Photochemistry of 124 kilodalton Avena phytochrome in vitro

The photochemical properties of purified 124 kilodalton (kD) Avena cv Garry phytochrome are examined and compared with those of the proteolytically degraded 118/114 kD species. The proportion of the chromoprotein in the far red absorbing form, Pfr, following saturating red irradiation is 0.86 for 124 kD phytochrome, substantially higher than the values of 0.79 determined here and 0.75 reported in the literature for 118/114 kD preparations. The ratio of the quantum yields for Pr to Pfr phototransformation and for Pfr to Pr phototransformation (r/fr) is 1.76 for the 124 kD molecule and 0.98 for the 118/114 kD species. Based on extinction coefficients determined using the Lowry assay as a measure of protein weight, the individual phototransformation quantum yields for 124 kD phytochrome are 0.17 for Pr → Pfr (r) and 0.10 for Pfr → Pr (fr). Comparison of these quantum yields with those of the 118/114 kD species (where r = fr = ~0.11) indicates that proteolytic degradation of the 124 kD molecule to the 118/114 kD species significantly affects only r. Therefore, the lower proportion of Pfr at photoequilibrium observed for 118/114 kD preparations is explained mainly in terms of a reduced efficiency of Pr → Pfr phototransformation. The absolute Pfr absorbance spectrum for 124 kD phytochrome obtained by correcting the measured spectrum for residual Pr exhibits a maximum at 730 nm and differs from previous absolute Pfr spectra for both `120' kD and 60 kD phytochrome in that it lacks a shoulder in the red region of the spectrum.     

Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro

Light-mediated conformational changes in highly purified 124-kDa phytochrome preparations from etiolated oat seedlings have been identified by steric exclusion high performance liquid chromatography and limited proteolytic studies. Steric exclusion high performance liquid chromatography studies of oat and rye phytochromes show photoreversible changes in retention times, with the red absorbing form of phytochrome (Pr form) eluting later than the far red absorbing form of phytochrome produced by saturating red light illumination of Pr (Pfr form) in a variety of different mobile phase buffers. Molecular mass calibration with globular protein standards in Tris-glycol buffers provides estimates of 318-349 and 363-366 kDa for the molecular sizes of the Pr and Pfr forms, respectively. These analyses support earlier studies that phytochrome is a nonglobular homodimer of 124-kDa subunits in vitro. Limited proteolytic dissection of phytochrome in nondenaturing buffers with seven different endoproteases provides evidence for two "operational" domains within the 124-kDa subunit with molecular mass values of 69-72 and 52-55 kDa. The larger 69-72-kDa domain contains the site for the chromophore attachment as shown by gel electrophoresis derived enzyme-linked immunosorbent assay utilizing site-directed rabbit antiserum to a synthetic undecapeptide which is homologous with the chromophore binding site on oat phytochrome. This chromophore domain exhibits a compact structure, resistant to further proteolysis except near its N terminus. By contrast, the 52-55-kDa nonchromophore domain contains multiple sites for further proteolytic cleavage as revealed by rapid cleavage to smaller polypeptide fragments. Detailed kinetic analyses of the limited proteolytic cleavage of phytochrome with four endoproteases, subtilisin BPN', thermolysin, trypsin, and clostripain, has mapped specific regions within the 124-kDa subunit that participate in light-induced conformational changes. These include a 4-10-kDa region near the N terminus of the chromophore binding domain and at least two regions within the nonchromophore domain. A comprehensive peptide map of the oat phytochrome subunit is presented, which incorporates the results of these proteolytic studies with the recent, yet unpublished sequence analyses of Avena phytochrome cDNA clones which show the N-terminal localization of the chromophore binding site (Hershey, H. P., Colbert, J. T., Lissemore, J. L., Barker, R. F., and Quail, P. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2332-2336).     

Differences in the physical properties of native and partially degraded phytochrome as probed by their differential sensitivity to permanganate oxidation

The differential sensitivities to permanganate oxidation of the red and far-red forms of native phytochrome from Avena sativa L. cv Mulaga (isolated as Pfr from red-irradiated tissue) and of partially degraded phytochrome (isolated as Pr from nonirradiated tissue) were determined. The far-red absorbing form of partially degraded phytochrome was 5 times more sensitive than its red-absorbing form, while both the far-red and red forms of native phytochrome exhibited identical sensitivity. The present data obtained with partially degraded phytochrome are in apparent agreement with the data and model of Hahn, Kang, and Song (1980 Biochem Biophys Res Commun 97: 1317-1323). Their model suggests that the chromophore of the red-absorbing form of phytochrome is buried in a hydrophobic crevice in the protein, while that of the far-red form is exposed. The data obtained with native phytochrome, however, are at variance with their model. Our data obtained with native phytochrome suggests that the chromophore of the red and the far-red absorbing forms of native phytochrome both are in a relatively protected environment and that only following partial proteolytic degradation of the phytochrome does the chromophore of its far-red form become relatively more exposed. The protective influence of the labile peptide could either be direct, because of its close physical proximity to the chromophore, or indirect, resulting in an alteration in chromophore-protein interaction.     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification

Accompanying the phototransformation of native 124-kilodalton (kDa) oat phytochrome from red-absorbing form (Pr) to far-red-absorbing form (Pfr), there is a photoreversible change in circular dichroism (CD) in the far-UV region indicative of a 3% increase in alpha-helical folding of apoprotein. To elucidate the conformational change involved in the phytochrome phototransformation, several monoclonal antibodies have been used as epitope-specific probes. Monoclonal antibody oat-25 suppressed the photoreversible CD spectral change using phytochrome with an A666/A280 as Pr of 1.13. Monoclonal antibodies oat-22, oat-13, and oat-31 did not significantly affect the CD spectral change of phytochrome. Oat-25 requires an epitope near the N-terminus of phytochrome. Oat-22, oat-13, and oat-31 recognize epitopes on the N-terminus, chromophore-containing half of phytochrome, albeit further removed from the N-terminus than that recognized by oat-25. Interestingly, oat-13 and oat-31 did, however, induce a time-dependent decrease in the far-UV CD, apparently due to aggregation of phytochrome (both Pr and Pfr forms). Monoclonal antibodies oat-26 and oat-28, which recognize epitopes on the C-terminus half of phytochrome, also did not suppress the photoreversible CD change, although oat-26 and oat-28 slightly inhibited it. The photoreversible CD spectral change can also be inhibited by sodium borohydride, which bleaches the chromophore by reducing it, and by tetranitromethane, which oxidizes the chromophore of phytochrome. Although explanations of these results based on indirect interactions between the chromophore and the N-terminus segment are possible, we propose that an additional alpha-helical folding of the Pfr form of the phytochrome may result from a photoreversible interaction between the Pfr form of the chromophore and the N-terminus segment.     

Chromophore topography and secondary structure of 124-kilodalton Avena phytochrome probed by Zn2(+)-induced chromophore modification

The relative extent of chromophore exposure of the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of 124-kDa oat phytochrome and the secondary structure of the phytochrome apoprotein have been investigated by using zinc-induced modification of the phytochrome chromophore. The absence of bleaching of Pr in the presence of a 1:1 stoichiometric ratio of zinc ions, in contrast to extensive spectral bleaching of the Pfr form, confirms previous reports of differential exposure of the Pfr chromophore relative to the Pr chromophore [Hahn et al. (1984) Plant Physiol. 74, 755-758]. The emission of orange fluorescence by zinc-chelated Pfr indicates that the Pfr chromophore has been modified from its native extended/semi-extended conformation to a cyclohelical conformation. Circular dichroism (CD) analyses of native phytochrome in 20 mM Tris buffer suggests that the Pr-to-Pfr phototransformation is accompanied by a photoreversible change in the far-UV region consistent with an increase in the alpha-helical folding of the apoprotein. The secondary structure of phytochrome in Tris buffer, as determined by CD, differs slightly from that of phytochrome in phosphate buffer, suggesting that phytochrome is a conformationally flexible molecule. Upon the addition of a 1:1 molar ratio of zinc ions to phytochrome, a dramatic change in the CD of the Pfr form is observed, while the CD spectrum of the Pf form is unaffected. Analysis of the bleached Pfr CD spectrum by the method of Chang et al. (1978) reveals that chelation with zinc ions significantly alters the secondary structure of the phytochrome molecule, specifically by increasing the beta-sheet content primarily at the expense of alpha-helical folding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Phosphorylation of Avena phytochrome in vitro as a probe of light-induced conformational changes

A polycation-dependent protein kinase was found to be associated with purified phytochrome preparations from etiolated Avena seedlings. This kinase and three mammalian protein kinases, the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and a Ca2+-activated phospholipid-dependent protein kinase, were used to probe light-induced conformational changes in 124-kilodalton Avena phytochrome in vitro. The red absorbing form of phytochrome (Pr) was found to be a substrate for all four protein kinases. Although the far-red absorbing form of phytochrome (Pfr) was as good a substrate as Pr with the cAMP-dependent protein kinase, the Pfr form was poorly phosphorylated by the other three protein kinases. Serine is the major amino acid residue phosphorylated on phytochrome regardless of the form of phytochrome used as substrate. Peptide mapping revealed that the sites of phosphorylation catalyzed by the cAMP-dependent protein kinase differ for Pr and Pfr forms of phytochrome. For the Pr form, the preferred site(s) of phosphorylation was near the amino terminus of the 124-kilodalton subunit. Upon photo-conversion to Pfr, this site can no longer be phosphorylated easily and a new phosphorylation site in the COOH-terminal nonchromophore domain of the molecule becomes accessible to the cAMP-dependent protein kinase. These studies of the phosphorylation of phytochrome provide a new means to study the effect of light absorption by phytochrome on the molecular conformation of the protein. The potential physiological implications of differential phosphorylation of Pr and Pfr await elucidation.     

Interactions between native oat phytochrome and tetrapyrroles

The suggestion, that the increase in the far-UV CD signal of the 124 kDa oat phytochrome upon phototransformation of the Pr to Pfr form is possibly due to the chromophore interaction with the N-terminus segment of the phytochrome protein in the Pfr from (Chai, Y.G., Song, P.S., Cordonnier, M.-M. and Pratt, L.H. (1987) Biochemistry 26, 4947-4952), has been investigated by measuring the circular dichroism in the absence of exogenous tetrapyrrolic chromophores (bilirubin, biliverdin, chlorophyllin and hemin). Open tetrapyrrolic chromophores (bilirubin and biliverdin) did not have any significant effect on the phototransformability of the far-UV CD signal of the phytochrome, whereas closed tetrapyrroles (chlorophyllin and hemin) almost completely blocked the increase in the far-UV CD signal upon Pr to Pfr phototransformation. However, closed tetrapyrroles had no effect on the decrease in the CD signal upon Pfr to Pr photoconversion. Secondary structure analysis showed that the alpha-helix content of both Pr and Pfr forms of phytochrome (with 53 and 56% alpha-helical content, respectively) increased to 62% when a 50-fold molar excess of chlorophyllin was added to them separately. Spectral phototransformation of phytochrome was not affected in the presence of tetrapyrroles, except in the case of hemin. A 50-fold molar mass of hemin caused a significant bleaching of the Pfr form of phytochrome but not that of the Pr form. These results suggest that the chromophore-protein interaction is significantly altered during the phototransformation of phytochrome.     

Molecular topography of phytochrome as deduced from the tritium-exchange method

The hydrogen-tritium-exchange measurements on phytochrome have been performed to detect the conformational differences between the red-absorbing (Pr) and the far-red absorbing (Pfr) forms of phytochrome. The large and small Pfr molecules revealed more exchangeable protons that did the corresponding Pr molecules by 96 and 70 protons, respectively. These results suggest that the Pr leads to Pfr phototransformation is accompanied by an additional exposure of the peptide chains in the Pfr molecule. Of 1682 theoretically exchangeable hydrogens in undegraded phytochrome, only 442 (26%) and 346 (21%) protons were found to be exchangeable (excluding instantaneously exchangeable protons that cannot be determined by the present method). Thus, the phytochrome protein appears to be compact and highly folded. The kinetic analyses of the tritium exchange-out curves indicate that two kinetically different groups are responsible for the conformational differences between the Pr and Pfr forms of phytochrome. These components are due to (1) the exposure of hydrogen-bonded peptide segments (alpha helix and/or beta-pleated sheet) in the chromophore vicinity of Pfr and (2) the exposure of hydrogen-bonded peptide segments on the chromophore peptide domain as well as on the chromophore-free tryptic domain of undegraded phytochrome.     

Identification with Monoclonal Antibodies of a Second Antigenic Domain on Avena Phytochrome that Changes upon Its Photoconversion

An enzyme-linked immunosorbent assay that revealed an antigenic difference between the red-absorbing and far-red-absorbing forms of phytochrome (Pr and Pfr, respectively) near its amino terminus (Cordonnier M-M, H Greppin, LH Pratt 1985 Biochemistry 24: 3246-3253) was used to screen eight additional monoclonal antibodies directed to phytochrome from etiolated oats. While six of these antibodies detected Pr and Pfr with equal affinity, two of them, designated Oat-9 and Oat-16, bound to Pfr 1.6 to 2.3 times better than to Pr. Competitive enzyme-linked immunosorbent assays indicate (a) that Oat-9 and Oat-16 probably bind to the same domain on phytochrome and (b) that this domain is at least 3.5 nanometers away from the epitope near its amino terminus that was shown earlier to change upon phototransformation. Neither the absorbance spectra of Pr and Pfr, nor the rate of dark reversion of Pfr to Pr, was influenced by the presence of Oat-9. Immunoblotting of sodium dodecyl sulfate polyacrylamide gels after electrophoretic separation of phytochrome fragments obtained by endogenous proteolytic digestion indicates that Oat-16 binds to an epitope located on the chromophore half of this chromoprotein. The observation that the epitope recognized by Oat-9 and Oat-16 is also present on at least some of the immunochemically distinct phytochrome that is obtained from green oat shoots (Shimazaki Y, LH Pratt 1985 Planta 164: 333-344), together with the evidence that this epitope undergoes a change upon photoransformation, indicates that it may play an important role in phytochrome function.     

Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.     

Effect of malformin on phytochrome reactions in Vigna and Avena

The rate of destruction of the far red absorbing form of phytochrome(Pfr) in green or etiolated cuttings of Vigna radiata was slowerin the presence of malformin than in its absence. Malforminhad no effect on the accumulation of total phytochrome in thedark, or on the reaccumulation of phytochrome after destructionin red light. The amount of photoconversion of the red absorbingform of phytochrome (Pr) to Pfr or Pfr to Pr by given dosesof red or far red radiation was slightly but consistently lessin malformin-treated cuttings of V. radiata than in controls.Malformin had no effect on the rate of destruction or photoconversionof phytochrome in etiolated shoots of Avena sativa. The decreasein destruction rate of Pfr by malformin in V. radiata may contributeto the inhibition of dark abscission by malformin after lighttreatment. (Received October 3, 1979; )     

Modifications of Sulfhydryl Groups on Phytochrome and Their Influence on Physicochemical Differences between the Red- and Far-Red-Absorbing Forms

Phytochrome extracted from shoots of dark-grown rye (Secale cereale cv Rymin) and oat (Avena sativa cv Garry) as the far-red-form (Pfr) and/or under conditions conducive to oxidation exhibited a blue shift in the visible absorption maximum of its red-light-absorbing form (Pr) relative to that measured in vivo. This spectral alteration could not be reversed but could be prevented by inclusion of 10 millimolar diethyldithiocarbamate and 140 millimolar 2-mercaptoethanol in homogenization buffers. Similar blue shifts were induced in purified rye phytochrome by addition of the sulfhydryl-modifying reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). In spectrally normal phytochrome (i.e., no detectable blue shift), Pfr had three to four more sulfhydryls available for rapid reaction with DTNB than did Pr. This difference was maintained over a 2.5-hour time course. Phytochrome purified under conditions resulting in a blue-shifted Pr absorption maximum exhibited a decreased short-term reactivity of Pfr to DTNB. Comparison of the binding and elution of altered and unaltered phytochrome from agarose-immobilized Cibacron blue 3GA confirmed that the Pfr form of spectrally normal phytochrome had a greater affinity for the dye than did the Pr form but that spectral alteration of phytochrome was accompanied by a loss of this difference as evidenced by an increased binding of Pr to the dye. It was concluded that phytochrome has highly reactive sulfhydryl residues located on the portion of the protein that undergoes conformational changes on interconversion of Pr and Pfr and that these residues require rigorous protection in order to extract the native form of the protein from plant tissue.     

Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L

A spectral, immunochemical, and proteolytic characterization of native 120-kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified (~200-fold) in the phytochrome—far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr λ_max at 730 nanometers, a spectral change ratio (ΔA_r/ΔA_fr) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH₂ terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH₂ terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr λ_max to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH-terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.     

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.     

Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation.

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.     

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome.

Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.     

Chromophore: apoprotein interactions in phytochrome A*

Phytochromes are molecular light switches by virtue of their photochromic red/far-red reversibility. The His-324 residue next to the chromophore-linked Cys-323 plays a critical role in conferring photochromism to the tetrapyrrole chromophore in native phytochrome A. The chromophore appears to be enclosed between the amphiphilic α-helical chains in a hydrophobic pocket. The absorbance maxima of both the Pr and the Pfr forms of pea phytochrome A are blue-shifted by 10 and 20 nm, respectively, upon C-terminal truncation. We speculate that the quaternary structure of the phytochrome A molecule involves some interactions of the C-terminal half with the chromophore domain. The Pfr conformation of phytochrome includes an amphiphilic α-helix of the amino terminal chain, which occurs in 113 ms after picosecond photoisomerization of the Pr form. Compared to α-helical folding, unfolding of the α-helix occurs faster in about 310 μs upon phototransformation of the Pfr form of phytochrome A. The photochromic transformation of phytochrome A modulates protein kinase-catalysed phosphorylation sites in vivo and in vitro, but only a subtle local change in conformation is detectable in the phosphorylated phytochromes. This suggests that the post-translational modification serves as a surface label, rather than a transducer-activating trigger, for the recognition of a putative phytochrome receptor.     

Sterically locked synthetic bilin derivatives and phytochrome Agp1 from Agrobacterium tumefaciens form photoinsensitive Pr- and Pfr-like adducts

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.