Exploring the Calcium-Binding Site in Photosystem II Membranes by Solid-State 113Cd NMR

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Abstract
Calcium (Ca2+) is an essential cofactor for photosynthetic oxygen evolution. Although the involvement of Ca2+ at the oxidizing side of photosystem II of plants has been known for a long time, its ligand interactions and mode of action have remained unclear. In the study presented here, 113Cd magic-angle spinning solid-state NMR spectroscopy is used to probe the Ca2+-binding site in the water-oxidizing complex of 113Cd2+-substituted PS2. A single NMR signal 142 ppm downfield from Cd(ClO4)2·2H2O was recorded from Cd2+ present at the Ca2+-binding site. The anisotropy of the signal is small, as indicated by the absence of spinning side bands. The signal intensity is at its maximum at a temperature of −60 °C. The line width of the proton signal in a WISE (wide-line separation) two-dimensional 1H−113Cd NMR experiment demonstrates that the signal arises from Cd2+ in a solid and magnetically undisturbed environment. The chemical shift, the small anisotropy, and the narrow line of the 113Cd NMR signal provide convincing evidence for a 6-fold coordination, which is achieved partially by oxygen and partially by nitrogen or chlorine atoms in otherwise a symmetric octahedral environment. The absence of a 113Cd signal below −70 °C suggests that the Ca2+-binding site is close enough to the tetramanganese cluster to be affected by its electron spin state. To our knowledge, this is the first report for the application of solid-state NMR in the study of the membrane-bound PS2 protein complex.