Francisco Velazquez Escobar

Cyanochromes are blue/green light photoreversible photoreceptors defined by a stable double cysteine linkage to a phycoviolobilin-type chromophore.

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C31 carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.

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 His(260) in the chromophore-binding pocket such that either the delta-nitrogen (M-HSD) or the epsilon-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His(260) 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.

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

Background: The Pr and Pfr parent states of prototypical and bathy bacteriophytochromes exhibit different thermal stabilities. Results: Unlike bathy phytochromes, the biliverdin cofactor of prototypical phytochromes displays distinct conformational heterogeneity in Pfr. Conclusion: This heterogeneity enables thermal Pfr to Pr conversion in prototypical phytochromes. Significance: Understanding thermal deactivation of the signaling Pfr state is essential for elucidating the molecular function of 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.

Photoconversion mechanism of the second GAF domain of cyanobacteriochrome AnPixJ and the cofactor structure of its green-absorbing state.

Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies concomitant to the hypsochromically shifted absorption maximum. However, the cofactor in Pg is protonated and adopts a conformation very similar to the Pfr state of classical phytochromes. The spectral differences are probably related to an increased solvent accessibility of the chromophore which may reduce the π-electron delocalization in the phycocyanobilin and thus raise the energies of the first electronic transition and the methine bridge stretching modes. Molecular dynamics simulations suggest that the Z → E photoisomerization of the chromophore at the C-D methine bridge alters the interactions with the nearby Trp90 which in turn may act as a gate, allowing the influx of water molecules into the chromophore pocket. Such a mechanism of color tuning AnPixJg2 is unique among the cyanobacteriochromes studied so far.

A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

Unusual spectral properties of bacteriophytochrome Agp2 result from a deprotonation of the chromophore in the red-absorbing form Pr.

Background: Typical phytochromes include a protonated chromophore in the parent states (Pr and Pfr) that transiently deprotonates during photoconversion. Results: In Agp2, the pKa of the chromophore is lowered from >11 to 7.6 during the conversion from Pfr to Pr. Conclusion: Chromophore protonation affects light-induced and thermal Pr to Pfr conversion. Significance: Agp2 can act as integrated light and pH sensor. 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 pKa is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pKa 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.

Structural parameters controlling the fluorescence properties of phytochromes.

Phytochromes constitute a class of photoreceptors that can be photoconverted between two stable states. The tetrapyrrole chromophore absorbs in the red spectral region and displays fluorescence maxima above 700 nm, albeit with low quantum yields. Because this wavelength region is particularly advantageous for fluorescence-based deep tissue imaging, there is a strong interest to engineer phytochrome variants with increased fluorescence yields. Such targeted design efforts would substantially benefit from a deeper understanding of those structural parameters that control the photophysical properties of the protein-bound chromophore. Here we have employed resonance Raman (RR) spectroscopy and molecular dynamics simulations for elucidating the chromophore structural changes in a fluorescence-optimized mutant (iRFP) derived from the PAS-GAF domain of the bacteriophytochrome RpBphP2 from Rhodopseudomas palustris . Both methods consistently reveal the structural consequences of the amino acid substitutions in the vicinity of the biliverdin chromophore that may account for lowering the propability of nonradiative excited state decays. First, compared to the wild-type protein, the tilt angle of the terminal ring D with respect to ring C is increased in iRFP, accompanied by the loss of hydrogen bond interactions of the ring D carbonyl function and the reduction of the number of water molecules in that part of the chromophore pocket. Second, the overall flexibility of the chromophore is significantly reduced, particularly in the region of rings D and A, thereby reducing the conformational heterogeneity of the methine bridge between rings A and B and the ring A carbonyl group, as concluded from the RR spectra of the wild-type proteins.

A Red/Green Cyanobacteriochrome Sustains Its Color Despite a Change in the Bilin Chromophore’s Protonation State

The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.

Conformational heterogeneity of the Pfr chromophore in plant and cyanobacterial phytochromes

Phytochromes are biological photoreceptors that can be reversibly photoconverted between a dark and photoactivated state. The underlying reaction sequences are initiated by the photoisomerization of the tetrapyrrole cofactor, which in plant and cyanobacterial phytochromes are a phytochromobilin (PΦB) and a phycocyanobilin (PCB), respectively. The transition between the two states represents an on/off-switch of the output module activating or deactivating downstream physiological processes. In addition, the photoactivated state, i.e., Pfr in canonical phytochromes, can be thermally reverted to the dark state (Pr). The present study aimed to improve our understanding of the specific reactivity of various PΦB- and PCB-binding phytochromes in the Pfr state by analysing the cofactor structure by vibrational spectroscopic techniques. Resonance Raman (RR) spectroscopy revealed two Pfr conformers (Pfr-I and Pfr-II) forming a temperature-dependent conformational equilibrium. The two sub-states—found in all phytochromes studied, albeit with different relative contributions—differ in structural details of the C-D and A-B methine bridges. In the Pfr-I sub-state the torsion between the rings C and D is larger by ca. 10° compared to Pfr-II. This structural difference is presumably related to different hydrogen bonding interactions of ring D as revealed by time-resolved IR spectroscopic studies of the cyanobacterial phytochrome Cph1. The transitions between the two sub-states are evidently too fast (i.e., nanosecond time scale) to be resolved by NMR spectroscopy which could not detect a structural heterogeneity of the chromophore in Pfr. The implications of the present findings for the dark reversion of the Pfr state are discussed.

Protonation-Dependent Structural Heterogeneity in the Chromophore Binding Site of Cyanobacterial Phytochrome Cph1

Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV-visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.5, respectively. The protonation pattern and vibrational properties of these states were further characterized by means of hybrid quantum mechanics/molecular mechanics calculations. From this combined experimental-theoretical study, we were able to identify His260 as the key residue controlling pH-dependent equilibria. This residue is not only responsible for the conformational heterogeneity of CBP in the Pr state of prokaryotic phytochromes, discussed extensively in the past, but it constitutes the sink and source of protons in the proton release/uptake mechanism involving the tetrapyrrole chromophore which finally leads to the formation of the Pfr state. Thus, this work provides valuable information that may guide further experiments toward the understanding of the specific role of protons in controlling structure and function of phytochromes in general.