Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization–MS, that porcine Cox17 binds co-operatively four Cu⁺ ions. Cu₄Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu₄Cox17 contains a Cu₄S₆-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu₄Cox17, and an apparent copper dissociation constant K_Cu=13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu⁺ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu₄Cox17. Cox17 binds non-co-operatively two Zn²⁺ ions, but does not bind Ag⁺ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu⁺ or Zn²⁺ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.