Mechanism of photoinactivation in photosynthetic systems I. The dark reaction in photoinactivation

1)General features of photoinactivation of chloroplast reactionsunder moderate light intensity were investigated. 2)The effects of temperature and of intermittent illuminationupon photoinactivation indicated that the process is not a simplephotochemical reaction but includes some dark reactions. 3)The action spectrum of the photoinactivation indicated thatthe energy absorbed by photosynthetic pigments is used for theprocess. 4)The relative sensitivity and different time courses seen inthe inactivation of various photochemical activities suggestedthe presence of different kinds of photoinactivation. ¹This study was aided by grants from the Ministry of Education(407, 160-1965, 91, 402-1966, 1967, 4, 103-1968). Financialsupport from Sanyo Broadcasting Scientific Foundation is alsoacknowledged with cordial thanks (Received July 30, 1969; )     

Mechanism of photoinactivation in photosynthetic systems III. Site and mode of photoinactivation in photosystem I

The presence of ferredoxin, PMS, pyocyanine or FMN in the preincubationmixture completely protected chloroplast fragments from photoinactivationof the activity associated with photosystem I of photosynthesis.Neither ferredoxin, plastocyanin, ferredoxin-NADP reductasenor the cytochromes contained in chloroplast fragments werethe site of photoinactivation. The site and mode of photoinactivationin system I of photosynthesis are discussed. (Received September 3, 1969; )     

Mechanism of photoinactivation in photosynthetic systems II. The occurrence and properties of two different types of photoinactivation

Photoinactivation of the activity of NADP photoreduction withreduced DPIP or with reduced TMPD as the electron donor wasinhibited by the absence of oxygen in the atmosphere or by thepresence of photosynthetic inhibitors (CMU, DCMU, o-phenanthroline)in the preillumination mixture. Photoinactivation of the photoreductionof NADP or DPIP with water as the electron donor was not affected,or even accelerated, by these conditions of preillumination.The concentrations of inhibitors required for maximum inhibitionin the former case corresponded to those required for inhibitionof photosynthetic electron transport. The results indicatedthe occurrence of 2 different types of photoinactivation, eachspecifically affecting photosystems I and II, and differingin behaviours; including their requirement for oxygen in theatmosphere and their responses toward the presence of photosyntheticinhibitors during the preillumination period. (Received July 30, 1969; )     

Fluorescence and photoinactivation of indoleacetic acid

These results indicate quite clearly that the induction of the photoinactivation of indoleacetic acid (IAA) is by no means a peculiarity of riboflavin but is a property common to many fluorescent substances. It is not essential that the compounds be colored. Colorless materials are also able to bring about IAA photoinactivation provided that they absorb ultraviolet light.After exposure to light, the reaction mixture becomes turbid and develops a light-pink coloration. Experiments are being performed to elucidate the nature of the reaction product.It is suggested that besides riboflavin other naturally occurring fluorescent substances in plants may play a role in light-induced growth reactions.     

Merocyanine-sensitized photoinactivation of enveloped viruses.

A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.     

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β^5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β^6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.     

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double beta(5.7)-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded beta(6.3)-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.     

Parameterization of photosystem II photoinactivation and repair

The photoinactivation (also termed photoinhibition or photodamage) of Photosystem II (PSII) and the counteracting repair reactions are fundamental elements of the metabolism and ecophysiology of oxygenic photoautotrophs. Differences in the quantification, parameterization and terminology of Photosystem II photoinactivation and repair can erect barriers to understanding, and particular parameterizations are sometimes incorrectly associated with particular mechanistic models. These issues lead to problems for ecophysiologists seeking robust methods to include photoinhibition in ecological models. We present a comparative analysis of terms and parameterizations applied to photoinactivation and repair of Photosystem II. In particular, we show that the target size and quantum yield approaches are interconvertible generalizations of the rate constant of photoinactivation across a range of incident light levels. Our particular emphasis is on phytoplankton, although we draw upon the literature from vascular plants. This article is part of a Special Issue entitled: Photosystem II.     

Phototoxicity and photoinactivation of blebbistatin in UV and visible light

Blebbistatin was recently identified as a selective, cell-permeant inhibitor of myosin II. Because blebbistatin is likely to be used extensively with fluorescence imaging in studies of cytoskeletal dynamics, its compatibility with common excitation wavelengths was examined. Illumination of blebbistatin-treated bovine aortic endothelial cells at 365 and 450-490 nm, but not 510-560 or 590-650 nm, caused dose-dependent cell death. Illumination of blebbistatin alone at 365 and 450-490 nm changed its absorption and emission spectra, but the resultant compounds were not toxic. In addition, photoreacted blebbistatin no longer disrupted myosin distribution in cells, indicating loss of pharmacological activity. Fluorescence microscopy showed that upon illumination, blebbistatin became bound to cells and to protein-coated glass, suggesting that toxicity may arise from light-induced reaction of blebbistatin with cell proteins. Blebbistatin should be used only with careful consideration of these photochemical effects.     

Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms

Diatoms host chlorophyll a/c chloroplasts distinct from green chloroplasts. Diatoms now dominate the eukaryotic oceanic phytoplankton, in part through their exploitation of environments with variable light. We grew marine diatoms across a range of temperatures and then analyzed their PSII function and subunit turnover during an increase in light to mimic an upward mixing event. The small diatom Thalassiosira pseudonana initially responds to increased photoinactivation under blue or white light with rapid acceleration of the photosystem II (PSII) repair cycle. Increased red light provoked only modest PSII photoinactivation but triggered a rapid clearance of a subpool of PsbA. Furthermore, PsbD and PsbB content was greater than PsbA content, indicating a large pool of partly assembled PSII repair cycle intermediates lacking PsbA. The initial replacement rates for PsbD (D2) were, surprisingly, comparable to or higher than those for PsbA (D1), and even the supposedly stable PsbB (CP47) dropped rapidly upon the light shift, showing a novel aspect of rapid protein subunit turnover in the PSII repair cycle in small diatoms. Under sustained high light, T. pseudonana induces sustained nonphotochemical quenching, which correlates with stabilization of PSII function and the PsbA pool. The larger diatom Coscinodiscus radiatus showed generally similar responses but had a smaller allocation of PSII complexes relative to total protein content, with nearly equal stiochiometries of PsbA and PsbD subunits. Fast turnover of multiple PSII subunits, pools of PSII repair cycle intermediates, and photoprotective induction of nonphotochemical quenching are important interacting factors, particularly for small diatoms, to withstand and exploit high, fluctuating light.     

Target theory and the photoinactivation of Photosystem II

Application of target theory to the photoinactivation of Photosystem II in pea leaf discs (Park et al. 1995, 1996a,b) reveals that there is a critical light dosage below which there is complete photoprotection and above which there is photoinactivation (i.e a light-induced loss of oxygen flash yield). The critical dosage is about 3 mol photons m^–2 for medium and high light-grown leaves and 0.36 mol photons m^–2 for low light-grown leaves. Photoinactivation is a one-hit process with an effective cross-section of 0.045 m² mol^–1 photons which does not vary with growth irradiance, unlike the cross-section for oxygen evolution which increases with decreasing growth irradiance. The cross-section for oxygen evolution increased by about 20% following exposure to 6.8 mol photons m^–2 which may be due to energy transfer from photoinactivated units to functional Photosystem II units. We propose that the photoinactivation of PS II begins when a small group of PS II pigment molecules whose structure is uninfluenced by growth irradiance, becomes uncoupled energetically from the rest of the photosynthetic unit and thus no longer transfers excitions to P680. De-excitation of this group of pigment molecules provides the energy which leads to the damage of Photosystem II. Treatment of pea leaves with dithiothreitol, an inhibitor of the xanthophyll cycle, decreases the critical dosage i.e. decreases photoprotection but has no effect on the PS II photoinactivation cross-section. Treatment with 1 M nigericin increased the photoinactivation cross-section of PS II as did exposure to lincomycin which inhibits D1 protein synthesis and thus the repair of PS II reaction centres.Abbreviations DTT- dithiothreitol - PS II- Photosystem II - Fm- maximum fluorescence - Fv- variable fluorescence - LHCIIb- main light harvesting pigment-protein complex of PS II - D1 protein- psbA gene product - P680- reaction centre chlorophyll of Photosystem II - Qa- first quinone electron acceptor of Photosystem II - (o₂)- cross-section for oxygen evolution - (pi)- cross-section for photoinactivation     

Compensation for PSII photoinactivation by regulated non-photochemical dissipation influences the impact of photoinactivation on electron transport and CO2 assimilation

The extent to which PSII photoinactivation affects electron transport (PhiPSII) and CO2 assimilation remains controversial, in part because it frequently occurs alongside inactivation of other components of photosynthesis, such as PSI. By manipulating conditions (darkness versus low light) after a high light/low temperature treatment, we examined the influence of different levels of PSII inactivation at the same level of PSI inactivation on PhiPSII and CO2 assimilation for Arabidopsis. Furthermore, we compared PhiPSII at high light and optimum temperature for wild-type Arabidopsis and a mutant (npq4-1) with impaired capacities for energy dissipation. Levels of PSII inactivation typical of natural conditions (< 50%) were not associated with decreases in PhiPSII and CO2 assimilation at photon flux densities (PFDs) above 150 micromol m(-2) s(-1). At higher PFDs, the light energy being absorbed was in excess of the energy that could be utilized by downstream processes. Arabidopsis plants downregulate PSII activity to dissipate such excess in accordance with the level of PSII photoinactivation that also serves to dissipate absorbed energy. Therefore, the overall levels of non-photochemical dissipation and the efficiency of photochemistry were not affected by PSII inactivation at high PFD. Under low PFD conditions, such compensation is not necessary, because the amount of light energy absorbed is not in excess of that needed for photochemistry, and inactive PSII complexes are dissipating energy. We conclude that moderate photoinactivation of PSII complexes will only affect plant performance when periods of high PFD are followed by periods of low PFD.     

Dye mediated photoinactivation of bacteriophages by nitrogen laser

The nitrogen laser (λ = 337.1 nm) was documented to have photosensitized inactivation of bacteriophages P1 and phage A havingEscherichia coli andSalmonella typhi as their respective hosts. Methylene blue and crystal violet had a direct virucidal effect whereas toluidine blue revealed accentuated lethal effect on photosensitization with N₂ laser for both of the bacteriophages taken in the study. The other dyes such as congo red, neutral red, auramin O and safranine showed differences in their virucidal activity among the two bacteriophages. However, malachite green did not show any change for the two viruses both by itself and on irradiation with the laser. A possibility of photosensitizing effect of N₂ laser for the therapy of viral infections needs to be explored.     

Factors influencing the haematoporphyrin-sensitized photoinactivation of Candida albicans

Photosensitizing activity of haematoporphyrin (Hp) on Candida albicans cells is mainly promoted by unbound dye molecules in the bulk aqueous medium. Moreover, the death of photosensitized cells is dependent on the dye concentration, irradiation time, irradiation temperature, and the composition of the growth media. Morphological and biochemical studies indicate that the photoprocess involves an initial limited alteration of the cytoplasmic membrane, which allows the penetration of the dye into the cell with consequent photodamage of intracellular targets. In this respect, the Hp-sensitized photoinactivation of eukaryotic microbial cells differs from that in prokaryotic cells.     

Photodynamic antimicrobial chemotherapy in aquaculture: photoinactivation studies of Vibrio fischeri

Background

Photodynamic antimicrobial chemotherapy (PACT) combines light, a light-absorbing molecule that initiates a photochemical or photophysical reaction, and oxygen. The combined action of these three components originates reactive oxygen species that lead to microorganisms'' destruction. The aim was to evaluate the efficiency of PACT on Vibrio fischeri: 1) with buffer solution, varying temperature, pH, salinity and oxygen concentration values; 2) with aquaculture water, to reproduce photoinactivation (PI) conditions in situ.

Methodology/Principal Findings

To monitor the PI kinetics, the bioluminescence of V. fischeri was measured during the experiments. A tricationic meso-substituted porphyrin (Tri-Py⁺-Me-PF) was used as photosensitizer (5 µM in the studies with buffer solution and 10–50 µM in the studies with aquaculture water); artificial white light (4 mW cm⁻²) and solar irradiation (40 mW cm⁻²) were used as light sources; and the bacterial concentration used for all experiments was ≈10⁷ CFU mL⁻¹ (corresponding to a bioluminescence level of 10⁵ relative light units - RLU). The variations in pH (6.5–8.5), temperature (10–25°C), salinity (20–40 g L⁻¹) and oxygen concentration did not significantly affect the PI of V. fischeri, once in all tested conditions the bioluminescent signal decreased to the detection limit of the method (≈7 log reduction). The assays using aquaculture water showed that the efficiency of the process is affected by the suspended matter. Total PI of V. fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of Tri-Py⁺-Me-PF.

Conclusions/Significance

If PACT is to be used in environmental applications, the matrix containing target microbial communities should be previously characterized in order to establish an efficient protocol having into account the photosensitizer concentration, the light source and the total light dose delivered. The possibility of using solar light in PACT to treat aquaculture water makes this technology cost-effective and attractive.     

Ultraviolet photoinactivation of galactosyltransferase. Protection by substrates.

Galactosyltransferase was irreversibly inactivated upon exposure to ultraviolet light and the rate of inactivation followed apparent first-order kinetics. Significant protection against inactivation was observed in the presence of various combinations of substrates. UDPgalactose and Mn2+ together gave the most protection. Amino acid analyses revealed the loss of 1 mol of tryptophan per mol of galactosyltransferase upon ultraviolet photoinactivation. Further evidence for an essential trypotphan was provided by difference spectra and by inactivation with 2-hydroxy-5-nitrobenzyl bromide and protection against this reagent by Mn2+ and UDPgalactose. The protection by UDPgalactose and Mn2+ was greater than that provided by UDPgalactose alone. Since Mn2+ provided no protection by itself, this suggested that the formation of the galactosyltransferase-Mn2+-UDPgalactose complex caused a conformational change which was responsible for the observed protection of the essential tryptophanyl residue.