Investigating the Role of Zinc and Copper Binding Motifs of Trafficking Sites in the Cyanobacterium Synechocystis PCC 6803

[Image: see text] Although zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial (Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaA(N) and PacS(N), respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to PacS, but differ in the residues found in their M/IXCXXC metal-binding motifs. To investigate the role of the nonconserved residues in this region on metal binding, the sequence from ZiaA(N) has been introduced into Atx1 and PacS(N), and the motifs of Atx1 and PacS(N) swapped. The motif sequence can tune Cu(I) affinity only approximately 3-fold. However, the introduction of the ZiaA(N) motif (MDCTSC) dramatically increases the Zn(II) affinity of both Atx1 and PacS(N) by up to 2 orders of magnitude. The Atx1 mutant with the ZiaA(N) motif crystallizes as a side-to-side homodimer very similar to that found for [Cu(I)(2)–Atx1](2) ( Badarau et al. Biochemistry2010, 49, 779820726513). In a crystal structure of the PacS(N) mutant possessing the ZiaA(N) motif (PacS(N)(ZiaA(N))), the Asp residue from the metal-binding motif coordinates Zn(II). This demonstrates that the increased Zn(II) affinity of this variant and the high Zn(II) affinity of ZiaA(N) are due to the ability of the carboxylate to ligate this metal ion. Comparison of the Zn(II) sites in PacS(N)(ZiaA(N)) structures provides additional insight into Zn(II) trafficking in cyanobacteria.