Although copper is a redox-active metal ion, the cupric oxidation state is generally considered to be unreactive to dioxygen. However, based on our fluorescence quenching experiments, clear differences in the two titration curves are observed following addition of copper(II) to F4W in the presence and absence of dioxygen. Therefore, it is possible that the Trp4 emission is indicating a secondary copper(II) binding site of decreased affinity. However, since small changes were also observed for Syn1-4, this explanation seems less probable. Another more likely explanation is that Trp4 is sensing a contribution from copper(I), the more reactive redox state. The “soft” CuI ion would likely have a lower affinity for the N-terminal binding site due to the “hard” N–amide backbone resulting in a higher Kd value. In the presence of O2, re-oxidation of CuI-Syn could occur leading to the enhanced affinity.
Indeed, the reduction of copper(II) to copper(I) has been suggested for Aβ, the amyloidogenic peptide implicated in Alzheimer's disease [1
]. For Aβ, the Met35 residue has been proposed as an electron donor for the reduction of CuII
-Aβ to CuI
-Aβ, therefore a similar electron transfer pathway is feasible for CuII/I
-Syn. We anticipate that if Met oxidation (Met → Met-O) as well as metal reduction (CuII
) is occurring then the metal-protein affinity would be altered. This would possibly account for the differences in the Kd
values reported herein. However, more work on α-syn is necessary before considering the specific involvement of Met1 or Met5 as an endogenous antioxidant and/or the role of copper(I) ion.
Since the primary copper(II) coordination site of α-syn is within the first four N-terminal residues, the effect of Met1 oxidation on the CuII
binding affinity as well as the peptide conformation was probed using the Syn1-4
peptides. Our CD spectroscopic and Trp4 fluorescence quenching data demonstrate that Met1 oxidation had little effect on the CuII
conformation. Generally, replacement of Met with M* leads to a decrease in side-chain flexibility, an increase in side-chain polarity and steric bulk as well as a potential H-bond acceptor site [25
]. As a result, it is surprising that a conformational change was not observed upon CuII
formation. However, a greater effect may result during aggregation and fibrillation. For example, early reports from Fink and coworkers suggested that methionine oxidation inhibits fibrillation of α-synuclein at neutral pH except in the presence of certain metals [26
], while Leong et. al.
recently reported that Met oxidation is required for dopamine to generate soluble α-syn oligomers [27
The spectral features observed within the CD spectra do suggest, however, the existence of a cation-π interaction between the CuII
ion and the Phe and Trp residue. Cation-π interactions are a recent addition to conventional copper-protein interactions as a result of the X-ray crystal structural characterization of the copper chaperone CusF [28
]. The suggested role of the CuI
-Trp interaction within CusF is to protect the metal ion from unwanted removal or oxidation so perhaps this proposal can be extended to the role of Phe4 (or Trp4) in α-syn.
Although oxidation of Met1 had little effect on the copper(II) binding affinity, it is expected that a larger effect would be observed upon copper(I) binding. Copper(I) has a greater potential of producing ROS in the presence of O2 or H2O2 and in turn may lead to enhanced oxidative stress in vivo. As a result, future experiments will be directed at examining the effect of dioxygen on copper(I) binding to α-syn and the possible role of Met1 as a ligand will be evaluated.