The photoreactions of recombinant phytochrome from the cyanobacterium Synechocystis: a low-temperature UV-Vis and FT-IR spectroscopic study

The interconvertible photoreactions of recombinant phytochrome from Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below -100 degrees C for the transformation of red-absorbing form of phytochrome (Pr)-->far-red-absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at -140 degrees C (lumi-R) was observed. Two intermediates could be stabilized below -40 degrees C and between -40 and -20 degrees C, and were denoted as meta-Ra and meta-Rc, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The FT-IR difference spectra of the late Pr-->Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-Ra were observed. A large band around 1700 cm-1 in the difference spectra between the intermediates and Pr which is tentatively assigned to the C19=O group of the prosthetic group indicates the Z,E isomerization around the C15=C16-methine bridge of the chromophore during the formation of meta-Ra. In the difference spectra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of 2H2O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm-1 in all 2H2O spectra suggests an assignment to an N-2H bending vibration of the chromophore.     

Conformation-changing aggregation in hydroxyacetone: a combined low-temperature FTIR, jet, and crystallographic study

Aggregation in hydroxyacetone (HA) is studied using low-temperature FTIR, supersonic jet expansion, and X-ray crystallographic (in situ cryocrystallization) techniques. Along with quantum chemical methods (MP2 and DFT), the experiments unravel the conformational preferences of HA upon aggregation to dimers and oligomers. The O-H···O═C intramolecular hydrogen bond present in the gas-phase monomer partially opens upon aggregation in supersonic expansions, giving rise to intermolecular cooperatively enhanced O-H···O-H hydrogen bonds in competition with isolated O-H···O═C hydrogen bonds. On the other hand, low-temperature IR studies on the neat solid and X-ray crystallographic data reveal that HA undergoes profound conformational changes upon crystallization, with the HOCC dihedral angle changing from ~0° in the gas phase to ~180° in the crystalline phase, hence giving rise to a completely new conformation. These conclusions are supported by theoretical calculations performed on the geometry derived from the crystalline phase.     

Regulation of cAMP-mediated photosignaling by a phytochrome in the cyanobacterium Anabaena cylindrica

Changes in cellular adenosine 3',5'-cyclic monophosphate (cAMP) content induced by monochromatic light of various wavelengths were determined in the cyanobacterium Anabaena cylindrica. Irradiation with monochromatic red light caused a rapid decrease in cAMP content. In contrast, far-red light caused a rapid increase in its content. The effects of red and far-red light were reversible, suggesting the involvement of a prototype phytochrome as the photoreceptor for cAMP-mediated light-responsive signal transduction.     

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.     

Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris

The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris. Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively). Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore. The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min. Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved. The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms.     

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.     

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.     

Analysis of the interaction of a hybrid system consisting of bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119

The mitochondrial steroid-hydroxylating system in vertebrates and the NADPH producing electron transfer chain in photosynthetic organisms contain structurally and functionally similar components. Examination of a potential hybrid reconstitution of the electron transfer chain between different components of both systems could help to improve our knowledge on protein-protein interaction and subsequent electron transfer. Here we analyzed the interaction between bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119. Optical biosensor as well as steady state and fast kinetic experiments showed their ability to form distinct productive complexes. Compared with the corresponding physiological systems the electron transfer is rather slow, probably due to the lack of specificity at the interaction surface.     

Phototaxis in the cyanobacterium Synechocystis sp. PCC 6803: role of different photoreceptors

The second cyanobacterial phytochrome Cph2 from Synechocystis sp. PCC 6803 was suggested as a part of a light-stimulated signal transduction chain inhibiting movement toward blue light. Cph2 has the two bilin binding sites, cysteine-129 and cysteine-1022, that might be involved in sensing of red/far-red and blue light, respectively. Here, we present data on wavelength dependence of the phototaxis inhibition under blue light, indicating that Cph2 itself is the photoreceptor for this blue light response. We found that inhibition of blue-light phototaxis in wild-type cells occurred below the transition point of about 470 nm. Substitution of cysteine-1022 with valine led to photomovement of the cells toward blue light (cph2(-) mutant phenotype). Analysis of mutants lacking cysteine-129 in the N-terminal chromophore binding domain indicated that this domain is also important for Cph2 function or folding of the protein. Furthermore, putative blue-light and phytochrome-like photoreceptors encoded by the Synechocystis sp. PCC 6803 genome were inactivated in wild-type and cph2 knockout mutant background. Our results suggest that none of these potential photoreceptors interfere with Cph2 function, although inactivation of taxD1 as well as slr1694 encoding a BLUF protein led to cells that reversed the direction of movement under blue light illumination in mutant strains of cph2.     

Retrieval of the physiological state of human skin from UV-Vis reflectance spectra - a feasibility study

We test the feasibility of using an accurate radiative transfer model for the coupled air-tissue system in conjunction with a classic inversion scheme based on Bayesian optimal estimation theory for retrieval of parameters describing the physiological state of human skin. To that end, we analyse ultraviolet and visible reflectance spectra from human skin measured before, immediately after, and on each day for two weeks after photodynamic treatment with the hexyl ester of ALA and exposure to red light (632 nm). For the first time, we show that it is possible to perform a simultaneous retrieval of the melanosome concentration in both the basal and the upper layers of the epidermis.     

The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production

Protein film electrochemistry (PFE) was utilized to characterize the catalytic activity and oxidative inactivation of a bidirectional [NiFe]-hydrogenase (HoxEFUYH) from the cyanobacterium Synechocystis sp. PCC 6803. PFE provides precise control of the redox potential of the adsorbed enzyme so that its activity can be monitored under changing experimental conditions as current. The properties of HoxEFUYH are different from those of both the standard uptake and the "oxygen-tolerant" [NiFe]-hydrogenases. First, HoxEFUYH is biased toward proton reduction as opposed to hydrogen oxidation. Second, despite being expressed under aerobic conditions in vivo, HoxEFUYH is clearly not oxygen-tolerant. Aerobic inactivation of catalytic hydrogen oxidation by HoxEFUYH is total and nearly instantaneous, producing two inactive states. However, unlike the Ni-A and Ni-B inactive states of standard [NiFe]-hydrogenases, both of these states are quickly (<90 s) reactivated by removal of oxygen and exposure to reducing conditions. Third, proton reduction continues at 25-50% of the maximal rate in the presence of 1% oxygen. Whereas most previously characterized [NiFe]-hydrogenases seem to be preferential hydrogen oxidizing catalysts, the cyanobacterial enzyme works effectively in both directions. This unusual catalytic bias as well as the ability to be quickly reactivated may be essential to fulfilling the physiological role in cyanobacteria, organisms expected to experience swings in cellular reduction potential as they switch between aerobic conditions in the light and dark anaerobic conditions. Our results suggest that the uptake [NiFe]-hydrogenases alone are not representative of the catalytic diversity of [NiFe]-hydrogenases, and the bidirectional heteromultimeric enzymes may serve as valuable models to understand the diverse mechanisms of tuning the reactivity of the hydrogen activating site.     

Remarkable wavelength-dependent photoreactions of the bis(diazo) ketone having inequivalent diazo groups: studies in fluid solutions and in low-temperature matrixes

The photoreactions of the bis(diazo) ketone 11, which has two inequivalent diazo groups, have been investigated in solutions at room temperature and in matrixes at 12 K. Irradiation of 11 in benzene containing methanol gave a mixture of the spironorcaradiene 13 and the diazo ketone 17 as primary isolable photoproducts. The former 13 originated from the diazo ketene 20, which was formed from the initial extrusion of N(2) from the 2-position of 11, while the latter product 17 was derived from the diazo ketene 24 which was generated by the initial decomposition of the diazo group at the 4-position of 11. The product distribution was remarkably dependent upon the excitation wavelength: 13 was predominantly obtained in the photolysis with light of >350 nm, while the irradiation with long-wavelength light (>420 nm) exclusively gave 17. The consistent wavelength effects were observed in photoreactions in an Ar matrix at 12 K. The irradiation of 11 matrix-isolated in Ar with light of >350 nm afforded 20 in preference to the isomer 24 as the first-formed intermediates, while 24 was mainly obtained in the long-wavelength irradiation (>420 nm). On the basis of these experimental data, we conclude that the short-wavelength irradiation of 11 causes a preferential cleavage of the diazo group at the 2-position and that the selective extrusion of N(2) from the 4-position is practically achieved by the irradiation with long-wavelength light. The reason for the selective cleavage of the two inequivalent diazo groups of 11 is discussed on the basis of theoretical calculations with the PM3 CI method.     

FTIR microspectroscopy of melanocytic skin lesions: a preliminary study

Fourier transform infrared (FTIR) microspectroscopy has been employed to investigate benign (ordinary dermal and Reed nevi), dysplastic and malignant (invasive melanoma) skin lesions through the analysis of spectral changes of melanocytes as well as in the evaluation of the presence of melanin. Hierarchical cluster analysis and principal component analysis led to a satisfactory separation of malignant from dysplastic and normal melanocytes. Also, on enlarging the clustering with spectra from Reed and dermal nevi, the multivariate analysis segregated well the spectral data into discrete clusters, allowing the obtaining of reliable average spectra for analysis at the molecular level of the main groups or components responsible for the biological and biochemical changes. The most significant spectral characteristics appear to be related to differences in secondary protein structures, in nucleic acid conformation, in intra- and intermolecular bonding. In all cases, supervised and unsupervised spectral analyses resulted in satisfactory agreement with histopathological findings.     

一种超分子纳米管的变温红外光谱研究

利用一对互补的分子组分5－（4－十二烷氧基苯乙烯基）－2,4,6－（1H,3H）－嘧啶三酮和4－胺基－2,6－二－十二烷基胺基－1,3,5－三嗪的自组装过程构筑了一种直径约为5nm的超分子纳米管。变温付立叶变温红外光谱研究表明,在纳米管的形成过程中,氢键、π－π相互作用和范德华力等非共价键相互作用导致了超分子纳米管的形成。     

Relative ground and excited-state pKa values of phytochromobilin in the photoactivation of phytochrome: a computational study

The conversion of the plant photoreceptor phytochrome from an inactive (Pr) to an active form (Pfr) is accomplished by a red-light induced Z --> E photoisomerization of its phytochromobilin chromophore. In recent years, the question whether the photoactivation involves a change in chromophore protonation state has been the subject of many experimental studies. Here, we have used quantum chemical methods to calculate relative ground and excited-state pKa values of the different pyrrole moieties of phytochromobilin in a protein-like environment. Assuming (based on experimental data) a Pr ZaZsZa chromophore and considering isomerizations at C15 and C5, it is found that moieties B and C are the strongest acids both in the ground state and in the bright first singlet excited state, which is rationalized in simple geometric and electronic terms. It is also shown that neither light absorption nor isomerization increases the acidity of phytochromobilin relative to the reference Pr state with all pyrrolenic nitrogens protonated. Hence, provided that the subset of chromophore geometries under investigation is biologically relevant, there appears to be no intrinsic driving force for a proton-transfer event. In a series of benchmark calculations, the performance of ab initio and time-dependent density functional theory methods for excited-state studies of phytochromobilin is evaluated in light of available experimental data.     

Temperature-dependent FTIR study of a supramolecular mesophase from the self-assembly of melamine and barbituric acid derivatives

A supramolecular mesophase was prepared from molecular recognition directed self- assembly of two complementary molecular components, namely 5-(4-dodecyloxy- benzylidene)-2,4,6-(1H,3H)-pyrimidinetrione and 4-amino-2,6-didodecylamino-1,3,5triazine. Differential scanning calorimetry measurements indicate a mesophase having liquid crystalline properties. Infrared studies suggest that not only hydrogen bonds but also pi-aromatic stacking and van der Waals interactions direct the formation of the mesophase.     

Solar PAR and UV radiation affects the physiology and morphology of the cyanobacterium Anabaena sp. PCC 7120

Solar UV radiation (280-400 nm) may affect morphology of cyanobacteria, however, little has been evidenced on this aspect while their physiological responses were examined. We investigated the impacts of solar PAR and UVR on the growth, photosynthetic performance and morphology of the cyanobacterium Anabaena sp. PCC7120 while it was grown under three different solar radiation treatments: exposures to (a) constant low PAR (photosynthetic active radiation, 400-700 nm), (b) natural levels of solar radiation with and (c) without UV radiation (290-400 nm). When the cells were exposed to solar PAR or PAR+UVR, the photochemical efficiency was reduced by about 40% and 90%, respectively, on day one and recovered faster under the treatment without UVR over the following days. Solar UVR inhibited the growth up to 40%, reduced trichome length by up to 49% and depressed the differentiation of heterocysts. Negligible concentrations of UV-absorbing compounds were found even in the presence of UVR. During the first 2 d of exposure to natural levels of PAR, carotenoid concentrations increased but no prolonged increase was evident. Heterocyst formation was enhanced under elevated PAR levels that stimulated quantum yield and growth after an initial inhibition. Higher concentrations of carotenoids and a twofold increase in the carotenoid to chlorophyll a ratio provided protection from the high levels of solar PAR. Under radiation treatments with UVR the relatively greater decrease in chlorophyll a concentrations compared with the increase in carotenoids was responsible for the higher carotenoid: chlorophyll a ratio. Heterocyst formation was disrupted in the presence of solar UVR. However, the longer term impact of heterocyst disruption to the survival of Anabaena sp. requires further study.     

Reactions of quinones with some amino alcohols,thiols and a UV-Vis study

Abstract

In the present study, the reactions of 2,3-dichloro-1,4-naphthoquinone (DCNQ) with amino-1,2-propanediol and some thiols were investigated. Novel N-, N,S-, and S,O- substituted derivatives were obtained and the structures of all compounds were characterized by spectroscopic methods (FT-IR, ¹H NMR, ¹³ Baker, R. A.; Tatum, J. H.; Nemec, S. Antimicrobial Activity of Naphthoquinones from Fusaria. Mycopathologia 1990, 111, 9–15. DOI: 10.1007/BF02277294.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]C NMR, Mass spectroscopy) and microanalysis. The absorption behaviors of novel compounds were also investigated with UV-Vis spectroscopy in different solvents, such as ethanol, tetrahydrofuran and chloroform.     

Fractionation and identification of metalloproteins from a marine cyanobacterium

Trace metals are essential for the growth of marine cyanobacteria, being required for key cellular processes such as photosynthesis and respiration. Despite this, the metalloproteomes of marine cyanobacteria are at present only poorly defined. In this study, we have probed the major cobalt, iron, manganese, and nickel-binding proteins in the marine cyanobacterium Synechococcus sp. WH8102 by using two different fractionation approaches combined with peptide mass fingerprinting. For the identification of intact metalloproteins, multidimensional native chromatography was used to fractionate the proteome, followed by inorganic mass spectrometry to identify metal-enriched fractions. This approach led to the detection of nickel superoxide dismutase together with its predicted cofactor. We also explored the utility of immobilized metal affinity chromatography (IMAC) to isolate subpopulations of proteins that display affinity for a particular metal ion. We conclude that low-resolution 2D liquid chromatography is a viable fractionation technique to correlate relatively low-abundance metal ions with their few cellular destinations (e.g. Ni), but challenges remain for more abundant metals with multiple destinations such as iron. IMAC has been shown as a useful pre-fractionation technique to screen for proteins with metal-binding capacity, and may become a particularly valuable tool for the identification of metal-trafficking proteins.