Characterization of two copper-binding components of the fungus, Dactylium dendroides

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Abstract Two copper-binding components of the fungus, Dactylium dendroides, have been isolated and characterized. Cells grown in culture media containing from 10 n m to 1 m m copper synthesize a copper-binding protein (Mr, ~7.5 × 103) which contains 2 g atoms of copper/mol when saturated. The levels of saturation in vivo are 20 and 100% at 10 n m and 100 μ m medium copper, respectively. The amount of this protein present in the cells is independent of medium copper concentration. This protein appears to be cytosolic. The purity of this copper-binding protein has been established by column chromatography, polyacrylamide gel electrophoresis, amino acid analysis, and peptide mapping. The cysteine content of 4.5% is similar to that of copper-binding proteins from rat and human liver, but lower than that found in chelatins or copper thioneins from rat liver and yeast. The copper-saturated protein exhibits no visible absorption or electron paramagnetic resonance spectrum, and a featureless near-ultraviolet absorption spectrum with a shoulder at ca. 260 nm. These properties are suggestive of mercaptide sulfur coordination. The cytosol of cells grown in 1 m m medium copper is blue, apparently due to the binding of copper to a second proteinacious ligand. This Cu-peptide complex(es) has an apparent molecular weight of 1350 as determined by gel permeation chromatography and contains 1 g atom of copper/mol. This metal is not removed by EDTA but is removed by treatment with Chelex 100 or diethyldithiocarbamic acid. Up to two additional copper atoms may be loosely bound, but these can be removed by gel permeation chromatography. The peptide contains no cysteine, histidine, or aromatic amino acids, nor does it contain any carbohydrate. The complex exhibits a visible absorption maximum at 740 nm (ϵ = 60). The principal spin Hamiltonian parameters (g∥ = 2.345 and A¦ = 145 G) together with the visible absorption spectrum indicate that the ligands binding to the copper are fairly ionic (N and/or O) and that the ligand field is close to square planar. When isolated from cells grown at 0.1 μ m medium copper, the ligand contains only trace amounts of metal. The absence of cysteine and carbohydrate makes this ligand a new type of metal binding species unlike the thioneins, chelatins, or bleomycin. The possible biological function of this ligand is discussed as are the differences between the copper-binding protein described and metal-binding species in other organisms.