A Cu-bis(imidazole) Substrate Intermediate Is the Catalytically Competent Center for Catechol Oxidase Activity of Copper Amyloid-β

Abstract

Interaction of copper ions with Aβ peptides alters the redox activity of the metal ion and can be associated with neurodegeneration. Many studies deal with the characterization of the copper binding mode responsible for the reactivity. Oxidation experiments of dopamine and related catechols by copper(II) complexes with the N-terminal amyloid-β peptides Aβ 16 and Aβ 9 , and the Aβ 16 [H6A] and Aβ 16 [H13A] mutant forms, both in their free amine and N-acetylated forms show that efficient reactivity requires the oxygenation of a Cu I -bis(imidazole) complex with a bound substrate. Therefore, the active intermediate for catechol oxidation differs from the proposed “in-between state” for the catalytic oxidation of ascorbate. During the catechol oxidation process, hydrogen peroxide and superoxide anion are formed but give only a minor contribution to the reaction.

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

“…Taken together, these data confirm the reaction mechanism previously proposed for the oxidation of catechols by copper complexes with other neuronal peptides [ 22 , 27 , 28 ]: [Cu 2+ –Aβ] + catechol ⇄ [Cu 2+ –Aβ–catechol] [Cu 2+ –Aβ–catechol] → [Cu + –Aβ] + semiquinone + [Cu + –Aβ] + catechol ⇄ [Cu + –Aβ–catechol] [Cu + –Aβ–catechol] + O 2 → [Cu–Aβ–catechol–O 2 ] [Cu–Aβ–catechol–O 2 ] → [Cu 2+ –Aβ] + quinone 2 semiquinone + → catechol + quinone …”

“…DA oxidation is slower than MC oxidation because of the lower semiquinone/catechol redox potential [ 21 ] and leads to the formation of mixtures of insoluble oligomeric products. As observed in a previous study [ 22 ], at high catechol concentration, the presence of Aβ 1−x peptides increases the rate of copper(II)-mediated oxidation, whereas the redox cycling of copper(II) is disfavored at low substrate concentration.…”

“…This difference is probably due to a different conformation of the Aβ 4−x peptide, which makes the coordination of copper(I) by histidines 13 and 14 less flexible and prone to leave access by catechol and molecular oxygen. Indeed, we showed previously [ 22 ] that the linear copper(I) coordination through His13 and His14 by Aβ 1−x must be distorted upon catechol coordination to allow a fast oxygenation reaction.…”

“…We also extended the catechol oxidation experiments to the copper(II) complexes with the longer Aβ 1–28 and Aβ 4–28 peptides because they better mimic the behavior of the full-length Aβ 1–40/42 and Aβ 4–40/42 peptides but, lacking the highly hydrophobic C -terminal tail, their aggregation is much slower [ 22 , 23 , 24 , 25 ]. In addition, the studies of copper(II) bound to the ATCUN motif were limited so far to the Aβ 4–16 peptide [ 9 , 26 ].…”

“…The lifetime of this [Cu II –Aβ–catechol] species is not related to an interaction with dioxygen, as verified by the initial 10 s of the kinetic traces obtained for [Cu–Aβ 4–16 ] in the presence of atmospheric oxygen or pure O 2 (1 atm) ( Figure S7 ). On the other hand, oxygen saturation affects the efficiency of the subsequent steps of the reaction, suggesting that the conversion from Cu I to Cu II remains the rate-limiting step of the oxidation of catechols [ 22 , 27 , 28 ]. Furthermore, the addition of ascorbate during the first seconds of the reaction leads to the quenching of the absorption band at 300 nm ( Figure S8 ).…”