Abstract

Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type ATPases CtaA and PacS, and the metallochaperone Atx1. We have determined the Cu(I) affinities of all of the soluble proteins and domains in this pathway. The Cu(I) affinities of the trafficking proteins range from 5 × 10(16) to 5 × 10(17) M(-1) at pH 7.0, consistent with values for homologues. Unusually, Atx1 binds Cu(I) significantly tighter than the metal-binding domains (MBDs) of CtaA and PacS (CtaA(N) and PacS(N)), and equilibrium copper exchange constants of approximately 0.2 are obtained for transfer to the MBDs. Dimerization of Atx1 increases the affinity for Cu(I), but the loop 5 His61 residue has little influence. The MBD of the zinc exporter ZiaA (ZiaA(N)) exhibits an almost identical Cu(I) affinity, and Cu(I) exchange with Atx1, as CtaA(N) and PacS(N), and the relative stabilities of the complexes must enable the metallochaperone to distinguish between the MBDs. The binding of potentially competing zinc to the trafficking proteins has been studied. ZiaA(N) has the highest Zn(II) affinity and thermodynamics could be important for zinc removal from the cell. Plastocyanin has a Cu(I) affinity of 2.6 × 10(17) M(-1), 15-fold tighter than that of the Cu(A) site of cytochrome c oxidase, highlighting the need for specific mechanisms to ensure copper delivery to both of these targets. The narrow range of Cu(I) affinities for the cytoplasmic copper proteins in Synechocystis will facilitate relocation when copper is limiting.