Photogeneration and quenching of singlet molecular oxygen by bacterial C40 carotenoids with long chain of conjugated double bonds.

Measurement of photosensitized luminescence of singlet oxygen has been applied to studies of singlet oxygen generation and quenching by C40 carotenoids (neurosporene, lycopene, rhodopin, and spirilloxanthin) with long chain of conjugated double bonds (CDB) using hexafluorobenzene as a solvent. It has been found that neurosporene, lycopene, and rhodopin are capable of the low efficient singlet oxygen generation in aerated solutions upon photoexcitation in the spectral region of their main absorption maxima. The quantum yield of this process was estimated to be (1.5-3.0) × 10-2. This value is near the singlet oxygen yields in solutions of ζ-carotene (7 CDB) and phytoene (3 CDB) and many-fold smaller than in solutions of phytofluene (5 CDB) (Ashikhmin et al. Biochemistry (Mosc) 85:773-780, https://doi.org/10.1134/S0006297920070056 , 2020, Biochemistry (Mosc) 87:1169-1178, 2022, https://doi.org/10.1134/S00062979221001082022 ). Photogeneration of singlet oxygen was not observed in spirilloxanthin solutions. A correlation was found between the singlet oxygen yields and the quantum yields and lifetimes of the fluorescence of the carotenoid molecules. All carotenoids were shown to be strong physical quenchers of singlet oxygen. The rate constants of 1O2 quenching by the carotenoids with long chain of CDB (9-13) were close to the rate constant of the diffusion-limited reactions ≈1010 M-1 s-1, being many-fold greater than the rate constants of 1O2 quenching by the carotenoids with the short chain of CDB (3-7) phytoene, phytofluene, and ζ-carotene studied in prior papers of our group (Ashikhmin et al. 2020, 2022). To our knowledge, the quenching rate constants of rhodopin and spirilloxanthin have been obtained in this paper for the first time. The mechanisms of 1O2 photogeneration by carotenoids in solution and in the LH2 complexes of photosynthetic cells, as well as the efficiencies of their protective action are discussed.

Activation of oxygen molecules by 1070 nm laser radiation in aerated solvents.

Population of the chemically active singlet 1Δg(0) state of molecular oxygen occurring due to direct laser excitation of the 1Δg(1)←3Σg-(0) transition has been observed for the first time, to the best of our knowledge, in oxygen molecules dissolved in organic solvents saturated with air under natural conditions (room temperature and normal atmospheric pressure). The data were obtained in 1 cm spectrophotometric cells due to the application of a set of high-power IR fiber and diode lasers. The rate of laser generation of the singlet (1Δg(0)) states in oxygen molecules was monitored by a chemical trapping method. It was found that the action spectra of singlet oxygen generation have one distinct band with a maximum at 1070 nm and half-width of ∼10nm. The absorption coefficients at 1070 nm were shown to be 100-110-fold lower than those at the main oxygen absorption peak (1273 nm) corresponding to the 1Δg(0)←3Σg-(0) transition. Under excitation at 810-1061 nm, very low trapping rates were observed, which did not depend on excitation wavelengths being probably caused by thermal effects. There was no reliable increase in the trapping rate under irradiation at 810 and 920 nm corresponding to the 1Δg(2,3)←3Σg-(0) transitions. This fact suggests that absorbance corresponding to these transitions is much lower than that at 1070 nm. The obtained results are important for both spectroscopy of oxygen and mechanistic studies of biological and therapeutic action of laser radiation.

Phytofluene as a Highly Efficient UVA Photosensitizer of Singlet Oxygen Generation.

Phytoene and phytofluene - uncolored C40 carotenoids with short chain of conjugated double bonds (3 and 5, respectively) - are known to be universal precursors in biosynthesis of colored carotenoids in photosynthesizing organisms. It is commonly recognized that C40 carotenoids are photoprotectors of cells and tissues. We have shown that phytofluene is an exception to this rule. By measuring photosensitized phosphorescence of singlet oxygen (1O2) we found out that phytofluene was very effective photosensitizer of 1O2 formation in aerated solutions under UVA irradiation (quantum yield of 85 ± 5%), whereas phytoene was almost inactive in this process. It was demonstrated that both carotenoids quench singlet oxygen in the dark. The obtained quenching rate constants [(4 ± 1) × 106 M-1·s-1 for phytoene and (2 ± 0.5) × 107 M-1·s-1 for phytofluene] were smaller than the rate constant of the diffusion-controlled reactions by 3-4 orders of magnitude. Thus, both carotenoids displayed rather weak protector properties. Moreover, phytofluene due to its high photosensitizing activity might be considered as a promoter of cell photodamage and a promising UVA photosensitizer for medical purposes.

Kinetic Measurements of Singlet Oxygen Phosphorescence in Hydrogen-Free Solvents by Time-Resolved Photon Counting.

Solvents lacking hydrogen atoms are very convenient models for elucidating the properties of singlet oxygen, since the lifetime of singlet oxygen in these solvents reaches tens milliseconds. Measuring intrinsic infrared (IR) phosphorescence of singlet oxygen at 1270 nm is the most reliable method of singlet oxygen detection. However, efficient application of the phosphorescence method to these models requires an equipment allowing reliable measurement of the phosphorescence kinetic parameters in the millisecond time range at low rates of singlet oxygen generation, which is a technically difficult problem. Here, we describe a highly sensitive LED (laser) spectrometer recently constructed in our laboratory for the steady-state and time-resolved measurements of the millisecond phosphorescence of singlet oxygen. In the steady-state mode, this spectrometer allows detection of singlet oxygen phosphorescence upon direct excitation of oxygen molecules in the region of dark-red absorption bands at 690 and 765 nm. For kinetic measurements, we used phenalenone as a photosensitizer, microsecond pulses of violet (405 nm) LED for excitation (irradiance intensity, ≤50 µW/cm2), a photomultiplier and a computer multichannel scaler for time-resolved photon counting. The decays of singlet oxygen in air-saturated CCl4, C6F6, and Freon 113 and quenching of singlet oxygen by phenalenone and dissolved molecules of triplet oxygen were measured. The relative values of the radiative rate constants of singlet oxygen in these media were determined. The results were compared with the absorption coefficients of oxygen measured by our group using the methods of laser photochemistry. Critical discussion of the obtained results and the data of other researchers is presented.

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants.

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ~1.9, ~1.5, and 1.1-1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments - carotenoids, Chl b, and phycobilins - were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.

[measurement of optical density in infrared absorption maxima of oxygen molecules based on their photochemical activity upon direct laser excitation].

Generation of singlet oxygen upon excitation of oxygen molecules by infrared diode lasers has been studied in organic media (carbon tetrachloride and acetone) saturated by air under normal pressure and temperature. A new approach to analysis of the experimental data has been developed taking into account a degree of overlapping of the spectral bands of oxygen and laser radiation. Optical density, molar absorption coefficient and the cross section of light absorption were determined for the main absorption maxima of O2 at 765 and 1273 nm. The results are compared with the data of previous studies. A significance of the obtained results for elucidation of photophysics and photochemistry of oxygen molecules and investigation of biological action of laser radiation is discussed.

Photochemical investigation of the IR absorption bands of molecular oxygen in organic and aqueous environment.

It is shown that the weak IR absorption bands corresponding to the forbidden triplet-singlet transitions in oxygen molecules can be reliably studied in air-saturated solvents under ambient conditions using measurements of the photooxygenation rates of singlet oxygen traps (1,3-diphenylisobenzofuran or uric acid) upon direct excitation of oxygen molecules by IR diode lasers. The best results were obtained from comparison of the oxygenation rates upon direct and photosensitized singlet oxygen excitation. In the present paper, this method was applied to estimation of the absorbance (A(ox)) and molar absorption coefficients (ε(ox)) corresponding to the oxygen absorption bands at 765 and 1273 nm in carbon tetrachloride, acetone, alcohols and water. In carbon tetrachloride, the band at 1073 nm was also investigated. Correlation of the obtained data with the luminescence spectra and radiative rate constants of singlet oxygen, contribution of oxygen dimols and biological significance of the studied effects are discussed.

Primary mechanisms of photoactivation of molecular oxygen. History of development and the modern status of research.

This review starts from a brief historical account devoted to the principles of the Bach-Engler peroxidation theory and experiments and ideas which led A. N. Bach to its creation. Then, the discovery of photodynamic action is described, which was shown to result from pigment photosensitized activation of molecular oxygen. The dramatic history of mechanistic studies of oxygen photoactivation is reviewed starting from the Bach-Engler peroxidation theory to the hypothesis of moloxide, discovery of singlet oxygen and free radicals and, then, to modern views on the primary photoactivation processes. The origin of widely used division of photodynamic processes into type I and type II and the relation of these processes to the nature of the primary photochemical reactions of photosensitizers are discussed. New definitions of these reactions are proposed on the basis of the mechanisms of oxygen photoactivation. Photographs of the scientists who greatly contributed to the development of this field of research are presented.

Activation of molecular oxygen by infrared laser radiation in pigment-free aerobic systems.

With the goal of mimicking the mechanisms of the biological effects of low energy laser irradiation, we have shown that infrared low intensity laser radiation causes oxygenation of the chemical traps of singlet oxygen dissolved in organic media and water saturated by air at normal atmospheric pressure. The photooxygenation rate was directly proportional to the oxygen concentration and strongly inhibited by the singlet oxygen quenchers. The maximum of the photooxygenation action spectrum coincided with the maximum of the oxygen absorption band at 1270 nm. The data provide unambiguous evidence that photooxygenation is determined by the reactive singlet (1)Delta(g )state formed as a result of direct laser excitation of molecular oxygen. Hence, activation of oxygen caused by its direct photoexcitation may occur in natural systems.

Photosensitization of singlet oxygen formation by pterins and flavins. Time-resolved studies of oxygen phosphorescence under laser excitation.

To elucidate the biochemical roles of singlet molecular oxygen (1(O2)) in the light-dependent reactions photosensitized by biological blue-light photoreceptors, time-resolved measurements of photosensitized 1O2 phosphorescence (1270 nm) were performed in air-saturated aqueous ((D2)O) solutions of pterins (2-amino-4-hydroxy-6,7-dimethylpteridine (DMP) and 2-amino-4-hydroxy-6-tetrahydroxybutyl-(D-arabo)pteridine (TOP)) and flavins (riboflavin and flavin mononucleotide (FMN)) under excitation with nitrogen laser (337.1 nm) pulses. The 1(O2) quantum yields were found to be 0.16, 0.20, 0.50, and 0.50 for DMP, TOP, riboflavin, and FMN, respectively. The data indicate that pterins and flavins are rather efficient photosensitizers of 1(O2) production that might be important for their photobiological functions.

Singlet molecular oxygen in photobiochemical systems: IR phosphorescence studies.

Singlet molecular oxygen (1O2) is one of the most active intermediates involved in photosensitized oxygenation reactions in chemical and biological systems. Deactivation of singlet oxygen is accompanied by infrared phosphorescence (1270 nm) which is widely employed for 1O2 detection and study. This review considers techniques for phosphorescence detection, phosphorescence spectra, quantum yields and kinetics under laser excitation, the radiative and real 1O2 lifetimes in organic solvents and water, 1O2 quenching by biomolecules, and estimation of singlet oxygen lifetimes, diffusion lengths and phosphorescence quantum yields in blood plasma, cell cytoplasm, erythrocyte ghosts, retinal rod outer segments and chloroplast thylakoids. The experiments devoted to 1O2 phosphorescence detection in photosensitizer-containing living cells are discussed in detail. Information reviewed is important for understanding the mechanisms of photodestruction in biological systems and various applied problems of photobiology and photomedicine.

Quenching of singlet molecular oxygen by carnosine and related antioxidants. Monitoring 1270-nm phosphorescence in aqueous media.

In order to elucidate the biochemical roles of imidazol-containing dipeptides, we have studied quenching of singlet molecular oxygen (1O2) by carnosine (beta-alanyl-L-histidine), its structural components (L-histidine, imidazole, and beta-alanine), and related natural free-radical scavengers-L-anserine (beta-alanyl-1-methyl-histidine), ergothioneine (2-thiol-L-histidine-betaine), and taurine (2-aminoethanesulfonic acid) in aqueous (D2O, pD 7) solutions by using monitoring of 1O2-phosphorescence (1270-nm). The rate constants of 1O2 quenching (Kq) by carnosine, anserine, and ergothioneine were shown to be similar [(3 +/- 1) x 10(7) M-1s-1]. Their values resembled those of free-L-histidine [Kq = (4 +/- 1) x 10(7) M-1s-1] and imidazole [Kq = (2 +/- 1) x 10(7) M-1s-1]. Non-aromatic amino acids-taurine and beta-alanine-showed very low quenching activities (Kq < 3 x 10(3) M-1c-1). The Kq values did not correlate with the literature data on abilities of the tested compounds to stimulate muscle working capacities and inhibit myeloperoxidase-catalyzed oxygenation. Thus, the dipeptides can be used as potent water-soluble protectors against 1O2 attack whereas their natural biochemical functions are most probably determined by the processes of different nature.

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes.

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for (1)O2 quenching by BChl molecules (kq (M)) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the kq (M) values coincide with the real values for (1)O2 quenching rate constants by BChl molecules. Aggregation weakly influenced the kq (M) values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the kq (M) value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus (1)O2 quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of (1)O2 formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as (1)O2 physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.

Metal as a novel type of the enzyme substrate. Metallic cadmium photogenerated in the system CdS-formate as a substrate of the NAD-dependent hydrogenase.

The process of NAD+ photoreduction under the coupled action of CdS semiconductor and NAD-dependent hydrogenase from hydrogen-oxidizing bacterium Alcaligenes eutrophus may be divided into light and dark stages. At the first stage, illumination of the system leads to the photooxidation of the sacrificial electron donor and results in the reduction of the semiconductor surface. At the second dark stage NAD+ is reduced to NADH in the presence of hydrogenase. Atoms of metallic Cd(0) are shown to be the true substrate of the enzymatic reaction. The prerequisite for the electron transfer from Cd(0) to hydrogenase is enzyme adsorption on the semiconductor surface. The redox center of the hydrogenase reacting with Cd(0) atoms resides on the flavin-containing heterodimer of the protein. The activity of the hydrogenase immobilized on CdS in the reaction of NAD+ reduction by metallic Cd is close to the enzyme activity with the physiological substrates in solution. Thus, the first example of a metal being the substrate of the enzymatic process is presented.

The photophysics of monomeric bacteriochlorophylls c and d and their derivatives: properties of the triplet state and singlet oxygen photogeneration and quenching.

Measurements of pigment triplet-triplet absorption, pigment phosphorescence and photosensitized singlet oxygen luminescence were carried out on solutions containing monomeric bacteriochlorophylls (Bchl) c and d, isolated from green photosynthetic bacteria, and their magnesium-free and farnesyl-free analogs. The energies of the pigment triplet states fell in the range 1.29-1.34 eV. The triplet lifetimes in aerobic solutions were 200-250 ns; they increased to 280 +/- 70 microseconds after nitrogen purging in liquid solutions and to 0.7-2.1 ms in a solid matrix at ambient or liquid nitrogen temperatures. Rate constants for quenching of the pigment triplet state by oxygen were (2.0-2.5) x 10(9) M-1 s-1, which is close to 1/9 of the rate constant for diffusion-controlled reactions. This quenching was accompanied by singlet oxygen formation. The quantum yields for the triplet state formation and singlet oxygen production were 55-75% in air-saturated solutions. Singlet oxygen quenching by ground-state pigment molecules was observed. Quenching was the most efficient for magnesium-containing pigments, kq = (0.31-1.2) x 10(9) M-1 s-1. It is caused mainly by a physical process of singlet oxygen (1O2) deactivation. Thus, Bchl c and d and their derivatives, as well as chlorophyll and Bchl a, combine a high efficiency of singlet oxygen production with the ability to protect photochemical and photobiological systems against damage by singlet oxygen.

Photogeneration of NADH under coupled action of CdS semiconductor and hydrogenase from Alcaligenes eutrophus without exogenous mediators.

Photoreduction of NAD has been accomplished by a system consisting of the NAD-dependent hydrogenase from Alcaligenes eutrophus immobilized on CdS particles with formate as artificial electron donor. Enzymatically active NADH is formed under illumination of this system by visible light. Accumulation of the coenzyme dimer (NAD)2 was not detected. NAD photoreduction is supposed to proceed via the direct electron transfer from the semiconductor to the enzyme electron transport chain. However, NADH formation as a result of hydrogenase interaction with anion-radicals (CO2.-) formed in the course of formate photooxidation cannot at present be excluded.

Quenching of singlet molecular oxygen by phthalocyanines and naphthalocyanines.

Using the direct measurement of the photosensitized luminescence of singlet molecular oxygen (1O2) the rate constants (kq) have been determined for 1O2 quenching by the monomeric molecules of the following phthalocyanines and naphthalocyanines in chloroform: tetra-(4-tert-butyl) phthalocyanine (I); octa-(3,6-butoxy) phthalocyanine (II), tetra-(6-tert-butyl)-2,3 naphthalocyanine (III), aluminium tetra-(1-tert-phenyl)-2,3 naphthalocyanine (IV), tri-(n-hexyl-siloxy) derivatives of silicon- (V), tin- (VI), aluminium- (VII) and gallium- (VIII) 2,3 naphthalocyanine. The following kq values were obtained (kq x 10(-8) M-1 s-1): 2.9 (I), 59 (II), 100 (III), 20 (IV), 3.9 (V), 53 (VI), 33 (VII), 110 (VIII). As most of the quenchers have the low-lying triplet levels, a contribution of the quenching mechanism based on the energy transfer from 1O2 to these levels has been analysed. A formula is proposed describing the relation between kq values caused by this mechanism, and photophysical constants of the quencher triplet state. This formula was applied to phthalocyanines, naphthalocyanines, beta-carotene and bacterochlorophyll a. The data suggest that the energy transfer can fully explain the activity of V and strongly contributes into the activities of II, III and VI-VIII. A charge transfer interaction might be an additional mechanism involved in 1O2 quenching by compounds studied. As some phthalocyanines and naphthalocyanines are strong physical quenchers of singlet oxygen they can be used as efficient inhibitors for photodestructive processes in photochemical systems.

Excited chlorophyll and related problems.

A historical outline is presented of the primary light energy conversion in photosynthesis studied by our research group. We found that photoexcited chlorophylls, pheophytins and porphyrins are capable of reversible and irreversible oxido-reduction. The mechanism of the photosensitized electron transfer from donor to acceptor molecule is based on the reversible photochemical oxido-reduction of the pigment-sensitizer. This property of the excited pigments is realized in the reaction centres of photosynthetic cells when photooxidation of bacteriochlorophyll(s) or chlorophyll of Photosystem II is coupled to pheophytin reduction leading to the final charge separation.The studies of the state and function of pigments in the course of chlorophyll biosynthesis in cellular and non-cellular systems revealed different monomeric and aggregated forms of pigments and the phenomenon of self-assembly of various forms of chlorophylls, bacteriochlorophylls and protochlorophylls. The discovery of protochlorophyll photoreduction in non-cellular system allowed the study of the molecular mechanisms of this reaction.In order to construct models of photosynthetic charge separation, we used inorganic photocatalysts-semiconductors, mainly titanium dioxide, and pigments incorporated into detergent micelles or lipid vesicles. To prevent back reactions we used heterogeneous systems where primary unstable products were spatially separated; coupling of solubilized chlorophylls or semiconductor particles with bacterial hydrogenase led to molecular hydrogen photoproduction. Light excitation of some coenzymes, mainly NADH and NADPH, was considered from the point of view of early events of chemical evolution.Now we are interested in the creation of photobiochemical systems using principles of photosynthesis for the conversion and storage of solar energy.

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves : Assignment of spectral bands.

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2-20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990-1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955-960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.