Structural basis and mechanism for metallochaperone-assisted assembly of the Cu A center in cytochrome oxidase.

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+center dot Cu1.5+ redox center (Cu-A) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB center dot Cu-A center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones Scol and PcuC. A previously unknown, highly stable Scol center dot Cu2+center dot CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuC center dot Cu1+center dot Cu2+ can release CoxB center dot Cu2+ from the Scol center dot Cu2+center dot CoxB complex. The CoxB center dot Cu-A center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuC center dot Cu1+center dot Cu2+ to CoxB center dot Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for Cu-A center formation and the order of their delivery to CoxB.