Understanding the Chemistry of Metallothionein-3 in Copper Homeostasis and Neurodegeneration

Copper is an essential trace element present in high concentrations in the central nervous system (CNS), where it plays critical roles as an enzyme co-factor, structural agent, and signaling ion. Due to its ability to redox-cycle between the Cu(I) and Cu(II) states in physiological conditions, copper requires tight regulation to prevent aberrant redox-cycling that leads to the formation of toxic reactive oxygen species (ROS). Thus, cells evolved a series of copper uptake and distribution pathways to prevent the presence of free copper in the cytosol. A key protective role is played by copper storage proteins called metallothioneins (MTs). Mammalian MTs are small (6–7 kDa), cysteine-rich proteins that play key functions in metal homeostasis, storage, delivery, detoxification, and in the defense against oxidative stresses. Through the conserved array of 20 coordinating cysteines comprising a third of their amino acid sequence, MTs bind d10 metal ions with high affinity in two solvent-shielded thiolate cores in their N-terminal β-domain and Cterminal α-domain. Out of the four human isoforms (MTs 1–4), the MT-3 isoform possesses the highest copper-binding character and is the only isoform primarily expressed in the CNS. However, the sequence, structural, and reactivity determinants that give MT-3 its copper-specific character remain elusive. By conducting mutational studies and spectroscopic characterization of MT-3 and MT-2, the isoform with highest zinc character, isoform-specific non-coordinative residues that are critical for the specific metal selectivity bias of each isoform were identified. Specific roles were assigned to these isoform-specific MT-3 residues in conferring faster metal exchange kinetics, increased Cu(I)-thiolate cluster stability, and decreased zinc selectivity in the β-domain, confirming that MT-3 is indeed fine-tuned for its copper-related roles in the CNS. The ability of MT-3 to store copper stems from its ability to bind Cu(I) in a stable cluster in its βdomain, which resists redox-cycling in the presence of molecular oxygen and reductants. While the coordination and reactivity of the cluster is established, its pathway of assembly is still unknown. Using rapid mixing electronic absorption, electron paramagnetic resonance, and luminescence spectroscopies to follow the reaction of Zn7MT-3 with Cu(II), evidence is provided, for the first time, of a long-lived and oxygen-stable copper-coupled disulfide radical anion intermediate. Thus, a concerted electron transfer mechanism which involves a stabilized intermediate was proposed, leading to the formation of stable Cu(I)4Zn4MT-3 species. Due to unique copper-binding properties, MT-3 has been proposed to protect against aberrant copper-protein interactions in neurodegenerative diseases. In Parkinson’s disease, copper levels are dysregulated resulting in aberrant binding to the α-synuclein (α-Syn) protein, potentiating toxic redox activities and aggregation. This work reveals that the physiologically relevant membranebound N-terminally acetylated α-Syn-Cu(II) complex catalyzes ROS and dityrosine cross-link formation and exacerbates the dopamine oxidase activity of the complex compared to soluble forms. Zn7MT-3, by scavenging Cu(II) from these complexes, reducing it to Cu(I), and binding it in a redox-stable core in the β-domain, silences all these toxic reactivities.