Mechanistic Studies of Cu(II) Binding to Amyloid-β Peptides and the Fluorescence and Redox Behaviors of the Resulting Complexes

Abstract

Due in large part to the lack of crystal structures of the amyloid-beta (Abeta) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal-Abeta complex has been difficult. In this work, we investigated the complexation of Cu(II) by Abeta through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to Abeta can be reduced to Cu(I) using sodium borohydride and that both Abeta-Cu(II) and Abeta-Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the Abeta fluorescence, commonly reported to be quenched upon Abeta-Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of Abeta-Cu(II) complexes provides evidence that the sole tyrosine residue in Abeta is not one of the four equatorial ligands bound to Cu(II), but remains close to the metal center, and its fluorescence is sensitive to the copper oxidation state and perturbations in the coordination sphere. Further analysis of the quenching and Cu(II) binding behaviors at different Cu(II) concentrations and in the presence of the competing ligand glycine offers evidence supporting the operation of two binding regimes which demonstrate different levels of fluorescence recovery upon addition of the reducing agent. We provide results that suggest the fluorescence quenching is likely caused by charge transfer processes. Thus, by using tyrosine to probe the coordination site, fluorescence spectroscopy provides valuable mechanistic insights into the oxidation state of copper ions bound to Abeta, the binding heterogeneity, and the influence of solution conditions on complex formation.

pH-dependent kinetics of copper ions binding to amyloid-β peptide

“…The results imply that acidic conditions could aggravate the oxidative stress in the presence of Cu 2+ and the weak affinities of Aβ−Cu 2+ under mild pH of 5.0-6.0 could further enhance the oxidative damage. The present researches are Journal of Inorganic Biochemistry 119 (2013) [21][22][23][24][25][26][27] beneficial for furthering our understanding of the role of Aβ−Cu 2+ complexes in AD neuropathology.…”

“…Atwood et al observed that Aβ significantly aggregates at neutral and mildly acidic pH values and a change in the Cu 2+ binding mode occurs [12]. The kinetic behaviors of Cu 2+ binding to Aβ have been investigated by isothermal titration calorimetry and fluorescence spectroscopy [20][21][22][23][24]. However, these results were obtained only under neutral or mildly acidic conditions, and the affinity of Aβ to Cu 2+ below pH 6.0 remains unclear.…”

Dual role of Cu2+ ions on the aggregation and degradation of soluble Aβ oligomers and protofibrils investigated by fluorescence spectroscopy and AFM

“…The origin of this remaining fluorescence is not clearly explained as yet [28], and certainly represents a matter of debate as it would indicate that the Cu 2+ ions are not directly bound to tyrosine phenolic groups [26][27][28]33]. Recently, Zhou and co-workers have shown that tyrosine fluorescence is retrieved (although not totally) when Cu 2+ bound to Aβ(1-42) is reduced to Cu + [29], thus indicating that at least part of these copper ions (those involved in fluorescence quenching) are indeed located in the close proximity of tyrosine units.…”

“…In 2005, Szalai and co-workers reported the apparent binding affinities of Cu 2+ for Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), Aβ and Aβ(1-40) that were determined by tyrosine fluorescence, which is known to be quenched when Cu 2+ is coordinated to the protein [28]. Subsequently, Zhou and co-workers showed that the tyrosine fluorescence could be recovered by reduction of Cu 2+ to Cu + with NaBH 4 [29].…”

“…In the present study, the intrinsic fluorescence of Aβ imparted by its tyrosine residue (Tyr, Scheme 1) [29] has been used to investigate the Cu-and Zn-mediated formation of amyloid aggregates from human Aβ . Indeed, it is recognized that the fluorescence properties of Aβ are altered upon binding to Cu or Zn [24].…”