Structure and Metal Loading of a Soluble Periplasm Cuproprotein

A copper-trafficking pathway was found to enable Cu2+ occupancy of a soluble periplasm protein, CucA, even when competing Zn2+ is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu2+, but not Zn2+, quenches the fluorescence of Trp(165), which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn2+ following exposure to equimolar Zn2+ and Cu2+. Cu2(2+)-CucA is more thermodynamically stable than Zn2+-CucA but k((Zn -> Cu) exchange) is slow, raising questions about how the periplasm contains solely the Cu2+ form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu2+-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of Delta ctaA cells, but the abundance of cucA transcripts was unaltered. Crucially, Delta ctaA cells overaccumulate low M-r copper complexes in the periplasm, and purified apoCucA can readily acquire Cu2+ from Delta ctaA periplasm extracts, but in vivo apoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm.