Chelator-Induced Disappearance of Carboxylate Stretching Vibrational Modes in S2/S1 FTIR Spectrum in Oxygen-Evolving Complex of Photosystem II

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Abstract
Fourier transform infrared (FTIR) spectroscopy has been applied toward studies of photosynthetic oxygen evolution, especially on the effects of Ca2+ depletion and chelating agents using S2/S1 FTIR difference spectrum in the mid-IR region. Ca2+ depletion showed little influences on the symmetric (1365/1404 cm-1) and the asymmetric (1587/1562 cm-1) stretching bands of a carboxylate, which are typical of the S2/S1 vibrational features induced by the oxidation of the Mn-cluster; however, minor changes were observed in the amide regions. Addition of a chelating agent (EDTA or EGTA) to the Ca2+-depleted membranes resulted in the disappearance of the carboxylate bands concurrent with large modifications of the amide bands with an apparent Kd value of approximately 0.49 mM (for EDTA). The carboxylate bands and the greater part of the amide bands were restored by the replenishment of CaCl2, and the chelators did not affect the spectrum in the nondepleted control membranes, indicating that the effects of the chelator are reversible and manifest only in the cases in which the Ca2+ site is unoccupied by Ca2+. Ca2+-depleted membranes showed the normal S2QA- thermoluminescence band, and further addition of EDTA did not show any effects on the peak temperature and peak intensity. Moreover, the Ca2+-depleted membranes in the presence of EDTA exhibited the S2 multiline EPR signal with nearly the normal hyperfine splittings. These results demonstrated that the Mn-cluster is oxidized to the S2 state with normal redox and magnetic properties in the presence of the chelator despite the loss of the carboxylate bands in the FTIR spectra. The results are interpreted as indicating that the chelator interacts with the Mn-cluster as a replacement of the native carboxylate ligand. This prevents the structural changes of the Mn-cluster and protein backbone which are induced upon the oxidation of the Mn-cluster up to the S2 state, but preserve the redox and magnetic properties of the S2 state Mn-cluster. The roles of Ca2+ in the photosynthetic oxygen evolution are also discussed.