N. P. Grishanova

The influence of structural-dynamic organization of RC from purple bacterium rhodobacter sphaeroides on picosecond stages of photoinduced reactions

Effects of the hydrogen bond network on the rate constants of energy migration (km), charge separation (ke), electron transfer to QA (kQ) and P+I- recombination in RC of Rhodobacter sphaeroides were analysed in control and modified RC preparations at different temperatures. Modification of RC were made by the addition of 40% v/v DMSO. The rate constants km, ke, kQ were evaluated from pump-and-probe measurements of the absorption difference kinetics at 665 nm corresponding to BPhL- formation and subsequent electron transfer to QA. For the investigation of P+I- recombination a primary quinone acceptor was pre-reduced in the dark by adding of 1 mg/ml of dithionite and 1 mM sodium ascorbate. Recombination kinetics were measured at 665 and 870 nm. The numerical analysis of the temperature dependence of ke and kQ was performed on the basis of the model proposed by Kakitani and Kakitani (T. Kakitani and H. Kakitani (1981), Biochim. Biophys. Acta, 635, 498-514). It was found that: (a) in control samples the molecular rate constants km, ke and kQ were about (3.4 ps)-1, (4.5 ps)-1 and (200 ps)-1, respectively; (b) under modification by DMSO these rates decrease up to (5.3 ps)-1, (10.3 ps)-1 and (500 ps)-1, respectively; (c) as the temperature drops from 300 K to 77 K the rate constant km decreases by 1.8 times in control and by 3.2 times in modified samples. In contrast to the observed km changes the increase in ke and kQ values by 2 and more times under cooling was found in control and modified RC; (d) in control preparations with QA acceptor pre-reduced in the dark the lowering of the temperature caused the increase in the time of P+I- recombination from 10 to 20 ns. After DMSO modification the kinetics of charge recombination in RC was biexponential at room temperature with tau=10 ns and tau1=0.8 ns, and at 77 K with tau=20 ns and tau1=0.6 ns, correspondingly. The results obtained reveal that in RC isolated from Rb. sphaeroides the processes of energy migration, charge separation, electron transfer to QA and ion-radical pair P+I- recombination depend on the state of hydrogen bonds of water-protein structure. Fast relaxation processes in RC structure including polarization of H-containing molecules in the surrounding of electron carriers can accept electron energy dissipated at the initial steps of energy and electron transfer. Copyright 1998 Elsevier Science B.V. All rights reserved.

Modification of protein hydrogen bonds influences the efficiency of picosecond electron transfer in bacterial photosynthetic reaction centers

Picosecond absorption spectroscopy was used to monitor laser‐induced oxidation‐reductions of reaction center (RC) bacteriochlorophyll (P) and bacteriopheophytin (I) in Rhodopseudomonas sphaeroides RC preparations on exposure to different chemicals. The D2O isotope substitution of H2O or partial substitution of water by organic solvents (ethylene glycol, glycerol, propylene glycol, dimethyl sulfoxide) causes the appearance of a fast, nanosecond component of P+ reduction, the result of an increased probability of recombination of the primary ion‐radical products P+I− → PI. The effect is accompanied by a noticeable slowing down of electron transfer from photoreduced bacteriopheophytin to the primary quinone acceptor QA. The effect of the organic solvents, known as cryoprotectors, is correlated with their degree of hydrophobicity, i.e. the ability to penetrate the RC protein and interact with bound water and protein hydrogen bonds. The conclusion drawn from the data is that the dielectric relaxation processes through which the intermediate energy levels of the carriers in the PIQA system are lowered to levels necessary for the stabilization of the photochemically separated charges proceed with the involvement of protons of the nearest water‐protein surrounding of the RC pigments and electron transport cofactors.

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.

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.

The nature of oscillations in the kinetics of electron transfer in the reaction center of purple bacteria.

87 The reaction centers (RCs) of purple bacteria are natural nanostructures able to transform electron excitation energy into the energy of separated charges with a high efficiency (~100%). The RC main unit is formed of two protein subunits, L and M, with four bacteriochlorophyll (BCl) molecules, two bacteriopheophytin ( H A and H B ) molecules, and two quinone ( Q A and Q B ) molecules attached to them. An iron atom is localized between the quinones. In turn, two of the four BCl molecules form a special pair, the primary electron donor P. Spatial organization of the Rhodobacter sphaeroides RC has been determined with a resolution of 2.65 A [1]. Study of the interaction between the excited states and the charge transfer states of RC cofactors provided for discovering oscillations in the kinetics of stimulated luminescence in the R. sphaeroides RC at the excitation at Q y absorption band of the special pair [2, 3]. Shuvalov et al. studied the oscillations in the absorption band of the reduced intermediate acceptor [4, 5]. These oscillations were explained by a wave packet formed on the potential energy surface for the interaction between the special pair and BCl in the active photosynthesis chain during electron transfer. The oscillation data were described by the Redfield super

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.

A comparison of the temperature dependence of charge recombination in the ion-radical pair P870+QA - and tryptophan fluorescence in the photosynthetic reaction centers of Rhodobacter sphaeroides

The temperature dependences of the charge-recombination rate in the ion-radical pair P870+QA- in photosynthetic reaction centers of Rhodobacter sphaeroides were investigated. Recombination kinetics were measured in the individual absorption bands of the donor (600 nm) and an electron acceptor (335 and 420–450 nm) for the reaction center in the water–glycerol and trehalose environment after freezing preparations to–180°С in the dark and on the actinic light and after their subsequent heating. In similar conditions the fluorescence lifetime of tryptophanyls in reaction centers (λreg = 325 and 345 nm), which is an internal indicator of the dynamic state of the protein matrix, was measured. A correlation between the temperature dependences of functional and dynamic parameters of reaction centers in different solvents was shown. The differences in the average fluorescence lifetime of tryptophanyls in reaction centers of preparations frozen in the dark or on the actinic light were found. These results are explained due to transitions of reaction centers between different conformational states and processes of proton relaxation in the structure of the hydrogen bonds in the environment of reaction-center cofactors.

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 lowintensity 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.

The influence of structural phase transition on the temperature dependence of the rate of charge recombination P+QA(-)-->PQA in Rhodobacter sphaeroides reaction centers.

Studies of the temperature dependence of the rate constants of the initial stages of light energy transformation in photosynthetic reaction centers (RCs) provide valuable information about the physical mechanisms of these processes. It was shown in our earlier works [1, 2] that the temperature dependence of the reaction of dark recombination between photooxidized bacteriochlorophyll (P + ) and reduced primary quinone

Role Played by Water–Protein Surrounding of Cofactors in Picosecond Electron Transport Steps in Photosynthesis Reaction Centers

Effect of the water–protein environment of cofactors on the rates and efficiency of conversion of light energy into the energy of a photochemical potential in reaction centers of purple bacterium Rhodobacter sphaeroides is studied. The environment is modified by isotopic replacement D2O → H2O or by adding glycerol and dimethyl sulfoxide (DMSO). The replacement D → H is shown to makes no impact on the midpoint potential Em of the electron donor, whereas addition of 70 vol % of glycerol or 35 vol % of DMSO raises Em by 30 and 45 mV, respectively. Rate constants of charge separation ke and electron transport onto quinone kQ remained unchanged following glycerol addition, while deuteration and DMSO addition diminished ke and kQ by two to three times. In addition to the known component with a characteristic time of about 10 ns, a component with a duration of 0.5–0.8 ns appears in the recombination kinetics of charges of deuterated and DMSO-treated preparations of reaction centers. The mechanism of the environment response on the emergence of nonequilibrium states of cofactors is analyzed theoretically. The energy model proposed for primary processes of photosynthesis accounts for the contribution made by the environment in the realization of a highly effective electron transport.