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The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus 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; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension 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 II), 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 vernicifera, 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 III 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.

Electron spin echo envelope modulation spectroscopy identified two ligand 14N interactions with the mixed-valence, Fe(II/III) diiron center of methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath). Characteristic features of the spectra obtained at 9 and 10 GHz were analyzed and fit by simulation. One of the nitrogens possessed superhyperfine parameters (Aiso = 0.8 MHz, reff = 3.2 A, e2Qq = 1.8 MHz, eta = 0.35) consistent with a non-coordinating amino nitrogen of a histidine imidazole ligand to a Fe(III). The second, more strongly interacting nitrogen (Aiso = 5.0 MHz, reff = 2.2 A, e2Qq = 3.0 MHz, eta = 0.3) corresponds to the N delta directly bound to the effective Fe(II). These findings extend the previous electron nuclear double resonance results on the Methylosinus trichosporium hydroxylase (Hendrich, M.P., Fox, B.G., Andersson, K.K., Debrunner, P.G., and Lipscomb, J.D. (1992) J. Biol. Chem. 267, 261-269), which identified the N delta-Fe(II) interaction but failed to quantify its magnitude. Measurement of the linear electric field g shift of this mixed-valence species indicated that the site is charge-polarized on to one of the iron atoms, and its symmetry suggests that either charge is shifted away from the Fe-Fe axis (if gmax is defined by the Fe-Fe axis) or that gmid and gmax are perpendicular to the Fe-Fe axis (charge strongly localized at Fe(III) and axis taken as gmin).