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H. S. Mchaourab, W. E. Antholine, H. A. Koteiche, M. Bortolus, J. McCracken; The Cu2+ Binding Site of -Crystallin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2109.
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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+.
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.
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.
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.
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