Selenocysteine positional variants reveal contributions to copper binding from cysteine residues in domains 2 and 3 of human copper chaperone for superoxide dismutase.

The human copper chaperone for superoxide dismutase binds copper both in an Atx1-like MTCQSC motif in domain 1 and via a multinuclear cluster formed by two CXC motifs at the D3 dimer interface. The composition of the Cu(I) cluster has been investigated previously by mutagenesis of the CXC motif, and by construction of a CXU selenocysteine derivative, which has permitted XAS studies at both Cu and Se absorption edges. Here, we report the semisynthesis and spectroscopic characterization of a series of derivatives with the sequences 243-CACA, 243-CAUA, 243-UACA, and 243-UAUA in the D1 double mutant (C22AC25A) background, prepared by expressed protein ligation of Sec-containing tetrapeptides to an hCCS-243 truncation. By varying the position of the Se atom in the CXC motif, we have been able to show that Se is always bridging (2 Se-Cu) rather than terminal (1 Se-Cu). Substitution of both D3 Cys residues by Sec in the UAUA variant does not eliminate the Cu-S contribution, confirming our previous description of the cluster as most likely a Cu(4)S(6) species, and suggesting that D2 Cys residues contribute to the cluster. As predicted by this model, when Cys residues C141, C144, and C227 are mutated to alanine either individually or together as a triple mutant, the cluster nuclearity is dramatically attenuated. These data suggest that Cys residues in D2 of hCCS are involved in the formation, stability, and redox potential of the D3 cluster. The significance of these finding to the SOD1 thiol/disulfide oxidase activity are discussed in terms of a model in which a similar multinuclear cluster may form in the CCS-SOD heterodimer.