Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu²⁺ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu²⁺ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of β-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu²⁺ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu²⁺ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu²⁺ ions to Cu⁺ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu⁺ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu²⁺ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.