Coherent phenomena of charge separation in reaction centers of LL131H and LL131H/LM160H/FM197H mutants of Rhodobacter sphaeroides.

Primary stage of charge separation and transfer of charges was studied in reaction centers (RCs) of point mutants LL131H and LL131H/LM160H/FM197H of the purple bacterium Rhodobacter sphaeroides by differential absorption spectroscopy with temporal resolution of 18 fsec at 90 K. Difference absorption spectra measured at 0-4 psec delays after excitation of dimer P at 870 nm with 30 fsec step were obtained in the spectral range of 935-1060 nm. It was found that a decay of P* due to charge separation is considerably slower in the mutant RCs in comparison with native RCs of Rba. sphaeroides. Coherent oscillations were found in the kinetics of stimulated emission of the P* state at 940 nm. Fourier analysis of the oscillations revealed a set of characteristic bands in the frequency range of 20-500 cm(-1). The most intense band has the frequency of ~130 cm(-1) in RCs of mutant LL131H and in native RCs and the frequency of ~100 cm(-1) in RCs of the triple mutant. It was found that an absorption band of bacteriochlorophyll anion B(A)(-) which is registered in the difference absorption spectra of native RCs at 1020 nm is absent in the analogous spectra of the mutants. The results are analyzed in terms of the participation of the B(A) molecule in the primary electron transfer in the presence of a nuclear wave packet moving along the inharmonic surface of P* potential energy.

Primary processes of charge separation in reaction centers of YM210L/FM197Y and YM210L mutants of Rhodobacter sphaeroides.

Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll B(A)(-) at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* --> P(+)B(A)(-) and to the absence of stabilization of separated charges in the state P(+)B(A)(-). Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule P(B) of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm(-1) in the single mutant YM210L to ~100 cm(-1) in the double mutant. Oscillations with 100-150 cm(-1) frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P(+)B(A)(-) state of both mutants reflect a motion of the P(B) molecule relatively to P(A) in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the B(A)(-) absorption band are conserved within a shorter time ~0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.

Mutant reaction centers of Rhodobacter sphaeroides I(L177)H with strongly bound bacteriochlorophyll a: structural properties and pigment-protein interactions.

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.

Pigment organization and their interactions in reaction centers of photosystem II: optical spectroscopy at 6 K of reaction centers with modified pheophytin composition.

Photosystem II reaction centers (RC) with selectively exchanged pheophytin (Pheo) molecules as described in [Germano, M., Shkuropatov, A. Ya., Permentier, H., Khatypov, R. A., Shuvalov, V. A., Hoff, A. J., and van Gorkom, H. J. (2000) Photosynth. Res. 64, 189-198] were studied by low-temperature absorption, linear and circular dichroism, and triplet-minus-singlet absorption-difference spectroscopy. The ratio of extinction coefficients epsilon(Pheo)/epsilon(Chl) for Q(Y) absorption in the RC is approximately 0.40 at 6 K and approximately 0.45 at room temperature. The presence of 2 beta-carotenes, one parallel and one perpendicular to the membrane plane, is confirmed. Absorption at 670 nm is due to the perpendicular Q(Y) transitions of the two peripheral chlorophylls (Chl) and not to either Pheo. The "core" pigments, two Pheo and four Chl absorb in the 676-685 nm range. Delocalized excited states as predicted by the "multimer model" are seen in the active branch. The inactive Pheo and the nearby Chl, however, mainly contribute localized transitions at 676 and 680 nm, respectively, although large CD changes indicate that exciton interactions are present on both branches. Replacement of the active Pheo prevents triplet formation, causes an LD increase at 676 and 681 nm, a blue-shift of 680 nm absorbance, and a bleach of the 685 nm exciton band. The triplet state is mainly localized on the Chl corresponding to B(A) in purple bacteria. Both Pheo Q(Y) transitions are oriented out of the membrane plane. Their Q(X) transitions are parallel to that plane, so that the Pheos in PSII are structurally similar to their homologues in purple bacteria.

Nuclear wavepacket motion producing a reversible charge separation in bacterial reaction centers.

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P*, which lead to the fs oscillations in stimulated emission from P* [M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.-C. Lambry and J.-L. Martin (1994) Biochemistry 33, 6750-6757] and in Qy absorption band of the primary electron acceptor, bacteriochlorophyll monomer B(A) [A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293-7298] with a set of fundamental frequencies in the range of 10-300 cm(-1). We have found that in pheophytin-modified RCs, the fs oscillations with frequency around 130 cm(-1) observed in the P*-stimulated emission as well as in the B(A) absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer B(A)-. These results are discussed in terms of a reversible electron transfer between P* and B(A) induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+B(A)-, when a maximal value of the Franck-Condon factor is created.

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. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC(1x)) or twice (RC(2x)). HPLC analysis and Pheo Q(X) absorption at 543 nm show that in RC(1x) about 50% of Pheo is replaced and in RC(2x) about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo(-) is unmodified in RC(1x) and decreased only in RC(2x), suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption 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 absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC(1x) or RC(2x), showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength.

Formation of a long-lived P+BA- state in plant pheophytin-exchanged reaction centers of Rhodobacter sphaeroides R26 at low temperature.

Femtosecond transient absorption spectroscopy in the range of 500-1040 nm was used to study electron transfer at 5 K in reaction centers of Rhodobacter sphaeroides R26 in which the bacteriopheophytins (BPhe) were replaced by plant pheophytin a (Phe). Primary charge separation took place with a time constant of 1.6 ps, similar to that found in native RCs. Spectral changes around 1020 nm indicated the formation of reduced bacteriochlorophyll (BChl) with the same time constant, and its subsequent decay in 620 ps. This observation identifies the accessory BChl as the primary electron acceptor. No evidence was found for electron transfer to Phe, indicating that electron transfer from BA- occurs directly to the quinone (QA) through superexchange. The results are explained by a model in which the free energy level of P+Phe- lies above that of P+BA-, which itself is below P*. Assuming that the pigment exchange does not affect the energy levels of P* and P+BA-, our results strongly support a two-step model for primary electron transfer in the native bacterial RC, with no, or very little, admixture of superexchange.

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II.

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.

Excitation energy transfer between ß-carotene and chlorophyll-a in various systems.

The excitation energy transfer from ß-carotene to chlorophyll-a in several seminatural systems such as liposomes, lipid layers and PSI complex has been studied at room and liquid nitrogen temperature. Only in a case of PSI complex an efficient energy transfer (about 30%) from ß-carotene to chlorophyll-a has been observed. The results of energy transfer were discussed on the ground of Dexter's mechanism by taking into account the recently discovered energy level ((1)Ag) of ß-carotene.