CopC Protein from <i>Pseudomonas fluorescens</i> SBW25 Features a Conserved Novel High-Affinity Cu(II) Binding Site

Copper homeostasis in the bacterium Pseudomonas fluorescens SBW25 appears to be mediated mainly via chromosomal <i>cue</i> and <i>cop</i> systems. Under elevated copper levels that induce stress, the <i>cue</i> system is activated for expression of a P_1B-type ATPase to remove excess copper from the cytosol. Under copper-limiting conditions, the <i>cop</i> system is activated to express two copper uptake proteins, <i>Pf</i>-CopCD, to import this essential nutrient. <i>Pf</i>-CopC is a periplasmic copper chaperone that may donate copper to the inner membrane transporter <i>Pf</i>-CopD for active copper importation. A database search revealed that <i>Pf</i>-CopC belongs to a new family of CopC proteins (designated Type B in this work) that differs significantly from the known CopC proteins of Type A that possess two separated binding sites specific for Cu­(I) and Cu­(II). This article reports the isolation and characterization of <i>Pf</i>-CopC and demonstrates that it lacks a Cu­(I) binding site and possesses a novel Cu­(II) site that binds Cu­(II) with 100 times stronger affinity than do the Type A proteins. Presumably, this is a requirement for a copper uptake role under copper-limiting conditions. The Cu­(II) site incorporates a highly conserved amino terminal copper and nickel (ATCUN) binding motif, NH₂-Xxx-Xxx-His, but the anticipated ATCUN binding mode is prevented by a thermodynamically more favorable binding mode comprising His1 as a key bidentate ligand and His3 and His85 as co-ligands. However, upon His1 mutation, the ATCUN binding mode is adopted. This work demonstrates how a copper chaperone may fine tune its copper binding site to meet new challenges to its function