Specific Copper Transfer from the Cox17 Metallochaperone to Both Sco1 and Cox11 in the Assembly of Yeast Cytochrome c Oxidase

Yih-Chern Horng,Paul A Cobine,Andrew B Maxfield,Heather S Carr,Dennis R Winge

doi:10.1074/jbc.m404747200

Abstract

The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins.

Highlights

Four proteins (Cox11, Cox17, Cox19 and Sco1) have been implicated in copper ion delivery and insertion into Cytochrome c oxidase (CcO)
Cox17 was postulated to function as a copper donor to Sco1 as an intermediate step in the copper metallation of the CuA in Cox2 during assembly of cytochrome c oxidase [7,17, 29]
In the present studies we directly confirm this postulate and extend the functional significance of Cox17 to show for the first time that it is a copper donor to Cox11

Summary

Introduction

Four proteins (Cox, Cox, Cox and Sco1) have been implicated in copper ion delivery and insertion into CcO (reviewed in 5). Among a collection of yeast respiratory deficient mutants defective in cytochrome c oxidase activity, only cox cells were suppressed by exogenous copper suggesting a role for Cox in copper ion metallation of CcO. Yeast cells lacking Cox are respiratory deficient, but this phenotype is not reversed by the addition of exogenous copper salts. The roles of Cox, Sco and Cox in copper metallation of CcO were substantiated by the observation that each protein is a Cu(I) binding protein and that mutations that abrogate in vivo function attenuate Cu(I) binding [11,13,14,15]. Mutation of any of these Cys residues reduces Cu(I) binding and abrogates CcO activity

Methods

Results

Conclusion