Highly efficient spectral hole-burning in oxygen-evolving photosystem II preparations

We present the first report of highly efficient persistent spectral hole-burning in active (oxygen-evolving) Photosystem II (PSII) preparations. Samples are poised in the S₁ state of the Kok cycle, with the primary quinone (QA) either neutral or photoreduced to Q_A ^- via a low-temperature pre-illumination. Remarkably efficient hole-burning is observed within the chlorophyll Q_y(0,0) absorption envelope in the wavelength range of 676-695 nm. The hole-burning action spectrum of a sample poised in the S₁(Q_A^-) state is dominated by a narrow feature (∼40 cm^-1) at 684 nm, where hole depths of 30% are attainable. The photoproduct for spectral holes burnt in this region is distributed across the ∼50 cm^-1 absorption feature centered at 683.5 nm, independent of the excitation wavelength within this band. Saturated hole-burning experiments indicate weak electron-phonon coupling near 684 nm but stronger coupling for holes burnt near 690 nm. Selective excitation near 690 nm of samples in the S₁(Q _A) state also results in efficient Q_A^- formation. Negligible hole-burning activity is observed at higher energies (<676 nm). Holewidths extrapolated to zero fluence and temperature are 2.0 ± 0.5 GHz near 685 nm for PSII samples in the S₁(Q _A^-) state. Holewidths are twice as large and hole-burning quantum efficiencies are up to an order of magnitude greater (approaching 1%) for samples in the S₁(Q_A) state. We ascribe hole-burning near 684 nm to slow (40-210 ps) excitation transfer from a CP43 chlorophyll to the PSII reaction center, and we ascribe hole-burning at ∼690 nm to excitation transfer from a chlorophyll in CP47. The unusually high hole-burning efficiency that we observe is attributed to a mechanism that involves charge separation in the reaction center that follows excitation transfer from these "slow transfer" states in CP43 and CP47. A key result of this work is the observation that selective excitation in the range 685-695 nm leads to efficient charge separation, as indicated by Q_A^- formation. This indicates the presence of (a relatively weak) P680 absorption in a native PSII, extending to low energy and underlying the CP47 chlorophyll trap absorption.