Temperature effect on the luminescence of synthetized Yt base in PVA film

The mean phosphorescence lifetimes, fluorescence yields and phosphorescence-to-fluorescence quantum yields ratios of Y_t base in PVA films were measured in the temperature range of 100–250°K. The quantum yield of triplet state formation was found to be temperature independent at a wide range of lower temperatures. The radiative and nonradiative rate constants for triplet state were measured.     

Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence

Regulation of nonradiative dissipation of absorbed light energy in PSII is an indispensable process to avoid photoinhibition in plants. To dissect molecular mechanisms of the regulation, we identified Arabidopsis mutants with reduced quenching of Chl fluorescence using a fluorescence imaging system. By analyses of Chl fluorescence induction pattern in the light and quantum yield of both photosystems, 37 mutants were classified into three groups. The first group was characterized by an extremely high level of minimum Chl fluorescence at the open PSII center possibly due to a defect in PSII. Mutants with significant reduction in the nonphotochemical quenching formation but not in quantum yield of both photosystems were classified into the second group. Mutants in the third group showed reduction in quantum yield of both photosystems possibly due to a defect in the electron transport activity. Mutants in the second and third groups were further characterized by light intensity dependence of Chl fluorescence parameters and steady state redox level of P700.     

Changes in P-700 Oxidation during the Early Stages of the Induction of Photosynthesis

Following dark adaptation, the response to irradiance of chlorophyll (Chl) fluorescence, the light-induced absorbance change around 820 nm (to measure reaction center Chl of photosystem I [PSI] P-700 oxidation), and CO2 fixation were examined in pea (Pisum sativum L.) leaves under a range of conditions. Initially, P-700 oxidation is restricted by a lack of regeneration of PSI electron acceptors, and the increase of oxidized P-700 (P-700+) that occurs during approximately the first 60 s of irradiation is largely independent of the resistance to electron flow between the two photosystems. Under these conditions, the quantum efficiency for linear electron flow is directly positively related to P-700+ accumulation, which is in contrast to the direct negative correlation that is the most frequently reported relationship between P-700+ accumulation and the quantum efficiency for linear electron flow.     

Absorption studies of primary reactions in Photosystem I. Yield and rate of formation of the P-700 triplet state

The quantum yield of P-700 triplet state formation has been found from flash-induced absorption studies in the microsecond time range to be 0.45 and 0.35 at 294 K and 6–20 K, respectively, in CP1-SDS particles which lack the secondary acceptors. From these quantum yield measurements, yields of formation of the P-700 triplet state from the primary biradical (P-700⁺–A⁻₀) were calculated to be around 0.6 at both temperatures, whereas double-laser flash experiments allowed us to derive upper limits for this yield (0.84 at 294 K and 0.79 at 20 K). These values agree with the high values that have been previously calculated from an earlier absorption study (Sétif, P., Hervo, G. and Mathis, P. (1981) Biochim. Biophys. Acta 638, 257–267) but appear significantly higher than the yield calculated from EPR experiments (5–10%) (Gast, P., Swarthoff, T., Ebskamp, F.C.R. and Hoff, A.J. (1983) Biochim. Biophys. Acta 722, 163–175). Possible explanations for this discrepancy are discussed. From absorption studies in the submicrosecond time range as well as from double-laser flash experiments, the lifetime of the biradical (P-700⁺–A⁻₀), from which the P-700 triplet state is formed by recombination, has been measured to increase from 30–50 ns at room temperature up to 120–130 ns between 10 and 110K.     

Energy transfer and charge separation in photosystem I: P700 oxidation upon selective excitation of the long-wavelength antenna chlorophylls of Synechococcus elongatus.

Photosystem I of the cyanobacterium Synechococcus elongatus contains two spectral pools of chlorophylls called C-708 and C-719 that absorb at longer wavelengths than the primary electron donor P700. We investigated the relative quantum yields of photochemical charge separation and fluorescence as a function of excitation wavelength and temperature in trimeric and monomeric photosystem I complexes of this cyanobacterium. The monomeric complexes are characterized by a reduced content of the C-719 spectral form. At room temperature, an analysis of the wavelength dependence of P700 oxidation indicated that all absorbed light, even of wavelengths of up to 750 nm, has the same probability of resulting in a stable P700 photooxidation. Upon cooling from 295 K to 5 K, the nonselectively excited steady-state emission increased by 11- and 16-fold in the trimeric and monomeric complexes, respectively, whereas the quantum yield of P700 oxidation decreased 2.2- and 1.7-fold. Fluorescence excitation spectra at 5 K indicate that the fluorescence quantum yield further increases upon scanning of the excitation wavelength from 690 nm to 710 nm, whereas the quantum yield of P700 oxidation decreases significantly upon excitation at wavelengths longer than 700 nm. Based on these findings, we conclude that at 5 K the excited state is not equilibrated over the antenna before charge separation occurs, and that approximately 50% of the excitations reach P700 before they become irreversibly trapped on one of the long-wavelength antenna pigments. Possible spatial organizations of the long-wavelength antenna pigments in the three-dimensional structure of photosystem I are discussed.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of porphyridium cruentum which had been frozen to -196 degrees C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at -196 degrees C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Energy transfer from phycobiliproteins to photosystem I in vegetative cells and heterocysts of Anabaena variabilis

The presence of phycobilins in heterocysts of Anabaena variabilis is established on the basis of absorption and fluorescence spectroscopy. At 77 K heterocysts exhibit fluorescence emission bands at 645 and 661 nm indicative of phycocyanin and allophycocyanin, respectively. Both allophycocyanin levels and fluorescence emission at 695 nm were low in heterocysts relative to whole filaments. In situ fluorescence microscopy confirmed the presence of phycobilins in individual heterocysts, but the pigment levels varied considerably among cells. Heterocysts exhibited Photosystem I activity, as evidenced by photooxidation of P-700, but no Photosystem II activity. The quantum efficiency of phycobilins in sensitizing P-700 photooxidation was 50-70% that of chlorophyll a. Phycoibins were also effective in promoting light-dependent reduction of acetylene to ethylene. The results are discussed in terms of the role of the heterocyst in nitrogen fixation and of the significance of energy transfer from phycobilins to Photosystem I in heterocysts.     

Evidence against proton gradient formation being the cause of chlorophyll fluorescence quenching by N-methylphenazonium methosulfate.

In strong illumination, 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while P-700 turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undectectable. When 10muM N-methylphenazonium methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 muM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in P-700 turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline. When 10muM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), P-700 turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurrs by a mechanism unrelated to the generation of a phosphyorylation potential. With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.     

Relationship between the Quantum Efficiencies of Photosystems I and II in Pea Leaves

The irradiance dependence of the efficiencies of photosystems I and II were measured for two pea (Pisum sativum [L.]) varieties grown under cold conditions and one pea variety grown under warm conditions. The efficiencies of both photosystems declined with increasing irradiance for all plants, and the quantum efficiency of photosystem I electron transport was closely correlated with the quantum efficiency of photosystem II electron transport. In contrast to the consistent pattern shown by efficiency of the photosystems, the redox state of photosystem II (as estimated from the photochemical quenching coefficient of chlorophyll fluorescence) exhibited relationships with both irradiance and the reduction of P-700 that varied with growth environment and genotype. This variability is considered in the context of the modulation of photosystem II quantum efficiency by both photochemical and nonphotochemical quenching of excitation energy.     

Structure property analysis of pentamethine indocyanine dyes: identification of a new dye for life science applications

A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.     

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature

Fluorescence emission spectra of chloroplasts, initially frozen to--196 degrees C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at--196 degrees C than at--78 degrees C. The initial rate of photooxidation of P-700 was also measured at--196 degrees C and--78 degrees C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at--78 degrees C P-700 traps approx. 20 times more energy than C-705 while, at--196 degrees C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to--196 degrees C is attributed to traps in Photosystem II which form only on cooling to temperatures near--196 degrees C.     

Evidence against proton gradient formation being the cause of chlorophyll fluorescence quenching by N-methylphenazonium methosulfate

In strong illumination, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while $\text{P-700}$ turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undetectable. When 10 μM $\text{N-}\text{methylphenazonium}$ methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 μM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in $\text{P-700}$ turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline.When 10 μM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), $\text{P-700}$ turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurs by a mechanism unrelated to the generation of a phosphorylation potential.With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.     

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.     

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Ribosome function determined by fluorescence.

Five different fluorescence phenomena are considered in relation to their use to study the structure and function of ribosomes. These are: quantum yield or emission intensity; emission wavelength maximum; fluorescence anisotropy; collisional quenching; and nonradiative energy transfer. Results from a number of studies in which these techniques were used are described and summarized in relation to the movement and conformation of tRNA, the nascent peptide, and mRNA in a ribosome during the reaction steps of peptide elongation.     

The study of the primary photoprocesses in Photosystem I of chloroplasts Recombination luminescence,chlorophyll triplet state and triplet-triplet annihilation

The dependence of the delayed luminescence of Photosystem I on the state of the reaction centers has been studied. Light flash induces a charge separation in the centers: $\text{P-700 · P-430}\textcolor{red}{}\text{P-700}{}^{\mathrm{+}}\text{· P-430}{}^{\mathrm{?}}$. Dark recombination of charges is accompanied by the recombination luminescence with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{ms}$.If the centers are in the P-700 · P-430^? state or if P-430 is inactivated by heat, then flashing of Photosystem I generates the triplet state chlorophyll with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 0.5}\text{ms}$. The triplet state has been measured by the delayed fluorescence of chlorophyll at 20 °C and 77 °K and by the chlorophyll phosphorescence at 77 °K. The delayed fluorescence at 20 °C arises from the thermal activation of the triplet state up to the excited singlet level of chlorophyll and at 77 °K it is due to triplet-triplet annihilation. The quantum yield of the triplet formation, estimated by a comparison of the light saturation curves of delayed fluorescence at 20 °C and of P-700 photooxidation under the same experimental (optical) conditions, is ≈ 0.9 of the P-700⁺ yield. Only one triplet of chlorophyll can be generated per P-700. Under heat inactivation of P-430 the triplet formation is not observed when P-700 is oxidized.It is assumed that the triplet-triplet annihilation at 77 °K is related with the strong interaction between the chlorophyll molecules in the pigment complex of Photosystem I. The possibility of a triplet participation in the primary processes of photosynthesis is discussed.     

Effects of O(2) and CO(2) Concentrations on Quantum Yields of Photosystems I and II in Tobacco Leaf Tissue

The interactive effects of irradiance and O₂ and CO₂ levels on the quantum yields of photosystems I and II have been studied under steady-state conditions at 25°C in leaf tissue of tobacco (Nicotiana tabacum). Assessment of radiant energy utilization in photosystem II was based on changes in chlorophyll fluorescence yield excited by a weak measuring beam of modulated red light. Independent estimates of photosystem I quantum yield were based on the light-dark in vivo absorbance change at 830 nanometers, the absorption band of P700⁺. Normal (i.e. 20.5%, v/v) levels of O₂ generally enhanced photosystem II quantum yield relative to that measured under 1.6% O₂ as the irradiance approached saturation. Photorespiration is suspected to mediate such positive effects of O₂ through increases in the availability of CO₂ and recycling of orthophosphate. Conversely, at low intercellular CO₂ concentrations, 41.2% O₂ was associated with lower photosystem II quantum yield compared with that observed at 20.5% O₂. Inhibitory effects of 41.2% O₂ may occur in response to negative feedback on photosystem II arising from a build-up in the thylakoid proton gradient during electron transport to O₂. Covariation between quantum yields of photosystems I and II was not affected by concentrations of either O₂ or CO₂. The dependence of quantum yield of electron transport to CO₂ measured by gas exchange upon photosystem II quantum yield as determined by fluorescence was unaffected by CO₂ concentration.     

Radiationless deactivation of 6-aminocoumarin from the S1-ICT state in nonspecifically interacting solvents

This paper presents the results of a spectral (absorption and emission) and photophysical study of 6-aminocoumarin (6AC) in the solvents with which this molecule interacts only nonspecifically (n-alkanes, tetrachloromethane and 1-chloro-n-alkanes) and in nitriles. The strong effects of the solvents on the emission spectra, fluorescence quantum yield and lifetime of 6AC were observed. The results of the steady-state and time-resolved photophysical study suggest the presence of very fast nonradiative deactivation processes. It is concluded that besides fluorescence, the efficient S(1)-ICT → S(0) internal conversion in nonpolar aprotic solvents arises from vibronic interactions between close-lying S(1)-ICT(π,π*) and S(2)(n,π*) states. Moreover, unexpectedly efficient triplet state formation occurs. In nitriles the intermolecular hydrogen-bonding interactions with solvent molecules also facilitate the nonradiative decay process involving the S(1)-exciplex.