phycocyanobilin and phytochromobilin

The geometric relaxation following light absorption of the biliverdin, phycocyanobilin and phytochromobilin tetrapyrrole chromophores of bacterial, cyanobacterial and plant phytochromes has been investigated using density functional theory methods. Considering stereoisomers relevant for both red-absorbing Pr and far-red-absorbing Pfr forms of the photoreceptor, it is found that the initial excited-state evolution is dominated by torsional motion at the C10-C11 bond. This holds true for all three chromophores and irrespective of which configuration the chromophores adopt. This finding suggests that the photochromic cycling of phytochromes between their Pr and Pfr forms, which is known to be governed by Z/E photoisomerizations at the C15-C16 bond, relies on interactions between the chromophore and the protein to prevent photoisomerizations at C10-C11. Further, it is found that the uneven distribution of positive charge between the pyrrole rings is a major factor for the photochemical reactivity of the C10-C11 bond.

Topics: Bile Pigments; Biliverdine; Models, Molecular; Phycobilins; Phycocyanin; Phytochrome; Quantum Theory; Stereoisomerism; Thermodynamics

The resonance Raman spectra of the Pr state of the N-terminal 65-kDa fragment of plant phytochrome phyA have been measured and analyzed in terms of the configuration and conformation of the tetrapyrroles methine bridges. Spectra were obtained from phyA adducts reconstituted with the natural chromophore phytochromobilin as well as phycocyanobilin and its isotopomers labeled at the terminal methine bridges through (13)C/(12)C and D/H substitution. Upon comparing the resonance Raman spectra of the various phyA adducts, it was possible to identify the bands that originate from normal modes dominated by the stretching coordinates of the terminal methine bridges A-B and C-D. Quantum chemical calculations of the isolated tetrapyrroles reveal that these modes are sensitive indicators for the methine bridge configuration and conformation. For all phyA adducts, the experimental spectra of Pr including this marker band region are well reproduced by the calculated spectra obtained for the ZZZasa configuration. In contrast, there are substantial discrepancies between the experimental spectra and the spectra calculated for the ZZZssa configuration, which has been previously shown to be the chromophore geometry in the Pr state of the bacterial, biliverdin-binding phytochrome from Deinococcus radiodurans (Wagner, J. R., J. S. Brunzelle, K. T. Forest, R. D. Vierstra. 2005. Nature. 438:325-331). The results of this work, therefore, suggest that plant and bacterial (biliverdin-binding) phytochromes exhibit different structures in the parent state although the mechanism of the photoinduced reaction cycle may be quite similar.

Topics: Biliverdine; Biophysics; Deinococcus; Light; Models, Chemical; Molecular Conformation; Photochemistry; Phycobilins; Phycocyanin; Phytochrome; Plants; Quantum Theory; Spectrophotometry; Spectrum Analysis, Raman

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.

Topics: Bacteria; Bile Pigments; Biliverdine; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Heme Oxygenase (Decyclizing); Phycobilins; Phycocyanin; Phycoerythrin; Phytochrome; Protein Engineering; Tetrapyrroles

The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.

Topics: Apoproteins; Bacterial Proteins; Biliverdine; Cyanobacteria; Genetic Engineering; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Phytochrome; Protein Kinases; Pyrroles; Tetrapyrroles

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C(15)-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-)(1) in P(r) and P(fr), respectively, are assigned as C(15)-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-)(1) in P(r) and at 672 and 654 cm(-)(1) in P(fr) are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximately 1300 cm(-)(1) in P(r) are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P(r) and P(fr) are C(15)-Z,syn and C(15)-E,anti, respectively. The intensities of the C(15)-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P(r) and P(fr) suggest that the initial photochemistry involves simultaneous bond rotations at the C(15)-methine bridge coupled to C(15)-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C(15)-E,anti P(fr) chromophore structure indicated by the intense 814 cm(-1) C(15) HOOP mode suggest that the excited state of P(fr) and its photoproduct states are strongly coupled.

Topics: Avena; Biliverdine; Deuterium; Ethylenes; Hydrogen; Light; Photochemistry; Phycobilins; Phycocyanin; Phytochrome; Plant Growth Regulators; Pyrroles; Recombinant Proteins; Spectrum Analysis, Raman; Tetrapyrroles

The red/far-red light absorbing phytochromes play a major role as sensor proteins in photomorphogenesis of plants. In Arabidopsis the phytochromes belong to a small gene family of five members, phytochrome A (phyA) to E (phyE). Knowledge of the dynamic properties of the phytochrome molecules is the basis of phytochrome signal transduction research. Beside photoconversion and destruction, dark reversion is a molecular property of some phytochromes. A possible role of dark reversion is the termination of signal transduction. Since Arabidopsis is a model plant for biological and genetic research, we focussed on spectroscopic characterization of Arabidopsis phytochromes, expressed in yeast. For the first time, we were able to determine the relative absorption maxima and minima for a phytochrome C (phyC) as 661/725 nm and for a phyE as 670/724 nm. The spectral characteristics of phyC and E are strictly different from those of phyA and B. Furthermore, we show that both phyC and phyE apoprotein chromophore adducts undergo a strong dark reversion. Difference spectra, monitored with phycocyanobilin and phytochromobilin as the apoprotein's chromophore, and in vivo dark reversion of the Arabidopsis phytochrome apoprotein phycocyanobilin adducts are discussed with respect to their physiological function.

Topics: Apoproteins; Arabidopsis; Arabidopsis Proteins; Biliverdine; Darkness; Half-Life; Kinetics; Multigene Family; Photoreceptor Cells; Phycobilins; Phycocyanin; Phytochrome; Phytochrome A; Phytochrome B; Pyrroles; Signal Transduction; Spectrophotometry; Tetrapyrroles; Transcription Factors; Yeasts

The photoconversion of phytochrome (phytochrome A from Avena satina) from the inactive (Pr) to the physiologically active form (Pfr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic temperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D exchange on the resonance Raman spectra. For the first time, reliable spectra of the "bleached" intermediates meta-R(A) and meta-R(C) were obtained. The vibrational bands in the region 1300-1700 cm(-)(1), which is particularly indicative of structural changes in tetrapyrroles, were assigned on the basis of recent calculations of the Raman spectra of the chromophore in C-phycocyanin and model compounds [Kneip, C., Hildebrandt, P., Németh, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental resonance Raman spectra Pr are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the chromophore structure in this parent state of phytochrome. Furthermore, marker bands could be identified that are of high diagnostic value for monitoring structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states Pr and Pfr but also in all intermediates the chromophore is protonated at the pyrroleninic nitrogen. The spectral changes observed for lumi-R confirm the view that the photoreaction of Pr is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-R(A) includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R(C) is accompanied by structural adaptations of the pyrrole rings B and C in the protein pocket. The far-reaching similarities between the chromophores of meta-R(A) and Pfr suggest that in the step meta-R(A) --> Pfr the ultimate structural changes of the protein matrix occur.

Topics: Avena; Biliverdine; Deuterium; Light; Photochemistry; Phycobilins; Phycocyanin; Phytochrome; Phytochrome A; Protons; Pyrroles; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Temperature; Tetrapyrroles

The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [Pr]/far-red-absorbing phytochrome [Pfr]) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the Pr-->Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.

Topics: Avena; Biliverdine; Bryopsida; Chromatography, Affinity; Dimerization; DNA, Plant; Immunosorbent Techniques; Kinetics; Open Reading Frames; Photolysis; Phycobilins; Phycocyanin; Phytochrome; Pyrroles; Recombinant Proteins; Saccharomyces cerevisiae; Spectrophotometry, Atomic; Tetrapyrroles

The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.

Topics: Biliverdine; Blotting, Western; Cloning, Molecular; Gene Expression; Kinetics; Molecular Structure; Mutation; Photolysis; Phycobilins; Phycocyanin; Phytochrome; Pichia; Plant Proteins; Polymerase Chain Reaction; Pyrroles; Recombinant Proteins; Saccharomyces cerevisiae; Sequence Deletion; Solanum tuberosum; Spectrophotometry; Tetrapyrroles

The recombinant 65-kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (PphiB) and phycocyanobilin (PCB), respectively. The Pr and Pfr states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA oat phytochrome demonstrating that the deletion of the C-terminal half of phyA does not alter the structure of the chromophore site within the N-terminal half. Most of the RR spectral changes observed upon replacing PphiB by PCB can be attributed exclusively to altered vibrational mode compositions due to the different ring D substitutions (vinyl vs. ethyl), implying that the chromophore structures are largely the same for PphiB- and PCB-assembled phytochromes. Only in the Pr state may the RR spectral changes also reflect subtle differences of the PphiB and PCB conformations in the 65-kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups.

Topics: Avena; Biliverdine; Binding Sites; Molecular Structure; Phycobilins; Phycocyanin; Phytochrome; Phytochrome A; Pichia; Protein Conformation; Pyrroles; Recombinant Proteins; Spectrum Analysis, Raman; Tetrapyrroles

Compared with phytochromes isolated from etiolated higher plant tissues and a number of lower plant species, the absorption spectrum of phytochrome isolated from the unicellular green alga Mesotaenium caldariorum is blue-shifted (Kidd, D. G., and Lagarias, J. C. (1990) J. Biol. Chem. 265, 7029-7035). The present studies were undertaken to determine whether this blue shift is due to a chromophore other than phytochromobilin or reflects a different protein environment for the phytochromobilin prosthetic group. Using reversed phase high performance liquid chromatography, we show that soluble protein extracts prepared from algal chloroplasts contain the enzyme activities for ferredoxin-dependent conversions of biliverdin IXalpha to (3Z)-phytochromobilin and (3Z)-phytochromobilin to (3Z)-phycocyanobilin. In vitro assembly of recombinant algal apophytochrome was undertaken with (3E)-phytochromobilin and (3E)-phycocyanobilin. The difference spectrum of the (3E)-phycocyanobilin adduct was indistinguishable from that of phytochrome isolated from dark-adapted algal cells, while the (3E)-phytochromobilin adduct displayed red-shifted absorption maxima relative to purified algal phytochrome. These studies indicate that phycocyanobilin is the immediate precursor of the green algal phytochrome chromophore and that phytochromobilin is an intermediate in its biosynthesis in Mesotaenium.

Topics: Biliverdine; Chlorophyta; Chloroplasts; Chromatography, High Pressure Liquid; Isomerism; Kinetics; Models, Chemical; Phycobilins; Phycocyanin; Phytochrome; Phytochrome A; Pyrroles; Tetrapyrroles

Incubation of recombinant apophytochrome with the phycobiliprotein chromophore precursor phycoerythrobilin produces a covalent adduct that exhibits a fluorescence excitation maximum at 576 nm and an emission maximum at 586 nm. Using these fluorescence parameters, we have developed a kinetic assay for quantitative analysis of the assembly of the plant photoreceptor phytochrome in real time. Kinetic measurements performed with different phycoerythrobilin concentrations confirm that bilin attachment to apophytochrome involves two steps, an initial formation of a reversible non-covalent complex followed by thioether bond formation. The kinetic constants for both steps of phycoerythrobilin attachment to apophytochrome were estimated with this assay. Methodology for determining the kinetic constants for the assembly of both the natural phytochrome chromophore precursor, phytochromobilin, and the analog phycocyanobilin is also described. Since the latter two bilins yield covalent, nonfluorescent adducts with apophytochrome, their co-incubation with phycoerythrobilin reduces the rate of formation of the fluorescent phycoerythrobilin adduct in an irreversible, competitive manner. Competition experiments were also performed with biliverdin, a structurally related bilin which does not form a covalent adduct with apophytochrome. Such measurements show that biliverdin reversibly binds to apophytochrome with a submicromolar binding constant, an affinity which is very similar to that of phytochromobilin. The utility of this fluorescence assay for identification of novel inhibitors of phytochrome assembly and for characterization of the structural features of both bilin and apophytochrome necessary for photoreceptor assembly is discussed.

Topics: Apoproteins; Biliverdine; Binding, Competitive; Fluorometry; Kinetics; Models, Chemical; Phycobilins; Phycocyanin; Phycoerythrin; Phytochrome; Pyrroles; Recombinant Proteins; Tetrapyrroles

The biological activity of the plant photoreceptor phytochrome requires the specific association of a linear tetrapyrrole prosthetic group with a large apoprotein. As an initial step to develop an in vivo assay system for structure-function analysis of the phytochrome photoreceptor, we undertook experiments to reconstitute holophytochrome in the yeast Saccharomyces cerevisiae. Here we show that yeast cells expressing recombinant oat apophytochrome A can take up exogenous linear tetrapyrroles, and, in a time-dependent manner, these pigments combine with the apoprotein to form photoactive holophytochrome in situ. Cell viability measurements indicate that holophytochrome assembly occurs in living cells. Unlike phytochrome A in higher plant tissue, which is rapidly degraded upon photoactivation, the reconstituted photoreceptor appears to be light stable in yeast. Reconstitution of photoactive phytochrome in yeast cells should enable us to exploit the power of yeast genetics for structure-function dissection of this important plant photoreceptor.

Topics: Apoproteins; Avena; Biliverdine; Photoreceptor Cells; Phycobilins; Phycocyanin; Phytochrome; Pyrroles; Recombinant Proteins; Saccharomyces cerevisiae; Spectrum Analysis; Tetrapyrroles; Time Factors

The phytochrome chromophore precursor, 3E-phytochromobilin, and the phycobiliprotein chromophore precursors, 3E-phycocyanobilin and 3E-phycoerythrobilin, are enzymatically converted to novel rubinoid products by purified rat liver biliverdin reductase. Phytochromobilin and phycocyanobilin are particularly good substrates for biliverdin reductase with Km and Vmax values very similar to those of the natural substrate, biliverdin IX alpha. Phycoerythrobilin is the least preferred of the three bilin substrates. 1H NMR spectroscopy of phycocyanorubin, the product of phycocyanobilin catalysis by biliverdin reductase, and comparison of absorption spectra of all three rubinoid products reveal that the C10 methine bridge is selectively reduced by biliverdin reductase without altering the A-ring ethylidene substituent. In vitro phytochrome assembly experiments demonstrate that the phytorubin products do not form photoactive adducts with recombinant apophytochrome. These results suggest that ectopic expression of biliverdin reductase in plants will prevent assembly of the functional photoreceptor and thus will potentially alter light-mediated plant growth and development.

Topics: Animals; Apoproteins; Biliverdine; Catalysis; Eukaryota; Liver; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Phycobilins; Phycocyanin; Phycoerythrin; Phytochrome; Plants; Pyrroles; Rats; Substrate Specificity; Tetrapyrroles