Victor V. Fadeev

A kinetic model of non-photochemical quenching in cyanobacteria

High light poses a threat to oxygenic photosynthetic organisms. Similar to eukaryotes, cyanobacteria evolved a photoprotective mechanism, non-photochemical quenching (NPQ), which dissipates excess absorbed energy as heat. An orange carotenoid protein (OCP) has been implicated as a blue-green light sensor that induces NPQ in cyanobacteria. Discovered in vitro, this process involves a light-induced transformation of the OCP from its dark, orange form (OCP(o)) to a red, active form, however, the mechanisms of NPQ in vivo remain largely unknown. Here we show that the formation of the quenching state in vivo is a multistep process that involves both photoinduced and dark reactions. Our kinetic analysis of the NPQ process reveals that the light induced conversion of OCP(o) to a quenching state (OCP(q)) proceeds via an intermediate, non-quenching state (OCP(i)), and this reaction sequence can be described by a three-state kinetic model. The conversion of OCP(o) to OCP(i) is a photoinduced process with the effective absorption cross section of 4.5 × 10(-3)Ų at 470 nm. The transition from OCP(i) to OCP(q) is a dark reaction, with the first order rate constant of approximately 0.1s(-1) at 25°C and the activation energy of 21 kcal/mol. These characteristics suggest that the reaction rate may be limited by cis-trans proline isomerization of Gln224-Pro225 or Pro225-Pro226, located at a loop near the carotenoid. NPQ decreases the functional absorption cross-section of Photosystem II, suggesting that formation of the quenched centers reduces the flux of absorbed energy from phycobilisomes to the reaction centers by approximately 50%.

Valence band of liquid water raman scattering: some peculiarities and applications in the diagnostics of water media

A short analysis of the results of our research on peculiarities of water Raman scattering spectra and of their applications in the diagnostics of water media is presented. The results of using a new approach for research on water Raman scattering spectra, i.e. the method of artificial neural networks for spectra treatment and for the solution of inverse problems in water media diagnostics, are also presented. This technique allowed us to improve the accuracy of water temperature determination (error 0.3 °C) by exploiting the temperature dependence of valence band parameters. However, no peculiarities of this dependence in the vicinity of 20, 36 and 76 °C, reported by others, were discovered. Copyright

Saturation spectroscopy as a method for determining the photophysical parameters of complicated organic compounds

A fundamentally new approach to the fluorescence diagnostics of complicated organic compounds and complexes has been proposed. The method is based on in vivo and in situ measurements of the photophysical parameters of organic molecules from saturation curves. The results obtained for the solution of the relevant inverse problem are presented.

Fluorescence diagnostics of oil pollution in coastal marine waters by use of artificial neural networks

We discuss the problems with and the real possibilities of determining oil pollution in situ in coastal marine waters with fluorescence spectroscopy and of using artificial neural networks for data interpretation. In general, the fluorescence bands of oil and aquatic humic substance overlap. At oil concentrations in water from a few to tens of micrograms per liter, the intensity of oil fluorescence is considerably lower than that of humic substances at concentrations that typically are present in coastal waters. Therefore it is necessary to solve the problem of separating the small amount of oil fluorescence from the humic substance background in the spectrum. The problem is complicated because of possible interactions between the components and variations in the parameters of the fluorescence bands of humic substances and oil in water. Fluorescence spectra of seawater samples taken from coastal areas of the Black Sea, samples prepared in the laboratory, and numerically simulated spectra were processed with an artificial neural network. The results demonstrate the possibility of estimating oil concentrations with an accuracy of a few micrograms per liter in coastal waters also in cases in which the contribution from other organic compounds, primarily humic substances, to the fluorescence spectrum exceeds that of oil by 2 orders of magnitude and more.

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.

The effect of hydration of ions of inorganic salts on the shape of the raman stretching band of water

The shape of the Raman stretching band of water molecules in aqueous solutions of electrolytes KBr, KCl, KI, NaCl, and NaI is studied. It is confirmed that the characteristics of the stretching band strongly depend on the concentration and type of salt. The behavior of different parameters of the band is explained in terms of the theory of hydration of salts.

New possibilities of laser fluorescence spectroscopy for diagnostics of petroleum hydrocarbons in natural water

New possibilities of fluorescence spectroscopy for diagnostics of oil pollution in natural water are discussed. It is shown that the choice of shorter excitation wavelengths than is accepted in the standard UNESCO technique for the determination of petroleum hydrocarbons concentration significantly improves the sensitivity and universality of the method and allows one to perform oil classification into basic classes. The authors have also considered the principal possibilities of direct (without extraction) fluorescence diagnostics under excitation at 266 nm. The results obtained may be considered as the next step to solve the very difficult problems of laser remote sensing of oil pollution in natural waters.<<ETX>>

Determination of Fluorescence Quantum Yields Using a Spontaneous Raman Scattering Line of the Solvent as Internal Standard

Abstract A method for determination of molecular quantum yields of fluorescent compounds in solution by internal calibration with the signal of spontaneous Raman scattering of the solvent is proposed. An experimental verification of the method is carried out for substances with known quantum yields and satisfactory agreement between experimental results and literature values is obtained.

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo : assessment of blood capillaries and structural proteins localization

The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

Laser fluorimetry of proteins containing one and two tryptophan residues

The true molecular photophysical parameters’ values of tryptophan residues in single-tryptophan-containing and two-tryptophan-containing proteins (by the example of human serum albumin and bovine serum albumin) have been determined for the first time with the use of the authors’ suggested algorithm. The algorthm is based on the simultaneous use of nonlinear and kinetic-laser fluorimetry. The obtained result opens up new approaches in the monitoring of living systems and other objects, containing single fluorophores and localized pairs of fluorophores.