The Two Faces of Copper Capture Driven by the Internal His/Met-Rich and N-Terminal His-Rich Domains of CopI.

While copper is an essential redox cofactor, excess copper, particularly Cu(I), is highly toxic and therefore requires precise control of metal speciation in the bacterial periplasm. CopI is a cupredoxin-like protein implicated in copper tolerance under microaerobic conditions and contains multiple histidine- and methionine-rich regions, yet its metal-binding properties remain poorly understood. Here, we deconstructRubrivivax gelatinosusCopI into its constituent metal-binding modules and provide the first quantitative, domain-resolved analysis of their Cu(II) coordination chemistry. Two peptide domains, an N-terminal His-rich region and an internal His/Met-rich motif, form mononuclear 1:1 Cu(II) complexes but exhibit strikingly different binding modes. The His-rich domain displays higher Cu(II) affinity and a rigid, predominantly nitrogen-based coordination environment, whereas the His/Met-rich motif binds Cu(II) more weakly and adopts a heterogeneous, N/S/O-donor coordination sphere indicative of structural flexibility. Neither domain forms stable secondary structure upon metal binding, indicating that intrinsic disorder is a functional feature rather than a structural deficiency. Comparative Zn(II) studies reveal substantially weaker binding, underscoring copper selectivity. Collectively, these results identify CopI as a chemically modular copper buffer and establish a coordination-chemical framework for understanding copper handling by periplasmic cupredoxin-like proteins.