Domains I and III of the Human Copper Chaperone for Superoxide Dismutase Interact via a Cysteine-Bridged Dicopper(I) Cluster

Abstract

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.

Solution Structure of Cox11, a Novel Type of β-Immunoglobulin-like Fold Involved in CuB Site Formation of Cytochrome c Oxidase

“…3 S at 2.24 Å and 1 Cu at 2.70 Å) has been observed in yeast Cox11 for which Cys residues are involved in copper binding (15). The distance of 2.7 Å is in accordance with Cu-Cu distances observed in doubly bridging sulfur complexes, which range from 2.7 to 3.0 Å, whereas the Cu-Cu distances in singly bridged sulfur clusters are around 3.3 Å (45,46). Similar EXAFS data were also found for Cu(I)-CopZ samples, indicating a common copper binding motif (47).…”

“…4). Indeed, the observed Cu-Cu distance of 2.7 Å is typical of doubly bridging sulfur complexes (56,57) and was found in other copper(I) chaperones with similar rearrangements (45,58). The N-terminal Cys-9, which is located far from the CFCF binding site and in an unstructured region only five/four residues of the inner membrane, would not be involved in copper binding but rather could be involved in an intermolecular disulfide bond with the corresponding cysteine of the other monomeric unit (Fig.…”

A redox switch in CopC: An intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites

“…A number of studies have shown that the Cu(I) coordination number and͞or geometry are correlated with the intensity of this transition (38,39). The height of the 1s-4p transition present in the edge of the Cu(I)-CopC spectrum suggests 3-or 4-coordination for Cu(I) (39,41).…”

The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status

“…Whereas the mechanism by which SOD1 acquires Zn(II) is not fully understood, several aspects of the copper insertion by the copper chaperone for SOD1 (CCS) are well established (7)(8)(9)(10)(11)(12). More recently, Furukawa et al (13) have shown that the intrasubunit disulfide bond is correctly introduced in yeast SOD1 by the copper-bound form of yeast CCS.…”