Comparative Time-Resolved Photosystem II Chlorophyll a Fluorescence Analyses Reveal Distinctive Differences between Photoinhibitory Reaction Center Damage and Xanthophyll Cycle-Dependent Energy Dissipation*

Abstract— The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important pho-toprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (ΔpH) and the carotenoid pigment interconversions of the xanthophyll cycle. Co-incidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chi a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chi a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhi-bition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from ～2 ns to ～0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonra-diative decay path for the charge-separated state of the PSII reaction center. In contrast, the ΔpH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et ah, 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the ΔpH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.     

Seasonal changes in the excess energy dissipation from Photosystem II antennae in overwintering evergreen broad-leaved trees Quercus myrsinaefolia and Machilus thunbergii

We monitored chlorophyll (Chl) fluorescence, pigment concentration and the de-epoxidation state of the xanthophyll cycle (DPS(1)) in two warm temperate broad-leaved evergreen species (Quercus myrsinaefolia and Machilus thunbergii). Reduction of the maximal quantum yield of Photosystem II (PSII) (calculated from Fv/Fm, variable to maximal Chl a fluorescence) and retention of a high DPS were observed in both species in the winter, and can be interpreted as acclimation to winter. In particular, the acclimation of PSII in these species can be chiefly attributed to thermal dissipation, which is correlated with the retention of high zeaxanthin. Furthermore, we attempted to divide the fate of the absorbed light energy by the PSII antennae into three components: (i) PSII photochemistry (represented by its quantum yield, ΦPSII), (ii) dissipation by down-regulation via non-photochemical quenching (ΦNPQ) and (iii) other non-photochemical processes (ΦONP). The estimated energy allocation of the absorbed light indicated that the proportion of ΦPSII decreased, whereas that of ΦNPQ+ΦONP increased during winter. This result suggests that the excess energy absorbed in the PSII complexes is safely dissipated from the PSII antennae. Based on these results, we conclude that thermal dissipation from the PSII antennae plays an important role in two overwintering broad-leaved evergreen trees growing in Japan.     

pH-Dependent Extraction of Ca 2+ from Photosystem II Membranes and Thylakoid Membranes: Indication of a Ca 2+-Sensitive Site on the Acceptor Side of Photosystem II

The action of low pH treatment (pH 3.6) known to release Ca²⁺ from the oxygen-evolving complex in photosystem II (PSII) membranes and to induce Ca²⁺-revers-ible inhibition of electron transport at the acceptor side of PSII in thylakoid membranes (TM) was compared in PSII membranes and TM. The rate of the inactivation of electron transport by low pH was four times higher in TM than in PSII membranes. Ferricyanide accelerated the inactivation of PSII membranes but decreased it in the case of TM. Low pH treatment also greatly modified the fluorescence induction kinetics in both preparations, but significant differences have been found in the fluorescence induction kinetics of treated TM and PSII membranes. Calcium restored the electron transport activity and fluorescence induction kinetics in PSII membranes and TM, whereas diphenylcarbazide restored these functions only in PSII membranes. The reactivation of Ca-depleted PSII membranes was more effective in the dark, whereas the reactivation of TM required weak light. In the case of PSII membranes subjected to low pH citrate buffer, maximal reactivation was observed at 60 mM Ca²⁺ but for TM about 10 mM Ca²⁺ was required and 60 mM fully inhibited electron transport in TM during reactivation. These results indicate that the Ca-dependent inactivation of the acceptor side of PSII in TM after low pH treatment cannot be explained by the extraction of Ca²⁺ from the oxygen-evolving complex. It is rather suggested that the Ca²⁺ involved in this inhibition is bound to the acceptor side of the photosystem near to the Q_A-non-heme iron binding site and may participate in the binding of a polypeptide of the PSII light antenna complex to the PSII reaction center.     

Regulation of the excitation energy utilization in the photosynthetic apparatus of chlorina f2 barley mutant grown under different irradiances

Acclimation of the photosynthetic apparatus of chlorophyll b-less barley mutant chlorina f2 to low light (100 micromolm(-2)s(-1); LL) and extremely high light level (1000 micromolm(-2)s(-1); HL) was examined using techniques of pigment analysis and chlorophyll a fluorescence measurements at room temperature and at 77 K. The absence of chlorophyll b in LL-grown chlorina f2 resulted in the reduction of functional antenna size of both photosystem II (by 67%) and photosystem I (by 21%). Chlorophyll fluorescence characteristics of the LL-grown mutant indicated no impairment of the utilization of absorbed light energy in photosystem II photochemistry. Thermal dissipation of excitation energy estimated as non-photochemical quenching of minimal fluorescence (SV(0)) was significantly higher as compared to the wild-type barley grown under LL. Despite impaired assembly of pigment-protein complexes, chlorina f2 was able to efficiently acclimate to HL. In comparison with chlorina f2 grown under LL, HL-grown chlorina f2 was characterized by unaffected maximal photochemical efficiency of photosystem II (F(V)/F(M), doubled content of both beta-carotene and the xanthophyll cycle pigments and considerably reduced efficiency of excitation energy transfer from carotenoids to chlorophyll a. The enormous xanthophyll cycle pool size was however associated with reduced SV(0) capacity. We suggest that the substantial part of the xanthophyll cycle pigments is not bound to the remaining pigment-protein complexes and acts as filter for excitation energy, thereby contributing to the efficient photoprotection of chlorina f2 grown under HL.     

Comparative study of the water oxidizing reactions and the millisecond delayed chlorophyll fluorescence in photosystem II at different pH

Water splitting activity, the multiline EPR signal associated with S(2)-state of the CaMn(4)-cluster and the fast and slow phases of the induction curve of the millisecond delayed chlorophyll fluorescence from photosystem II (PSII) in the pH range of 4.5-8.5 were studied in the thylakoid membranes and purified PSII particles. It has been found that O(2) evolution and the multiline EPR signal were inhibited at acidic (pK approximately 5.3) and alkaline (pK approximately 8.1) pH values, and were maximal at pH 6.0-7.0. Our results indicate that the loss of O(2) evolution and the S(2)-state multiline EPR signal associated with the decrease of the millisecond delayed chlorophyll fluorescence only in alkaline region (pH 7.0-8.5). Possible correlations of the millisecond delayed chlorophyll fluorescence components with the donor side reactions in PSII are discussed.     

Redirecting electron transfer in photosystem II from water to redox-active metal complexes

A negatively charged region on the surface of photosystem II (PSII) near Q(A) has been identified as a docking site for cationic exogenous electron acceptors. Oxygen evolution activity, which is inhibited in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), is recovered by adding Co(III) complexes. Thus, a new electron-transfer pathway is created with Co(III) as the new terminal electron acceptor from Q(A)(-). This binding site is saturated at ～2.5 mM [Co(III)], which is consistent with the existence of low-affinity interactions with a solvent-exposed surface. This is the first example of a higher plant PSII in which the electron-transfer pathway has been redirected from the normal membrane-associated quinone electron acceptors to water-soluble electron acceptors. The proposed Co(III) binding site may enable efficient collection of electrons generated from photochemical water oxidation by PSII immobilized on an electrode surface.     

Rational design, synthesis, and spectroscopic and photophysical properties of a visible-light-excitable, ratiometric, fluorescent near-neutral pH indicator based on BODIPY

A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Φ(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4×10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Φ(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-deprotonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of the added buffer and salt concentration. In aqueous solution in the absence of buffer and in the pH range 5.20-7.45, dual exponential fluorescence decays are obtained with decay time τ(1)=4.3 ns for the cationic and τ(2)=3.3 ns for the neutral form of II. The excited-state proton exchange of II at near-neutral pH becomes reversible on addition of phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, and a pH-dependent change of the fluorescence decay times is induced. Global compartmental analysis of fluorescence decay traces collected as a function of pH and phosphate buffer concentration was used to recover values of the deactivation rate constants of the excited cationic (k(01)=2.4×10(8) s(-1)) and neutral (k(02)=3.0×10(8) s(-1)) forms of II.     

Photosystem II fluorescence lifetime imaging in avocado leaves: contributions of the lutein-epoxide and violaxanthin cycles to fluorescence quenching

Lifetime-resolved imaging measurements of chlorophyll a fluorescence were made on leaves of avocado plants to study whether rapidly reversible ΔpH-dependent (transthylakoid H(+) concentration gradient) thermal energy dissipation (qE) and slowly reversible ΔpH-independent fluorescence quenching (qI) are modulated by lutein-epoxide and violaxanthin cycles operating in parallel. Under normal conditions (without inhibitors), analysis of the chlorophyll a fluorescence lifetime data revealed two major lifetime pools (1.5 and 0.5 ns) for photosystem II during the ΔpH build-up under illumination. Formation of the 0.5-ns pool upon illumination was correlated with dark-retention of antheraxanthin and photo-converted lutein in leaves. Interconversion between the 1.5- and 0.5-ns lifetime pools took place during the slow part of the chlorophyll a fluorescence transient: first from 1.5 ns to 0.5 ns in the P-to-S phase, then back from 0.5 ns to 1.5 ns in the S-to-M phase. When linear electron transport and the resulting ΔpH build-up were inhibited by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the major fluorescence intensity was due to a 2.2-ns lifetime pool with a minor faster contribution of approximately 0.7 ns. In the presence of DCMU, neither the intensity nor the lifetimes of fluorescence were affected by antheraxanthin and photo-converted lutein. Thus, we conclude that both antheraxanthin and photo-converted lutein are able to enhance ΔpH-dependent qE processes that are associated with the 0.5-ns lifetime pool. However, unlike zeaxanthin, retention of antheraxanthin and photo-converted lutein may not by itself stabilize quenching or cause qI.     

Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude-modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII-photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 micromol m(-2) s(-1)) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 micromol m(-2) s(-1)). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.     

Excited-State Energy Level Does Not Determine the Differential Effect of Violaxanthin and Zeaxanthin on Chlorophyll Fluorescence Quenching in the Isolated Light-Harvesting Complex of Photosystem II

Abstract— The mechanism of action of xanthophyll cycle carotenoids in controlling the quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II (LHCIIb) has been investigated. Auroxanthin, a diepoxy carotenoid with 7 conjugated carbon double bonds, violaxanthin (9 conjugated double bonds) and zeaxanthin (11 conjugated double bonds) have been compared with regard to their effects in vitro on fluorescence quenching and LHCIIb oligomerization. It was found that auroxanthin stimulated fluorescence quenching, similar to the effect of zeaxanthin and in contrast to the inhibition caused by violaxanthin. Auroxanthin caused an increase in the oligomerization of LHCIIb and an increase in relative emission of long-wavelength fluorescence at 77 K. It is concluded that auroxanthin can mimic the effect of zeaxanthin on LHCII, strongly suggesting that the xanthophyll cycle carotenoids control quenching in vitro by an indirect structural effect and not by direct quenching of chlorophyll excited states.     

INFLUENCE OF THE POOL SIZE OF THE XANTHOPHYLL CYCLE ON THE EFFECTS OF LIGHT STRESS IN A DIATOM: COMPETITION BETWEEN PHOTOPROTECI'ION AND PHOTOINHIBITION

Abstract In a study of the relationship between nonphotochemical quenching of fluorescence and the xanthophyll cycle, we show that the diatom Phaeodactylum tricornutum exhibits several interesting characteristics. This xanthophyll cycle consists of only one reversible epoxidating/deepoxidating step (diadinoxanthiddiatoxanthin). Diadinoxan-thin, which increases from 8 to 17 molecules/100 chlorophyll a (Chl a ) during the ageing of the culture, was present as two separate pools, with a portion (of about 5 molecules/100 Chl a) which was never deepoxidated. Under a defined irradiance, the time necessary to abolish net photosynthesis increases with the pool size of diadinoxanthin available for deepoxidation. A close correlation is found between nonphotochemical quenching and the relative ratio of diatoxanthin until the photosytem II center is inactivated. The photoprotective effect of diadinoxanthin deepoxidation is limited to the phase during which quenching of the minimum fluorescence (F₀) develops.     

Effects of water deficit on photosystem II photochemistry and photoprotection during acclimation of lyreleaf sage (Salvia lyrata L.) plants to high light

Acclimation of photosynthetic light reactions to high light requires adjustments in photosystem II (PSII) photochemistry and may be affected by environmental stresses, such as water deficit. In this study, we examined the effects of this stress on PSII photochemistry and photoprotection, with an emphasis on the role of carotenoids and tocopherols, during acclimation of lyreleaf sage (Salvia lyrata L.) plants to high light. Violaxanthin was rapidly converted to zeaxanthin under high light, the de-epoxidation state of the xanthophyll cycle reaching maximum levels of 0.97 after 10 days of high light exposure. Under a higher photoprotective demand caused by water deficit, plants showed significant decreases in beta-carotene and enhanced oxidation of alpha-tocopherol to alpha-tocopherol quinone, which was followed by decreases in the F(v)/F(m) ratio. The levels of beta-carotene decreased more in water-stressed than irrigated plants during acclimation to high light, being particularly degraded (up to 73%) after 14 days of water deficit. Tocopherol levels increased significantly during acclimation to high light, particularly under water deficit, which caused 6.6- and 10-fold increases in alpha-tocopherol and alpha-tocopherol quinone, respectively. We conclude that when xanthophyll cycle-dependent excess energy dissipation could not afford further protection during high light acclimation and the photoprotective demand increased in lyreleaf sage plants by water deficit, enhanced oxidation of alpha-tocopherol and beta-carotene occurred. As stress persisted, enhanced formation of reactive oxygen species might ultimately damage the PSII, as indicated by the reductions in the F(v)/F(m) ratio.     

Interaction of albumin with polysaccharides containing ionic groups

It is very important to clarify the mechanisms of the interaction between components of organisms. In this report, the interaction between bovine serum albumin (BSA) and ionic polysaccharides were discussed. The fluorescence spectrum of tryptophan (Trp) in BSA changed as its conformation changed. On adding polysaccharide containing sulfonic acid residues (sulfonic‐type) at pH 7.4, a remarkable blue shift of the emission maximum (λ_em) of Trp was observed. This blue shift was decreased by adding NaCl. In contrast, polysaccharide containing carboxylic acid residues (carboxy‐type) scarcely changed Trp fluorescence at the same pH. At a pH lower than the isoelectric point (PI = 4.7–4.9) of BSA, a remarkable blue shift was observed not only by adding sulfonic‐type polysaccharide but also by adding carboxy‐type polysaccharide. Moreover, using the energy transfer method, in the pH region higher than the PI of BSA, carboxylic‐type polysaccharides may interact relatively weakly with the binding site II of BSA, but sulfonic‐type ones can selectively interact with binding site I weakly and with binding site II strongly. And in the pH region lower than the PI of BSA, carboxylic‐type polysaccharides interact with binding site II strongly. On the other hand, sulfonic‐type polysaccharides are bound to both binding site I and binding site II very strongly. Copyright © 1999 John Wiley & Sons, Ltd.     

Acetate binding at the photosystem II oxygen evolving complex: an S(2)-state multiline signal ESEEM study

Previously, using acetate deuterated in the methyl hydrogen positions, we showed that acetate binds in close proximity to the Mn cluster/Y(.)(z) tyrosine dual spin complex in acetate-inhibited photosystem II (PSII) preparations exhibiting the "split" EPR signal arising from the S(2)-Y(.)(z) interaction [Force, D. A.; Randall, D. W.; Britt, R. D. Biochemistry 1997, 36, 12062-12070]. By using paramagnetic NO to quench the paramagnetism of Y(.)(z), we are able to observe the ESEEM spectrum of deuterated acetate interacting with only the Mn cluster. A good fit of the ESEEM data indicates two (2)H dipolar hyperfine couplings of 0.097 MHz and one of 0.190 MHz. Modeling of these dipolar interactions, using our "dangler" 3 + 1 model for the S(2)-state of the Mn cluster, reveals distances consistent with direct ligation of acetate to the Mn cluster. As acetate inhibition is competitive with the essential cofactor Cl(-), this suggests that Cl(-) ligates directly to the Mn cluster. The effect of acetate binding on the structure of the Mn cluster is investigated by comparing the Mn-histidine coupling in NO/acetate-treated PSII and untreated PSII using ESEEM. We find that the addition of acetate and NO does not affect the histidine ligation to the Mn cluster. We also investigate the ability of acetate to access Y(.)(z) in Mn-depleted PSII, a PSII preparation expected to be more solvent accessible than intact PSII. We detect no coupling between Y(.)(z) and acetate. We have previously shown that small alcohols such as methanol can ligate to the Mn cluster with ease, while larger alcohols such as 2-propanol, as well as DMSO, are excluded [Force, D. A.; Randall, D. W.; Lorigan, G. A.; Clemens, K. L.; Britt, R. D. J. Am. Chem. Soc. 1998, 120, 13321-13333]. We probe the effect of acetate binding on the ability of methanol and DMSO to bind to the Mn cluster. We find that methanol is able to bind to the Mn cluster in the presence of acetate. We detect no DMSO binding in the presence of acetate. Thus, acetate binding does not increase the affinity or accessibility for DMSO binding at the Mn cluster. We also explore the possibility that the acetate binding site is also a binding site for substrate water. By comparing the ratioed three-pulse ESEEM spectra of a control, untreated PSII sample in 50% D(2)O to an NO/acetate-treated PSII sample in 50% D(2)O, we find that the binding of acetate to the oxygen evolving complex of photosystem II displaces deuterons bound very closely to the Mn cluster.     

Site-selective binding and dual mode recognition of serum albumin by a squaraine dye

With the objective of developing small molecule based probes for proteins, interactions of polyhydroxyl-substituted squaraine dye (SQ) with bovine serum albumin (BSA) have been investigated by absorption, steady-state and time-resolved fluorescence, circular dichroism (CD), cyclic voltammetry (CV), 1H NMR, scanning electron, and tapping mode atomic force microscopic techniques. Increase in addition of BSA resulted in increase in absorbance and fluorescence quantum yields (80-fold) of SQ, along with significant bathochromic shifts in the absorption and fluorescence maxima. Half-reciprocal analysis of the absorption data gave a 1:1 stoichiometry for the complex between BSA and SQ with high association (Kass) constant of (1.4 +/- 0.1) x 106 M-1 and change in free energy of -35 kJ/mol. The complex formation was further confirmed by observation of induced CD signal corresponding to the SQ chromophore at 610 nm, upfield shift (about Deltadelta 0.1 ppm) of aromatic protons of SQ in 1H NMR spectra, and decrease in current intensity (CV) of SQ when bound to BSA. The picosecond time-resolved fluorescence studies indicated that the BSA-SQ complex exhibits biexponential decay with significantly enhanced lifetimes of 0.5 and 1.5 ns when compared to the lifetime of SQ (tau = 121 ps) in the absence of BSA. Employing displacement cum fluorimetry using site-specific binding ligands, such as dansylproline and dansylamide, indicated that SQ binds with protein selectively at site II involving hydrophobic, hydrogen bonding, and electrostatic interactions. The uniqueness of this molecular system is that it interacts with BSA selectively at site II and signals the binding event through dual mode recognition of "visual color" change and "turn on" fluorescence mechanism.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

pH‐ and Thiol‐Responsive BODIPY‐Based Photosensitizers for Targeted Photodynamic Therapy

A diiodo distyryl boron dipyrromethene (BODIPY) core was conjugated to two ferrocenyl quenchers through acid‐labile ketal and/or thiol‐cleavable disulfide linkers, of which the fluorescence and photosensitizing properties were significantly quenched through a photoinduced electron‐transfer process. The two symmetrical analogues that contained either the ketal or disulfide linkers could only be activated by a single stimulus, whereas the unsymmetrical analogue was responsive to dual stimuli. Upon interaction with acid and/or dithiothreitol (DTT), these linkers were cleaved selectively. The separation of the BODIPY core and the ferrocenyl moieties restored the photoactivities of the former in phosphate buffered saline and inside the MCF‐7 breast cancer cells, rendering these compounds as potential activable photosensitizers for targeted photodynamic therapy. The dual activable analogue exhibited the greatest enhancement in intracellular fluorescence intensity in both an acidic environment (pH 5) and the presence of DTT (4 mm ). Its photocytotoxicity against MCF‐7 cells also increased by about twofold upon preincubation with 4 mm of DTT. The activation of this compound was also demonstrated in nude mice bearing a HT29 human colorectal carcinoma. A significant increase in fluorescence intensity in the tumor was observed over 9 h after intratumoral injection.     

The primary photophysics of the Avena sativa phototropin 1 LOV2 domain observed with time-resolved emission spectroscopy

The phototropins are blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine in light, oxygen or voltage (LOV) domains. The primary reactions of the Avena sativa phototropin 1 LOV2 domain were investigated by means of time-resolved and low-temperature fluorescence spectroscopy. Synchroscan streak camera experiments revealed a fluorescence lifetime of 2.2 ns in LOV2. A weak long-lived component with emission intensity from 600 to 650 nm was assigned to phosphorescence from the reactive FMN triplet state. This observation allowed determination of the LOV2 triplet state energy level at physiological temperature at 16600 cm(-1). FMN dissolved in aqueous solution showed pH-dependent fluorescence lifetimes of 2.7 ns at pH 2 and 3.9-4.1 ns at pH 3-8. Here, too, a weak phosphorescence band was observed. The fluorescence quantum yield of LOV2 increased from 0.13 to 0.41 upon cooling the sample from 293 to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K in the steady-state emission.     

流动注射在线氧化荧光法结合透析采样研究盐酸硫利达嗪与牛血清白蛋白的结合作用

利用流动注射在线氧化结合在线透析采样技术建立了荧光法测定血浆中游离盐酸硫利达嗪的新方法, 并将其应用于盐酸硫利达嗪与牛血清白蛋白作用机制的研究. 将盐酸硫利达嗪与牛血清白蛋白以不同比例混合并在37 ℃下培养, 以流动注射透析采样技术从混合液中分离出游离盐酸硫利达嗪, 在线氧化为强荧光化合物后测定其浓度; 应用Scrathard方程分析所测数据, 求得盐酸硫利达嗪和牛血清白蛋白的结合常数为1.2×104 L/mol, 结合位点数为0.98; 根据热力学参数推断, 盐酸硫利达嗪与牛血清白蛋白之间以疏水作用力为主; 进一步基于荧光猝灭现象和Fster非辐射能量转移理论, 得到盐酸硫利达嗪小分子与牛血清白蛋白之间的能量转移效率(E=0.497)和结合距离(r0=3.27 nm).     

Phosphatidylcholine-induced reactivation of photosystem II membranes pretreated with Triton X-100

Triton X-100-induced inactivation and phosphatidylcholine-induced reactivation of photosystem II (PSII) membranes were investigated using oxygen electrode, variable fluorescence and spectroscopic techniques including absorption and circular dichroism spectroscopy. Incubation of the PSII membrane with Triton X-100 reduced the oxygen-evolving rate, modified the variable chlorophyll fluorescence kinetics, changed the protein secondary structures, altered the chlorophyll binding state to proteins and decreased the excitonic interaction of chlorophyll molecules. Phosphatidylcholine addition did not change the protein secondary structures, but could partially reactivate the reduced oxygen-evolving rate, and partly reversed the variable fluorescence parameters, the chlorophyll binding state and the excitonic interaction of the chlorophyll molecules. The results indicate that the phosphatidylcholine environment can optimize the tertiary structures of PSII.