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Phosphorescence measurements at 77 K and light-induced FTIR difference spectroscopy at 95 K were applied to study of the triplet state of chlorophyll a ((3)Chl) in photosystem II (PSII) core complexes isolated from spinach. Using both methods, (3)Chl was observed in the core preparations with doubly reduced primary quinone acceptor QA. The spectral parameters of Chl phosphorescence resemble those in the isolated PSII reaction centers (RCs). The main spectral maximum and the lifetime of the phosphorescence corresponded to 955±1 nm and of 1.65±0.05 ms respectively; in the excitation spectrum, the absorption maxima of all core complex pigments (Chl, pheophytin a (Pheo), and β-carotene) were observed. The differential signal at 1667(-)/1628(+)cm(-1) reflecting a downshift of the stretching frequency of the 13(1)-keto C=O group of Chl was found to dominate in the triplet-minus-singlet FTIR difference spectrum of core complexes. Based on FTIR results and literature data, it is proposed that (3)Chl is mostly localized on the accessory chlorophyll that is in triplet equilibrium with P680. Analysis of the data suggests that the Chl triplet state responsible for the phosphorescence and the FTIR difference spectrum is mainly generated due to charge recombination in the reaction center radical pair P680(+)PheoD1(-), and the energy and temporal parameters of this triplet state as well as the molecular environment and interactions of the triplet-bearing Chl molecule are similar in the PSII core complexes and isolated PSII RCs.

Difference absorption spectroscopy with temporal resolution of approximately 20 fsec was used to study the primary phase of charge separation in isolated reaction centers (RCs) of Chloroflexus aurantiacus at 90 K. An ensemble of difference (light-minus-dark) absorption spectra in the 730-795 nm region measured at -0.1 to 4 psec delays relative to the excitation pulse was analyzed. Comparison with analogous data for RCs of HM182L mutant of Rhodobacter sphaeroides having the same pigment composition identified the 785 nm absorption band as the band of bacteriopheophytin Phi(B) in the B-branch. By study the bleaching of this absorption band due to formation of Phi(B)(-), it was found that a coherent electron transfer from P* to the B-branch occurs with a very small delay of 10-20 fsec after excitation of dimer bacteriochlorophyll P. Only at 120 fsec delay electron transfer from P* to the A-branch occurs with the formation of bacteriochlorophyll anion B(A)(-) absorption band at 1028 nm and the appearance of P* stimulated emission at 940 nm, as also occurs in native RCs of Rb. sphaeroides. It is concluded that a nuclear wave packet motion on the potential energy surface of P* after a 20-fsec light pulse excitation leads to the coherent formation of the P(+)Phi(B)(-) and P(+)B(A)(-) states.

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The native pheophytin a (Pheo a) in isolated reaction centers of photosystem II (PSII RCs) has been chemically exchanged with extraneous 7-deformyl-7-hydroxymethyl-Pheo b (7(1)-OH-Pheo b) which differs from Pheo a by the C-7 substituent (hydroxymethyl instead of methyl). The two pigments have similar reduction potentials in vitro [M. Meyer, Dissertation, Universität München, 1997], while their absorption spectra show small but distinct differences in the visible region. The resulting 7(1)-OH-Pheo b-modified reaction center preparations were characterized by high-performance liquid chromatography, electronic absorption and light-induced Fourier transform infra red absorption difference spectroscopies, together with photoaccumulation of the reduced pheophytin electron acceptor and NaBH4-treatment. About 70% of the total Pheo a molecules are found to be replaced by 7(1)-OH-Pheo b molecules in modified preparations, indicating that both the photochemically active (PheoD1) and inactive (PheoD2) binding sites were subjected to pigment exchange. The 7(1)-OH-Pheo b molecule located at the PheoD1 site is able to functionally replace the native Pheo a, participating in primary charge separation as an electron acceptor. The Qx absorption band of this modified pheophytin molecule is localized at ~546nm; its Qy band is blue-shifted with respect to the absorption of other reaction center core pigments, being located at ~665nm. The Qy and Qx optical transitions of the 7(1)-OH-Pheo b molecule exchanged into the PheoD2 site are identified at 677 and 543.5nm, respectively. The photochemically active double-modified PSII RCs additionally containing 7-deformyl-7-hydroxymethyl-13(1)-deoxo-13(1)-hydroxy-Pheo b at the PheoD2 site were obtained by treatment of the 7(1)-OH-Pheo b-modified RCs with NaBH4.