Determination of the Miss Probabilities of Individual S-State Transitions during Photosynthetic Water Oxidation by Monitoring Electron Flow in Photosystem II Using FTIR Spectroscopy

Abstract

Water oxidation by plants and cyanobacteria is performed via a light-driven cycle of five intermediates called S states (S(0)-S(4)) at the water oxidizing center (WOC) in photosystem II (PSII). The information about misses, i.e., the probabilities that the S-state transitions failed to advance, is crucial for detailed analysis of various spectroscopic data in investigations of the water oxidation mechanism. In this study, we have determined the miss probabilities of the individual S-state transitions using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The extent of S-state transitions in the WOC upon each saturating flash was monitored by detecting the flow of electrons from the WOC to ferricyanide, an exogenous electron acceptor, using the CN stretching bands of ferricyanide and ferrocyanide. Simulation of the oscillation pattern of the flash-number dependence of the signal amplitude provided the miss probabilities for the S(0) → S(1), S(1) → S(2), S(2) → S(3), and S(3) → S(0) transitions (α(0)-α(3), respectively) without any assumption about fitting parameters. The results for PSII preparations from Thermosynechococcus elongatus and spinach showed a general tendency of misses in the order, α(0) ≤ α(1) < α(2) < α(3), indicating that a more oxidized WOC has a higher miss probability. A very similar result observed for the Y(D)-less mutant (D2-Y160F) of T. elongatus confirmed that Y(D) does not affect the estimated misses. It was further shown that NO(3)(-) treatment specifically increased α(3), consistent with inactivation of the S(3) state reported previously. These results demonstrate the usefulness of this FTIR method for estimating individual miss probabilities in the S-state cycle in elucidation of the molecular mechanism of photosynthetic water oxidation.