Copper Redox Cycling in the Prion Protein Depends Critically on Binding Mode

Abstract

The prion protein (PrP) takes up four to six equivalents 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., 127, 12647–12656, 2005). 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 Pathogens, 5(4): e1000390, 2009). 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.

The rich electrochemistry and redox reactions of the copper sites in the cellular prion protein

“…In the absence of metal ions, both PrP and PrP-derived peptides are not reducible or oxidizable over a wide range of potential[16–18], suggesting that their amino acid residues remain stable under most circumstances. However, PrP and PrP peptides containing copper display rich electrochemistry, which is dependent on a variety of factors.…”

“…8 is rather dramatic (0.47 V for the OP–Cu(II) complex), implying that Cu(I) is bound rather weakly. At such a negative potential, Cu(I) would further be reduced to Cu(0) [13], which would be released from the complex and deposit onto the electrode [18]. The same group later extended their studies of the binding of Cu(II) by PEG (polyethylene glycol)-tagged tetrarepeats of OP (i.e., OP 4 or OR)[19] and the influence of nitric oxide (NO) on mono- and dinuclear Cu(II) complexes with OP and OP 2 [20].…”

Prion infection in cells is abolished by a mutated manganese transporter but shows no relation to zinc

“…The association between metals and prion diseases is now well established (Brown et al, 1997;Davies et al, 2011;Jackson et al, 2001;Jones et al, 2005;Kralovicova et al, 2009;Liu et al, 2011;Stevens et al, 2009;Stockel et al, 1998;Walter et al, 2009;Wells et al, 2006), but the exact role, if any, that the metals play in the disease process is not fully understood. Initially, PrP was identified as a copper binding protein in vivo (Brown et al, 1997).…”

Copper(II) complexation to 1-octarepeat peptide from a prion protein: Insights from theoretical and experimental UV-visible studies

“…At the N-terminal of PrP C , there is a region with four or five repeats of the amino acid sequence PHGGGWGQ, designated as the octarepeat region, which has the notable feature of complexing copper ions in its structure [10,11]. This complexation is also related to the metal concentration and copper-binding mode in the protein center [12,13] because the protein forms a wrap-around cover by linking several histidines in the sequence at low concentrations. Because the pH is determined by the metal concentration under normal conditions, up to four copper ions can bind to the deprotonated nitrogen atoms in the imidazole groups in the HGGG domain of the octarepeat's histidine region [14], and two copper ions can connect to histidines 96 and 111 in the peptide sequence [15].…”

“…Through mechanisms that are not yet well understood, free copper ions can have a toxic effect on the complexation of copper with PrP C when they are present at high concentrations in the cellular medium. Under these conditions, 4 or 5 copper(II) ions can be complexed in the octarepeat region, changing the octarepeat region conformation and alter the redox potential of copper [12]. In fact, copper exists in two redox states, Copper(I) and Copper(II), and it represents an excellent catalyst for redox cycling in the presence of oxygen, which generates partially reduced and highly reactive O 2 derivatives, the socalled "reactive oxygen species" (ROS) [16].…”