Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 8, resolution. The crystallographic R-value for all 18,488 reflections ((T > 0) between 50-1.6 8, is 0.195. The overall fold is organized in two P-sheets, both with four P-strands. Two cy-helices are found in loop regions between P-strands. The P-sheets form a P-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the P-barrel is more flattened, there is an extra N-terminal a-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sariwus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; 11, a 109 amino acid copper-binding domain; 111, a 26 amino acid hydroxyproline-and serine-rich peptide characteristic of motifs found in the extensin family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain 11), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus verniciferu, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline-and serine-rich domain I11 suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

The crystal structure of the basic blue protein (plantacyanin) from spinach (SBP) has been solved to a resolution of 2.05 A by molecular replacement using the homologous protein from cucumber (CBP) as a model. Although the sequence identity of 58% between both proteins is only moderate, the three-dimensional structures turned out to be highly similar and the buried residues, which form the hydrophobic core of the protein, are almost completely conserved. However, the redox potentials of both proteins differ by 40 mV, and a comparison of the two structures leads to a single lysine replacing a proline in the cucumber sequence, which causes a shift of the peptide chain and thus a subtle distortion of the copper ligand geometry in respect to CBP. The crystal contained three monomers of SBP in the asymmetric unit which show considerable variations in outer loop regions owing to crystal packing, but not in the regions presumed to be essential for redox partner recognition and redox potential fine tuning of the copper centers. Still, bond length variations at the copper site are at the same scale between the monomers of SBP as they are in respect to CBP, indicating that in the oxidized state the protein does not impose a high conformational strain on the copper.

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