The Mitochondrial Copper Metallochaperone Cox17 Exists as an Oligomeric, Polycopper Complex

Abstract

Cox17 is the candidate copper metallochaperone for delivery of copper ions to the mitochondrion for assembly of cytochrome c oxidase. Cox17 purified as a recombinant molecule lacking any purification tag binds three Cu(I) ions per monomer in a polycopper cluster as shown by X-ray absorption spectroscopy. The CuCox17 complex exists in a dimer/tetramer equilibrium with a 20 microM k(d). The spectroscopic data do not discern whether the dimeric complex forms a single hexanuclear Cu(I) cluster or two separate trinuclear Cu(I) clusters. The Cu(I) cluster(s) exhibit(s) predominantly trigonal Cu(I) coordination. The cluster(s) in Cox17 resemble(s) the polycopper clusters in Ace1 and the Cup1 metallothionein in being pH-stable and luminescent. The physical properties of the CuCox17 complex purified as an untagged molecule differ from those reported previously for a GST-Cox17 fusion protein. The CuCox17 cluster is distinct from the polycopper cluster in Cup1 in being labile to ligand exchange. CuCox17 localized within the intermitochondrial membrane space appears to be predominantly tetrameric, whereas the cytosolic CuCox17 is primarily a dimeric species. Cys-->Ser substitutions at Cys23, Cys24, or Cys26 abolish the Cox17 function and prevent tetramerization, although Cu(I) binding is largely unaffected. Thus, the oligomeric state of Cox17 may be important to its physiological function.

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

“…Protein Purification-Recombinant Cox17 was purified as described previously by Heaton et al (14). E. coli BL21 (DE3) transformants with COX17 on a pAED3 vector were used for the Cox17 purification using conventional chromatography on an AKTA fast protein liquid chromatography unit (Amersham Biosciences).…”

“…The roles of Cox17, Sco1, and Cox11 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). Cox17 is a soluble protein that localizes within the IMS and also within the cytoplasm (8).…”

“…Multiple Cu(I) ions bind to Cox17 within a polycuprous-thiolate cluster that stabilizes a mixture of dimers and tetramers (14). A second functional domain in Cox17 lies near the C terminus.…”

Visualizing Biological Copper Storage: The Importance of Thiolate‐Coordinated Tetranuclear Clusters

“…The scarcity of Cu I clusters bound by organothiolates in biological systems, such as those that drive Mt Csp3 Cu I core formation, is surprising given their rich coordination chemistry18, 19 and the thiophilic nature of Cu I . Proteins involved in copper homeostasis, particularly in Saccharomyces cerevisiae , have been found to bind tetranuclear Cu I clusters in vitro using Cys residues, but no crystal structures are available 20, 21, 22, 23, 24. Extended X‐ray absorption fine structure data for these are similar to that of [Cu 4 (SPh) 6 ] 2− 20, 21, 23, 25.…”

XIAP: Cell death regulation meets copper homeostasis

“…Cox17 is found in both the cytosol and mitochondria. It was originally thought that Cox 17 binds copper in the cytosol, translocates into mitochondria and delivers copper to Cox1 and Cox2 [48][49][50]. However, further studies in yeast showed that in Cox 17 deficient cells, CcO activity can be fully restored by mitochondrial restricted expression of Cox17 [51].…”