Oxidase Reactivity of CuII Bound to N-Truncated Aβ Peptides Promoted by Dopamine

Abstract

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-β peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aβ sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu-Aβ_4-x] and [Cu-Aβ_1-x] complexes toward dopamine and other catechols. The results show that the Cu^II-ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [Cu^II-Aβ-catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the Cu^I complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu-Aβ_4-x reveal that the His-tandem is able to bind Cu^II ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of Cu^II bound to the secondary site.

Keywords: Alzheimer’s disease; amyloid-β peptides; copper; dopamine; neurodegeneration; oxidative stress.

Figure 1

Kinetic profiles of DA (3 mM, panel (a)) and MC (3 mM, panel (b)) oxidation with time in 50 mM HEPES buffer at pH 7.4 and 20 °C in the presence of Cu^II alone (25 µM, green trace) and with 1 equiv. Aβ_1–16 (25 µM, pink), 2 equiv. Aβ_1–16 (50 µM, grey), 1 equiv. Aβ_4–16 (25 µM, light blue), and 2 equiv. Aβ_4–16 (50 µM, orange).

Figure 2

Kinetic profiles of DA (0.3 mM, panel (a)) and MC (0.3 mM, panel (b)) oxidation with time in 50 mM HEPES buffer at pH 7.4 and 20 °C in the presence of Cu^II alone (25 µM, green trace) and with 1 equiv. Aβ_1–16 (25 µM, pink), 2 equiv. Aβ_1–16 (50 µM, grey), 1 equiv. Aβ_4–16 (25 µM, light blue), and 2 equiv. Aβ_4–16 (50 µM, orange).

Figure 3

Kinetic profiles of DA (3 mM, panel (a)) and MC (3 mM, panel (b)) oxidation with time in 50 mM HEPES buffer at pH 7.4 and 20 °C in the presence of Cu^II alone (25 µM, green trace) and with 1 equiv. Aβ_1–28 (25 µM, pink), 2 equiv. Aβ_1–28 (50 µM, grey), 1 equiv. Aβ_4–28 (25 µM, light blue), and 2 equiv. Aβ_4–28 (50 µM, orange).

Figure 4

Kinetic profiles of DA (0.3 mM, panel (a)) and MC (0.3 mM, panel (b)) oxidation with time in 50 mM HEPES buffer at pH 7.4 and 20 °C in the presence of Cu^II alone (25 µM, green trace) and with 1 equiv. Aβ_1–28 (25 µM, pink), 2 equiv. Aβ_1–28 (50 µM, grey), 1 equiv. Aβ_4–28 (25 µM, light blue), and 2 equiv. Aβ_4–28 (50 µM, orange).

Figure 5

Kinetic profiles of MC (3 mM) oxidation with time in 50 mM HEPES buffer at pH 7.4 and 20 °C in the presence of Cu^II alone (25 μM, green trace). The same experiments were also performed upon the addition of [Cu–Aβ_4–16] at 1:1 molar ratio (25 µM, light blue) and [Ni–Cu–Aβ_4–16] at 1:1:1 molar ratio (25 µM, red).

Figure 6

Kinetic profiles of MC (0.3 mM) oxidation with time in 50 mM HEPES buffer at pH 7.4 and 20 °C in the presence of Cu^II alone (25 μM, green trace). The same experiments were also performed upon the addition of Aβ_4–16 complexes: [Cu–Aβ_4–16] at 1:1 molar ratio (25 µM, light blue), [Ni–Cu–Aβ_4–16] at 1:1:1 molar ratio (25 µM, red) and at 2:1:1 molar ratio (25 µM, violet).

Figure 7

Titration of nickel(II)–Aβ_4–16 complex (blue spectrum, at 1:1 ratio, 0.5 mM) with 0.0–0.55 mM copper(II) (final point as violet spectrum) in 5 mM phosphate buffer solution at pH 7.4. The pink spectrum corresponds to the absorption of free nickel(II).

Figure 8

Far-UV CD spectra of 1.1 equiv. Aβ_4–16 peptide (10 µM, panel a) and 1.1 equiv. Aβ_4–28 peptide (5.5 µM, panel b) in 5 mM phosphate buffer solution at pH 7.4 (blue traces) and upon the addition of copper(II) (1 equiv., light blue) and DA (1 equiv., pink).

Figure 9

Far-UV CD spectra of Aβ_4–16 peptide (10 µM) in 5 mM phosphate buffer solution at pH 7.4 (blue trace) and upon the addition of Ni^II (9.5 µM), after few seconds of incubation (light blue) and 15 min (green). Then, 1 (pink) and 2 equiv. of copper(II) (grey) were added to the Ni–Aβ_4–16 complex.

Figure 10

(a) Vis-CD spectra of of copper(II) (2 mM) alone in 5 mM phosphate buffer solution at pH 7.4 (grey trace) and upon the addition of 1 equiv. Aβ_4–16 peptide (2 mM, pink) and further addition of 1 equiv. of Ni^II to the initial complex (blue). (b) Vis-CD spectra of nickel(II) (2 mM) alone (grey trace) and upon the addition of 1 equiv. Aβ_4–16 peptide (2 mM, pink) and further addition of 1 equiv. of Cu^II (blue).