High Cu(I) and low proton affinities of the CXXC motif of Bacillus subtilis CopZ

Abstract

CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pK(a) values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5-8.0. From these data, a pH-corrected formation constant, beta(2)= approximately 6 x 10(22) M(-2), was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein-chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.

Functional Role of Two Interhelical Disulfide Bonds in Human Cox17 Protein from a Structural Perspective

“…The reaction progress was monitored by UV/visible spectroscopy by following the increase in the absorption band of Cu I (BCA) 2 at 562 nm. The data were fitted using the program Origin 8.1 following an approach previously reported (33,34). ␤ 2 for the formation of the Cu(I)(BCA) 2 complex was taken as 2.0 ϫ 10 17 M Ϫ1 (35).…”

Bacterial copper storage proteins

“…3B, Fig. 4, B and C, and range (13,26,40,(68)(69)(70)(71). Faster Cu(I) unloading by small molecule Cu(I) ligands occurs for Csp1s (35), but a kinetic barrier to removal is present in Csp3s (36).…”

“…Copper homeostasis has been extensively investigated in certain Csp3-possessing non-methanotrophs, with probably the best example being Bacillus subtilis (21,28,68,76,77). Therefore, the Csp3 from this model Gram positive bacterium has been studied in vitro (36).…”

“…Comparative Cu(I) removal studies have also been carried out with well-characterized chromophoric Cu(I) chelating molecules such as bicinchoninic acid, and particularly bathocuproine disulfonate (BCS). These ligands have routinely been used to measure how tightly Cu(I) binds to a range of proteins (26,(68)(69)(70)(71), including determination of the Cu(I) affinities of Mbns (52,54) and the average values for the Csps (35,36). They have also been implemented as model acceptors for investigating Cu(I) removal from homeostasis proteins (68,72).…”

“…These ligands have routinely been used to measure how tightly Cu(I) binds to a range of proteins (26,(68)(69)(70)(71), including determination of the Cu(I) affinities of Mbns (52,54) and the average values for the Csps (35,36). They have also been implemented as model acceptors for investigating Cu(I) removal from homeostasis proteins (68,72). Using a large excess of the higher-affinity ligand BCS results in complete removal of Cu(I) from MtCsp1 in ~1 h (35), but only ~20% from MtCsp3 in 85 h (36).…”