Switching sides-Reengineered primary charge separation in the bacterial photosynthetic reaction center.

We report 90% yield of electron transfer (ET) from the singlet excited state P* of the primary electron-donor P (a bacteriochlorophyll dimer) to the B-side bacteriopheophytin (HB) in the bacterial photosynthetic reaction center (RC). Starting from a platform Rhodobacter sphaeroides RC bearing several amino acid changes, an Arg in place of the native Leu at L185-positioned over one face of HB and only ∼4 Šfrom the 4 central nitrogens of the HB macrocycle-is the key additional mutation providing 90% yield of P+HB- This all but matches the near-unity yield of A-side P+HA- charge separation in the native RC. The 90% yield of ET to HB derives from (minimally) 3 P* populations with distinct means of P* decay. In an ∼40% population, P* decays in ∼4 ps via a 2-step process involving a short-lived P+BB- intermediate, analogous to initial charge separation on the A side of wild-type RCs. In an ∼50% population, P* → P+HB- conversion takes place in ∼20 ps by a superexchange mechanism mediated by BB An ∼10% population of P* decays in ∼150 ps largely by internal conversion. These results address the long-standing dichotomy of A- versus B-side initial charge separation in native RCs and have implications for the mechanism(s) and timescale of initial ET that are required to achieve a near-quantitative yield of unidirectional charge separation.

Pigment-modified reaction centers of Chloroflexus aurantiacus: chemical exchange of bacteriopheophytins with plant-type pheophytins.

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.

Electrostatic profiling of photosynthetic pigments: implications for directed spectral tuning.

Photosynthetic pigment-protein complexes harvest solar energy with a high quantum efficiency. Protein scaffolds are known to tune the spectral properties of embedded pigments principally through structured electrostatic environments. Although the physical nature of electrostatic tuning is straightforward, the precise spatial principles of electrostatic preorganization remain poorly explored for different protein matrices and incompletely characterized with respect to the intrinsic properties of different photosynthetic pigments. In this work, we study the electronic structure features associated with the lowest excited state of a series of eight naturally occurring (bacterio)chlorophylls and pheophytins to describe the precise topological differences in electrostatic potentials and hence determine intrinsic differences in the expected mode and impact of electrostatic tuning. The difference electrostatic potentials between the ground and first excited states are used as fingerprints. Both the spatial profile and the propensity for spectral tuning are found to be unique for each pigment, indicating spatially and directionally distinct modes of electrostatic tuning. The results define a specific partitioning of the protein matrix around each pigment as an aid to identify regions with a maximal impact on spectral tuning and have direct implications for dimensionality reduction in protein design and engineering. Thus, a quantum mechanical basis is provided for understanding, predicting, and ultimately designing sequence-modified or pigment-exchanged biological systems, as suggested for selected examples of pigment-reconstituted proteins.

Formulation of Ethyl Cellulose Microparticles Incorporated Pheophytin A Isolated from Suaeda vermiculata for Antioxidant and Cytotoxic Activities.

BACKGROUND: This study is designed to discover a method for delivering an efficient potent pheophytin a (pheo-a) into more absorbed and small polymeric ethyl cellulose (EC) microparticles. METHODS: Silica gel and Sephadex LH-20 columns were used to isolate pheo-a from the chloroform extract of the edible plant, Suaeda vermiculata. Pheo-a was incorporated into EC microparticles using emulsion-solvent techniques. The antioxidant activity of pheo-a microparticles was confirmed by the level of superoxide radical (SOD), nitric oxide (NO), and reducing power (RP) methods. Meanwhile, the cytotoxic effect of the product was investigated on MCF-7 cells using MTT assay. RESULTS: Pheo-a was isolated from S. vermiculata in a 12% concentration of the total chloroform extract. The structures were confirmed by NMR and IR spectroscopic analysis. The formulated microparticles were uniform, completely dispersed in the aqueous media, compatible as ingredients, and had a mean diameter of 139 ± 1.56 µm as measured by a particle size analyzer. Pheo-a demonstrated a valuable antioxidant activity when compared with ascorbic acid. The IC50 values of pheo-a microparticles were 200.5 and 137.7 µg/mL for SOD, and NO respectively. The reducing power of pheo-a microparticles was more potent than ascorbic acid and had a 4.2 µg/mL for IC50 value. Pheo-a microparticles did not show notable cytotoxicity on the MCF-7 cell line (IC50 = 35.9 µg/mL) compared with doxorubicin (IC50 = 3.2 µg/mL). CONCLUSIONS: the results showed that water-soluble pheo-a microparticles were prepared with a valuable antioxidant activity in a wide range of concentrations with a noteworthy cytotoxic effect.

Modelling of the cathodic and anodic photocurrents from Rhodobacter sphaeroides reaction centres immobilized on titanium dioxide.

As one of a number of new technologies for the harnessing of solar energy, there is interest in the development of photoelectrochemical cells based on reaction centres (RCs) from photosynthetic organisms such as the bacterium Rhodobacter (Rba.) sphaeroides. The cell architecture explored in this report is similar to that of a dye-sensitized solar cell but with delivery of electrons to a mesoporous layer of TiO2 by natural pigment-protein complexes rather than an artificial dye. Rba. sphaeroides RCs were bound to the deposited TiO2 via an engineered extramembrane peptide tag. Using TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) as an electrolyte, these biohybrid photoactive electrodes produced an output that was the net product of cathodic and anodic photocurrents. To explain the observed photocurrents, a kinetic model is proposed that includes (1) an anodic current attributed to injection of electrons from the triplet state of the RC primary electron donor (PT) to the TiO2 conduction band, (2) a cathodic current attributed to reduction of the photooxidized RC primary electron donor (P+) by surface states of the TiO2 and (3) transient cathodic and anodic current spikes due to oxidation/reduction of TMPD/TMPD+ at the conductive glass (FTO) substrate. This model explains the origin of the photocurrent spikes that appear in this system after turning illumination on or off, the reason for the appearance of net positive or negative stable photocurrents depending on experimental conditions, and the overall efficiency of the constructed cell. The model may be a used as a guide for improvement of the photocurrent efficiency of the presented system as well as, after appropriate adjustments, other biohybrid photoelectrodes.

Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus.

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P(+)QB(-) are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 µs. The resulting ranking of mutants for their yield of P(+)QB(-) from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P(+)HB(-)→P(+)QB(-) electron transfer or initial P*→P(+)HB(-) conversion highlight unmet challenges of optimizing both processes simultaneously.

Factors controlling the reactivity of divalent metal ions towards pheophytin a.

In this study, we evaluate the factors which determine the reactivity of divalent metal ions in the spontaneous formation of metallochlorophylls, using experimental and computational approaches. Kinetic studies were carried out using pheophytin a in reactions with various divalent metal ions combined with non- or weakly-coordinative counter ions in a series of organic solvents. To obtain detailed insights into the solvent effect, the metalations with the whole set of cations were investigated in three solvents and with Zn2+ in seven solvents. The reactions were monitored using electronic absorption spectroscopy and the stopped-flow technique. DFT calculations were employed to shed light on the role of solvent in activating the metal ions towards porphyrinoids. This experimental and computational analysis gives detailed information regarding how the solvent and the counter ion assist/hinder the metalation reaction as activators/inhibitors. The metalation course is dictated to a large extent by the reaction medium, via either the activation or deactivation of the incoming metal ion. The solvent may affect the metalation in several ways, mainly via H-bonding with pyrrolenine nitrogens and the activation/deactivation of the incoming cation. It also seems to affect the activation enthalpy by causing slight conformational changes in the macrocyclic ligand. These new mechanistic insights contribute to a better understanding of the "metal-counterion-solvent" interplay in the metalation of porphyrinoids. In addition, they are highly relevant to the mechanisms of metalation reactions catalyzed by chelatases and explain the differences between the insertion of Mg2+ and other divalent cations.

Spectroscopic properties of photosystem II reaction center revisited.

Photosystem II (PSII) is the only biological system capable of splitting water to molecular oxygen. Its reaction center (RC) is responsible for the primary charge separation that drives the water oxidation reaction. In this work, we revisit the spectroscopic properties of the PSII RC using the complex time-dependent Redfield (ctR) theory for optical lineshapes [A. Gelzinis et al., J. Chem. Phys. 142, 154107 (2015)]. We obtain the PSII RC model parameters (site energies, disorder, and reorganization energies) from the fits of several spectra and then further validate the model by calculating additional independent spectra. We obtain good to excellent agreement between theory and calculations. We find that overall our model is similar to some of the previous asymmetric exciton models of the PSII RC. On the other hand, our model displays differences from previous work based on the modified Redfield theory. We extend the ctR theory to describe the Stark spectrum and use its fit to obtain the parameters of a single charge transfer state included in our model. Our results suggest that ChlD1+PheoD1- is most likely the primary charge transfer state, but that the Stark spectrum of the PSII RC is probably also influenced by other states.

Anti-HIV Activity of Ocimum labiatum Extract and Isolated Pheophytin-a.

Ocimum plants are traditionally used to manage HIV/AIDS in various African countries. The effects of Ocimum labiatum extract on HIV-1 protease (PR) and reverse transcriptase (RT) is presented here along with characterization of an identified bioactive compound, achieved through ¹H- and 13C-NMR. The extract's effect on HIV-1 replication was assessed by HIV-1 p24 antigen capture. Cytotoxicity of samples was evaluated using tetrazolium dyes and real-time cell electronic sensing (RT-CES). Ocimum labiatum inhibited HIV-1 PR with an IC50 value of 49.8 ± 0.4 µg/mL and presented weak inhibition (21%) against HIV-1 RT. The extract also reduced HIV-1 replication in U1 cells at a non-cytotoxic concentration (25 µg/mL). The CC50 value of the extract in U1 cells was 42.0 ± 0.13 µg/mL. The HIV-1 PR inhibiting fraction was purified using prep-HPLC and yielded a chlorophyll derivative, pheophytin-a (phy-a). Phy-a inhibited HIV-1 PR with an IC50 value of 44.4 ± 1.5 µg/mL (51 ± 1.7 µM). The low cytotoxicity of phy-a in TZM-bl cells was detected by RT-CES and the CC50 value in U1 cells was 51.3 ± 1.0 µg/mL (58.9 ± 1.2 µM). This study provides the first in vitro evidence of anti-HIV activity of O. labiatum and isolated phy-a, supporting further investigation of O. labiatum for lead compounds against HIV-1.

Electron Transfer in Bacterial Reaction Centers with the Photoactive Bacteriopheophytin Replaced by a Bacteriochlorophyll through Coordinating Ligand Substitution.

The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-called ß-type mutants (in which the HA cofactor is called ßA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques. Similar to that of the previously characterized M214LH (ß mutant), the strong energetic mixing of the P(+)BA(-) and P(+)ßA(-) states (the mixed anion is denoted I(-)) increased the rate of charge recombination between P(+) and I(-) in competition with the I(-) → QA forward reaction. This reduced the overall yield of charge separation forming the P(+)QA(-) state. While the kinetics of the primary electron transfer forming P(+)I(-) were essentially identical in all three ß mutants, the rates of the ßA(-) (I(-)) → QA electron transfer in M214LQ and M214LH were very similar but quite different from that of the M214LN mutant. The observed yield changes and the differences in kinetics are correlated more closely with the volume of the mutated amino acid than with their charge characteristics. These results are consistent with those of previous studies of a series of M214 mutants with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan, J., et al. (2013) J. Phys. Chem. B 117, 7179-7189]. Both studies indicate that protein relaxation in this region of the reaction center plays a key role in stabilizing charge-separated states involving the HA or ßA cofactor. The effect is particularly pronounced for reactions occurring on time scales of tens and hundreds of picoseconds (forward transfer to the QA and charge recombination).

Bacteriopheophytin triplet state in Rhodobacter sphaeroides reaction centers.

It is well established that photoexcitation of Rhodobacter sphaeroides reaction centers (RC) with reduced quinone acceptors results in the formation of a triplet state localized on the primary electron donor P with a significant yield. The energy of this long-lived and therefore potentially damaging excited state is then efficiently quenched by energy transfer to the RC spheroidenone carotenoid, with its subsequent decay to the ground state by intersystem crossing. In this contribution, we present a detailed transient absorption study of triplet states in a set of mutated RCs characterized by different efficiencies of triplet formation that correlate with lifetimes of the initial charge-separated state P(+)H A (-) . On a microsecond time scale, two types of triplet state were detected: in addition to the well-known spheroidenone triplet state with a lifetime of ~4 µs, in some RCs we discovered a bacteriopheophytin triplet state with a lifetime of ~40 µs. As expected, the yield of the carotenoid triplet increased approximately linearly with the lifetime of P(+)H A (-) , reaching the value of 42 % for one of the mutants. However, surprisingly, the yield of the bacteriopheophytin triplet was the highest in RCs with the shortest P(+)H A (-) lifetime and the smallest yield of carotenoid triplet. For these the estimated yield of bacteriopheophytin triplet was comparable with the yield of the carotenoid triplet, reaching a value of ~7 %. Possible mechanisms of formation of the bacteriopheophytin triplet state are discussed.

Pheophytin a isolated from the seagrass Syringodium isoetifolium plausibly blocks umuC proteins of select bacterial pathogens, in silico.

AIMS: This investigation is designed to evaluate the antibacterial efficiency of the noodle grass Syringodium isoetifolium, which is commonly found in the Indian coastal waters. Also, this study characterizes the active compound and predicts the mode of action in silico. METHODS AND RESULTS: Human pathogenic bacteria were treated with crude metabolites of S. isoetifolium. The potent fraction b was analysed by UV/VIS, Spectroscopy RP-HPLC, FT-IR, ESI-Mass and 1 H and 13 C NMRs and determined to be a hydrate of pheophytin a (C55 H74 N4 O6 ). The isolated compound Pheo had MIC values of 6·2 ± 0·7 (Salmonella typhi) and 12·5 ± 0·8 (Escherichia coli and Pseudomonas aeruginosa) µg ml-1 . Molecular docking studies of the compound were done to find the binding sites on the pathogens using a Molegro Virtual Docker platform. Pheo targets umuC proteins by binding compactly to five amino acid residues with interaction energy of -3·66 and a Moldock score of -160·175. CONCLUSIONS: Hence, we conclude that pheophytin a, besides being an accessory photosynthetic pigment, also has proven to be antibacterial against human pathogens. Lesser MIC values with definite binding sites predicted in silico are suggestive of a precise of action for this compound. SIGNIFICANCE AND IMPACT OF THE STUDY: Easy extraction methods of the active compound that has a definite target render this under-explored seagrass a good source of antibacterial compound against human pathogenic bacteria. This learning may favour more researches in this unexplored area to build up Pheo-based natural products as antibiotic therapies.

Modification of the pheophytin redox potential in Thermosynechococcus elongatus Photosystem II with PsbA3 as D1.

In Photosystem II (PSII) of the cyanobacterium Thermosynechococcus elongatus, glutamate 130 in the high-light variant of the D1-subunit (PsbA3) was changed to glutamine in a strain lacking the two other genes for D1, psbA1 and psbA2. The resulting PSII (PsbA3/Glu130Gln) was compared with those from the "native" high-light (PsbA3-PSII) and low-light (PsbA1-PSII) variants, which differ by 21 amino acid including Glu130Gln. H-bonding from D1-Glu130Gln to the primary electron acceptor, PheophytinD1 (PheoD1), is known to affect the Em of the PheoD1/PheoD1(-) couple. The Gln130 mutation here had little effect on water splitting, charge accumulation and photosensitivity but did slow down S2QA(-) charge recombination and up-shift the thermoluminescence while increasing its yield. These changes were consistent with a ≈-30mV shift of the PheoD1/PheoD1(-)Em, similar to earlier single site-mutation results from other species and double the ≈-17mV shift seen for PsbA1-PSII versus PsbA3-PSII. This is attributed to the influence of the other 20 amino-acids that differ in PsbA3. A computational model for simulating S2QA(-) recombination matched the experimental trend: the S2QA(-) recombination rate in PsbA1-PSII differed only slightly from that in PsbA3-PSII, while in Glu130-PsbA3-PSII there was a more pronounced slowdown of the radical pair decay. The simulation predicted a major effect of the PheoD1/PheoD1(-) potential on (1)O2 yield (~60% in PsbA1-PSII, ~20% in PsbA3-PSII and ~7% in Gln130-PsbA3-PSII), reflecting differential sensitivities to high light.

Extensive fragmentation of pheophytin-a by infrared multiphoton dissociation tandem mass spectrometry.

RATIONALE: In a previous tandem mass spectrometry (MS/MS) study on chlorophyll-a, infrared multiphoton dissociation (IRMPD) was demonstrated as a more effective fragmentation method than collision-induced dissociation (CID) and electron-induced dissociation (EID), where odd-electron product ions were observed ubiquitously in CID and IRMPD. To further understand the role of the macrocycle and the central Mg atom in the MS/MS process, the fragmentation behaviour of pheophytin-a, the Mg-free chlorophyll-a, was investigated. METHODS: CID, IRMPD, and EID were applied to the singly protonated pheophytin-a using an ultra-high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The result is compared with the MS/MS study of chlorophyll-a. RESULTS: For pheophytin-a, some different fragmentation patterns from chlorophyll-a were obtained by all three MS/MS methods, but IRMPD still appears the most efficient method of generating product ions. The detection of odd-electron fragments in the CID and IRMPD spectra of protonated pheophytin-a suggests that the macrocyclic structure effectively stabilizes radicals, and these radical ions seem to have a relatively higher abundance in the presence of the central Mg atom. CONCLUSIONS: The strong absorption in the infrared region of pheophytin-a and secondary free radical rearrangement are proposed to explain the extensive frgmentation in IRMPD spectra. In addition, a comparison of the IRMPD spectra of chlorophyll-a and pheophytin-a shows that the macrocycle in the absence of the Mg atom is much more fragile.

Production of bacteriopurpurin-18 phytyl ester from bacteriopheophytin a via allomerization by contact with titanium oxides in the presence of molecular oxygen.

Incubation of bacteriopheophytin (BPhe) a, which was a demetalated pigment of bacteriochlorophyll a in photosynthetic bacteria, in CH2Cl2 in the presence of TiO2 particles with bubbling O2 in the dark produced a pigment absorbing 814nm. Detailed characterization of the novel pigment isolated from the CH2Cl2 suspension revealed that bacteriopurpurin-18 phytyl ester possessing an anhydride-type six-membered exocyclic E-ring was majorly formed by the treatment with TiO2 particles under oxygenic conditions. Oxidation of the bacteriochlorin ring in BPhe a, namely formations of derivatives of 3-acetyl pheophytin a and 3-acetyl protopheophytin a, can barely be detected through the conversion processes.

Fluorescence and Docking Studies of the Interaction between Human Serum Albumin and Pheophytin.

In the North of Brazil (Pará and Amazonas states) the leaves of the plant Talinum triangulare (popular: cariru) replace spinach as food. From a phytochemical point of view, they are rich in compounds of the group of pheophytins. These substances, related to chlorophyll, have photophysical properties that give them potential application in photodynamic therapy. Human serum albumin (HSA) is one of the main endogenous vehicles for biodistribution of molecules by blood plasma. Association constants and thermodynamic parameters for the interaction of HSA with pheophytin from Talinum triangulare were studied by UV-Vis absorption, fluorescence techniques, and molecular modeling (docking). Fluorescence quenching of the HSA's internal fluorophore (tryptophan) at temperatures 296 K, 303 K, and 310 K, resulted in values for the association constants of the order of 104 L∙mol(-1), indicating a moderate interaction between the compound and the albumin. The negative values of ΔG° indicate a spontaneous process; ΔH° = 15.5 kJ∙mol(-1) indicates an endothermic process of association and ΔS° = 0.145 kJ∙mol(-1)∙K(-1) shows that the interaction between HSA and pheophytin occurs mainly by hydrophobic factors. The observed Trp fluorescence quenching is static: there is initial non-fluorescent association, in the ground state, HSA:Pheophytin. Possible solution obtained by a molecular docking study suggests that pheophytin is able to interact with HSA by means of hydrogen bonds with three lysine and one arginine residues, whereas the phytyl group is inserted in a hydrophobic pocket, close to Trp-214.

Lipoxygenase inhibitors derived from marine macroalgae.

The solvent extracts from the algae Sargassum thunbergii (Sargassaceae) and Odonthalia corymbifera (Rhodomelaceae) were subjected to soybean lipoxygenase inhibitory screening. Two hydrophobic inhibitors were obtained from the extracts of S. thunbergii through inhibitory assay-guided fractionation. The inhibitors were identified as known exo-methylenic alkapolyenes (6Z,9Z,12Z,15Z)-1,6,9,12,15-henicosapentaene (1) and (6Z,9Z,12Z,15Z,18Z)-1,6,9,12,15,18-henicosahexaene (2). The alkapolyenes 1 and 2 showed higher inhibitory activity than the known inhibitor nordihydroguaiaretic acid (NDGA). Pheophytin a (3) was obtained from the extract of O. corymbifera. The inhibitor 3 also showed higher inhibitory activity than NDGA. This is the first report on lipoxygenase inhibition of exo-methylenic alkapolyenes and a chlorophyll a-related substance.

Primary radical ion pairs in photosystem II core complexes.

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700-710 nm at 278 K. It was found that the initial charge separation between P680* and ChlD1 (Chl-670) takes place with a time constant of ~1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680*((1-δ)) (P680(δ+)ChlD1(δ-)), where δ ~ 0.5. The subsequent electron transfer from P680(δ+)ChlD1(δ-) to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (ChlD1(δ-)) and bleaching of the PheoD1 bands at 420, 545, and 680 nm with development of the Pheo(-) band at 460 nm. Further electron transfer to QA occurs within 250 ps in accordance with earlier data. The spectra of P680(+) and Pheo(-) formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ~1 and ~13 ps due to the formation of P680(δ+)ChlD1(δ-) and P680(+)PheoD1(-), respectively.

Toxicity of imine-iminium dyes and pigments: electron transfer, radicals, oxidative stress and other physiological effects.

Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer-reactive oxygen species-oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer-reactive oxygen species-oxidative stress arising from imine-iminium.

Temporal dynamics of natural product biosynthesis in marine cyanobacteria.

Sessile marine organisms are prolific sources of biologically active natural products. However, these compounds are often found in highly variable amounts, with the abiotic and biotic factors governing their production remaining poorly understood. We present an approach that permits monitoring of in vivo natural product production and turnover using mass spectrometry and stable isotope ((15)N) feeding with small cultures of various marine strains of the natural product-rich cyanobacterial genus Lyngbya. This temporal comparison of the amount of in vivo (15)N labeling of nitrogen-containing metabolites represents a direct way to discover and evaluate factors influencing natural product biosynthesis, as well as the timing of specific steps in metabolite assembly, and is a strong complement to more traditional in vitro studies. Relative quantification of (15)N labeling allowed the concurrent measurement of turnover rates of multiple natural products from small amounts of biomass. This technique also afforded the production of the neurotoxic jamaicamides to be more carefully studied, including an assessment of how jamaicamide turnover compares with filament growth rate and primary metabolism and provided new insights into the biosynthetic timing of jamaicamide A bromination. This approach should be valuable in determining how environmental factors affect secondary metabolite production, ultimately yielding insight into the energetic balance among growth, primary production, and secondary metabolism, and thus aid in the development of methods to improve compound yields for biomedical or biotechnological applications.