Time-resolved fluorometry of reaction center (RC) preparations from Rhodopseudomonas sphaeroides. wild strain 1760-1, shows that the lifetime of the excited state of bacteriochlorophyll P870* is T = 6 k 1.5 ps and independent of temperature within the range 293-77 K. This value was found to coincide well with the time (7k3 ps) of the RC porphyrin pigment transition into the ion-radical pair state PF, as measured by picosecond absorption spectroscopy of the same preparations.
In a direct experiment, the rate constants of photochemical k and non-photochemical k quenching of the chlorophyll fluorescence have been determined in spinach photosystem II (PS II) membrane fragments, oxygen-evolving PS II core, as well as manganese-depleted PS II particles using pulse fluorimetry. In the dark-adapted reaction center(s) (RC), the fluorescence decay kinetics of the antenna were measured at low-intensity picosecond pulsed excitation. To create a "closed" P680Q state, RCs were illuminated by high-intensity actinic flash 8 ns prior to the measuring flash. The obtained data were approximated by the sum of two decaying exponents. It was found that the antennae fluorescence quenching efficiency by the oxidized photoactive pigment of RC P680 was about 1.5 times higher than that of the neutral P680 state. These results were confirmed by a single-photon counting technique, which allowed to resolve the additional slow component of the fluorescence decay. Slow component was assigned to the charge recombination of P680Pheo in PS II RC. Thus, for the first time, the ratio k /k ≅ 1.5 was found directly. The mechanism of the higher efficiency of non-photochemical quenching comparing to photochemical quenching is discussed.
The reaction center (RC) of purple bacteria is formed by three protein subunits (L, M, and H) bound with four bacteriochlorophyll (Bchl) molecules, two bacteriopheophytin (Bph) molecules, two quinone ( Q A and Q B ) molecules, and one atom of nonheme iron. Two of the four Bchl molecules for a special pair P (Bchl dimer), which is a primary electron donor.After excitation of the P electron, it is transferred along the active A-chain, leading to consecutive formation of the anion radicals Bchl -, Bph -, and during 3, 0.9, and 200 ps, respectively [1][2][3]. In the excited state, P forms a dipole, the base of which ( d ) is approximately 0.6 Å; the base of the dipole (Bchl -Bchl + )*, the charge-transfer state of the dimer P, is 5-7 Å; and the base of the ion-radical pair P + Bchl -is approximately 17 Å. Dipoles or individual charges are sources of electric field; the latter interacts with charged atoms or atomic groups of water-protein environment and induces its rearrangement. It is known that the duration of rearrangement of hydrogen bonds is 10 -13 -10 -12 s [4,5]. It can be expected that the protons of the water-protein environment are primary messengers in the interaction between the electronegative (positive) state of cofactors and the medium within the femto-to picosecond time interval.In RCs isolated from the purple bacterium Rhodobacter sphaeroides , the excitation of the primary electron donor P is accompanied by bleaching of absorption bands at 870 and 600 nm. The exposure of the singlet excited state P + to an probing pulse resulted in a stimulated emission at 920 nm. Separation of charges P* Bchl → P + Bchl -is accompanied by a simultaneous decrease in the intensity of stimulated emission at 920 nm, bleaching of the absorption band of Bchl at Q A -800 nm, and appearance of a new absorption band at 1020 nm, ascribed to Bchl - [6,7]. Further transfer of the electron from Bchl -to Bph is monitored by the decrease in the absorption band at 760 nm, increase in the absorption band at 665 nm (Bph → Bph -), partial relaxation of absorption at 800 nm (Bchl -→ Bchl), and almost complete disappearance of absorption at 1020 nm (Bchl − → Bchl). It may be expected that the formation of states P*, P + , Bchl -, and Bph -will be reflected on the time course of spectral and amplitude characteristics of differential absorption spectra of these components of RC, including the femto-and picosecond time intervals, reflecting the interaction between unsteady states of cofactors and protons of water-protein environment. Figure 1 shows time-resolved differential absorption spectra of native preparations of Rb. sphaeroides RCs suspended in 10 mM sodium-phosphate buffer (pH 8.0), which were excited at room temperature at 600 nm with 70-fs light pulses at different temporal delays of the outgoing light pulse relative to the excitatory one. The time course of differential absorption spectra was analyzed for transitions P → P* → P + (830-890 nm) and Bph → Bph -(730-790 nm). As seen from Fig. 1, spectral position of the maximum of ...
Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of photosynthetic electron transport chain in the bacterial chromatophores of Rba. sphaeroides was investigated. DIP was found to affect efficiency of energy transfer from the light-harvesting complex LH2 to the LH1–reaction center core complex and to produce the long-wavelength (“red”) shift of the absorption band of light-harvesting bacteriochlorophyll molecules in the IR spectral region at 840-900 nm. This shift is associated with the membrane transition to the energized state. It was shown that DIP is able to reduce the photooxidized bacteriochlorophyll of the reaction center, which accelerated electron flow along the electron transport chain, thereby stimulating generation of the transmembrane potential on the chromatophore membrane. The results are important for clarifying possible mechanisms of DIP influence on the activity of membrane-bound functional proteins. In particular, they might be significant for interpreting numerous therapeutic effects of DIP.
In direct experiments, rate constants of photochemical (kP) and non-photochemical (kP(+)) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the "dark" reaction center (RC) state, antenna fluorescence decay kinetics were measured under low-intensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a "closed" [P680(+)QA(-)] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680(+)QA(-)] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680(+)) state was found to be ~1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ1 obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680(+)Pheo(-) charge recombination. Thus, for the first time the ratio of kP(+)/kP = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.
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