Fourier-transform infrared spectroscopic studies on the coordination of the side-chain COO^- groups to Ca²⁺ in equine lysozyme

Interactions between Ca²⁺ and the Asp side chains in the Ca²⁺-binding site of equine lysozyme were investigated by Fourier-transform infrared (FT-IR) spectroscopy. In the spectrum of equine lysozyme, the intensities of the bands at about 1595 cm^-1 and 1578 cm^-1 in the region of the COO^- antisymmetric stretches increased upon Ca²⁺ binding. In the region of the COO^- symmetric stretches, the loss of intensity at about 1388 cm^-1 and gains of intensities at about 1423 cm^-1 and 1403 cm^-1 were observed due to Ca²⁺ binding to equine lysozyme. The spectral changes for equine lysozyme indicate that the COO^- groups of Asp85, Asp90 and Asp91 in the Ca²⁺-binding site coordinate to Ca²⁺ in the pseudo-bridging mode, where divalent metal cation is bound to one of the two oxygens in the COO^- group and a water molecule is hydrogen bonded to the other oxygen. The results presented here provide further evidence for a high degree of similarity between Ca²⁺-binding lysozyme and a-lactalbumin. The effects of Ca²⁺ binding on the main-chain conformation of equine lysozyme were compared with those of bovine α-lactalbumin and hen egg-white lysozyme.