RESUMO
The temperature dependence of the dark recombination rate in photooxidized bacteriochlorophyll (P) and photoreduced quinone acceptors (ubiquinones) QA and QB of photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides (Rb. sphaeroides) was studied. Photoinduced changes in the absorption were detected in the Qx absorption band of photooxidized bacteriochlorophyll at 600â¯nm and in the bands corresponding to the redox changes of ubiquinones at 335 and 420-450â¯nm. Kinetic analysis was used to evaluate the activation energy and the characteristic time of the transient process of relaxation accompanying electron stabilization at the final quinone acceptor. A comparative study of the kinetics of oxidation-reduction reactions of photoactive bacteriochlorophyll RC purple bacteria and quinone acceptors in their individual absorption bands is an informative approach to studying the mechanisms of this stabilization. The analysis of the revealed kinetic differences makes it possible to estimate the activation energy and the characteristic times of the transition relaxation processes associated with the stabilization of the electron in the quinone acceptor part of RC. Purple bacterial reaction centers have fundamental similarities with PSII reaction centers. Such a similarity represents evolutional closeness between the two types of RC. So it is possible that the photoinduced charge separation in PSII RC, as well as in purple bacteria RC, is also accompanied by definite conformational changes. The possible role of hydrogen bonds of surrounding protein in the relaxation processes accompanying the electron transfer to quinone acceptors is discussed.
Assuntos
Elétrons , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Ligação de Hidrogênio , Cinética , Oxirredução , Quinonas , Temperatura , TermodinâmicaRESUMO
The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1 M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5-417.5 nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from -60 to +10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wavelength B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.
Assuntos
Fluorescência , Temperatura , Triptofano/química , Glicerol/química , Meia-Vida , Cinética , Soluções , Espectrometria de Fluorescência , Trealose/química , ÁguaRESUMO
The study of the effect of vasodilator, antiplatelet agent, and inhibitor P-glycoprotein dipyridamole (DIP) on the functioning of the transmembrane protein of the reaction center (RC) of Rb. sphaeroides showed that the activation of RC by constant light generates the DIP radical cation, which significantly affects the kinetics of recombination of charges divided between photoactive bacteriochlorophyll and quinone acceptors. Thus, the antioxidant properties of DIP may affect the functional activity of membrane proteins, and this apparently should be taken into account in the studies of the mechanisms of therapeutic action of this drug.
Assuntos
Dipiridamol/metabolismo , Luz , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/efeitos da radiação , Radicais Livres/metabolismo , Cinética , Rhodobacter sphaeroides/enzimologiaRESUMO
The effect of heating at 65°C for 20 min on the absorption spectra and kinetics of the dark recombination of charges separated between photoactive bacteriochlorophyll and quinone acceptors was studied in dry films of bacterial photosynthetic reaction centers (RCs), RC films in polyvinyl alcohol, and trehalose. A pronounced protective effect of trehalose against pheophytinizaiton of molecules bacteriochlorophylls in RC structure and in maintaining their higher photochemical activity was found.
Assuntos
Temperatura Alta , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos dos fármacos , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Trealose/farmacologia , Cinética , Rhodobacter sphaeroides/citologia , Rhodobacter sphaeroides/enzimologiaRESUMO
Effect of chromate ions on the culture of a marine diatom Phaeodactylum tricornutum was studied using an M-PEA-2 fluorimeter, which carries out simultaneous measurement of fluorescence induction and redox transformations of the P700 pigment within a millisecond range. Chromate ions were shown to inhibit electron transport in PS II and decrease the rate of QÐ reduction. This results in decreased values of the quantum yield of electron transport in PS II (ÏEo) and performance index (PI ABS), lower rates of P700 reduction, and increased energy (DI0/RC) and ΔpH-dependent nonphotochemical quenching (q E ). Emergence of the slow component of P700 reduction was observed, indicating the activation of cyclic transport in the presence of chromate. Performance index (PI ABS), which was the most sensitive parameter, may be recommended for detection of chromate ions at early stages of their toxic action. The fluorescence parameter F O is promising application in biotesting to assess the algal growth rates.
Assuntos
Clorofila/metabolismo , Cromatos/metabolismo , Diatomáceas/metabolismo , Cromatos/farmacologia , Diatomáceas/crescimento & desenvolvimento , Fluorescência , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , OxirreduçãoRESUMO
The present work is related to the investigation of slow kinetics of electron transport in the reaction centers (RCs) of Rhodobacter sphaeroides. Experimental data on the absorption kinetics of aqueous solutions of reaction centers at different modes of photoexcitation are given. It is shown that the kinetics of oxidation and reduction of RCs are well described by the sum of three exponential functions. This allows to suggest a two-level kinetic model for electron transport in the RC as a system of four electron-conformational states which correspond to three balance differential equations combined with state equation. The solution of inverse problem made it possible to obtain the rate constant values in kinetic equations for different times and intensities of exciting light. Analysis of rate constant values in different modes of RC excitation allowed to suggest that two mechanisms of structural changes are involved in RC photo-oxidation. One mechanism leads to the increment of the rate of electron return, another one-to its drop. Structural changes were found out to occur in the RCs under incident light. After light was turned off, the reduction of RCs was determined by the second mechanism.
RESUMO
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.
Assuntos
Biotecnologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Pontos Quânticos/metabolismo , Rhodobacter sphaeroides/metabolismo , Transporte de Elétrons , Transferência de Energia , Estabilidade Proteica , TrealoseRESUMO
Quantum dots (QDs) can absorb ultraviolet and long-wavelength light energy much more efficiently than natural light-harvesting proteins and transfer the excitation energy to photosynthetic reaction centers (RCs). Inclusion into liposomes of RC membrane pigment-protein complexes combined with QDs as antennae opens new opportunities for using such hybrid systems as a basis for artificial energy-transforming devices that potentially can operate with greater efficiency and stability than devices based only on biological components. RCs from Rhodobacter sphaeroides and QDs with fluorescence maximum at 530 nm (CdSe/ZnS with hydrophilic covering) were embedded in lecithin liposomes by extrusion of a solution of multilayer lipid vesicles through a polycarbonate membrane or by dialysis of lipids and proteins dispersed with excess detergent. The dimensions of the resulting hybrid systems were evaluated using dynamic light scattering and by transmission cryoelectron microscopy. The efficiency of RC and QD interaction within the liposomes was estimated using fluorescence excitation spectra of the photoactive bacteriochlorophyll of the RCs and by measuring the fluorescence decay kinetics of the QDs. The functional activity of the RCs in hybrid complexes was fully maintained, and their stability was even increased.
Assuntos
Lipossomos/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Pontos Quânticos/química , Rhodobacter sphaeroides/metabolismo , Bacterioclorofilas/química , Lecitinas , Lipossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Processos FotoquímicosRESUMO
Acute toxicity of silver nanoparticle (AgNP) for photosynthesis in Chlamydomonas reinhardtii was studied using an M-PEA2 fluorimeter. Analysis of the fluorescence induction curves in the presence of AgNP at low concentrations revealed inhibited electron transport in the PS2 photosystem and increased content of Q(B)-nonreducing centers. No direct effect of AgNP on the reactions of P700 oxidation in PS1 was found, while energization of the photosynthetic membranes was affected. Investigation of the parameters of the prompt and delayed fluorescence is proposed as a method for early detection of AgNP in the environment.
Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Chlamydomonas reinhardtii/metabolismo , Clorofila , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Fluorescência , Complexo de Proteína do Fotossistema II/metabolismo , Testes de Toxicidade AgudaAssuntos
Elétrons , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Quinonas/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinética , Complexo de Proteínas do Centro de Reação Fotossintética/química , Ligação Proteica , Rhodobacter sphaeroides/químicaAssuntos
Chlorella vulgaris/efeitos dos fármacos , Temperatura Baixa , Luz , Compostos de Metilmercúrio/toxicidade , Complexo de Proteína do Fotossistema II/antagonistas & inibidores , Chlorella vulgaris/fisiologia , Chlorella vulgaris/efeitos da radiação , Complexo de Proteína do Fotossistema II/fisiologia , Estresse FisiológicoRESUMO
The time evolution of the photoinduced differential absorption spectrum of isolated Rhodobacter sphaeroides photosynthetic reaction centers was investigated. The measurements were carried out in the spectral region of 400-500 nm on the time scale of up to 200 microseconds. The spectral changes observed can be interpreted in terms of the effects of proton shift along hydrogen bonds between the primary quinone acceptor and the protein. A theoretical analysis of the spectrum time evolution was performed, which is based on the consideration of the kinetics of proton tunneling along the hydrogen bond. It was shown that the stabilization of the primary quinone electronic state occurs within the first several tens of microseconds after quinone reduction. It slows down upon the deuteration of reaction centers as well as after adding 90% of glycerol; on the other hand, it accelerates as temperature rises up to 40 degrees C.
Assuntos
Benzoquinonas/química , Elétrons , Glicerol/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/enzimologia , Benzoquinonas/metabolismo , Sítios de Ligação , Temperatura Alta , Ligação de Hidrogênio , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Espectrofotometria UltravioletaRESUMO
Radial distribution functions were deduced by Fourier transform analysis of angular dependences of diffuse x-ray scattering intensities for the following proteins with different hydration degree: water-soluble a-protein myoglobin, water-soluble alpha+beta protein lysozyme, and transmembrane proteins of photosynthetic reaction centers from purple bacteria Rhodobacter sphaeroides and Blastochlorii viridis. The results of Fourier analysis of x-ray scattering intensities give the quantitative characteristics of the mechanisms underlying the influence of water on the formation of biomacromolecules. Water, on the one hand, weakens the intraglobular hydrogen bond net, loosens the protein structure, and increases the internal conformational dynamics. Concurrently water arranges the stability and ordering of the macromolecule. A sharp correlation is observed between the shift of the "first" peak of radial distribution functions, the weakening of the intraglobular hydrogen bond net, the increase in intraglobular mobility, and the appearance of functional activity in macromolecules. The behavior of the "first" peak is similar to that observed in transmembrane protein of reaction center and water-soluble proteins. The "first" peak for transmembrane protein of reaction center reaches its maximum value much faster (at smaller hydration degrees) than for water-soluble proteins. The fast transfer of reaction center protein to its native state during hydration is due to the fact that the dehydrated conformation of reaction center protein is very close to the native one. From a comparison of the radial distribution functions for water, water-soluble proteins and transmembrane proteins, one may conclude that water has the lowest packing density and the lowest order; water-soluble proteins have a larger packing density and are more ordered than water, and transmembrane proteins have the highest degree of packing density and ordering.
Assuntos
Análise de Fourier , Proteínas/química , Água/química , Animais , Conformação Proteica , Rhodobacter sphaeroides/química , Difração de Raios XRESUMO
High-resolution 1H-NMR spectra registered within a temperature range of 25-40 degrees C revealed a nonmonotonous dome-shaped temperature dependence of the ratio between integral NMR signal intensities determined at ppm intervals 2.5-4.5 and 0.2-2.5 with a maximum at 30 degrees C. This may be due to RC structural changes accompanying the temperature rise and accelerating the recombination reaction between oxidized bacteriochlorophyll and reduced primary quinone at temperatures above 30 degrees C.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/metabolismo , Temperatura , Transporte de Elétrons , Hidrogênio , Complexos de Proteínas Captadores de Luz/química , Espectroscopia de Ressonância MagnéticaRESUMO
It is shown that the addition of dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4d]py rim idine) (up to 10(-4) M) leads to a drastic acceleration of the dark recombination reaction between photooxidized bacteriochlorophyll and photoreduced primary quinone in reaction centers of Rhodobacter sphaeroides. The value of the acceleration is similar to that registered under cryogenic temperatures. The extent of the effect of dipyridamole derivatives depended on their structure. In wild-type bacteriorhodopsin and D96N mutant, dipyridamole slowed down the Schiff base reprotonation (the kinetics of M412 form decay was registered). It is suggested that dipyridamole can influence the structural and dynamic state of membrane proteins by affecting the system of their hydrogen-bonds and thus modify electron and proton transport processes.