The Cu2+ Binding Site of -Crystallin

Abstract

Purpose: : A number of studies have reported that lens proteins bind metal ions including redox active copper (Cu2⁺). Analysis of human lenses indicates the presence of copper at significant concentrations.Cu²⁺ catalyzes the production of oxygen reactive species and has been implicated as a catalyst for the autoxidation of SH groups in the lens. Here, we have used electron paramagnetic resonance spectroscopy (EPR) to characterize the Cu²⁺ binding site of A-crystallin and determine the identity of atoms coordinated to Cu²⁺.

Methods: : We prepared samples of recombinant rat A-crystallin mixed with varying concentrations of Cu²⁺ at pH 7.2. Continuous -wave EPR analysis was carried out at multiple microwave frequencies at 77 K. Pulse Electron Spin Echo Envelope Modulation spectroscopy (ESEEM) was carried out at 10 K.

Results: : The EPR spectrum of A-crystallin bound Cu²⁺ has with g=2.27 and A=172 G and is invariant with the amount of added Cu²⁺. These EPR parameters are consistent with three nitrogens and one oxygen donor atoms according to the Peisach-Blumberg plots, but no nitrogen superhyperfine lines were resolved at X-band (9.15 GHz). At a lower microwave frequency (S-band, 3.425 GHz), nitrogen superhyperfine lines were resolved in the g₁ region. Simulation of the EPR spectrum gave a best fit with three nitrogens and 1 oxygen donor atoms. In comparison, addition of Cu²⁺ to Hsp16.5 leads to an EPR spectrum characteristic of non specific binding. The ESEEM spectrum for A-bound Cu²⁺ is typical of one strongly bound, equatorial nitrogen donor atom from the imidazole of a histidine amino acid. No resolved peaks are observed between 2.3 and 3.2 MHz where combination lines would indicate that more than one imidazole is bound to the same Cu²⁺ ion.

Conclusions: : The binding of Cu²⁺ appears to be an evolved property of mammalian sHSP. Its likely function is to protect cells from oxidative damage. The age-related truncation of -crystallin in the lens may lead to loss of Cu²⁺ chelation and thus exposes the lens to oxidative damage.

Keywords: crystallins • chaperones • oxidation/oxidative or free radical damage