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

Abstract

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu^1.5+•Cu^1.5+ redox center (Cu_A) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•Cu_A center in vitro from <i>apo</i>-CoxB and the <i>holo</i>-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu²⁺•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•Cu¹⁺•Cu²⁺ can release CoxB•Cu²⁺ from the ScoI•Cu²⁺•CoxB complex. The CoxB•Cu_A center was then formed quantitatively by transfer of Cu¹⁺ from a second equivalent of PcuC•Cu¹⁺•Cu²⁺ to CoxB•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.