Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.     

Protonation state and structural changes of the tetrapyrrole chromophore during the Pr --> Pfr phototransformation of phytochrome: a resonance Raman spectroscopic study

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.     

FTIR study of the photoinduced processes of plant phytochrome phyA using isotope-labeled bilins and density functional theory calculations

Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global ¹³C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE→ZZZ), which involves only small protein structural changes.     

Chromophore Structure of Cyanobacterial Phytochrome Cph1 in the Pr State: Reconciling Structural and Spectroscopic Data by QM/MM Calculations

A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His²⁶⁰ in the chromophore-binding pocket such that either the δ-nitrogen (M-HSD) or the ɛ-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His²⁶⁰ with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.Abbreviations: Agp1, phytochrome from Agrobacterium tumefaciens; α-CPC, α-subunit of C-phycocyanin; BV, biliverdin IXα; B3LYP, three-parameter exchange functional according to Becke, Lee, Yang, and Parr; DFT, density functional theory; DrBphP, phytochrome from Deinococcus radiodurans; GAF, domain found in cGMP-specific phosphodiesterases; MM, molecular mechanics; MD, molecular dynamics; N-H ip, N-H in-plane bending; PCB, phycocyanobilin; PED, potential energy distribution; phyA, plant phytochrome; Pr, Pfr, red- and far-red absorbing parent states of phytochrome; PΦB, phytochromobilin; QM, quantum mechanics; RMSD, root mean-square deviation; RR, resonance Raman     

Light-induced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore

The Pr --> Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R(C) were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 micros and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R(C), respectively. Resonance Raman measurements of Meta-R(C) indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R(C) are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr --> Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.     

Resonance Raman spectroscopic study of the tryptic 39-kDa fragment of phytochrome

The 39-kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state P(r) reveals far-reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the P(r) phototransformation, denoted as P(bl), is more closely related to that of the P(fr) precursor of the native phytochrome, i.e. meta-R(C), rather than to that of P(fr) itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in P(bl) so that, unlike in P(fr), the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of P(fr), cannot be established in the 39-kDa fragment due to the lack of the major C-terminal part of the protein. These findings, furthermore, support the view that the meta-R(C)-->P(fr) transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome.     

Resonance Raman analysis of the Pr and Pfr forms of phytochrome

Resonance Raman vibrational spectra of the Pr and Pfr forms of oat phytochrome have been obtained at room temperature. When Pr is converted to Pfr, new bands appear in the C = C and C = N stretching region at 1622, 1599, and 1552 cm-1, indicating that a major structural change of the chromophore has occurred. The Pr to Pfr conversion results in an 11 cm-1 lowering of the N-H rocking band from 1323 to 1312 cm-1. Normal mode calculations correlate this frequency drop with a Z----E isomerization about the C15 = C16 bond. A line at 803 cm-1 in Pr is replaced by an unusually intense mode at 814 cm-1 in Pfr. Calculations on model tetrapyrrole chromophores suggest that these low-wavenumber modes are hydrogen out-of-plane (HOOP) wagging vibrations of the bridging C15 methine hydrogen and that both the intensity and frequency of the C15 HOOP mode are sensitive to the geometry around the C14-C15 and C15 = C16 bonds. The large intensity of the 814-cm-1 mode in Pfr indicates that the chromophore is highly distorted from planarity around the C15 methine bridge. If the Pr----Pfr conversion does involve a C15 = C16 Z----E isomerization, then the intensity of the C15 HOOP mode in Pfr argues that the chromophore has an E,anti conformation. On the basis of a comparison with the vibrational calculations, the low frequency (803 cm-1) and the reduced intensity of the C15 HOOP mode in Pr suggest that the chromophore in Pr adopts the C15-Z,syn conformation.     

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.     

Solid-state NMR spectroscopic study of chromophore-protein interactions in the Pr ground state of plant phytochrome A

Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1H-15N experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.     

Arabidopsis phytochromes C and E have different spectral characteristics from those of phytochromes A and B

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.     

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.     

Fourier transform resonance Raman spectroscopy of phytochrome.

The Pr and Pfr forms of phytochrome in H2O and D2O have been studied by Fourier transform resonance Raman spectroscopy with near-infrared excitation (1064 nm). It is demonstrated that this technique is a powerful method for analyzing the chromophore structures of photosensitive pigments. The high spectral quality allows discussion of vibrational assignments based on an empirical approach using previously published data obtained from model compounds. The reduction in intensity of a high-frequency band assigned to the ring-C/D methine bridge vibration is an indication for the non-coplanarity of the ring D in Pfr. The high intensity of a C-H out-of-plane vibration also supports this hypothesis. In Pr, a broad peak at approximately 1100 cm-1 is assigned to an out-of-plane vibration of a strongly hydrogen-bonded pyrrole C=NH+ group. It is missing in Pfr, suggesting deprotonation of the corresponding ring during the transformation from Pr to Pfr.     

The system of phytochromes: photobiophysics and photobiochemistry in vivo.

Phytochrome is a key photoregulation pigment in plants which determines the strategy of their development throughout their life cycle. The major achievement in the recent investigations of the pigment is the discovery of its structural and functional heterogeneity: existence of a family of phytochromes (phyA-phyE) differing by the apoprotein was demonstrated. We approach this problem by investigating the chromophore component of the pigment with the use of the developed method of in vivo low-temperature fluorescence spectroscopy of phytochrome. In etiolated plants, phytochrome fluorescence was detected and attributed to its red-light absorbing form (Pr) and the first photoproduct (lumi-R), and a scheme of the photoreaction in phytochrome, a distinction of which is the activation barrier in the excited state, was put forward. It was found that the spectroscopic and photochemical characteristics of Pr depend on the plant species and phytochrome mutants and overexpressors used, on localization of the pigment in organs and tissues, plant age, effect of preillumination and other physiological factors. This variability of the parameters was interpreted as the existence of at least two phenomenological Pr populations, which differ by their spectroscopic characteristics and activation parameters of the Pr --> lumi-R photoreaction (in particular, by the extent of the Pr --> lumi-R photoconversion at low temperatures, gamma1): the longer-wavelength major and variable by its content in plant tissues Pr' with gamma1 = 0.5 and the shorter-wavelength minor relatively constant Pr" with gamma1 < or = 0.05. The analysis of the phytochrome mutants and overexpressors allows a conclusion that phytochrome A (phyA), which dominates in etiolated seedlings, is presented by two isoforms attributed to Pr' and Pr" (phyA' and phyA", respectively). Phytochrome B (phyB) accounts for less than 10% of the total phytochrome fluorescence and belongs to the Pr" type. It is also characterized by the relatively low extent of the Pr photoconversion into the far-red-light absorbing physiologically active phytochrome form, Pfr. Fluorescence of the minor phytochromes (phyC-phyE) is negligible. The recently discovered phytochrome of the cyanobacterium Synechocystis also belongs to the phenomenological Pr" type. PhyA' is a light-labile and soluble fraction, while phyA" is a relatively light-stable and, possibly, membrane (protein)-associated. Experiments with transgenic tobacco plants overexpressing full-length and C- and N-terminally truncated oat phytochrome A suggest that phyA' and phyA" might differ by the post-translational modification of the small N-terminal segment (amino acid residues 7-69) of the pigment. PhyA' is likely to be active in the de-etiolation processes while phyA" together with phyB, in green plants as revealed by the experiments on transgenic potato plants and phytochrome mutants of Arabidopsis and pea with altered levels of phytochromes A and B and modified phenotypes. And finally, within phyA', there are three subpopulations which are, possibly, different conformers of the chromophore. Thus, there is a hierarchical system of phytochromes which include: (i) different phytochromes; (ii) their post-translationally modified states and (iii) conformers within one molecular type. Its existence might be the rationale for the multiplicity of the photoregulation reactions in plants mediated by phytochrome.     

Assembly of synthetic locked phycocyanobilin derivatives with phytochrome in vitro and in vivo in Ceratodon purpureus and Arabidopsis

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.     

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.     

Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings.

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.     

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.     

Phytochrome assembly. Defining chromophore structural requirements for covalent attachment and photoreversibility.

Assembly of holophytochrome in the plant cell requires covalent attachment of the linear tetrapyrrole chromophore precursor, phytochromobilin, to a unique cysteine in the nascent apoprotein. In this investigation we compare chromophore analogs with the natural chromophore precursor for their ability to attach covalently to recombinant oat apophytochrome and to form photoactive holoproteins. Ethylidene-containing analogs readily form covalent adducts with apophytochrome, whereas chromophores lacking this double bond are poor substrates for attachment. Kinetic measurements establish that although the chromophore binding site on apophytochrome is best tailored to phytochromobilin, apophytochrome will accommodate the two analogs with modified D-rings, phycocyanobilin and phycoerythrobilin. The phycocyanobilin-apophytochrome adduct is photoactive and undergoes a light-induced protein conformational change similar to the native holoprotein. By contrast, the phycoerythrobilin adduct is locked into a photochemically inactive protein conformation that is similar to the red light-absorbing Pr form of phytochrome. These results support the hypothesis that the photoconversion from Pr to Pfr, the far red light- absorbing form of phytochrome, involves the photoisomerization of the C15 double bond. Knowledge gained from these studies provides impetus for rational design of chromophore analogs whose insertion into apophytochrome should elicit profound changes in light-mediated plant growth and development.     

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.     

Surface-enhanced resonance Raman spectroscopy of phycocyanin and allophycocyanin

High quality surface-enhanced resonance Raman (SERR) spectra were recorded from native and denatured phycocyanin and allophycocyanin on ascorbic acid treated silver hydrosols. The visible-excited SERR and resonance Raman (RR) spectra of the phycobiliproteins were very similar, indicating a predominantly electromagnetic surface enhancement mechanism. Investigation of pH-induced denaturation ofx allophycocyanin has shown that even small differences in protein/chromophore conformational are sensitively reflected by the SERR spectra. Concerning the adsorption of the protein to the metal surface, the experiments have shown that: (i) there is limited possibility for changing protein conformation during the adsorption process, (ii) there are no changes after the protein has been adsorbed onto the silver surface and (iii) for each protein an optimal activation of the silver sol has to be found for recording proper SERR spectra. The results obtained on phycobiliproteins are also discussed in connection with the interpretation of phytochrome Raman spectra.