Structural Perturbation of the Carboxylate Ligands to the Manganese Cluster upon Ca2+/Sr2+ Exchange in the S-State Cycle of Photosynthetic Oxygen Evolution As Studied by Flash-Induced FTIR Difference Spectroscopy

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Abstract
A Ca2+ ion is an indispensable element in the oxygen-evolving Mn cluster in photosystem II (PSII). To investigate the structural relevance of Ca2+ to the Mn cluster, the effects of Sr2+ substitution for Ca2+ on the structures and reactions of ligands to the Mn cluster during the S-state cycle were investigated using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra representing the four S-state transitions, S1 → S2, S2 → S3, S3 → S0, and S0 → S1, were recorded by applying four consecutive flashes either to PSII core complexes from Thermosynechococcus elongatus or to PSII-enriched membranes from spinach. The spectra were also recorded using biosynthetically Sr2+-substituted PSII core complexes from T. elongatus and biochemically Sr2+-substituted PSII membranes from spinach. Several common spectral changes upon Sr2+ substitution were observed in the COO- stretching region of the flash-induced spectra for both preparations, which were best expressed in Ca2+-minus-Sr2+ double difference spectra. The significant intensity changes in the symmetric COO- peaks at ∼1364 and ∼1418 cm-1 at the first flash were reversed as opposite intensity changes at the third flash, and the slight shift of the ∼1446 cm-1 peak at the second flash corresponded to the similar but opposite shift at the fourth flash. Analyses of these changes suggest that there are at least three carboxylate ligands whose structures are significantly perturbed by Ca2+/Sr2+ exchange. They are (1) the carboxylate ligand having a bridging or unidentate structure in the S2 and S3 states and perturbed in the S1 → S2 and S3 → S0 transitions, (2) that with a chelating or bridging structure in the S1 and S0 states and perturbed also in the S1 → S2 and S3 → S0 transitions, and (3) that with a chelating structure in the S3 and S0 states and changes in the S2 → S3 and S0 → S1 transitions. Taking into account the recent FTIR studies using site-directed mutagenesis and/or isotope substitution [Chu et al. (2004) Biochemistry 43, 3152−3116; Kimura et al. (2005) J. Biol. Chem. 280, 2078−2083; Strickler et al. (2006) Biochemistry45, 8801−8811], it was concluded that these carboxylate groups do not originate from either D1-Ala344 (C-terminus) or D1-Glu189, which are located near the Ca2+ ion in the X-ray crystallographic model of the Mn cluster. It was thus proposed that if the X-ray model is correct, the above carboxylate groups sensitive to Sr2+ substitution are ligands to the Mn ions strongly coupled to the Ca2+ ion rather than direct ligands to Ca2+.