Two intermediate states I and J trapped at low temperature in the photocycles of two BLUF domain proteins of cyanobacteria Synechocystis sp. PCC6803 and Thermosynechococcus elongatus BP-1

We identified the two intermediate states, I and J, that are common in the photocycles of the cyanobacterial BLUF (sensor of Blue Light Using Flavin) domain proteins of Slr1694 of Synechocystis sp. PCC6803 and Tll0078 of Thermosynechococcus elongatus BP-1 by analyzing the absorption spectra at 5 K. Illumination at 5 K accumulated intermediate forms (designated as I5 and I9), which showed 5 and 9 nm redshifts of the absorption bands of flavin in the Tll0078 and Slr1694 proteins, respectively. I5 (I9) was converted into the next intermediate, which have 11 nm (14 nm) red-shifted absorption bands J11 (J14) after dark annealing at 230 K (240 K). Further dark annealing at 280 K (270 K) of J11 (J14) produced the signal-transmitting final form F490 (F495), with a small increase in the absorption at around 490 nm (495 nm). The results indicate that the BLUF proteins of Tll0078 and Slr1694 exhibit the common photocycle of D471 (D467) --> I5 (I9) --> J11 (J14) --> F490 (F495) at low temperature. The transition temperatures for these intermediate forms differ for two proteins. The amount of I5 (I9) accumulated at 5 K was small and increased at a higher temperature, suggesting heterogeneity of the protein structure that determines the reaction pathway.     

Function and association of CyanoP in photosystem II of Synechocystis sp. PCC 6803

The CyanoP protein is a cyanobacterial homolog of the PsbP protein, which is an extrinsic subunit of photosystem II (PSII) in green plant species. The molecular function of CyanoP has been investigated in mutant strains of Synechocystis but inconsistent results have been reported by different laboratories. In this study, we generated and characterized a Synechocystis mutant in which entire region of the CyanoP gene was eliminated. After repeated subculture in CaCl₂-depleted medium, growth retardation was clearly observed for a CyanoP knockout mutant of Synechocystis sp. PCC 6803 (?P). The PSII-mediated oxygen-evolving activity of the ?P cells was more susceptible to depletion of CaCl₂ than that of wild-type cells. The 77 K fluorescence emission spectra indicated that energy coupling between phycobilisome and PSII was perturbed in both wild-type and ?P cells under CaCl₂-depleted conditions, and was more evident for the ?P mutant. To examine the association of CyanoP with PSII complexes, we tested several detergents for solubilization of thylakoid membranes and showed that CyanoP was partly included in fractions containing large protein complexes in gel-filtration analysis. These results indicate that CyanoP constitutively stabilizes PSII functionality in vivo.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Absorption and emission spectroscopic characterization of blue-light receptor Slr1694 from Synechocystis sp. PCC6803

The BLUF protein Slr1694 from the cyanobacterium Synechocystis sp. PCC6803 is characterized by absorption and emission spectroscopy. Slr1694 expressed from E. coli which non-covalently binds FAD, FMN, and riboflavin (called Slr1694(I)), and reconstituted Slr1694 which dominantly contains FAD (called Slr1694(II)) are investigated. The receptor conformation of Slr1694 (dark adapted form Slr1694(r)) is transformed to the putative signalling state (light adapted form Slr1694(s)) with red-shifted absorption and decreased fluorescence efficiency by blue-light excitation. In the dark at 22 degrees C, the signalling state recovers back to the initial receptor state with a time constants of about 14.2s for Slr1694(I) and 17s for Slr1694(II). Quantum yields of signalling state formation of approximately 0.63+/-0.07 for both Slr1694(I) and Slr1694(II) were determined by transient transmission measurements and intensity dependent steady-state transmission measurements. Extended blue-light excitation causes some bound flavin conversion to the hydroquinone form and some photo-degradation, both with low quantum efficiency. The flavin-hydroquinone re-oxidizes slowly back (time constant 5-9 min) to the initial flavoquinone form in the dark. A photo-cycle dynamics scheme is presented.     

Tracking phototactic responses and modeling motility of Synechocystis sp. strain PCC6803

The unicellular cyanobacterium Synechocystis sp. Strain PCC 6,803 exhibits phototaxis by moving along a surface towards a light source. This process requires Type IV pili and a phytochrome-like photoreceptor coupled to a complex signal transduction pathway. Cells progress through different phases of interaction before the development of finger-like projections moving in the direction of the light that are characteristic of phototaxis. To probe the interaction between individual cells during the initial phase of phototaxis we tracked and analyzed a large number of cells. We observed that individual cells have limited motility, but when cells divide and/or aggregate to attain a certain minimal group size, enhanced motility and phototaxis is observed. At the later stages of motility, there is noticeable phototactic behavior which results in the appearance of the finger-like projections. Our results indicate that cells prefer to move over areas previously traversed by other cells and confine themselves to these areas and that cells alter local surface characteristics allowing for enhanced motility. Based on cell tracking data we present a preliminary random walk model showing the forces that might interact to create the typical phases of phototaxis and motility. In this model, we can simulate the formation of finger-like projections that are characteristic of phototaxis.     

Spectroscopic and photochemical characterization of the red-light sensitive photosensory module of Cph2 from Synechocystis PCC 6803

Cyanobacterial phytochromes are a diverse family of light receptors controlling various biological functions including phototaxis. In addition to canonical bona fide phytochromes of the well characterized Cph1/plant-like clade, cyanobacteria also harbor phytochromes that absorb green, violet or blue light. The Synechocystis PCC 6803 Cph2 photoreceptor, a phototaxis inhibitor, is unconventional in bearing two distinct chromophore-binding GAF domains. Whereas the C-terminal GAF domain is most likely involved in blue-light perception, the first two domains correspond to a Cph1-like photosensory module lacking the PAS domain. Biochemical and spectroscopic studies show that this region switches between red (P(r) ) and far-red (P(fr) ) absorbing states. Unlike Cph1, the P(fr) state of Cph2 decays rapidly in darkness. Mutations close to the PCB chromophore further destabilize the P(fr) state without drastically affecting the spectroscopic features such as the quantum efficiency of P(r) →P(fr) conversion, fluorescence, or the Resonance-Raman signature of the chromophore. Overall, the PAS-less photosensory module of Cph2 resembles Cph1 including its mode of isomerisation, but the P(fr) state is unstable.     

Frequency-domain spectroscopic study of the PS I-CP43' supercomplex from the cyanobacterium Synechocystis PCC 6803 grown under iron stress conditions

Absorption, fluorescence excitation, emission, and hole-burning (HB) spectra were measured at liquid helium temperatures for the PS I-CP43' supercomplexes of Synechocystis PCC 6803 grown under iron stress conditions and for respective trimeric PS I cores. Results are compared with those of room temperature, time-domain experiments (Biochemistry 2003, 42, 3893) as well as with the low-temperature steady-state experiments on PS I-CP43' supercomplexes of Synechococcus PCC 7942 (Biochim. Biophys. Acta 2002, 1556, 265). In contrast to the CP43' of Synechococcus PCC 7942, CP43' of Synechocystis PCC 6803 possesses two low-energy states analogous to the quasidegenerate states A and B of CP43 of photosystem II (J. Phys. Chem. B 2000, 104, 11805). Energy transfer between the CP43' and the PS I core occurs, to a significant degree, through the state A, characterized with a broader site distribution function (SDF). It is demonstrated that the low temperature (T = 5 K) excitation energy transfer (EET) time between the state A of CP43' (IsiA) and the PS I core in PS I-CP43' supercomplexes from Synechocystis PCC 6803 is about 60 ps, which is significantly slower than the EET observed at room temperature. Our results are consistent with fast (< or =10 ps) energy transfer from state B to state A in CP43'. Energy absorbed by the CP43' manifold has, on average, a greater chance of being transferred to the reaction center (RC) and utilized for charge separation than energy absorbed by the PS I core antenna. This indicates that energy is likely transferred from the CP43' to the RC along a well-defined path and that the "red antenna states" of the PS I core are localized far away from that path, most likely on the B7-A32 and B37-B38 dimers in the vicinity of the PS I trimerization domain (near PsaL subunit). We argue that the A38-A39 dimer does not contribute to the red antenna region.     

Protective effects of mycosporine-like amino acids of Synechocystis sp. PCC 6803 and their partial characterization

In this work, mycosporine-like amino acids (MAAs) of Synechocystis sp. PCC 6803 were characterized and were investigated on UV induction and protective ability. High performance liquid chromatographic (HPLC) studies revealed three major compounds in the MAAs. By UV absorption and mass spectra analysis, one of the compounds was tentatively identified as mycosporine-tau (M-tau). One novel compound similar to usujirene was tentatively named as dehydroxylusujirene, and the other novel compound was named as M-343 according to its absorption maximum. In vivo experiments indicated that M-tau was induced by both UV-A and UV-B, while dehydroxylusujirene and M-343 were only induced by UV-A, suggesting that different chromophores were involved in MAAs synthesis in Synechocystis sp. PCC 6803. It was also indicated that M-343 could be photochemically synthesized from some precursors. Under both UV and oxidation stresses, M-343 was more stable than dehydroxylusujirene and M-tau. Considering the reaction with H2O2, M-tau and dehydroxylusujirene might be potential antioxidants in reaction with physiological reactive oxygen species in vivo. In protection experiments, the MAAs exhibited efficient protective ability towards UV-B and H2O2 stresses, with maximal protection rates of 30% and 21.5%, respectively. These results indicate that the MAAs in Synechocystis sp. PCC 6803 act as both UV-screen and antioxidant.     

A synthetic DNA and fusion PCR approach to the ectopic expression of high levels of the D1 protein of photosystem II in Synechocystis sp. PCC 6803

A hybrid approach involving synthetic DNA, fusion PCR, and ectopic expression has been used to genetically manipulate the expression of the D1 protein of photosystem II (PSII) in the model cyanobacterium Synechocystis sp. PCC6803. Due to the toxicity of the full-length psbA gene in E. coli, a chimeric psbA2 gene locus was commercially synthesised and cloned in two halves. High-fidelity fusion PCR utilizing sequence overlap between the two synthetic gene halves allowed the production of a DNA fragment that was able to recombine the full-length psbA2 gene into the Synechocystis chromosome at an ectopic (non-native) location. This was accomplished by designing the synthetic DNA/fusion PCR product to have the psbA2 gene, with control sequences, interposed between chimeric sequences corresponding to an ectopic target chromosomal location. Additionally, a recipient strain of Synechocystis lacking all three psbA genes was produced by a combination of traditional marker replacement and markerless replacement techniques. Transformation of this multiple deletion strain by the synthetic DNA/fusion PCR product faithfully restored D1 expression in terms of its expression and PSII repair capacity. The advantages and potential issues for using this approach to rapidly introduce chimeric sequence characteristics as a general tool to produce novel genetic constructs are discussed.     

Assignment of the lowest Qy-state and spectral dynamics of the CP29 chlorophyll a/b antenna complex of green plants: a hole-burning study

Low-temperature absorption, fluorescence and persistent non-photochemical hole-burned spectra are reported for the CP29 chlorophyll (Chl) a/b antenna complex of photosystem II of green plants. The absorption-origin band of the lowest Qy-state lies at 678.2 nm and carries a width of approximately 130 cm-1 that is dominated by inhomogeneous broadening at low temperatures. Its absorption intensity is equivalent to that of one of the six Chl a molecules of CP29. The absence of a significant satellite hole structure produced by hole burning, within the absorption band of the lowest state, indicates that the associated Chl a molecule is weakly coupled to the other Chl and, therefore, that the lowest-energy state is highly localized on a single Chl a molecule. The electron-phonon coupling of the 678.2 nm state is weak with a Huang-Rhys factor S of 0.5 and a peak phonon frequency (omega m) of approximately 20 cm-1. These values give a Stokes shift (2S omega m) in good agreement with the measured positions of the absorption band at 678.2 nm and a fluorescence-origin band at 679.1 nm. Zero-phonon holes associated with the lowest state have a width of approximately 0.05 cm-1 at 4.2 K, corresponding to a total effective dephasing time of approximately 400 ps. The temperature dependence of the zero-phonon holewidth indicates that this time constant is dominated at temperatures below 8 K by pure dephasing/spectral diffusion due to coupling of the optical transition to the glass-like two-level systems of the protein. Zero-phonon hole-widths obtained for the Chl b bands at 638.5 and 650.0 nm, at 4.2 K, lead to lower limits of 900 +/- 150 fs and 4.2 +/- 0.3 ps, respectively, for the Chl b-->Chl a energy-transfer times. Downward energy transfer from the Chl a state(s) at 665.0 nm occurs in 5.3 +/- 0.6 ps at 4.2 K.     

Optical dynamics of the reaction center of photosystem II. A hole-burning and photon-echo study

Photon-echo and transient hole-burning experiments on the P-band of the reaction center of photosystem II are reported which show that the P-band exhibits a large homogeneous width. A photon-echo signal with a decay time of 500 ps is also observed in the same spectral region and assigned to extraneous chlorophyll. These observations are discussed with reference to the bacterial reaction center and photosystem I.     

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.     

How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem II in cryptophyte Rhodomonas CS24 cells

We report an investigation of energy migration dynamics in intact cells of the photosynthetic cryptophyte Rhodomonas CS24 using analyses of steady-state and time-resolved fluorescence anisotropy measurements. By fitting a specific model to the fluorescence data, we obtain three time scales (17, 58, and 113 ps) by which the energy is transferred from phycoerythrin 545 (PE545) to the membrane-associated chlorophylls (Chls). We propose that these time scales reflect both an angular distribution of PE545 around the photosystems and the relative orientations of the donor dihydrobiliverdin (DBV) bilin and the acceptor Chl. Contrary to investigations of the isolated antenna complex, it is demonstrated that energy transfer from PE545 does not occur from a single-emitting bilin, but rather both the peripheral dihydrobiliverdin (DBV) chromophores in PE545 appear to be viable donors of excitation energy to the membrane-bound proteins. The model shows an almost equal distribution of excitation energy from PE545 to both photosystem I (PSI) and photosystem II (PSII), whose trap times correspond well to those obtained from experiments on isolated photosystems.     

Photosystem II activity and turnover of the D1 protein are impaired in the psbA Y112L mutant of Synechocystis PCC6803 sp.

Site-directed psbA mutants at the tyrosine Y112 position have been generated in Synechocystis PCC6803 cells. The mutation Y112F does not affect photosystem II (PSII) activity as compared with control 4 delta 1K cells. However, the Y112L mutant exhibits a photosynthetically impaired phenotype. PSII activity is not detectable in this mutant when grown at 30 mumol photons m-2 s-1, while low levels of the D1 and D2 proteins and oxygen evolution activity are present in the mutant cells grown at a low light intensity (0.5-1 mumol m-2 s-1). The recombination of the QB-/S2,3 states of PSII in the Y112L mutant cells as detected by thermoluminescence (TL) is altered. The TL signal emission maximum of these cells due to charge recombination of the S2,3/QB- occurs at 20 degrees C as compared to 35-40 degrees C for the wild-type cells, indicating a possible change in the S2,3/Yz equilibrium. The Y112L mutant cells are rapidly photoinactivated and impaired in the recovery of the PSII activity. These results suggest that replacement of the aromatic residue at position Y112 by a hydrophobic amino acid may alter the function of the donor-side activity and affects the degradation and replacement of the PSII core proteins.     

Triplet states as non-radiative traps in multichromophoric entities: single molecule spectroscopy of an artificial and natural antenna system

Energy transfer in antenna systems, ordered arrays of chromophores, is one of the key steps in the photosynthetic process. The photophysical processes taking place in such multichromophoric systems, even at the single molecule level, are complicated and not yet fully understood. Instead of directly studying individual antenna systems, we have chosen to focus first on systems for which the amount of chromophores and the interactions among the chromophores can be varied in a systematic way. Dendrimers with a controlled number of chromophores at the rim fulfill those requirements perfectly. A detailed photophysical study of a second-generation dendrimer, containing eight peryleneimide chromophores at the rim, was performed 'J. Am. Chem. Soc., 122 (2000) 9278'. One of the most intriguing findings was the presence of collective on/off jumps in the fluorescence intensity traces of the dendrimers. This phenomenon can be explained by assuming a simultaneous presence of both a radiative trap (energetically lowest chromophoric site) and a non-radiative trap (triplet state of one chromophore) within one individual dendrimer. It was shown that an analogue scheme could explain the collective on/off jumps in the fluorescence intensity traces of the photosynthetic pigment B-phycoerythrin (B-PE) (Porphyridium cruentum). The different values of the triplet lifetime that could be recovered for a fluorescence intensity trace of B-PE were correlated with different intensity levels in the trace, suggesting different chromophores acting as a trap as function of time.     

Observation of rotamers of m-aminobenzoic acid: zero kinetic energy photoelectron and hole-burning resonantly enhanced multiphoton ionization spectroscopy

We report studies of supersonically cooled m-aminobenzoic acid using two-color resonantly enhanced multiphoton ionization (REMPI) and two-color zero kinetic energy (ZEKE) photoelectron spectroscopy. Two conformers have been identified and characterized using the hole-burning method in the REMPI experiment. With the aid of ab initio and density functional calculations, vibrational modes of the first electronically excited state (S(1)) of the neutral species and those of the ground state cation (D(0)) have been assigned, and the adiabatic ionization potentials have been determined for both conformers. The REMPI spectra are dominated by in-plane motions of the substituents and ring deformation modes. A propensity of Deltav=0, where Deltav is the change in vibrational quantum number from the S(1) to the D(0) state, is observed in the ZEKE spectra. The origin of this behavior is discussed in the context of electron back donation from the two substituents in the excited state and in the cationic state. Comparisons of these results with those of p-aminobenzoic acid will be analyzed.     

Isolation and characterization of a photosystem I-associated antenna (LHC I) and a photosystem I—core complex from the chlorophyll c-containing alga Pleurochloris meiringensis (Xanthophyceae)

A photosystem (PS) I holocomplex was isolated from Pleurochloris meiringensis Vischer (Xanthophyceae) using sucrose density centrifugation. This complex exhibited a fluorescence emission maximum at 715 nm, which is in accordance with the long wavelength emission of whole cells. The complex was further dissociated into a core complex and a light-harvesting protein (LHC I). The core protein contains mainly Chl a and β-carotene, is 8.25 times enriched in P700 and has its main emission maximum at 715 nm. Therefore, the longest wavelength emission of P. meiringensis is due to the PS I core, which is in contrast to higher plants. The LHC I differs from LHC II with regard to its polypeptide pattern as well as its spectral properties. The arrangement of antennae is discussed in relation to the regulation of energy transfer between the photosystems.     

Fluorescence from ion-pair and Rydberg states of I2

Absorption and fluorescence excitation spectra of I₂ have been recorded in the region 1100–2100 Å using synchrotron radiation. The strongest fluorescence is excited in the region 1730–2000 Å and is associated with the D(0_u⁺) ion-pair state: fluorescence in the region 1320–1500 Å is assigned in part to excited atoms formed by predissociation of Rydberg states.     

Time resolved spectroscopy of carbenepyridene ylides: Distinguishing carbenes from diazirine excited states

The photochemistry of diazirines and diazo compounds is not as simple as nitrogen extrusion and carbene formation. The C-H bonds adjacent to the diazo and diazirine moieties can migrate in the excited state and produce stable products without the benefit of a relaxed carbene intermediate. Additionally, cyclobutyl substituted systems exhibit carbon migration. It is unfortunate that the products of photochemical rearrangement of precursor excited states are identical to the products of thermal rearrangement of carbenes. This has prevented accurate measurement of the yield and absolute reactivity of alkylcarbenes. That pyridine reacts selectively with carbenes and not with the excited states of their nitrogenous precursors has allowed the separation of these two pathways and an appreciation of their relative importance with structural variation.