Temperature Dependence of Tryptophan Fluorescence Lifetime as an Indicator of Its Microenvironment Dynamics.

The spectral-kinetic characteristics of the fluorescence of the tryptophan molecule in an aqueous solution and in the composition of a protein (albumin) were studied in the temperature range from -170 to 25°C. To explain the observed changes in the spectra and the tryptophan fluorescence lifetime with temperature, a model of transitions between the excited and ground states involving a charge-transfer state was used, which takes into account the nonlinear nature of the dynamics of these transitions. In these processes, an important role is played by the interaction of tryptophan molecules with its microenvironment, as well as rearrangements in the system of hydrogen bonds of the water-protein matrix surrounding the tryptophan molecule.

Non-photochemical Fluorescence Quenching in Photosystem II Antenna Complexes by the Reaction Center Cation Radical.

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.

Temperature dependence of protein fluorescence in Rb. sphaeroides reaction centers frozen to 80 K in the dark or on the actinic light as the indicator of protein conformational dynamics.

The differences in the average fluorescence lifetime (τav) of tryptophanyls in photosynthetic reaction center (RC) of the purple bacteria Rb. sphaeroides frozen to 80 K in the dark or on the actinic light was found. This difference disappeared during subsequent heating at the temperatures above 250 K. The computer-based calculation of vibration spectra of the tryptophan molecule was performed. As a result, the normal vibrational modes associated with deformational vibrations of the aromatic ring of the tryptophan molecule were found. These deformational vibrations may be active during the nonradiative transition of the molecule from the excited to the ground state. We assume that the differences in τav may be associated with the change in the activity of these vibration modes due to local variations in the microenvironment of tryptophanyls during the light activation.

The efficiency of non-photochemical fluorescence quenching by cation radicals in photosystem II reaction centers.

In a direct experiment, the rate constants of photochemical k p and non-photochemical k p+ 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" P680+Q A- 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 P680+Pheo- in PS II RC. Thus, for the first time, the ratio k p+ /k p â‰… 1.5 was found directly. The mechanism of the higher efficiency of non-photochemical quenching comparing to photochemical quenching is discussed.

Investigation of Stability of Photosynthetic Reaction Center and Quantum Dot Hybrid Films.

The efficiency of interaction (efficiency of energy transfer) between various quantum dots (QDs) and photosynthetic reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides and conditions of long-term stability of functioning of such hybrid complexes in film preparations were investigated. It was found that dry films containing RCs and QDs and maintained at atmospheric humidity are capable to keep their functional activity for at least some months as judging by results of measurement of their spectral characteristics, efficiency of energy transfer from QDs to RCs, and RC electron-transport activity. Addition of trehalose to the films giving them still greater stability is especially expressed for films maintained at low humidity. These stable hybrid film structures are promising for further biotechnological studies for developing new phototransformation devices.

[Possible effect of structural phase transition in the reactive center of Rhodobacter sphaeroides on the rate of dark adaptation of photooxidized bacteriochlorophyll from primary quinone]. / Vozmozhnoe vliianie strukturnogo fazovogo perekhoda v reaktsionnykh tsentrakh Rhodobacter sphaeroides na skorost' temnovogo vosstanovleniia fotookislennogo bakteriokhlorofilla ot pervichnogo khinona.

The dependence of the rate of dark recombination between the photooxidized primary donor--dimer bacteriochlorophyll molecule (P) and reduced primary quinone acceptor (QA), P+QA(-)-->PQA was studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides in the temperature range of 100-320 K. Control RC preparations, RC species with the removed H-subunit as well as RC samples with the hydrogen bonds network modified by isotopic D2O-H2O substitution were investigated. An anomalous temperature dependence of the recombination time (tau rec) of dark reaction P+QA(-)-->PQA was found for all RC samples. It was found that upon heating from 120 to 290 K tau rec increased 2.5 fold. However, upon further heating to 320 K, tau rec decreased again. The temperature dependences of the P+QA(-)-->PQA recombination time were compared with those of the thermodepolarization current of RC preparations in the same temperature range. The temperature curve of the thermodepolarization current was also nonmonotonous. The theoretical interpretation of the temperature dependence of tau rec as well as of the thermodepolarization current was made in the framework of the theory of structural phase transitions within the hydrogen bond network in the water-protein surrounding of the redox centers participating in the electron transfer reactions.

[Photooxidation and light-induced transport of phenazine methosulfate in chromatophores of purple bacteria]. / Fotookislenie i fotoindutsirovannyi transport fenazinmetosul'fata v khromatoforakh purpurnykh bakterii.

The light-induced interaction of phenazine methosulfate (PMS) with chromatophores of the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas sphaeroides was studied, using an ion-specific electrode. Illumination caused an initial rapid increase in the concentration of methylphenazinium cation (MP+) and a subsequent slow (1-3 min) decrease of the MP+ concentration to a low steady level. The rapid phase of the light-induced MP+ concentration change is specifically enhanced by ascorbate. The slow phase (uptake of MP+ from the medium) is stimulated on addition of valinomycin, which is known to collapse the membrane potential of energized chromatophores, and is partly inhibited by NH4Cl, which enhances the membrane potential in chromatophores. The light-induced uptake of MP+ is sharply stimulated by dibromothymoquinone. It is concluded that the initial rapid increase of the MP+ concentration in the outer medium results from the oxidation of the reduced PMS by photooxidized reaction centers. The slow decrease of the external MP+ concentration is due to active transport of MP+ into the internal space of the chromatophores via a mechanism of a chemiosmotic type. The accumulation of MP+ is directly mediated by the redox reactions of PMS at the outer and inner surfaces of the photosynthetic membrane, which are involved in cyclic electron transport.

Photo-induced electron transport and water state in Rhodospirillum rubrum chromatophores.

It is shown that in bacterial chromatophores the pronounced changes in the free water content with a proton spin-spin relaxation time (T2) of 10(-3)--10(-2) s does not influence the efficiency of electron transfer from the photosynthetic reaction centre to the membrane pool of secondary acceptors. An abrupt inhibition of this process occurs only after the loss of the water with faster proton spin-spin relaxation time (T2 of 10(-4) s). The process is reversible. The water fraction in question is obviously bound to the chromatophore proteins and forms the primary hydration layer.