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. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1311–1329.     

The Fragments of the Photosynthetic Electron Transfer Chain in Model Systems

In this paper the recent research from our laboratory is reviewed. Short fragments of the photochemical electron transfer chain of photosynthesis were reproduced in aqueous detergent solutions or in organic solvents. The function of photosystem I is reproduced in a ternary system of chlorophylls, electron donors (dienols, sulfhydryl compounds, hydrazine, etc.), and electron acceptors (viologens, nicotinamide-adenine dinucleotide [NAD], flavines, etc.). Chlorophyll-photosensitized reduction of viologens in some cases is activated by oxygen at the expense of active reductants formed during the photosensitized oxidation of an initial electron donor (thiourea). Chlorophyll-photosensitized oxidoreduction of cytochromes is activated by flavines, viologens, vitamin K derivatives, and some other redox systems (cofactors of cyclic photophosphorylation). The primary mechanism of the reactions studied depends on the reversible chlorophyll photooxidoreduction. In binary systems, chlorophyll (monomeric or aggregated) and electron donor or electron acceptor, reversible photoreduction or photooxidation is observed. Irreversible bacteriochlorophyll oxidation leads to the formation of chlorophyll and protochlorophyll analogues; irreversible protochlorophyll photoreduction results in chlorophyll-like pigment appearance. The photodisaggregation of chlorophyll was observed. The models of photosystem II studied were the photochemical oxygen evolution in aqueous solutions of electron acceptors (ferric compounds, quinone), photosensitized in the near UV part of the spectrum by inorganic semiconductors (tungsten, titanium, and zinc oxides). All reactions described are based on electron (hydrogen) transfer photosensitized by pigment system.     

Mechanism of glutathione oxidation by photosensitized retinal

Sulphur radicals appearing in the retinal photosensitized glutathione oxidation have been recorded by spin trapping. There is no reaction in the absence of oxygen. That is why the single oxygen is supposed to take part in the reaction (IId type photosensitized reaction).     

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.     

Photosensitization of membrane components

The chemical mechanisms of the photosensitized peroxidation of unsaturated fatty acids and cholesterol are reviewed and the subsequent reactions of peroxide decomposition products with biological targets such as DNA and amino acids are analyzed. The importance of protein photooxidation and cross-linking in membrane function impairment is discussed.     

Carotenoids as cellular antioxidants.

Consumption of carotenoids is associated with an enhanced immune response and protection against neoplasia and atherosclerosis. Because these effects have been achieved using carotenoids with no pro-vitamin A activity, they are assumed to be due to the antioxidant properties of carotenoids. Carotenoids protect against photosensitized oxidation by quenching singlet oxygen. In addition, beta-carotene reacts chemically with peroxyl radicals to produce epoxide and apocarotenal products. To investigate the potential significance of these reactions to biological systems, we have used soybean lipoxygenase to generate peroxyl radical enzymatically. beta-Carotene inhibits the oxidation of linoleic acid by soybean lipoxygenase as well as the formation of the hydroperoxide product. In addition, the absorption of beta-carotene is diminished (bleached) by soybean lipoxygenase. The potential significance of these antioxidant reactions of carotenoids to biological function is discussed.     

Porphyrin-nucleic acid interactions: a review

Research involving three important interactions of synthetic cationic porphyrins with nucleic acids: DNA binding, oxidative-reductive strand scission and photosensitized strand scission, is examined retrospectively. The observation that these porphyrins as a class can associate with DNA by intercalative binding, outside binding and outside binding with self-stacking, i.e., the "three-mode binding model", is evaluated with regard to supporting data from several studies including recent evidence from NMR spectroscopy. Results from investigations into the "nuclease-like" activity of the metallo-derivatives of this class of porphyrins are surveyed for demonstrations of base specificity and the mechanism of the chemical interaction. The ability of cationic porphyrins to induce photosensitized damage in DNA is also reviewed with an emphasis on their strand scission activity via a singlet oxygen intermediate.     

Studies on the mechanism of the photosensitized inactivation of E. coli and reactivation phenomenon

In order to find a more satisfactory interpretation of the phenomenon of photosensitized inactivation of bacteria, studies were performed under various experimental conditions on methylene blue and E. coli. In summary the findings are as follow:— 1. The dye is absorbed by the bacteria according to the Langmuir isotherm and can be removed by ionic substitutions; the dye binding to the bacteria is predominantly ionic; the dye-bacteria complex produces a new absorption peak in the 610 mµ wave length region, and the action spectrum corresponds to the spectral absorption of the dye-bacteria complex. 2. There is an optimum dye concentration range for the photosensitized inactivation. 3. Photosensitized inactivation of bacteria can take place both in the frozen and liquid states and the presence of oxygen is essential to the inactivation process. 4. Hydrogen peroxide, formed by reoxidation of the reduced methylene blue, does not inactivate bacteria. 5. Following the photosensitized inactivation, E. coli lose their ability to reduce the methylene blue in the presence of various hydrogen donors, suggesting that enzymes are involved in the inactivation process. 6. Bacteria inactivated by photosensitization can be reactivated by prolonged storage after irradiation; the recovery rate increases with increasing temperature (maximum 37°), and is also influenced by the presence of various hydrogen donors. In view of collected experimental data, the basic reaction mechanisms are analyzed in photosensitized inactivation. The first step of the reaction seems to be excitation of the dye-bacteria, or dye-bacteria oxygen complex, by a photon which produces an activated complex. In such a state, molecular oxygen is capable of producing an oxidizing reaction, which results in the inactivation of the bacteria. Some aspects of the detailed reactions taking place at the cell surface are discussed.     

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.     

Retinal-sensitized photo-oxidation of rhodopsin

Kinetics of retinal photosensitized initiation of radicals of sulfhydryl groups of cysteine and rhodopsin is investigated by spin trapping. Photooxidation of both systems is the result of free radical mechanism. Photooxidation of SH-groups proceeds both with singlet oxygen participation and by direct interaction of photosensitizer with the substrate. The rate constants of these reactions are measured. The rate constant with oxygen participation (K0 = 1.1 X 10(9) M-1 S-1) is higher than the one without oxygen (K = 2.5 X 10(8) M-1 S-1) correspondingly. The lifetime of retinal triplet state in photoreceptor membrane is tau = 4 X 10(-6) s.     

Zeaxanthin in combination with ascorbic acid or alpha-tocopherol protects ARPE-19 cells against photosensitized peroxidation of lipids

The antioxidant action of carotenoids is believed to involve quenching of singlet oxygen and scavenging of reactive oxygen radicals. However, the exact mechanism by which carotenoids protect cells against oxidative damage, particularly in the presence of other antioxidants, remains to be elucidated. This study was carried out to examine the ability of exogenous zeaxanthin alone and in combination with vitamin E or C, to protect cultured human retinal pigment epithelium cells against oxidative stress. The survival of ARPE-19 cells, subjected to merocyanine 540-mediated photodynamic action, was determined by the MTT test and the content of lipid hydroperoxides in photosensitized cells was analyzed by HPLC with electrochemical detection. We found that zeaxanthin-supplemented cells, in the presence of either alpha-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. Cells with added antioxidants exhibited increased viability and accumulated less lipid hydroperoxides than cells without the antioxidant supplementation. Such a synergistic action of zeaxanthin and vitamin E or C indicates the importance of the antioxidant interaction in efficient protection of cell membranes against oxidative damage induced by photosensitized reactions.     

2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin

2,3-Butanedione sensitized the rapid photodestruction of free alpha-amino acids, and the photoinactivation of alpha-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of alpha-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.     

Photosensitized oxidation of phenyl and tert-butyl sulfides.

The photosensitized oxygenation of diphenyl (1), di-tert-butyl (2) and phenyl tert-butyl sulfide (3) was studied. Bimolecular rate constants of singlet oxygen quenching are low (1 to 5 x 10(4) M(-1)s(-1)) since the sulfides are poor nucleophiles due to sterical hindrance (2, 3) or the HOMO on the sulfur atom being a less accessible p(z) orbital (1). The quenching is mainly physical, but chemical reaction leading to sulfoxides also takes place in methanol and, to a lower degree, in acetonitrile. Catalysis by carboxylic acids considerably enhances the rate of sulfoxidation. Inefficiency in the chemical reaction is again due to the poor nucleophilicity of the sulfides, which limits oxygen transfer by electrophilic intermediates such as the protonated persulfoxide.     

Photo- and thermal-oxidation studies on methyl and phenyl linoleate: anti-oxidant behaviour and rates of reaction

Photo-peroxidation of methyl and phenyl linoleate in methanol solutions at 25 degrees C, in the presence of methylene blue or 5,10,15,20-tetra(4-pyridyl)-porphyrin (TPP) as sensitisers of singlet oxygen, was found to proceed at more than 30 times the rate of the same polyunsaturated fatty acid (PUFA) ester species undergoing thermal-peroxidation in the bulk phase at 50 degrees C. The addition of anti-oxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) quench the thermal-oxidation effectively but appear to only partially inhibit the photosensitized peroxidation reactions. The kinetics of the overall peroxidation reactions were followed by ultraviolet spectroscopy, measurements of hydroperoxide concentration and by high performance liquid chromatography (HPLC). The photo-peroxidation reaction proceeds more rapidly in chloroform solution as the lifetime of singlet oxygen is shown to be over ten times longer in chloroform than methanol. The initial fast reaction kinetics of the photo-peroxidation reactions were evaluated using a pulsed laser technique to show that singlet oxygen reacts competitively with both the anti-oxidants and the polyunsaturated fatty acid ester. Second order kinetic rate constants (in the range 10(5)-10(7) dm(3) mol(-1) s(-1)) were evaluated for the reactivity of singlet oxygen with a range of anti-oxidants and a singlet oxygen quencher, and the results used to explain the effect of anti-oxidants at different concentrations on the rate of the linoleate photo-peroxidation reaction.     

Isolation and activity of the photodynamic pigment hypericin

Abstract. Hypericin, a photodynamic pigment, occurring in members of the Hypericaceae, can induce photosensitivity in grazing animals. The pigment has been isolated from the glandular trichomes located on the calyx of Hypericum hirsulum. Hypericin is shown to be capable of sensitizing the photo-oxidation of methyl linolenate. This activity is reduced in the presence of crocin, a carotenoid. Evidence for the generation of singlet molecular oxygen by hypericin is provided by the monitoring of oxygen consumption during the photosensitized oxidation of imidazole. Rates of oxygen consumption were modified by deuterium oxide and sodium azide. The photodynamic action of hypericin on pea leaf discs results in the promotion of photo-oxidative damage, measured by pigment loss and ethane production. These results are discussed in relation to the possible function of hypericin within the plant and the role of photo-dynamic reactions in nature.     

Visible light damage to Escherichia coli in seawater: oxidative stress hypothesis

The effect of visible light on Escherichia coli H10407 in seawater microcosms was investigated. Light damage was estimated by loss of colony-forming ability. Illumination of E. coli suspended in oligotrophic seawater with visible light at an intensity of about 40 klux caused a drastic decrease of culturable bacteria which turned to a viable but non-culturable state. In seawater E. coli exhibited weak metabolic activity as estimated by ³H methyl-thymidine incorporation in the cell. Visible light did not significantly alter this metabolic activity and did not involve detectable oxidation of lipid membranes as evaluated by gas chromatography analysis of fatty acids. The involvement of oxygen and reactive oxygen species in phototoxicity was studied. A decrease of the toxic effect was observed when E. coli was exposed to visible light under anaerobic conditions. Scavengers of reactive oxygen species exhibited variable protective effects. β-Carotene, a singlet oxygen scavenger, and superoxide dismutase were equally ineffective. On the other hand, catalase, which eliminates hydrogen peroxide and thiourea, a hydroxyl radical scavenger, showed a net protection. In addition desferrioxamine B, an iron chelator, was also effective in reducing phototoxicity, probably by preventing hydroxyl radical generation by decomposition of hydrogen peroxide in the presence of iron (Fenton reaction). Therefore, hydrogen peroxide and hydroxyl radical seem to be reactive intermediates of oxygen-dependent (type II) photosensitized reactions.     

Inorganic semiconductors as photosensitizers in biochemical redox reactions.

The results of studies of biochemical redox reactions photosensitized by inorganic semiconductor particles are reviewed. The mechanisms of hydrogen photoproduction, NAD+ or NADP+ photoreduction, CO2 photofixation and photosynthesis of organic and amino acids under the coupled action of TiO2, ZnO, CdS, ZnS and enzymes or bacterial cells are considered. Studies on the photocatalytic activity of ferritin, a protein containing microcrystals of hydrous ferric oxide, are described. The data on biosynthesis of cadmium sulfide by microorganisms and plants are analyzed. The possibility of the participation of inorganic semiconductors in photoprocesses in vivo is discussed.     

Paradoxical effects of temperature and solar irradiance on the photodegradation state of killed phytoplankton

The aim of this paper was to study the effects of temperature and irradiance on the photodegradation state of killed phytoplankton cells. For this purpose, killed cells of the diatom Chaetoceros neogracilis RCC2022 were irradiated (photosynthetically active radiation) at 36 and 446 J · s^?1 · m^?2 (for the same cumulative dose of irradiation energy) and at two temperatures (7°C and 17°C). Analyses of specific lipid tracers (fatty acids and sterols) revealed that low temperatures and irradiances increased photooxidative damages of monounsaturated lipids (i.e., palmitoleic acid, cholesterol and campesterol). The high efficiency of type II photosensitized degradation processes was attributed to: (i) the relative preservation of the sensitizer (chlorophyll) at low irradiances allowing a longer production of singlet oxygen and (ii) the slow diffusion rate of singlet oxygen through membranes at low temperatures inducing more damages. Conversely, high temperatures and irradiances induced (i) a rapid degradation of the photosensitizer and a loss of singlet oxygen by diffusion outside the membranes (limiting type II photosensitized oxidation), and (ii) intense autoxidation processes degrading unsaturated cell lipids and oxidation products used as photodegradation tracers. Our results may explain the paradoxical relationship observed in situ between latitude and photodegradation state of phytoplankton cells.     

In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.     

Photodynamic activity and singlet oxygen

The primary mechanisms for the photodynamic action of pigments and dyes, the principles of their division into mechanisms of type I and type II, and the role of these processes in biological systems are reviewed. Singlet oxygen is considered to be an indicator of the mechanisms of photodynamic reactions. The methods of its detection are described, which are based on the use of chemical traps, measurements of infrared phosphorescance at 1270 nm, and the registration singlet oxygen-sensitized delayed fluorescence caused by the summation of the energy of two singlet oxygen molecules by one dye molecule.