Unique photosynthetic electron transport tuning and excitation distribution in heterokont algae.

Heterokont algae are significant contributors to marine primary productivity. These algae have a photosynthetic machinery that shares many common features with that of Viridiplantae (green algae and land plants). Here we demonstrate, however, that the photosynthetic machinery of heterokont algae responds to light fundamentally differently than that of Viridiplantae. While exposure to high light leads to electron accumulation within the photosynthetic electron transport chain in Viridiplantae, this is not the case in heterokont algae. We use this insight to manipulate the photosynthetic electron transport chain and demonstrate that heterokont algae can dynamically distribute excitation energy between the two types of photosystems. We suggest that the reported electron transport and excitation distribution features are adaptations to the marine light environment.

The plastoquinone pool of Nannochloropsis oceanica is not completely reduced during bright light pulses.

The lipid-producing model alga Nannochloropsis oceanica has a distinct photosynthetic machinery. This organism possesses chlorophyll a as its only chlorophyll species, and has a high ratio of PSI to PSII. This high ratio of PSI to PSII may affect the redox state of the plastoquinone pool during exposure to light, and consequently may play a role in activating photoprotection mechanisms. We utilized pulse-amplitude modulated fluorometry to investigate the redox state of the plastoquinone pool during and after bright light pulses. Our data indicate that even very intense (5910 µmol photons s-1m-2 of blue light having a wavelength of 440 nm) light pulses of 0.8 second duration are not sufficient to completely reduce the plastoquinone pool in Nannochloropsis. In order to achieve extensive reduction of the plastoquinone pool by bright light pulses, anaerobic conditions or an inhibitor of the photosynthetic electron transport chain has to be utilized. The implication of this finding for the application of the widely used saturating pulse method in algae is discussed.

Fluorescence and phosphorescence of tryptophan in peptides of different length and sequence.

To interpret accurately protein fluorescence and phosphorescence, it is essential to achieve a better understanding of the luminescence properties of tryptophan (Trp, or W) in peptides. In published literature data on luminescence of peptides of varied length are scarce. This article describes studies of fluorescence and phosphorescence properties of the eight Trp-containing synthetic peptides: WAK, AWK, SWA, KYLWE, AVSWK, WVSWAK, WAKLAWE, and AVSWAKLARE. The aim was to investigate which factors influence the fluorescence yield and phosphorescence-spectra and lifetimes. Absorption spectra, room temperature fluorescence emission and corresponding excitation spectra and time-resolved phosphorescence spectra (77K) have been recorded; the dependence of the fluorescence quantum yield on the specific peptide and its variation with the wavelength of excitation has been studied. The changes in fluorescence yield and shape of phosphorescence spectra are explained in terms of internal electron and proton transfer. The structured phosphorescence spectrum originates from proton transfer occurring upon excitation of Trp, while electron transfer gives rise to a non-structured luminescence spectrum. There is also electron transfer from higher vibronic S1 states. In the peptides there is higher probability of electron transfer than in Trp alone. The obtained data are interpreted in light of the peptides' sequence, length and conformation.

Ruthenium porphyrin-induced photodamage in bladder cancer cells.

Photodynamic therapy (PDT) is a noninvasive treatment for solid malignant and flat tumors. Light activated sensitizers catalyze photochemical reactions that produce reactive oxygen species which can cause cancer cell death. In this work we investigated the photophysical properties of the photosensitizer ruthenium(II) porphyrin (RuP), along with its PDT efficiency onto rat bladder cancer cells (AY27). Optical spectroscopy verified that RuP is capable to activate singlet oxygen via blue and red absorption bands and inter system crossing (ISC) to the triplet state. In vitro experiments on AY27 indicated increased photo-toxicity of RuP (20µM, 18h incubation) after cell illumination (at 435nm), as a function of blue light exposure. Cell survival fraction was significantly reduced to 14% after illumination of 20µM RuP with 15.6J/cm(2), whereas the "dark toxicity" of 20µM RuP was 17%. Structural and morphological changes of cells were observed, due to RuP accumulation, as well as light-dependent cell death was recorded by confocal microscopy. Flow cytometry verified that PDT-RuP (50µM) triggered significant photo-induced cellular destruction with a photoxicity of (93%±0.9%). Interestingly, the present investigation of RuP-PDT showed that the dominating mode of cell death is necrosis. RuP "dark toxicity" compared to the conventional chemotherapeutic drug cisplatin was higher, both evaluated by the MTT assay (24h). In conclusion, the present investigation shows that RuP with or without photoactivation induces cell death of bladder cancer cells.

Dissolution of copper mineral phases in biological fluids and the controlled release of copper ions from mineralized alginate hydrogels.

Here we investigate the dissolution behaviour of copper minerals contained within biocompatible alginate hydrogels. Copper has a number of biological effects and has most recently been evaluated as an alternative to expensive and controversial growth factors for applications in tissue engineering. Precise control and sustained release of copper ions are important due to a narrow therapeutic window of this potentially toxic ion, and alginate would appear to be a good material of choice for this purpose. We found that aqueously insoluble copper minerals could be precipitated during gelling within or mixed into alginate hydrogels in the form of microbeads prior to gelling to serve as depots of copper. These minerals were found to be soluble in a variety of biological fluids relevant to in vitro and in vivo investigations, and the alginate carrier served as a barrier to diffusion of these ions and therefore offered control over the rate and duration of release (Cu(2+) release rates observed between 10-750 µMol g(-1) h(-1) and duration for up to 32 d). Copper mineral and copper mineralized alginate microbeads were characterized using powder x-ray diffraction, FTIR, thermogravimetric analysis and scanning electron microscopy. Dissolution kinetics were studied based on measurements of copper ion concentrations using colourimetric methods. In addition we characterized the complexes formed between released copper ions and biological fluids by electron paramagnetic spectroscopy which offers an insight into the behaviour of these materials in the body.

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution.

Under strong light conditions, long-lived chlorophyll triplets ((3)Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench (3)Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet-triplet energy transfer and quenching of (3)Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching (3)Chl.

Pulsed-source time-resolved phosphorimetry: comparison of a commercial gated photomultiplier with a specially wired ungated photomultiplier.

A common problem encountered in recording delayed light emission is that the signal of interest is preceded by a much more intense signal arising from prompt fluorescence. When a photomultiplier tube (PMT) is used as the photosensor in a pulsed-source phosphorimeter, two options are open to an experimenter who finds mechanical shutters inconvenient or impracticable and photon counting inappropriate: apply an electronic gate that suppresses the PMT gain for a brief period, or use a wiring scheme that enables the PMT to quickly regain normal operation after an intense burst of prompt emission. The performance of a squirrel-cage PMT that operates in the latter mode is compared with a new gateable PMT (Hamamatsu H11526 series) with a minimum gate time of 100 ns. The two detectors are found to provide practically the same temporal record of the delayed emission, but the ungated PMT is slightly superior in terms of recovery time and signal-to-noise ratio.

Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants.

Plants are commonly subjected to several environmental stresses that lead to an overproduction of reactive oxygen species (ROS). As plants accumulate proline in response to stress conditions, some authors have proposed that proline could act as a non-enzymatic antioxidant against ROS. One type of ROS aimed to be quenched by proline is singlet oxygen ((1)O(2))-molecular oxygen in its lowest energy electronically excited state-constitutively generated in oxygenic, photosynthetic organisms. In this study we clearly prove that proline cannot quench (1)O(2) in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against (1)O(2).

Temporal profile of the singlet oxygen emission endogenously produced by photosystem II reaction centre in an aqueous buffer.

The temporal profile of the phosphorescence of singlet oxygen endogenously photosensitized by photosystem II (PSII) reaction centre (RC) in an aqueous buffer has been recorded using laser excitation and a near infrared photomultiplier tube. A weak emission signal was discernible, and could be fitted to the functional form a[exp(-t/τ(2) - exp(-t/τ(1)], with a > 0 and τ(2) > τ(1). The value of τ(2) decreased from 11.6 ± 0.5 µs under aerobic conditions to 4.1 ± 0.2 µs in oxygen-saturated samples, due to enhanced bimolecular quenching of the donor triplet by oxygen, whereas that of τ(1), identifiable with the lifetime of singlet oxygen, was close to 3 µs in both cases. Extrapolations based on the low amplitude of the emission signal of singlet oxygen formed by PSII RC in the aqueous buffer and the expected values of τ(1) and τ(2) in chloroplasts indicate that attempts to analyse the temporal profile of singlet oxygen in chloroplasts are unlikely to be rewarded with success without a significant advance in the sensitivity of the detection equipment.

Trolox, a water-soluble analogue of α-tocopherol, photoprotects the surface-exposed regions of the photosystem II reaction center in vitro. Is this physiologically relevant?

Can Trolox, a water-soluble analogue of α-tocopherol and a scavenger of singlet oxygen ((1)O(2)), provide photoprotection, under high irradiance, to the isolated photosystem II (PSII) reaction center (RC)? To answer the question, we studied the endogenous production of (1)O(2) in preparations of the five-chlorophyll PSII RC (RC5) containing only one ß-carotene molecule. The temporal profile of (1)O(2) emission at 1270 nm photogenerated by RC5 in D(2)O followed the expected biexponential behavior, with a rise time, unaffected by Trolox, of 13 ± 1 µs and decay times of 54 ± 2 µs (without Trolox) and 38 ± 2 µs (in the presence of 25 µM Trolox). The ratio between the total (k(t)) and chemical (k(r)) bimolecular rate constants for the scavenging of (1)O(2) by Trolox in aqueous buffer was calculated to be ~1.3, with a k(t) of (2.4 ± 0.2) × 10(8) M(-1) s(-1) and a k(r) of (1.8 ± 0.2) × 10(8) M(-1) s(-1), indicating that most of the (1)O(2) photosensitized by methylene blue chemically reacts with Trolox in the assay buffer. The photoinduced oxygen consumption in the oxygen electrode, when RC5 and Trolox were mixed, revealed that Trolox was a better (1)O(2) scavenger than histidine and furfuryl alcohol at low concentrations (i.e., <1 mM). After its incorporation into detergent micelles in unbuffered solutions, Trolox was able to photoprotect the surface-exposed regions of the D1-D2 heterodimer, but not the RC5 pigments, which were oxidized, together with the membrane region of the protein matrix of the PSII RC, by (1)O(2). These results are discussed and compared with those of studies dealing with the physiological role of tocopherol molecules as a (1)O(2) scavenger in thylakoid membranes of photosynthetic organisms.

Photo induced hexylaminolevulinate destruction of rat bladder cells AY-27.

Photodynamic therapy (PDT) is of increasing interest as a relevant treatment for human urinary bladder cancer. In the present experiments, the rat bladder transitional carcinoma cell line AY-27 was used as a model to study cell destruction mechanisms induced by PDT. Red LED light (630 nm) PDT with hexylaminolevulinate (HAL) as precursor for the photosensitizer protoporphyrin IX (PpIX) was used in treatment of the cells. Flow cytometry with fluorescent markers annexin V, propidium iodide and YO-PRO-1, as well as MTT assay and confocal microscopy, were used to map cell inactivation after PDT. Dark toxicity of HAL alone was low in these procedures and LD(50) (24 h, MTT assay) was approximately 1.6 J cm(-2) for standard red light (LED) irradiation (36 mW cm(-2)). Measurements done 1 h after HAL-PDT showed a maximum apoptotic level of about 10% at 6 J cm(-2), however the dominating mode of cell death was necrosis. Forward light scattering indicated an increase in cell size at low doses, possibly due to necrosis. Survival curves had a dual-slope shape, a fit to single hit, multi-target approximation gave a parameter estimate of n = 10 and D(0) about 2.6 J cm(-2). Replacing continuous light with fractionated light delivery (45 s light/60 s darkness) did not affect the treatment outcome.

Facile method for spectroscopic examination of radical ions of hydrophilic carotenoids.

Hydrophilic carotenoids, unusual members of an intrinsically hydrophobic family, and their radical ions are important reactants. An all-optical method for generating singly charged radical ions of a hydrophilic carotenoid (Car) is described. It relies on photolyzing an aqueous mixture of Car and a photoionizable auxiliary solute (A), and making conditions conducive to the capture, by Car, of the hydrated electron (e(aq)(-)) or the positive hole in A(*)(+) or both. When A is Trolox (TOH), only e(aq)(-) can be captured, since TOH (*)(+) deprotonates too rapidly to be a hole donor; when A is Trolox methyl ether (TOMe), both Car(*)(-) and Car(*)(+) are formed, since TOMe (+) lives long enough to transfer its positive hole to Car; formation of Car(*)(-) is prevented under aerobic conditions.

Triplet excited states of some thiophene and triazole substituted platinum(II) acetylide chromophores.

The photophysical properties of a series of platinum(II) acetylide compounds (trans-Pt(PBu(3))(2)(C[triple bond]C-R)(2)) with the R group consisting of two or three aryl rings (phenyl, phenyl/thiophenyl, phenyl/triazolyl) linked together with ethynyl groups were systematically investigated. Four new structurally similar compounds are reported with: (i) a bithiophene unit in the ligands, (ii) methyl or (iii) methoxy substituents on the aryl ring ligands that promote a more twisted conformation along the long axis of the molecule, and (iv) with two different alkynylaryl ligands giving rise to an asymmetric substitution with respect to the photoactive metal ion center. The spectroscopic studies include optical absorption, spectrally and time-resolved luminescence, as well as transient absorption spectra. The ground-state UV absorption between 300 and 420 nm gave rise to fluorescence with quantum efficiencies in the range of 0.1-1% and efficient intersystem crossing to triplet states. Phosphorescence decay times were in the order of 10-500 micros in oxygen-evacuated samples. The triplet states also lead to strong broadband triplet-triplet absorption between 400 and 800 nm. The complex with asymmetric substitution was found to populate two triplet states of different structure and energy.

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II.

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II(slow) and Signal II(fast) of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y(Z) and Y(D) of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043+/-0.0003 and a line width of 10+/-1G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q(A) in the absence of magnetic interaction with the non-heme Fe2+. We conclude that peroxynitrite impairs PSII electron transport in the Q(A)Fe2+ niche.

Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection.

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.

Reaction center of photosystem II with no peripheral pigments in D2 allows secondary electron transfer in D1.

A pigment-deficient reaction center of photosystem II (PSII)-with all the core pigments (two molecules of chlorophyll a and one of pheophytin a in each D protein) but with only one molecule each of peripheral chlorophyll a (Chlz) and beta-carotene (Car)-has been investigated by pump-probe spectroscopy. The data imply that Car and Chlz are both bound to D1. The absence of Car and Chlz in D2 allows the unprecedented observation of secondary electron transfer in D1 of PSII reaction centers at room temperature. The absorption band of the Car cation in D1 (Car(D1)(+*)) peaks around 910 nm (as against 990 nm for Car(D2)(+*)), and its positive hole is shared by ChlzD1, whereas Car(D2)(+*) can disappear by capturing an electron from ChlzD2.

Hydrophilic carotenoids: surface properties and aggregation of an astaxanthin-lysine conjugate, a rigid, long-chain, highly unsaturated and highly water-soluble tetracationic bolaamphiphile.

The surface and aggregation properties of a synthetic, highly water-soluble carotenoid, the tetracationic astaxanthin-lysine conjugate (Asly), have been examined through measurements of surface tension, optical absorption and dynamic light scattering. The following parameters were determined: critical aggregation concentration c(M), surface concentration Gamma, molecular area a(m), free energy of adsorption and aggregation (DeltaG(ad) degrees and DeltaG(M) degrees , respectively), and the aggregate size r(H). The compound forms true monomolecular solutions in water below c(M); aggregates emerge only at rather high concentrations (> or =2.18 mM).

Formation and geminate quenching of singlet oxygen in purple bacterial reaction center.

The phosphorescence of singlet oxygen ((1)X( *)) photosensitized by the carotenoidless reaction center (RC) of Rhodobacter sphaeroides R26.1 has been investigated, using H(2)O and D(2)O as the suspending media. To enhance (under neutral conditions) the triplet quantum yield of the special pair P(870) (P) by the radical pair mechanism, the quinone acceptor Q(A) was removed by means of a chemical treatment. The phosphorescence signal fits the functional form P(0)[exp (-t/tau)-exp(-t/zeta)], regardless of whether (1)X( *) is sensitized by P(dagger) or M(dagger) (where the dagger denotes triplet excitation and M is a water-soluble molecule). The time constant zeta was identified with the decay time of (1)X( *); when P(dagger) is the sensitizer, one finds zeta(P)((1))=3.3+/-0.3 micros, and zeta(P)((2))=34+/-3 micros, where the superscripts 1 and 2 refer to H(2)O and D(2)O, respectively; the corresponding values for sensitization by M(dagger) (in the absence of RC) are zeta(M)((1))=3.7+/-0.4 micros, and zeta(M)((2))=75+/-5 micros. The addition of RC's to the solution of M in D(2)O reveals that the RC is a quencher of (1)X( *); however, for equal concentrations of the RC, zeta(P)((2))

Electron- and energy-transfer properties of hydrophilic carotenoids.

The antioxidant activities-expressed as the electron-donating properties-of five hydrophilic carotenoids (carotenoid surfactants) and three related hydrophobic carotenoids were investigated by flash photolysis. The electron-transfer rates of the carotenoids to the triplet state of the sensitizer 2-nitronaphthalene and the energy transfer rates of triplet 2-nitronaphthalene to the carotenoids were determined. The results demonstrate that the electron-donating effects of the hydrophilic and hydrophobic carotenoids were comparable when evaluated in acetonitrile. In the presence of water, however, electron transfer (i.e., antioxidant efficiency) was enhanced by a factor of four for the hydrophilic carotenoids. The increased hydrophilicity of carotenoids, therefore, could expand their antioxidant properties, thus facilitating their use as aqueous-phase radical scavengers. At the same time, it was shown that supramolecular assembly ("aggregation") of the amphiphilic carotenoids prevented electron transfer, thus deactivating the antioxidant function. Modulation of the biophysical properties of carotenoids through synthetic modification is capable of increasing the biological and medical utility of this natural class of predominantly hydrophobic antioxidant compound.