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.

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.

Phosphorescence characterized by the main emission band at 952 ± 1 nm (1.30 eV), the lifetime of 1.5 ± 0.1 ms and the quantum yield nearly equal to that for monomeric chlorophyll a in aqueous detergent dispersions, has been detected in isolated reaction centers (RCs) of spinach photosystem II at 77 K. The excitation spectrum shows maxima corresponding to absorption bands of chlorophyll a, pheophytin a, and β-carotene. The phosphorescence intensity strongly depends upon the redox state of RCs. The data suggest that the phosphorescence signal originates from the chlorophyll triplet state populated via charge recombination in the radical pair [Formula: see text].

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H(A)(-). The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P(A) and P(B) in the P(+) state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P(A) and P(B) macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P(+)Q(-)/PQ spectra suggests the 17(3)-ester group of the bacteriochlorophyll P(A) to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B(A) and the bacteriopheophytin H(A). Structural changes are observed in the monomer bacteriochlorophyll B(B) binding site in the inactive chromophore branch of the mutant RCs.

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