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

Abstract

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 KCu=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.

Figure 1. Binding of Cu⁺ ions to Cox17

Mass spectra of Cox17 (1.4 μM) reconstituted with 1–6 equivalents of Cu⁺ ions in the presence of 20 mM ammonium acetate (pH 7.6) and 0.15–0.35 mM DTT at 25 °C. Charge state +6 ions are presented and numbers on the peaks denote the metal stoichiometry of the complex.

Figure 2. Binding of Cu⁺ ions to Cox17 in the presence of DTT

Mass spectra of Cox17 (2 μM) reconstituted with 4 equivalents of Cu⁺ ions in the presence of 20 mM ammonium acetate (pH 7.6) and DTT at various concentrations. Concentrations of DTT: (a) 0.3 mM, (b) 0.75 mM, (c) 3.0 mM, (d) 6 mM and (e) 10 mM. Charge state +6 ions are presented and numbers on the peaks denote the metal stoichiometry of the complex.

Figure 3. Determination of the dissociation constant K_d for the Cu₄Cox17 complex

Dependence of the fractional content of metallated Cu₄Cox17, Y=I_Cu4Cox17/(I_Cox17+I_Cu4Cox17), on the concentration of free Cu⁺ ions in the metal competition experiment [20 mM ammonium acetate (pH 7.6); 25 °C]. ——, Curve fitted for hyperbola with K_Cu=1.3×10⁻¹⁴ M is shown. Inset: the same results in Hill coordinates [Φ=Y/(1−Y)] and a linear fit (——) are shown; h (represented by n in the Figure), Hill coefficient.

Figure 4. Binding of Ag⁺ and bivalent metal ions by Cox17

Mass spectra of Cox17 (1.4 μM) reconstituted with 4 equivalents of Ag⁺ (b), Zn²⁺ (c), Cd²⁺ (d) and Fe²⁺ ions (e) in 20 mM ammonium acetate (pH 7.6) and 0.3 mM DTT at 25 °C. Charge state +6 ions are presented and numbers on the peaks denote the metal stoichiometry of the complex.

Figure 5. Charge-state analysis of different forms of Cox17

Mass spectra of (a) fully oxidized Cox17, (b) reduced Cox17, (c) reduced Cox17 reconstituted with 1 equivalent of Cu⁺ ions, (d) reduced Cox17 reconstituted with 4 equivalents of Cu⁺ ions and (e) partially oxidized Cox17 reconstituted with 4 equivalent of Cu⁺ ions. Conditions: 1.4 μM Cox17, 20 mM ammonium acetate (pH 7.6) and 0.3 mM DTT at 25 °C. Charge states of ions (underlined) are presented and numbers on the peaks denote the metal stoichiometry of the complex.

Figure 6. UV absorption and fluorescence studies

UV absorption spectra (a) and fluorescence spectra (b) of Cox17 after the addition of Cu⁺ ions. Conditions: 20 mM ammonium acetate (pH 7.6) at 25 °C. (a, b) Insets: absorption at 265 nm (a) or luminescence intensity at 585 nm as a function of the mole equivalents of Cu⁺ ions added.

Figure 7. Proposed structure of the metal-binding motif in Cox17

(a) Structure of the [Cu₄(S)₆]²⁻ model cluster proposed for the structure of the copper–thiolate cluster in Cu₄Cox17; (b) sequence alignment of yeast and mammalian Cox17.