V.Z. Paschenko

Probing the kinetics of Photosystem I and Photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.

Oriented purple-membrane films as a probe for studies of the mechanism of bacteriorhodopsin functioning. II: Photoelectric processes

Abstract Photoelectric processes have been investigated in dry orderly oriented preparations of purple membranes from Halobacterium halobium under both continuous light and flash excitation. An electrophoretic sedimentation on Al, Cu, Fe, Ni, Pt and SnO 2 substrates was used to obtain orderly oriented purple-membrane films. The photoelectric response of the purple-membrane film is the sum of a light-induced ‘displacement’ current and a constant steady-state current, the proportion between the two depending upon the chemical nature of the electrodes and humidity of the film. With a high humidity, the steady-state photocurrent is correlated with the reactivity of the cathode metal (toward H + reduction reaction. A correlation is found to exist between the kinetics of photopotential rise and decay and formation and decay of the K 630 and M 412 intermediates of the bacteriorhodopsin photocycle at temperatures ranging from 293 to 83 K, indicating the electrogenic nature of these intermediates. In purple-membrane films deposited on an SnO 2 substrate, a correlation exists between the ‘dark’ potential and electron work function of the second electrode.

Coupling of different isolated photosynthetic light harvesting complexes and CdSe/ZnS nanocrystals via Förster resonance energy transfer☆

The present work describes results obtained on hybrid systems formed in aqueous buffer solution by self-assembly of different CdSe quantum dots (QDs) surrounded by a ZnS shell and functionalized by covering the surface with anionic and cationic groups and various isolated pigment-protein complexes from the light-harvesting antennae of photosynthetic organisms (light-harvesting complexes 1 and 2 (LH1 and LH2, respectively) from purple bacteria, phycobiliproteins (PBPs) from cyanobacteria and the rod-shaped PBP from the cyanobacterium Acaryochloris marina). Excitation energy transfer (EET) from QDs to PBP rods was found to take place with varying and highly temperature-dependent efficiencies of up to 90%. Experiments performed at room temperature on hybrid systems with different QDs show that no straightforward correlation exists between the efficiency of EET and the parameter J/(R(12)(6)) given by the theory of Förster resonance energy transfer (FRET), where J is the overlap integral of the normalized QD emission and PBP absorption and R(12) the distance between the transition dipole moments of donor and acceptor. The results show that the hybrid systems cannot be described as randomly orientated aggregates consisting of QDs and photosynthetic pigment-protein complexes. Specific structural parameters are inferred to play an essential role. The mode of binding and coupling seems to change with the size of QDs and with temperature. Efficient EET and fluorescence enhancement of the acceptor was observed at particular stoichiometric ratios between QDs and trimeric phycoerythrin (PE). At higher concentrations of PE, a quenching of its fluorescence is observed in the presence of QDs. This effect is explained by the existence of additional quenching channels in aggregates formed within hybrid systems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Probing the fluorescence emission kinetics of the photosynthetic apparatus of Rhodopseudomonas sphaeroides, strain 1760-1, on a picosecond pulse fluorometer

Abstract Using the pulse picosecond fluorometric technique the fluorescence properties of intact cells, isolated chromatophores and photosynthetic reaction centres were studied in bacteria Rhodopseudomonas sphaeroides , strain 1760-1. The fluorescent emission from reduced reaction centres excited by 694.3 nm light has a biphasic character, the lifetimes of the components being τ 1 = 15±8 ps and τ 2 = 250 ps. The faster component, τ 1 , contributes to the integral fluorescence in the long wavelength region. It disappears with oxidation of the reaction centres and is attributed to photoactive bacteriochlorophyll P870. The slow component, τ, is apparently due to both bacteriochlorophyll P800 and bacteriopheophytin. The fluorescence from intact cells exhibits a monophasic pattern and decays with τ = 200 ps. The fluorescence emitted by chromatophores comprises two components with τ 3 = 200 ps and τ 4 = 4200 ps. The duration of fluorescence τ 3 increases to its maximum of 500–550 ps, as P870 is oxidized chemically or photochemically, while τ 4 remains unchanged. The fluorescence with a lifetime of 200 ps was ascribed to the photosystem and the 4200-ps fluorescence to bacteriochlorophyll which had lost its functional links with the photosystem. The rise time of the fluorescence emitted by chromatophores varies from 60 or 70 ps to 350 ps depending on the wavelength of the exciting light and the recorded spectral region. On the basis of our findings the rate for energy migration was estimated to be 10 9 s −1 .

Fluorescence quenching of the phycobilisome terminal emitter LCM from the cyanobacterium Synechocystis sp. PCC 6803 detected in vivo and in vitro

The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in vitro and in vivo, which suggests the crucial role of the OCP-L(CM) interaction in non-photochemical quenching (NPQ) of cyanobacteria. This implication was further supported (i) by low-temperature (77K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photoactivated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.

Spectroscopic investigations of fluorescence behaviour, role and function of the long-wavelength pigments of Photosystem I

A method was established for quantitative evaluation of fluorescence spectra of intact leaves without expensive sample preparation. Fluorescence induction and excitation effects were used to evaluate the contribution of Photosystem I to the long-wavelength fluorescence of intact leaves at room temperature which appeared to be about 25%. To characterize its spectral line-shape we studied low-temperature fluorescence. The connection of thesethree methods made it possible to determine the quantitative spectra of both photosystems in intact leaves. Investigations of fluorescence decay kinetics on isolated Photosystem I subchloroplast particles provided information about functional aspects of the long-wavelength pericentral antenna pigments. It appeared that these pericentral pigments are not essential for transferring excitation energy toward photochemically active reaction centres. Only if the reaction centres were closed the pericentral antenna would act as a protection guard against photodestruction.

Estimation of the rate of photochemical charge separation in Rhodopseudomonas sphaeroides reaction centers by fluorescence and absorption picosecond spectroscopy

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 τ = 6± 1.5 ps and independent of temperature within the range 293 – 77 K. This value was found to coincide well with the time (7 ± 3 ps) of the RC porphyrin pigment transition into the ion‐radical pair state PF, as measured by picosecond absorption spectroscopy of the same preparations.

The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures - from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmolphotonsm⁻²s⁻¹. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

PS II model based analysis of transient fluorescence yield measured on whole leaves of Arabidopsis thaliana after excitation with light flashes of different energies

Our recently presented PS II model (Belyaeva et al., 2008) was improved in order to permit a consistent simulation of Single Flash Induced Transient Fluorescence Yield (SFITFY) traces that were earlier measured by Steffen et al. (2005) on whole leaves of Arabidopsis (A.) thaliana at four different energies of the actinic flash. As the essential modification, the shape of the actinic flash was explicitly taken into account assuming that an exponentially decaying rate simulates the time dependent excitation of PS II by the 10 ns actinic flash. The maximum amplitude of this excitation exceeds that of the measuring light by 9 orders of magnitude. A very good fit of the SFITFY data was achieved in the time domain from 100 ns to 10s for all actinic flash energies (the maximum energy of 7.5 × 10¹⁶ photons/(cm²flash) is set to 100%, the relative energies of weaker actinic flashes were of ∼8%, 4%, ∼1%). Our model allows the calculation and visualization of the transient PS II redox state populations ranging from the dark adapted state, via excitation energy and electron transfer steps induced by pulse excitation, followed by final relaxation into the stationary state eventually attained under the measuring light. It turned out that the rate constants of electron transfer steps are invariant to intensity of the actinic laser flash. In marked contrast, an increase of the actinic flash energy by more than two orders of magnitude from 5.4×10¹⁴ photons/(cm²flash) to 7.5×10¹⁶ photons/(cm²flash), leads to an increase of the extent of fluorescence quenching due to carotenoid triplet (³Car) formation by a factor of 14 and of the recombination reaction between reduced primary pheophytin (Phe(-)) and P680(+) by a factor of 3 while the heat dissipation in the antenna complex remains virtually constant. The modified PS II model offers new opportunities to compare electron transfer and dissipative parameters for different species (e.g. for the green algae and the higher plant) under varying illumination conditions.

Photosystem 2 effective fluorescence cross-section of cyanobacterium Synechocystis sp. PCC6803 and its mutants

The effective fluorescence cross-section of photosystem 2 (PS2) was defined by measurements of chlorophyll a fluorescence induction curves for the wild type of the unicellular cyanobacterium Synechocystis sp. PCC6803, C-phycocyanin deficient mutant (CK), and mutant that totally lacks phycobilisomes (PAL). It was shown that mutations lead to a strong decrease of the PS2 effective fluorescence cross-section. For instance, the effective fluorescence cross-section of PS2 for wild type, CK and PAL mutants excited at λ(ex)=655 nm were found to be 896, 220 and 83 Å(2) respectively. Here we present an estimation of energy transfer efficiency from phycobilisomes to the pigment-protein complexes of PS2. It was shown that the PS2 fluorescence enhancement coefficient reaches a maximum value of 10.7 due to the energy migration from phycobilisomes. The rate constant of energy migration was found to be equal to 1.04 × 10(10) s(-1).