Identification of a Mn−O−Mn Cluster Vibrational Mode of the Oxygen-Evolving Complex in Photosystem II by Low-Frequency FTIR Spectroscopy

Abstract

We have developed conditions for recording the low-frequency S₂/S₁ Fourier transform infrared difference spectrum of hydrated PSII samples. By exchanging PSII samples with buffered ¹⁸O water, we found that a positive band at 606 cm^-1 in the S₂/S₁ spectrum in ¹⁶O water is clearly downshifted to 596 cm^-1 in ¹⁸O water. By taking double-difference (S₂/S₁ and ¹⁶O minus ¹⁸O) spectra, we assign the 606 cm^-1 mode to an S₂ mode and also identify a corresponding S₁ mode at about 625 cm^-1. In addition, by Sr and ⁴⁴Ca substitution experiments, we found that the 606 cm^-1 mode is upshifted to about 618 cm^-1 by Sr²⁺ substitution but that this mode is not affected by substitution with the ⁴⁴Ca isotope. On the basis of these results and also on the basis of studies of Mn model compounds, we assign the 625 cm^-1 mode in the S₁ state and the 606 cm^-1 mode in the S₂ state to a Mn−O−Mn cluster vibration of the oxygen-evolving complex (OEC) in PSII. This structure may include additional bridge(s), which could be another oxo, carboxylato(s), or atoms derived from an amino acid side chain. Our results indicate that the bridged oxygen atom shown in this Mn−O−Mn cluster is exchangeable and accessible by water. The downshift in the Mn−O−Mn cluster vibration as manganese is oxidized during the S₁ → S₂ transition is counterintuitive; we discuss possible origins of this behavior. Our results also indicate that Sr²⁺ substitution in PSII causes a small structural perturbation that affects the bond strength of the Mn−O−Mn cluster in the PSII OEC. This suggests that Sr²⁺, and by inference, Ca²⁺, communicates with, but is not integral to, the manganese core.