Selective detection of the structural changes upon photoreactions of several redox cofactors in photosystem II by means of light-induced ATR-FTIR difference spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was applied for the first time to detect the structural changes upon photoreactions of redox cofactors in photosystem II (PSII). The PSII-enriched membranes from spinach were adsorbed on the surface of a silicon prism, and FTIR measurements of various redox cofactors were performed for the same sample but under different conditions by exchanging buffers in a flow cell. Light-induced FTIR difference spectra upon redox reactions of the oxygen-evolving Mn cluster, the primary quinone electron acceptor QA, the redox-active tyrosine YD, the primary electron acceptor pheophytin, and the primary electron donor chlorophyll P680 were successively recorded in buffers including different redox reagents and inhibitors. All of these cofactors remained active in the PSII membranes on the silicon surface, and the resultant spectra were basically identical to those previously recorded by the conventional transmission method. These ATR-FTIR measurements enable accurate comparison between reactions of different active sites in a single PSII sample. The present results demonstrated that the ATR-FTIR spectroscopy is a useful technique for investigation of the reaction mechanism of PSII.