We present the first report of highly efficient persistent spectral hole-burning in active (oxygen-evolving) Photosystem 11 (PSII) preparations. Samples are poised in the S, state of the Kok cycle, with the primary quinone (Q(A)) 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 676695 nm. The hole-burning action spectrum of a sample poised in the SI(Q(A)(-)) state is dominated by a narrow feature (similar to40 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 similar to50 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 S-1 (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 SI(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 SI(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 similar to690 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.