Copper redox cycling in the prion protein depends critically on binding mode

Abstract

The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc. 2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches copper's inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP's putative role in cellular signaling.

Figure 1

Structures of OR–Cu²⁺ (low Cu²⁺ occupancy) and OR–Cu²⁺₄ (high Cu²⁺ occupancy).

Figure 2

Cyclic voltammograms of (A) OR–Cu²⁺, (B) OR–Cu²⁺₄, (C) non-OR–Cu²⁺ and (D) free Cu²⁺ in N₂-saturated (black curve) and O₂-purged solutions (red curve), respectively. The Cu²⁺ concentration in all cases was 90 μM, while the OR and non-OR concentrations were 100, 25, and 100 μM in panels (A), (B) and (C), respectively. The dotted line curve in (A) corresponds to the CV of OR. The scan rate was 5 mV/s.

Figure 3

Cyclic voltammograms of (A) OR–Cu²⁺, (B) OR–Cu²⁺₄, and (C) non-OR–Cu²⁺ at different pH values. In panel (A) the curves correspond to pH 7.4 (black), 6.5 (red) and 6.0 (blue) and in panels (B) and (C) the curves correspond to 7.4 (black), 7.0 (red) and 6.0 (blue). The concentrations of Cu²⁺ and peptides used are the same as those in Figure 2. Panel (D) contrasts the voltammograms of OR–Cu²⁺ at pH 5.5 in the presence (red curve) and absence (black curve) of O₂.

Figure 4

(A) Change of AA (200 μM) absorbance as a function of reaction time in the absence (green curve) and presence of different Cu²⁺-containing species: OR–Cu²⁺ (red curve), non-OR–Cu²⁺ (cyan), OR–Cu²⁺₄ (blue), and free Cu²⁺ (black). The magenta curve corresponds to the AA absorbance variation recorded after the addition of 100 μM OR–Cu²⁺ to 200 μM AA solution (B) Amounts of H₂O₂ generated by OR–Cu²⁺, Aβ(1–16)–Cu²⁺, α-syn(1–19)–Cu²⁺, OR–Cu²⁺₄, and non-OR–Cu²⁺. [Cu²⁺] was 5 μM, and concentrations of the peptide molecules were all 100 μM except for the solution of OR–Cu²⁺₄ wherein [OR] = 1.25 μM.

Figure 5

(A) Time-dependence of H₂O₂ generation in different solutions: OR–Cu²⁺ (black), OR–Cu²⁺₄ (red), non-OR–Cu²⁺ (blue), Cu²⁺/glutamine (green) and free Cu²⁺ (magenta). All solutions contained 5 μM Cu²⁺ and 200 μM AA and the ligand concentration was 100 μM except for the solution of OR–Cu²⁺₄ wherein [OR] = 1.25 μM. (B) Variations of [H₂O₂] generated by OR–Cu²⁺ in solutions of different pH values: 7.4 (black), 6.5 (red), 6.0 (green) and 5.5 (blue). Each data point is the average of three replicates and the error bars are the standard deviations.

Figure 6

Schematic representation of the possible roles of PrP–Cu²⁺ complexes in quenching the Cu²⁺ redox cycling or gradual production of H₂O₂ for signal transduction. PrP is tethered to cell membrane via the glycosylphosphatidylinositol (GPI) anchor (green) with its α-helices in the C terminus shown in orange, N-linked carbohydrates in purple, and the OR and non-OR domains near the N-terminus depicted in white. When [Cu²⁺] is at a low level (nM or lower) and at pH ranging between 5.5 and 7.4, Cu²⁺ (blue sphere) remains bound in the OR–Cu²⁺ mode (left), quenching the Cu²⁺ redox cycling. At higher [Cu²⁺] (μM) and a pH closer to the physiological value, the binding mode transitions to OR–Cu²⁺₄ (right), leading to a gradual and controlled production of H₂O₂. Coordinates for the PrP C-terminal domain, along with carbohydrates, GPI anchor and membrane were kindly provided by Professor Valerie Daggett (U. Washington).