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Isolated reaction centers of photosystem II with an altered pigment content were obtained by chemical exchange of the native pheophytin a molecules with externally added 13(1)-deoxo-13(1)-hydroxy-pheophytin a. Judged from a comparison of the absorption spectra and photochemical activities of exchanged and control reaction centers, 70-80% of the pheophytin molecules active in charge separation are replaced by 13(1)-deoxo-13(1)-hydroxy-pheophytin a after double application of the exchange procedure. The new molecule at the active branch was not active photochemically. This appears to be the first stable preparation in which a redox active chromophore of the reaction center of photosystem II was modified by chemical substitution. The data are compatible with the presence of an active and inactive branch of cofactors, as in bacterial reaction centers. Possible applications of the 13(1)-deoxo-13(1)-hydroxy-pheophytin a-exchanged preparation to the spectral and functional analysis of native reaction centers of photosystem II are discussed.

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH4-). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH4- in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13(1)-deoxo-13(1)-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo- and P680+. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Qy and Qx transitions of this molecule were determined to be located at 5 degrees C at 679.5-680 nm and 542 nm, respectively.