CopC is a copper chelator found in the bacterial species such as Pseudomonas fluorescens SBW25. CopC proteins are found in the periplasmic space where it has a role in maintaining Cu-homeostasis. PfCopC contain a mono-nuclear Cu-binding site. The binding site has a similar composition as the active-site histidine brace found in the Cu-dependent Lytic Polysaccharide Monooxygenases.
We characterized the WT PfCopC and generated mutants of this protein to gain valuable insights into which residues hold importance for Cu sequestration.
PfCopC protein and variants where expressed E.coli system and purified using a three-step chromatographic procedure. We characterized the Cu-binding directly by monitoring tryptophan emission. CopC contains a single tryptophan residue that changes fluorescent emission intensity upon Cu-protein binding. The emission intensity is gradually reduced until stabilized at Cu saturation. Orthogonally, we performed Isothermal calorimetric determination of the Cu binding to the variants. Stoichiometry and the thermodynamic parameter where extrapolated based on observed thermographs. We found that most variants do not change binding stoichiometry or release Cu. However, we found a second sphere interacting residue that showed retardation in Cu binding stoichiometry especially when exposed to redox environments. Furthermore, the conserved composition of amino acid ligands but change in geometry around the chelating site showed a very different profile with more release of Cu into the media.