Intermolecular Transfer of Copper Ions from the CopC Protein of Pseudomonas syringae. Crystal Structures of Fully Loaded Cu^ICu^II Forms

Abstract

CopC is a small soluble protein expressed in the periplasm of Pseudomonas syringae pathovar tomato as part of its copper resistance response (cop operon). Equilibrium competition reactions confirmed two separated binding sites with high affinities for Cu(I) (10(-7) > or = K(D) > or = 10(-13) M) and Cu(II) (K(D) = 10(-13(1)) M), respectively. While Cu(I)-CopC was converted cleanly by O2 to Cu(II)-CopC, the fully loaded form Cu(I)Cu(II)-CopC was stable in air. Variant forms H1F and H91F exhibited a lower affinity for Cu(II) than does the wild-type protein while variant E27G exhibited a higher affinity. Cation exchange chromatography detected each of the four different types of intermolecular copper transfer reactions possible between wild type and variant forms: Cu(I) site to Cu(II) site; Cu(II) site to Cu(I) site; Cu(I) site to Cu(I) site; Cu(II) site to Cu(II) site. The availability of an unoccupied site of higher affinity induced intermolecular transfer of either Cu(I) or Cu(II) in the presence of O2 while buffering concentrations of cupric ion at sub-picomolar levels. Crystal structures of two crystal forms of wild-type Cu(I)Cu(II)-CopC and of the apo-H91F variant demonstrate that the core structures of the molecules in the three crystal forms are conserved. However, the conformations of the amino terminus (a Cu(II) ligand) and the two copper-binding loops (at each end of the molecule) differ significantly, providing the structural lability needed to allow transfer of copper between partners, with or without change of oxidation state. CopC has the potential to interact directly with each of the four cop proteins coexpressed to the periplasm.

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

“…The preference of Cu(I) for soft bases (His, Cys, and Met) is manifest in the coordination environments of cytoplasmic metallochaperones (35)(36)(37) and the Met-rich sites found in periplasmic copperbinding proteins (38)(39)(40)(41). However, in the context of Cu(I) transport through membrane channels, kinetic lability may be more important than thermodynamic stability, and indeed high binding affinity is likely to inhibit transport, unless accompanied by energy input that can toggle high-and low-affinity states via conformational change, as has been proposed as a mechanism for intramembrane transport in P1B-type ATPases (42).…”

Cu(I) recognition via cation-π and methionine interactions in CusF

“…3), or two Cu(I) ions each bind to four methionines across a dimer interface 8 . In contrast, the Cu(I)-CusF and Ag(I)-CusF described here are discrete mononuclear 1:1 protein-metal complexes with protected trigonal Met 2 His copper coordination still poised at the protein surface.…”

Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11

“…Copper plasticity has been also been shown recently for the copper-dependent tyrosinase from Bacillus megaterium (37). Histidine-rich copper-binding motifs as in CuA and CuB of FetP is Cu(II)-specific in the copper homeostasis protein CopC, whereas a more methionine-rich binding motif is Cu(I)-specific (38,39 may preferentially bind Cu(I) and thus may not be detected in the ITC titrations. In addition to copper, FetP also bound iron (as shown by AAS) and manganese (as demonstrated by AAS and ITC).…”