Protein-Driven Copper Redox Regulation: Uncovering the Role of Disulphide Bonds and Allosteric Modulation.

Copper plays essential roles in enzymatic activity, redox reactions, and cellular signalling but becomes toxic when redox homeostasis is disrupted. While Cu(II) reduction is commonly attributed to unfolded or amyloid proteins, here we show that the well-folded plasma protein human serum albumin (HSA) intrinsically reduces Cu(II) to Cu(I) in the absence of external reductants. Using x-ray absorption spectroscopy (XAS), small-angle x-ray scattering (SAXS), and circular dichroism (CD), we propose a redox mechanism involving the disulphide bond Cys392-Cys438 in domain III of HSA. Cu binding at the high-affinity ATCUN (amino-terminal copper and nickel binding site) motif might trigger conformational changes that expose this disulphide bond, enabling thiol-mediated electron transfer and Cu(I) formation. Chelation with tetrathiomolybdate (TTM) impairs this reduction by restricting access to the reactive disulphide site. Comparative analysis with other globular proteins reveals that Cu reduction requires both accessible disulphide motifs and a native folded structure. Simulations and spectroscopy of SOD1 (Superoxide Dismutase 1) confirm that disulphide cleavage enhances Cu-thiolate interaction, supporting a generalisable two-site redox mechanism. These findings reveal a previously unrecognised mode of protein-mediated copper reduction and suggest broader physiological roles for disulphide-regulated redox switching in metal homeostasis.