Copper(I) stabilization by cysteine/tryptophan motif in the extracellular domain of Ctr4

•Ctr4 has a Cys- and Trp-rich sequence in the N-terminal extracellular domain.•Cu(II) is reduced and stabilized as Cu(I) by the Cys/Trp-motif.•Trp protects the thiolate-Cu(I) center against oxidation via cation–π interaction.•This sequence may be a novel Cu(I)-binding motif in an oxidizing environment.

Copper transporter Ctr4 of fission yeast has a quasi-palindromic sequence rich in cysteine and aromatic amino acid residues, CX4YWNWYX4C (where X represents any amino acid), in the N-terminal extracellular domain. A 24-mer peptide comprising this sequence is bound to Cu(I) through the cysteine thiolate coordination. Luminescence, UV absorption and resonance Raman spectra of the Cu(I)-peptide complex show that at least one of the two tryptophan side chains is located in close proximity to the thiolate-Cu(I) center and interacts with the Cu(I) ion via π-electrons of the indole ring. Although the thiolates and Cu(I) are oxidized to disulfide and Cu(II), respectively, only very slowly in air-saturated solutions, replacements of the tryptophan residues to phenylalanine significantly accelerate the oxidation reactions. The results obtained indicate that the interaction between Cu(I) and tryptophan via π-electrons plays a significant role in protecting the thiolate-Cu(I) center against the oxidation. The cysteine- and tryptophan-rich quasi-palindromic sequence may be a metal binding motif that stabilizes Cu(I) in the oxidizing extracellular environment.

Graphical abstractCopper transporter Ctr4 has a cysteine- and tryptophan-rich sequence in the N-terminal extracellular domain. This sequence reduces Cu(II) to Cu(I), and then stabilizes the Cu(I) by thiolate coordination and cation–π interaction with tryptophan. The Cys/Trp-rich sequence may be a novel metal binding motif that stabilizes Cu(I) in the oxidizing extracellular environment.Figure optionsDownload full-size imageDownload as PowerPoint slide