Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 13(1)-deoxo-13(1)-hydroxy-pheophytin a and comparison of their 6 K absorption spectra

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13 (1)-deoxo-13(1)-hydroxy-pheophytin a (13(1)-OH-Pheo). The absorption bands of 13(1)-OH-Pheo are blue-shifted and well separated from those of Pheo. Tw o kinds of modified reaction centre preparations can be obtained by applyin g the exchange procedure once (RC1x) or twice (RC2x). HPLC analysis and Phe o Q(X) absorption at 543 nm show that in RC1x about 50% of Pheo is replaced and in RC2x about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitativ e excitation transfer from the new pigment to the emitting chlorophylls. Ph otoaccumulation of Pheo(-) is unmodified in RC1x and decreased only in RC2x , suggesting that the first exchange replaces the inactive and the second t he active Pheo. Comparing the effects of the first and the second replaceme nt on the absorption spectrum at 6 K did not reveal substantial spectral di fferences between the active and inactive Pheo. In both cases, the absorpti on changes in the Q(Y) region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls abso rbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm re gion. No absorption decrease is observed at 670 nm for RC1x or RC2x, showin g that neither of the two reaction centre pheophytins contributes substanti ally to the absorption at this wavelength.