Cysteine Protonation States in Copper Tracking Proteins and its Influence on Biological Cu(I) Transfer Mechanism

Copper delivery from metallochaperones to P-type ATPases requires coordination by cysteine residues, yet the protonation states of these incoming thiols at the moment of first Cu(I) engagement remain undefined. The long-standing assumption that reactive cysteines exist as thiolates has not been rigorously tested under the conditions relevant to early protein-protein encounters, limiting mechanistic insight into the formation of the initial Cu(I)-S bond. In the Atx1-Ccc2a system, we assess whether the local protein environment can modulate cysteine acidity sufficiently to alter ligand protonation states and reactivity. MD simulations combined with DFT-based pKa analysis demonstrate that neither of the binding site cysteines of Ccc2a undergo substantial pKa reduction in their apo or pre-reactive states; the only perturbation occurs when Cu(I) is already in vicinity to the incoming cysteine thiol. QM/MM calculations further show that direct Cu(I)-thiol coordination is chemically feasible without prior deprotonation. We further analyze the initial Cu(I)-S bond formation within the framework of the Curtin-Hammett principle, explicitly evaluating whether rapid thiol-thiolate equilibration can govern the product distribution during early copper transfer. Our analysis reveals, thiol-thiolate interconversion is slower than Cu(I)-S ligation, indicating that early Cu(I) binding is dictated by the equilibrium population of thiol species rather than by transition-state energetics. Thus, early Cu(I) recognition is directed by transient conformational states and encounter geometries rather than by cysteine activation. These findings refine the mechanistic model of Cu(I) tracking and provide a framework for rational design of metalloprotein mimics and copper homeostasis therapies. † Equal contributions.