Dynamic Stark Effect in Two-Dimensional Spectroscopy Revealing Modulation of Ultrafast Charge Separation in Bacterial Reaction Centers by an Inherent Electric Field.

Despite extensive study, mysteries remain regarding the highly efficient ultrafast charge separation processes in photosynthetic reaction centers (RCs). In this work, transient Stark signals were found to be present in ultrafast two-dimensional electronic spectra recorded for purple bacterial RCs at 77 K. These arose from the electric field that is inherent to the intradimer charge-transfer intermediate of the bacteriochlorophyll pair (P), PA+PB-. By comparing three mutated RCs, a correlation was found between the efficient formation of PA+PB- and a fast charge separation rate. Importantly, the energy level of P* was changed due to the Stark shift, influencing the driving force for P* → P+BA- electron transfer and hence its rate. Furthermore, the orientation and amplitude of the inherent electric field varied in different ways upon different mutation, leading to contrasting changes in the rates. This mechanism of modulation provides a solution to a long-lasting inconsistency between experimental observations and activation energy theory.

High-Efficiency Excitation Energy Transfer in Biohybrid Quantum Dot-Bacterial Reaction Center Nanoconjugates.

Reaction centers (RCs) are the pivotal component of natural photosystems, converting solar energy into the potential difference between separated electrons and holes that is used to power much of biology. RCs from anoxygenic purple photosynthetic bacteria such as Rhodobacter sphaeroides only weakly absorb much of the visible region of the solar spectrum, which limits their overall light-harvesting capacity. For in vitro applications such as biohybrid photodevices, this deficiency can be addressed by effectively coupling RCs with synthetic light-harvesting materials. Here, we studied the time scale and efficiency of Förster resonance energy transfer (FRET) in a nanoconjugate assembled from a synthetic quantum dot (QD) antenna and a tailored RC engineered to be fluorescent. Time-correlated single-photon counting spectroscopy of biohybrid conjugates enabled the direct determination of FRET from QDs to attached RCs on a time scale of 26.6 ± 0.1 ns and with a high efficiency of 0.75 ± 0.01.

1H, 13C and 15N assignment of the paramagnetic high potential iron-sulfur protein (HiPIP) PioC from Rhodopseudomonas palustris TIE-1.

High potential iron-sulfur proteins (HiPIPs) are a class of small proteins (50-100 aa residues), containing a 4Fe-4S iron-sulfur cluster. The 4Fe-4S cluster shuttles between the oxidation states [Fe4S4]3+/2+, with a positive redox potential in the range (500-50 mV) throughout the different known HiPIPs. Both oxidation states are paramagnetic at room temperature. HiPIPs are electron transfer proteins, isolated from photosynthetic bacteria and usually provide electrons to the photosynthetic reaction-center. PioC, the HIPIP isolated from Rhodopseudomonas palustris TIE-1, is the smallest among all known HiPIPs. Despite their small dimensions, an extensive NMR assignment is only available for two of them, because paramagnetism prevents the straightforward assignment of all resonances. We report here the complete NMR assignment of 1H, 13C and 15N signals for the reduced [Fe4S4]2+ state of the protein. A set of double and triple resonance experiments performed with standardized parameters/datasets provided the assignment of about 72% of the residues. The almost complete resonance assignment (99.5% of backbone and ca. 90% of side chain resonances) was achieved by combining the above information with those obtained using a second set of NMR experiments, in which acquisition and processing parameters, as well as pulse sequences design, were optimized to account for the peculiar features of this paramagnetic protein.

Determination of the Complete Spin Density Distribution in 13C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory.

Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically 13C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary QA and secondary QB ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides 13C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected 13C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and pπ-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.

A new procedure for fast soft staining of BN-PAGEs on photosynthetic complexes.

We report a fast and sensitive procedure for blue native PAGE staining, in which the conventional staining step with CBB is avoided. After running, a short exposure to a mix of polar protic solvents (ethanol and acetic acid) leads to a fast and selective removal of the dye from the migration front and a specific binding to the protein bands, while the rest undergo a selective and complete background removal, leading to an intense contrast. This single-step staining-destaining technique is useful in protein samples that bind colored cofactors such as photosystems, which can be selectively discerned by their characteristic green color. After the staining of such samples, the green color persists, while the other unpigmented protein complexes and the molecular standard remain CBB stained, creating a useful reference system for the assignment of the bands. The advantages and chemical basis of this staining procedure are discussed.

Biophotoelectrochemistry of Photosynthetic Proteins.

This chapter presents biophotoelectrochemical systems where one of nature's photosynthetic proteins, such as photosystem 1 (PS1), photosystem 2 (PS2), or bacterial reaction centers, are employed to create devices for technological applications. We use recent advances in biophotoelectrodes for energy conversion and sensing to illustrate the fundamental approaches in half-cell design and characterization. The aim is to guide electrochemists and photosynthetic researchers in the development of hybrid systems interfacing photosynthetic proteins with electrodes ranging from biosensors to biophotovoltaic cells. The first part gives an overview of the photosynthetic electron transfer chain with details on photosynthetic proteins and on the properties relevant for technological applications. The second part describes and critically discusses the main applications of biophotoelectrochemical cells based on photosynthetic proteins and exposes the respective requirement in electrode design. The following and final parts present the standard methodologies for the characterization of the biophotoelectrochemical half-cells with the main objectives of enhancing our mechanistic understanding of electron transfer, charge recombination, overpotential in photocurrent generation and protein degradation processes in devices, and thus open the perspectives for novel biophotoelectrochemical concepts and their rational optimization toward practical efficiencies.

Arabidopsis plants grown in the field and climate chambers significantly differ in leaf morphology and photosystem components.

BACKGROUND: Plants exhibit phenotypic plasticity and respond to differences in environmental conditions by acclimation. We have systematically compared leaves of Arabidopsis thaliana plants grown in the field and under controlled low, normal and high light conditions in the laboratory to determine their most prominent phenotypic differences. RESULTS: Compared to plants grown under field conditions, the "indoor plants" had larger leaves, modified leaf shapes and longer petioles. Their pigment composition also significantly differed; indoor plants had reduced levels of xanthophyll pigments. In addition, Lhcb1 and Lhcb2 levels were up to three times higher in the indoor plants, but differences in the PSI antenna were much smaller, with only the low-abundance Lhca5 protein showing altered levels. Both isoforms of early-light-induced protein (ELIP) were absent in the indoor plants, and they had less non-photochemical quenching (NPQ). The field-grown plants had a high capacity to perform state transitions. Plants lacking ELIPs did not have reduced growth or seed set rates, but their mortality rates were sometimes higher. NPQ levels between natural accessions grown under different conditions were not correlated. CONCLUSION: Our results indicate that comparative analysis of field-grown plants with those grown under artificial conditions is important for a full understanding of plant plasticity and adaptation.

Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species.

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex.

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.

Photosynthetic characteristics of marine aerobic anoxygenic phototrophic bacteria Roseobacter and Erythrobacter strains.

A coastal Roseobacter strain of marine aerobic anoxygenic phototrophic bacteria (AAnPB) was isolated and phylogenetically determined. The strain OBYS 0001 was characterized by its physiological and biochemical properties with reference to the Erythrobacter longus type strain NBRC 14126. When grown in batch cultures, the growth curves of the both strains were similar. Cellular bacteriochlorophyll a concentrations of the strains reached the maxima in the stationary growth conditions. In vivo fluorescence excitation/optical density spectra between 470 and 600 nm for OBYS 0001 represented higher values than NBRC 14126. Variable fluorescence measurements revealed that the functional absorption cross section (σ) of the bacterial photosynthetic complexes for OBYS 0001 was significantly higher than that for NBRC 14126 under green excitation. These results suggest that Roseobacter can capture green light more efficiently than Erythrobacter for photosynthesis. The photochemical quantum efficiencies (F (v)/F (m)) of the bacterial photosynthetic complexes for OBYS 0001 were consistently lower than those for NBRC 14126. A relationship between the growth rate and F (v)/F (m) was significant for OBYS 0001, but that was not found for NBRC 14126. These results suggested that F (v)/F (m) for AAnPB could not be used as a proxy of the growth rate which is consistent with their mostly heterotrophic characters.

Self-assembly and sensor response of photosynthetic reaction centers on screen-printed electrodes.

Photosynthetic reaction centers were immobilized onto gold screen-printed electrodes (Au-SPEs) using a self-assembled monolayer (SAM) of mercaptopropionic acid (MPA) which was deliberately defective in order to achieve effective mediator transfer to the electrodes. The pure Photosystem II (PS II) cores from spinach immobilize onto the electrodes very efficiently but fair badly in terms of photocurrent response (measured using duroquinone as the redox mediator). The cruder preparation of PS II known as BBY particles performs significantly better under the same experimental conditions and shows a photocurrent response of 20-35 nA (depending on preparation) per screen-printed electrode surface (12.5mm(2)). The data was corroborated using AFM, showing that in the case of BBY particles a defective biolayer is indeed formed, with grooves spanning the whole thickness of the layer enhancing the possibility of mass transfer to the electrodes and enabling biosensing. In comparison, the PS II core layer showed ultra-dense organization, with additional formation of aggregates on top of the single protein layer, thus blocking mediator access to the electrodes and/or binding sites. The defective monolayer biosensor with BBY particles was successfully applied for the detection of photosynthesis inhibitors, demonstrating that the inhibitor binding site remained accessible to both the inhibitor and the external redox mediator. Biosensing was demonstrated using picric acid and atrazine. The detection limits were 1.15 nM for atrazine and 157 nM for picric acid.

[Ionization energies and infrared spectra studies of histidine using density functional theory].

Histidines provide axial ligands to the primary electron donors in photosynthetic reaction centers (RCs) and play an important role in the protein environments of these donors. In this paper the authors present a systematic study of ionization energies and vibrational properties of histidine using hybrid density functional theory (DFT). All calculations were undertaken by using B3LYP method in combination with four basis sets: 6-31G(d), 6-31G(df, p), 6-31+G(d) and 6-311+G(2d, 2p) with the aim to investigate how the basis sets influence the calculation results. To investigate solvent effects and gain a detailed understanding of marker bands of histidine, the ionization energies of histidine and the vibrational frequencies of histidine which are unlabeled and 13C, 15N, and 2H labeled in the gas phase, CCl4, protein environment, THF and water solution, which span a wide range of dielectric constant, were also calculated. Our results showed that: (1) The main geometry parameters of histidine were impacted by basis sets and mediums, and C2-N3 and N3-C4 bond of imidazole ring of histidine side chain display the maximum bond lengths in the gas phase; (2) single point energies and frequencies calculated were decreased while ionization energies increased with the increasing level of basis sets and diffuse function applied in the same solvent; (3) with the same computational method, the higher the dielectric constant of the solvent used, the lower the ionization energy and vibrational frequency and the higher the intensity obtained. In addition, calculated ionization energy in the gas phase and marker bands of histidine as well as frequency shift upon 13C and 15N labeling at the computationally more expensive 6-311+G(2d, 2p) level are in good agreement with experimental observations available in literatures. All calculations indicated that the results calculated by using higher level basis set with diffuse function were more accurate and closer to the experimental value. In conclusion, the results provide useful information for the further studies of the functional and vibrational properties of chlorophyll-a ligated to histidine residue in photosynthetic reaction center.

Artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria): morphological and physiological characterization.

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO2 assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40% of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.

Methodology of pulsed photoacoustics and its application to probe photosystems and receptors.

We review recent advances in the methodology of pulsed time-resolved photoacoustics and its application to studies of photosynthetic reaction centers and membrane receptors such as the G protein-coupled receptor rhodopsin. The experimental parameters accessible to photoacoustics include molecular volume change and photoreaction enthalpy change. Light-driven volume change secondary to protein conformational changes or electrostriction is directly related to the photoreaction and thus can be a useful measurement of activity and function. The enthalpy changes of the photochemical reactions observed can be measured directly by photoacoustics. With the measurement of enthalpy change, the reaction entropy can also be calculated when free energy is known. Dissecting the free energy of a photoreaction into enthalpic and entropic components may provide critical information about photoactivation mechanisms of photosystems and photoreceptors. The potential limitations and future applications of time-resolved photoacoustics are also discussed.

Internal electrostatic control of the primary charge separation and recombination in reaction centers from Rhodobacter sphaeroides revealed by femtosecond transient absorption.

We report the observation of two conformational states of closed RCs from Rhodobacter sphaeroides characterized by different P(+)H(A)(-) --> PH(A) charge recombination lifetimes, one of which is of subnanosecond value (700 +/- 200 ps). These states are also characterized by different primary charge separation lifetimes. It is proposed that the distinct conformations are related to two protonation states either of reduced secondary electron acceptor, Q(A)(-), or of a titratable amino acid residue localized near Q(A). The reaction centers in the protonated state are characterized by faster charge separation and slower charge recombination when compared to those in the unprotonated state. Both effects are explained in terms of the model assuming modulation of the free energy level of the state P(+)H(A)(-) by the charges on or near Q(A) and decay of the P(+)H(A)(-) state via the thermally activated P(+)B(A)(-) state.

Identification of the intermediate charge-separated state P+betaL- in a leucine M214 to histidine mutant of the Rhodobacter sphaeroides reaction center using femtosecond midinfrared spectroscopy.

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted betaL). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B(L) and B(M)), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P(L)/P(M))). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm(-1) and 1770 cm(-1), and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P* decayed on a timescale of approximately 6.3 ps to P+BL-. The decay of P+BL- occurred with a lifetime of approximately 2 ps, approximately 3 times slower than that observed in wild-type and R-26 RCs (approximately 0.7 ps). Further electron transfer to the betaL BChl resulted in formation of the P+betaL- state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P+BL- and P+betaL- showed clear differences related to the different environments of the two BChls in the mutant RC.

Purification and proteomic characterization of plastids from Brassica napus developing embryos.

Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.

Conserved role of proton gradient regulation 5 in the regulation of PSI cyclic electron transport.

There are at least two photosynthetic cyclic electron transport (CET) pathways in most C(3) plants: the NAD(P)H dehydrogenase (NDH)-dependent pathway and a pathway dependent upon putative ferredoxin:plastoquinone oxidoreductase (FQR) activity. While the NDH complex has been identified, and shown to play a role in photosynthesis, especially under stress conditions, less is known about the machinery of FQR-dependent CET. Recent studies indicate that FQR-dependent CET is dependent upon PGR5, a small protein of unknown function. In a previous study we found that overexpression of PGR5 causes alterations in growth and development associated with decreased chloroplast development and a transient increase in nonphotochemical quenching (NPQ) after the shift from dark to light. In the current study we examine the spatiotemporal expression pattern of PGR5, and the effects of overexpression of PGR5 in Arabidopsis under a host of light and stress conditions. To investigate the conserved function of PGR5, we cloned PGR5 from a species which apparently lacks NDH, loblolly pine, and overexpressed it in Arabidopsis. Although greening of cotyledons was severely delayed in overexpressing lines under low light, mature plants survived exposure to high light and drought stress better than wild-type. In addition, PSI was more resistant to high light in the PGR5 overexpressors than in wild-type plants, while PSII was more sensitive to this stress. These complex responses corresponded to alterations in linear and cyclic electron transfer, suggesting that over-accumulation of PGR5 induces pleiotropic effects, probably via elevated CET. We conclude that PGR5 has a developmentally-regulated, conserved role in mediating CET.

Computational analysis of photosynthetic systems.

The use of various computational techniques for the study of photosynthetic systems is described ranging from genome analysis to density functional simulations of the oxygen evolving complex of PSII. The use of simulations for analyzing protein structures can aid in clarifying ambiguous and incomplete experimental results to identifying underlying rules to create efficient light-initiated charge separation at high efficiency.

Two stereoisomers of spheroidene in the Rhodobacter sphaeroides R26 reaction center: a DFT analysis of resonance Raman spectra.

From a theoretical analysis of the resonance Raman spectra of 19 isotopomers of spheroidene reconstituted into the reaction center (RC) of Rhodobacter sphaeroides R26, we conclude that the carotenoid in the RC occurs in two configurations. The normal mode underlying the resonance Raman transition at 1239 cm(-1), characteristic for spheroidene in the RC, has been identified and found to uniquely refer to the cis nature of the 15,15' carbon-carbon double bond. Detailed analysis of the isotope-induced shifts of transitions in the 1500-1550 cm(-1) region proves that, besides the 15,15'-cis configuration, spheroidene in the RC adopts another cis-configuration, most likely the 13,14-cis configuration.