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

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Abstract
Due in large part to the lack of crystal structures of the amyloid-β (Aβ) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal−Aβ complex has been difficult. In this work, we investigated the complexation of Cu(II) by Aβ through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to Aβ can be reduced to Cu(I) using sodium borohydride and that both Aβ−Cu(II) and Aβ−Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the Aβ fluorescence, commonly reported to be quenched upon Aβ−Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of Aβ−Cu(II) complexes provides evidence that the sole tyrosine residue in Aβ 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 Aβ, the binding heterogeneity, and the influence of solution conditions on complex formation.