Human Sco1 functional studies and pathological implications of the P174L mutant

Abstract

The pathogenic mutant (P174L) of human Sco1 produces respiratory chain deficiency associated with cytochrome c oxidase (CcO) assembly defects. The solution structure of the mutant in its Cu(I) form shows that Leu-174 prevents the formation of a well packed hydrophobic region around the metal-binding site and causes a reduction of the affinity of copper(I) for the protein. K(D) values for Cu(I)WT-HSco1 and Cu(I)P174L-HSco1 are approximately 10(-17) and approximately 10(-13), respectively. The reduction potentials of the two apo proteins are similar, but slower reduction/oxidation rates are found for the mutant with respect to the WT. The mitochondrial metallochaperone in the partially oxidized Cu(1)(I)Cox17(2S-S) form, at variance with the fully reduced Cu(4)(I)Cox17, interacts transiently with both WT-HSco1 and the mutant, forming the Cox17/Cu(I)/HSco1 complex, but copper is efficiently transferred only in the case of WT protein. Cu(1)(I)Cox17(2S-S) indeed has an affinity for copper(I) (K(D) approximately 10(-15)) higher than that of the P174L-HSco1 mutant but lower than that of WT-HSco1. We propose that HSco1 mutation, altering the structure around the metal-binding site, affects both copper(I) binding and redox properties of the protein, thus impairing the efficiency of copper transfer to CcO. The pathogenic mutation therefore could (i) lessen the Sco1 affinity for copper(I) and hence copper supply for CcO or (ii) decrease the efficiency of reduction of CcO thiols involved in copper binding, or both effects could be produced by the mutation.

Fig. 1.

Binding of copper ions to human Sco1 and mutant P174LSco1. ESI-TOF mass spectra of WT-HSco1 (0.8 μM) and P174L-HSco1 (1.2 μM) reconstituted with Cu(I)DTT complex in 50 mM ammonium acetate, pH 7.5. (A) apoWT-HSco1. (B) WT-HSco1 + 1 eq of Cu(I) in the presence of 1.0 mM DTT. (C) apoP174L-HSco1. (D) P174L-HSco1 + 1 eq of Cu(I) in the presence of 1.0 mM DTT. (E) P174L-HSco1 + 1 eq of Cu(I) + 3 eq of Cox17_2S-S in the presence of 1.0 mM DTT. Charge state +8, +9, and +10 ions are presented and numbers on the peaks denote the metal stoichiometry of the complex.

Fig. 2.

Solution structure of Cu₁(I)P174L-HSco1 mutant. (A) Backbone atoms are represented as a tube with variable radius, proportional to the backbone rmsd value of each residue for Cu₁(I)P174L-HSco1 and Cu₁(I)WT-HSco1. The side chains of Cys-169, Cys-173, His-260, and the copper(I) ions are shown in yellow, green, and orange, respectively. The secondary structure elements also are indicated: β-strands are in cyan and α-helices in red. (B) Side-chain packing involving Pro-174 or Leu-174 (in yellow) and Tyr-216 and Phe-220 (in green) is shown on the WT and P174L mean minimized solution structures. The mutant protein was copper-saturated resulting in a 1:1 copper:protein complex.

Fig. 3.

Redox properties of WT-HSco1 and P174L-HSco1. (A) Fluorescence emission spectra of 5 μM WT-HSco1 (black) and P174L-HSco1 (blue) recorded under native conditions of the oxidized (50 mM phosphate buffer, pH 7.0, 0.01 mM GSSG; dotted lines) and the reduced (50 mM phosphate buffer, pH 7.0, 200 mM GSH; solid lines) protein after excitation at 280 nm. (B) Redox equilibrium of WT-HSco1 and P174L-HSco1 with different [GSH]²/GSSG ratios as followed by measuring fluorescence emission at 327 nm. After nonlinear regression, values of K_eq = 17.70 ± 2.23 mM and of 15.66 ± 1.86 mM (correlation coefficient: 0.986 and 0.996) were obtained for the WT-HSco1 and P174L-HSco1/glutathione systems, corresponding to a redox potentials of −0.277 ± 0.030 V and −0.275 ± 0.028 V, respectively, by using the glutathione standard potential of −0.240 V at pH 7.0 and 298 K.

Fig. 4.

The effect of the P174L-Sco1 mutation in the copper transfer with Cox17. The pathogenic human Sco1 mutation P174L, next to the copper-binding motif, determines a reduction of the efficiency of the copper transfer from the mitochondrial metallochaperone Cox17 to Sco1.