Which factors determine the acidity of the phytochromobilin chromophore of plant phytochrome?

Quantum chemical calculations aimed at identifying the factors controlling the acidity of phytochromobilin, the tetrapyrrole chromophore of the plant photoreceptor phytochrome, are reported. Phytochrome is converted from an inactive (Pr) to an active form (Pfr) through a series of events initiated by a Z --> E photoisomerization of phytochromobilin, forming the Lumi-R intermediate, and much controversy exists as to whether the protonation state of the chromophore (cationic in Pr with all nitrogens protonated) changes during the photoactivation. Here, relative ground (S0) and excited-state (S1) pKa s of all four pyrrole moieties of phytochromobilin in all 64 possible configurations with respect to the three methine bridges are calculated in a protein-like environment, using a recently benchmarked level of theory. Accordingly, the relationships between acidity and chromophore geometry and charge distribution, hydrogen bonding, and light absorption are investigated in some detail, and discussed in terms of possible mechanisms making a proton transfer reaction more probable along the Pr --> Pfr reaction than in the parent cationic Pr state. It is found that charge distribution in the cationic species, intra-molecular hydrogen bonding in the neutral, and hydrogen bonding with two highly conserved aspartate and histidine residues have a significant effect on the acidity, while overall chromophore geometry and electronic state are less important factors. Furthermore, based on the calculations, two processes that may facilitate a proton transfer by substantially lowering the pKa s relative to their Pr values are identified: (i) a thermal Z,anti --> Z,syn isomerization at C5, occurring after formation of Lumi-R; (ii) a perturbation of the hydrogen bonding network which in Pr comprises the nitrogens of pyrroles A, B and C and the two aspartate and histidine residues.     

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

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

Solid‐State NMR Spectroscopy to Probe Photoactivation in Canonical Phytochromes

The photoreceptor phytochrome switches photochromically between two thermally stable states called Pr and Pfr. Here, we summarize recent solid‐state magic‐angle spinning (MAS) NMR work on this conversion process and interpret the functional mechanism in terms of a nano‐machine. The process is initiated by a double‐bond photoisomerization of the open‐chain tetrapyrrole chromophore at the methine bridge connecting pyrrole rings C and D. The Pr‐state chromophore and its surrounding pocket in canonical cyanobacterial and plant phytochromes has significantly less order, tends to form isoforms and is soft. Conversely, Pfr shows significantly harder chromophore–protein interactions, a well‐defined protonic and charge distribution with a clear classical counterion for the positively charged tetrapyrrole system. The soft‐to‐hard/disorder‐to‐order transition involves the chromophore and its protein surroundings within a sphere of at least 5.5 Å. The relevance of this collective event for signaling is discussed. Measurement of the intermediates during the Pfr → Pr back‐reaction provides insight into the well‐adjusted mechanics of a two‐step transformation. As both Pr → Pfr and Pfr → Pr reaction pathways are different in ground and excited states, a photochemically controlled hyper‐landscape is proposed allowing for ratchet‐type reaction dynamics regulating signaling activity.     

Phytochrome structure: A new methodological approach

Higher plants use the protein phytochrome as a photosensor. In physiological temperatures phytochrome exists in two forms: Pr and Pfr. The chromophore of phytochrome is an open-chain tetrapyrrole. On the pathway from Pr to Pfr four intermediates (Lumi-R, Meta-Ra, Meta-Rb, and Meta-Rc) can be distinguished, while only two (Lumi-F and Meta-F) can be seen on the way back from Pfr to Pr. We have used the x-ray structure of the C-Phycocyanin protein Fremyella diplosiphon bacteria as a template to build a model (∼200 atoms) that includes only the chromophore and five amino acids of the phytochrome (Arg316–Cys321–His322–Leu323–Gln324) around it. Using the existing experimental evidences, we have proposed a three-dimensional (3D) structure for Pr, Pfr, and intermediates and a mechanism for the photoisomerization as well. Structures were fully optimized using AM1 (Unichem package on a Cray J90-NACAD). Using the INDO/S method of Zerner and co-workers, we calculated the absorption spectra of the model compounds and compared them with the experimental data. The oscillator strength ratio is an indicator of the chomophore conformation in biliproteins. The calculated spectra reproduces well the spectra of the phytochrome (Pr, Pfr, and intermediates) except for the lower energy band. This result is attributed to the small number of amino acids in the models. The calculated ratios (f_VIS/f_UV−f_osc of visible band over f_osc of UV band and f₂/f₁−f_osc of second absorption band over f_osc of first absorption band) for the models match very well the experimental ratios obtained for the phytochrome (Pr, Pfr, and intermediates). This supports the proposed mechanism for the photoisomerization process. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 70: 1145–1157, 1998     

ULTRAVIOLET RESONANCE RAMAN SPECTRA OF PHYTOCHROME: A COMPARISON OF THE ENVIRONMENTS OF TRYPTOPHAN SIDE CHAINS BETWEEN RED LIGHT-ABSORBING AND FAR-RED LIGHT-ABSORBING FORMS

Ultraviolet resonance Raman spectra of phytochrome in the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr) are reported. The spectra excited at 240-nm provide structural information about the protein part of phytochrome. The protein contains only a very small amount of β-sheet structure and most of the tyrosine side chains are located in hydrophobic environments. Indole rings of tryptophan (Trp) interact with neighboring groups in the Pr form and these interactions become weaker with the conversion from Pr to Pfr. Some Trp side chains of Pfr are surrounded by aliphatic groups but such is not the case in Pr. These changes in the environment occur at the same time as changes in orientation of Trp side chains. Our observations suggest that interactions between Trp residues and the tetrapyrrolic chromophore occur in the Pr form and that the strength of these interactions diminishes in the Pfr form.     

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome

Photoisomerization of a protein‐bound chromophore is the basis of light sensing and signaling in many photoreceptors. Phytochrome photoreceptors can be photoconverted reversibly between the Pr and Pfr states through photoisomerization of the methine bridge between rings C and D. Ground‐state heterogeneity of the chromophore has been reported for both Pr and Pfr. Here, we report ultrafast visible (Vis) pump–probe and femtosecond polarization‐resolved Vis pump–infrared (IR) probe studies of the Pfr photoreaction in native and ¹³C/¹⁵N‐labeled Cph1 phytochrome with unlabeled PCB chromophore, demonstrating different S₀ substates, Pfr‐I and Pfr‐II, with distinct IR absorptions, orientations and dynamics of the carbonyl vibration of ring D. We derived time constants of 0.24 ps, 0.7 ps and 6 ps, describing the complete initial photoreaction. We identified an isomerizing pathway with 0.7 ps for Pfr‐I, and silent dynamics with 6 ps for Pfr‐II. We discuss different origins of the Pfr substates, and favor different facial orientations of ring D. The model provides a quantum yield for Pfr‐I of 38%, in line with ~35% ring D rotation in the electronic excited state. We tentatively assign the silent form Pfr‐II to a dark‐adapted state that can convert to Pfr‐I upon light absorption.     

Formation of the early photoproduct lumi-R of cyanobacterial phytochrome cph1 observed by ultrafast mid-infrared spectroscopy

The photoreactions of the Pr ground state of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 have been investigated by picosecond time-resolved mid-infrared spectroscopy at ambient temperature. With femtosecond excitation of the Pr state at 640 nm, the photoisomerized Lumi-R product state is generated with kinetics and associated difference spectra indicative of vibrational cooling with tau(1) = 3 ps time constant and excited state decay with tau(1) = 3 ps, tau(2) = 14 ps, and tau(3) = 134 ps time constants. The Lumi-R state is characterized by downshifted absorption of three C=C modes assigned to C(15)=C(16), C(4)=C(6), and a delocalized C=C mode, in addition to the downshifted C(19)=O mode. The Lumi-R minus Pr difference spectrum is indicative of global restructuring of the chromophore on the ultrafast timescale, which is discussed in light of C(15) Z/E photoisomerization in addition to changes near C(5), which could be low bond order torsional angle changes.     

15N MAS NMR studies of cph1 phytochrome: Chromophore dynamics and intramolecular signal transduction

Solid-state nuclear magnetic resonance (NMR) is applied for the first time to the photoreceptor phytochrome. The two stable states, Pr and Pfr, of the 59-kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 containing a uniformly 15N-labeled phycocyanobilin cofactor are explored by 15N cross-polarization (CP) magic-angle spinning (MAS) NMR. As recently shown by 15N solution-state NMR using chemical shifts [Strauss, H. M.; Hughes, J.; Schmieder, P. Biochemistry 2005, 44, 8244], all four nitrogens are protonated in both states. CP/MAS NMR provides two additional independent lines of evidence for the protonation of the nitrogens. Apparent loss of mobility during photoactivation, indicated by the decrease of line width, demonstrates strong tension of the entire chromophore in the Pfr state, which is in clear contrast to a more relaxed Pr state. The outer rings (A and D) of the chromophore are significantly affected by the phototransformation, as indicated by both change of chemical shift and line width. On the other hand, on the inner rings (B and C) only minor changes of chemical shifts are detected, providing evidence for a conserved environment during phototransformation. In a mechanical model, the phototransformation is understood in terms of rotations between the A-B and C-D methine bridges, allowing for intramolecular signal transduction to the protein surface by a unit composed of the central rings B and C and its tightly linked protein surroundings during the highly energetic Pfr state.     

Steric Effects Govern the Photoactivation of Phytochromes

Phytochromes constitute a superfamily of photoreceptor proteins existing in two forms that absorb red (Pr) and far‐red (Pfr) light. Although it is well‐known that the conversion of Pr into Pfr (the biologically active form) is triggered by a Z→E photoisomerization of the linear tetrapyrrole chromophore, direct evidence is scarce as to why this reaction always occurs at the methine bridge between pyrrole rings C and D. Here, we present hybrid quantum mechanics/molecular mechanics calculations based on a high‐resolution Pr crystal structure of Deinococcus radiodurans bacteriophytochrome to investigate the competition between all possible photoisomerizations at the three different (AB, BC and CD) methine bridges. The results demonstrate that steric interactions with the protein are a key discriminator between the different reaction channels. In particular, it is found that such interactions render photoisomerizations at the AB and BC bridges much less probable than photoisomerization at the CD bridge.     

Real-time tracking of phytochrome's orientational changes during Pr photoisomerization

Photoisomerization of a protein bound chromophore is the basis of the light sensing and signaling responses of many photoreceptors. Z-to-E photoisomerization of the Pr Cph1Δ2 phytochrome has been investigated by polarization resolved femtosecond visible pump-infrared probe spectroscopy, which yields structural information on the Pr excited (Pr*), Pr ground, and lumi-R product states. By exhaustive search analysis, two photoreaction time constants of (4.7 ± 1.4) and (30 ± 5) ps were found. Ring D orientational change in the electronic excited state to the transition state (90° twist) has been followed in real-time. Rotation of ring D takes place in the electronically excited state with a time constant of 30 ± 5 ps. The photoisomerization is best explained by a single rotation around C(15)═C(16) methine bridge in the Pr* state and a diffusive interaction with its protein surrounding.     

The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV-Vis and FTIR study

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV–Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV–Vis and FTIR experiments. Three intermediates have been trapped at −25, −45 and −120°C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at −70 and −140°C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore–protein interactions.     

THE PHOTOTRANSFORMATION PATHWAY OF DIMERIC OAT PHYTOCHROME FROM THE RED-LIGHT-ABSORBING FORM TO THE FAR-RED-LIGHT-ABSORBING FORM AT PHYSIOLOGICAL TEMPERATURE IS COMPOSED OF FOUR INTERMEDIATES

Abstract— –Phototransformation of oat type I phytochrome in vitro from the red-light-absorbing form (Pr) to the far-red-light-absorbing form (Pfr) at physiological temperature (24°C) was investigated with a multichannel transient spectrum analyser. Four sequential intermediates were detected between Pr and Pfr. Absorption spectra of these intermediates suggested that three of them corresponded with the intermediates lumi-R, meta-Ra and meta-Rc detected earlier at low temperature spectroscopy. A new intermediate named meta-Rb was found in the pathway between meta-Ra and meta-Rc. The new intermediate is not identical with meta-Rb previously detected at low temperature. The rate constant of Pfr appearance in isolated oat phytochrome dissolved in buffer containing 5% (vol/vol) glycerol was similar to that of etiolated pea epicotyl tissue.     

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

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

THE DISTANCE BETWEEN THE PHYTOCHROME CHROMOPHORE AND THE N-TERMINAL CHAIN DECREASES DURING PHOTOTRANSFORMATION. A NOVEL FLUORESCENCE ENERGY TRANSFER METHOD USING LABELED ANTIBODY FRAGMENTS

A novel antibody-fluorescence method has been developed to elucidate the chromophore topography in phytochrome as it undergoes a photochromic transformation. Förster energy transfer from N-terminal bound, fluorescently labeled Oat-25 Fab antibody fragments to the phytochrome chromophore was measured. The results suggest that the chromophore moves relative to the N-terminus upon the Pr → Pfr phototransformation. This conclusion is consistent with previous models which have proposed a reorientation and an interaction of the Pfr chromophore with the N-terminus. The method described appears to be the first study of a Forster energy transfer measurement using a donor-label attached to a Fab fragment of a photosensor protein.     

Structural and Vibrational Characterization of the Chromophore Binding Site of Bacterial Phytochrome Agp1

Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red‐light‐absorbing (Pr) and a far‐red‐light‐absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin‐chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process. Although the three‐dimensional structure of Agp1 has been solved at high resolution, the precise position of hydrogen atoms and protonation pattern in the chromophore binding pocket has not been investigated yet. Here, we present protonated structure models of Agp1 in the Pr state involving appropriately placed hydrogen atoms that were generated by hybrid quantum mechanics/molecular mechanics‐ and electrostatic calculations and validated against experimental structural‐ and spectroscopic data. Although the effect of histidine protonation on the vibrational spectra is weak, our results favor charge neutral H250 and H280 both protonated at Nε. However, a neutral H250 with a proton at Nε and a cationic H280 may also be possible. Furthermore, the present QM/MM calculations of IR and Raman spectra of Agp1 containing isotope‐labeled BV provide a detailed vibrational assignment of the biliverdin modes in the fingerprint region.     

EVENTS IN THE PHYTOCHROME MOLECULE AFTER IRRADIATION

The photoconversion of Pr to Pfr has been investigated by a large number of investigators. We have previously demonstrated that Z, E isomerization of the tetrapyrrole chromophore is involved in the photoconversion. It is the best candidate for the primary photoreaction. Conformation and configuration of the Pr chromophore will be compared with that of chromophores in phycocyanin. The crystal structure of phycocyanin had been elucidated by x-ray analysis. Proton transfer and/or Z, E isomerization of the tetrapyrrole are probably involved in different steps of the photoconversion in phytochrome and in photoreversible phycobiliproteins. Fluorescence decay kinetics of irradiated Pr and intermediate formation show heterogeneity. Possible reasons for this heterogeneity will be discussed.     

Time-resolved thermodynamic analysis of the oat phytochrome A phototransformation. A photothermal beam deflection study.

The time-resolved enthalpy and the structural volume changes after excitation of native oat phytochrome A were studied in the micro- to milliseconds range by photothermal beam deflection (PBD), a technique that follows the time-resolved refractive index changes upon decay of the excited species. The first set of intermediates, I700(1) and I700(2), stores ca 83% of the energy of the first excited state, in agreement with previous optoacoustic data, whereas the second set stores only ca 18%. The temperature dependence of the amplitudes ratio for the optical absorbances of the (I700(1) + I700(2)) intermediates set is explained on the basis of the thermochromic equilibrium between Pr,657 and Pr,672, which also is in line with the present PBD data. These data were best fitted with a parallel mechanism (with equal yield in each branch) for the production of the first set of intermediates, I700(1) and I700(2), as well as the second set of intermediates, Ibl1 and Ibl2. Thus, the final steps toward Pfr should be largely driven by positive entropic changes brought about by protein movements, in line with previous resonance Raman data. For the production of the first set of intermediates (I700(1) and I700(2)) an expansion of 18 +/- 13 mL mol-1 was determined, and a further expansion > or = 7 mL mol-1 was estimated for the decay from I700(1) to the set of Ibl intermediates, indicating that the far red-absorbing form of phytochrome (Pfr) has a larger volume than the red-absorbing form of phytochrome. This is in agreement with previous chromatographic and circular dichroism data according to which Pfr shows a larger volume and the chromophore shows a higher accessibility, respectively, in the Pfr state.     

DIRECT MEASUREMENT OF PHYTOCHROME PHOTOCONVERSION INTERMEDIATES AT HIGH PHOTON FLUENCE RATES

A custom-built modulated split-beam spectrophotometer has been used to measure the absorbance of tissue samples and purified phytochrome whilst exposing the sample to actinic 633 nm laser radiation at fluence rates approaching those of daylight. This approach has allowed the direct observation of the accumulation of phytochrome photoconversion intermediates at high fluence rates. At ca 1250 μmol m^?2 s^?1 upwards of 35% of the total phytochrome was present in the form of photoconversion intermediates in tissues of maize, sunflower and tomato. In other tissues tested (wheat, bean and Amaranthus) and in purified oat phytochrome, rather smaller levels of intermediates accumulated. Upon “lights-off” only a proportion of the accumulated intermediates decayed to far-red absorbing phytochrome (Pfr), the remainder appearing as the red-absorbing form (Pr). Difference spectra suggested that, at high light levels, Pr may be reformed via a photochemical back-conversion of an intermediate in the Pr—Pfr pathway, although the involvement of intermediates in the Pfr—Pr pathway cannot be excluded. The implications of the results for the ecological function of phytochrome are discussed.     

PHOTOTRANSFORMATIONS OF PHYTOCHROME

Abstract— –Phytochrome is the photoreversible chromoprotein that controls many aspects of plant growth and development Phototransformations of the red absorbing form (Pr) and the far red absorbing form (Pfr) involve initial photoreactions followed by dark relaxation reactions. Techniques for the study of intermediates of phototransformation and the present picture of intermediates involved in the phototransformations of Pr and Pfr are outlined. The molecular natures of the phototransformations are reviewed in relationship to knowledge of the chemistry of the chromophore and apoprotein. The significance of phytochrome intermediates in understanding the physiology of phytochrome controlled responses is discussed.     

PHYTOCHROME and PROTEIN PHOSPHORYLATION

The molecular mode of signal transduction triggered by phytochrome is unknown. One characteristic structural/topographic feature of the physiologically active form (Pfr) of phytochrome is that its tetrapyrrole chromophore becomes preferentially exposed in the Pfr form (compared to the Pr form). Phytochrome in its Pfr form appears to affect phosphorylation of cellular proteins. The literature on the phytochrome-mediated protein phosphorylation has been reviewed in an attempt to search for the role of the chromophore topography of phytochrome in the signal transduction process. In order to initiate a dephosphorylation-phosphorylation cascade as a possible step for the signal transduction, it may interact with a cellular protein kinase to inhibit its activity. This hypothesis has been reviewed with results from phosphorylation inhibition assays by the Pfr form of phytochrome and in light of the inhibition of protein kinase activity by tetrapyrroles in general.