, room-temperature circular dichroism, and magnetic circular dichroism spectra of plastocyanin, stellacyanin, and azurin were studied355. As can be seen for plastocyanin in Fig. 12, the relative intensities (and signs, in the case of CD and MCD) of these transitions vary among the different types of spectra. This is a result of the difference in selection rules for absorption, CD, and MCD spectra, as mentioned in the Introduction. A careful comparison of the three types of spectra and the absorption bandshape temperature dependence (see moment analysis in Ref. 35, pp. 176-177)... 

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). 

Intriguingly, the blue copper sites, especiaUy those with a carbonyl oxygen at the axial coordination position, display high affinity for Zn + ions. Mutants in which the Met is replaced by Gin or Glu preferentiaUy bind Zn + when expressed in heterologous systems, e.g., Escherichia coli. Examples include azurin, amicyanin, nitrite reductase, and possibly also plastocyanin (Diederix et al., 2000 Hibino et al., 1995 Murphy et al., 1995 Nar et al., 1992a Romero et al., 1993). In the case of azurin it has been shown that both wild-type and the Met—Gin mutant have the same affinity for both Zn +and Cu + (Romero ci a/., 1993). In addition, EXAFS studies showed that some preparations of blue copper proteins purihed from their natural sources also contain small fractions of Zn derivatives (DeBeer George, personal communication). 

Fig. 3. Geometries of the type 1 copper sites of various blue copper proteins. The trigonal planar geometry is the type 1 site of laccase from Coprinus cinereus (PDB Code 1A65). The trigonal bipyramidal geometry shown is the copper site of azurin from Pseudomonas aeruginosa (PDB Code lAZU). The trigonal pyramidal/distorted tetrahedral sites are of the stellacyanin from Cucumis sativus (PDB Code IJER), NNSO site, and of the plastocyanin from Populus nigra (PDB Code IPLC) NNSS site.

For reference, the copper sites in plastocyanin, azurin, tomato plantacyanin, and cncnmber stellacyanin are shown in Figme 6. 

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