Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy.

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn(4) cluster are known in all S states except S(4). Many signals are insufficiently understood and the S(0), S(1), and S(3) states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S(0), S(1), and S(3) states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S(0), split S(1), split S(3) and S(2) state multiline EPR signals. We demonstrate that quantification of the S(1), S(3) and S(0) states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S(1) --> S(2) transition proceeds without misses following a saturating flash at 1 degrees C, whilst substantial misses occur in the S(2) --> S(3) transition following the second flash.

Enhancement of YD spin relaxation by the CaMn4 cluster in photosystem II detected at room temperature: a new probe for the S-cycle.

The long-lived, light-induced radical Y(D) of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn(4) cluster, situated approximately 30 A away. In this study we report a transient step-change increase in Y(D) EPR intensity upon the application of a single laser flash to S(1) state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y(D) by the OEC in the S(2) state, as a consequence of the single laser flash turnover. The post-flash decay reflected S(2)-->S(1) back-turnover, as confirmed by their correlations with independent measurements of S(2) multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y(D) as a probe.