Anthony G. Wedd

A manifestation of Alzheimer’s disease (AD) is the aggregation in the brain of Aβ peptides derived from the amyloid precursor protein (APP). APP has been linked to modulation of normal copper homeostasis while dysregulation of Aβ production and clearance has been associated with disruption of copper balance. However, quantitative copper chemistry on APP is lacking, in contrast to the plethora of copper chemistry available for Aβ peptides. The soluble extracellular protein domain sAPPα (molar mass including post-translational modifications ~ 100 kD) has now been isolated in good yield and high quality. It is known to feature several copper binding sites with different affinities. However, under copper-limiting conditions, it binds either Cu(I) or Cu(II) with picomolar affinity at a single site (labeled M1) that is located within the APP E2 subdomain. M1 in E2 was identified previously by X-ray crystallography as a Cu(II) site that features four histidine side-chains (H313, H386, H432, H436) as ligands. The presence of Cu^II(His)₄ is confirmed in solution at pH ≤ 7.4 while Cu(I) binding involves either the same ligands or a subset. The binding affinities are pH-dependent and the picomolar affinities for both Cu(I) and Cu(II) at pH 7.4 indicate that either oxidation state may be accessible under physiological conditions. Redox activity was observed in the presence of an electron donor (ascorbate) and acceptor (dioxygen). A critical analysis of the potential biological implications of these findings is presented.

Aberrant regulation of transition metals and the resultant disregulation of neuronal reactive oxygen species (ROS) are considered significant in the etiology of Alzheimer's disease (AD). We determined the solution structure of the D2 domain of APL-1 (APL1-D2), the Caenorhabditis elegans ortholog of the amyloid precursor protein domain 2 (APP-D2). The copper binding affinities of APL1-D2 and APP-D2 were estimated and the ability of their copper complexes to promote formation of ROS was tested. The two protein domains are isostructural, consisting of a three-stranded β-sheet packed against a short α-helix in a βαββ fold. A six-residue insert in APL1-D2, absent in APP-D2, forms an extended loop. The putative copper binding ligands in APP-D2 are not conserved in APL1-D2 and this delineates a clear difference between them. APL1-D2 and APP-D2 bind one equivalent of Cu(i) weakly, with dissociation constants K_D ∼10^−8.6 M and ∼10⁻¹⁰ M, respectively, and up to two equivalents of Cu(ii) with K_D values in the range 10⁻⁶ –10⁻⁸ M. The relative abilities of APL1-D2, APP-D2 and amyloid-β (Aβ) copper complexes to generate H₂O₂ correspond to their copper binding affinities. Copper affinities for Aβ (K_D ∼ 10⁻¹⁰ M for both Cu(i) and Cu(ii)) and APP-D2 are in a range allowing redox cycling to occur, albeit less efficiently for APP-D2. However, APL1-D2 binds Cu(i) and Cu(ii) too weakly to sustain catalysis which further suggests that it plays no significant role in copper handling in C. elegans. Overall, the data are consistent with a possible role in copper homeostasis for APP-D2, but not APL1-D2.

The human metallo-chaperone protein Atox1 features a high affinity CuI binding site Cys12GlyGlyCys15 (KD = 10−17.4 M at pH 7.0) and delivers copper to

S K-edge X-ray absorption spectroscopy (XAS) was performed on wild type Cp rubredoxin and its Cys → Ser mutants in both solution and lyophilized forms. For wild type rubredoxin and for the mutants where an interior cysteine residue (C6 or C39) is substituted by serine, a normal solvent effect is observed, that is, the S covalency increases upon lyophilization. For the mutants where a solvent accessible surface cysteine residue is substituted by serine, the S covalency decreases upon lyophilization which is an inverse solvent effect. Density functional theory (DFT) calculations reproduce these experimental results and show that the normal solvent effect reflects the covalency decrease due to solvent H-bonding to the surface thiolates and that the inverse solvent effect results from the covalency compensation from the interior thiolates. With respect to the Cys → Ser substitution, the S covalency decreases. Calculations indicate that the stronger bonding interaction of the alkoxide with the Fe relative to that of thiolate increases the energy of the Fe d orbitals and reduces their bonding interaction with the remaining cysteines. The solvent effects support a surface solvent tuning contribution to electron transfer, and the Cys → Ser result provides an explanation for the change in properties of related iron−sulfur sites with this mutation.

Bu₄N⁺ salts of [S₂W₁₈O₆₂]^5– and [S₂W₁₈O₆₂]^6– can be isolated by electro reduction of γ*-(Bu₄N)₄[S₂W₁₈O₆₂] in MeCN. α-(Bu₄N)₅[S₂W₁₈O₆₂]·4MeCN crystallizes in space group C2/c with a = 26.226(2) Å, b = 15.570(2) Å, c = 38.407(2) Å, β = 115.59(2)°, V = 14 145(2) ų, and Z = 8. The observations indicate that a γ* « α isomerization is associated with the [S₂W₁₈O₆₂]^4–– [S₂W₁₈O₆₂]^5– couple. However, the transformation could not be detected on the timescale of cyclic voltammetry up to a scan rate of 10 V s^–1. The available data suggest that either: (i) the γ* « α equilibrium favors one of the isomers in solution for both redox states, or (ii) the isomerization is slow on the cyclic voltammetric timescale but the differences in E_1/2 values for the α-[S₂W₁₈O₆₂]^4––[S₂W₁₈O₆₂]^5– and γ*-[S₂W₁₈O₆₂]^4––[S₂W₁₈O₆₂]^5– couples cannot be resolved by cyclic voltammetry.Key words: polyoxometalates, synthesis, electrochemistry, isomerization.