Vibrational couplings between protein and cofactor in bacterial phytochrome Agp1 revealed by 2D-IR spectroscopy.

Phytochromes are ubiquitous photoreceptor proteins that undergo a significant refolding of secondary structure in response to initial photoisomerization of the chromophoric group. This process is important for the signal transduction through the protein and thus its regulatory function in different organisms. Here, we employ two-dimensional infrared absorption (2D-IR) spectroscopy, an ultrafast spectroscopic technique that is sensitive to vibrational couplings, to study the photoreaction of bacterial phytochrome Agp1. By calculating difference spectra with respect to the photoactivation, we are able to isolate sharp difference cross-peaks that report on local changes in vibrational couplings between different sites of the chromophore and the protein. These results indicate inter alia that a dipole coupling between the chromophore and the so-called tongue region plays a role in stabilizing the protein in the light-activated state.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes via photoswitching between two states of different physiological activity, i.e. a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from Agrobacterium fabrum) together with the bathy-like phytochrome XccBphP from Xanthomonas campestris by resonance Raman and IR difference spectroscopy. In all three phytochromes, the photoinduced conversions display the same mechanistic pattern as reflected by the chromophore structures in the various intermediate states. We also observed in each case the secondary structure transition of the tongue, which is presumably crucial for the function of phytochrome. The three phytochromes differ in details of the chromophore conformation in the various intermediates and the energetic barrier of their respective decay reactions. The specific protein environment in the chromophore pocket, which is most likely the origin for these small differences, also controls the proton transfer processes concomitant to the photoconversions. These proton translocations, which are tightly coupled to the structural transition of the tongue, presumably proceed via the same mechanism along the Pr → Pfr conversion whereas the reverse Pfr → Pr photoconversion includes different proton transfer pathways. Finally, classification of phytochromes in prototypical and bathy (or bathy-like) phytochromes is discussed in terms of molecular structure and mechanistic properties.

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Distinct chromophore-protein environments enable asymmetric activation of a bacteriophytochrome-activated diguanylate cyclase.

Sensing of red and far-red light by bacteriophytochromes involves intricate interactions between their bilin chromophore and the protein environment. The light-triggered rearrangements of the cofactor configuration and eventually the protein conformation enable bacteriophytochromes to interact with various protein effector domains for biological modulation of diverse physiological functions. Excitation of the holoproteins by red or far-red light promotes the photoconversion to their far-red light-absorbing Pfr state or the red light-absorbing Pr state, respectively. Because prototypical bacteriophytochromes have a parallel dimer architecture, it is generally assumed that symmetric activation with two Pfr state protomers constitutes the signaling-active species. However, the bacteriophytochrome from Idiomarina species A28L (IsPadC) has recently been reported to enable long-range signal transduction also in asymmetric dimers containing only one Pfr protomer. By combining crystallography, hydrogen-deuterium exchange coupled to MS, and vibrational spectroscopy, we show here that Pfr of IsPadC is in equilibrium with an intermediate "Pfr-like" state that combines features of Pfr and Meta-R states observed in other bacteriophytochromes. We also show that structural rearrangements in the N-terminal segment (NTS) can stabilize this Pfr-like state and that the PHY-tongue conformation of IsPadC is partially uncoupled from the initial changes in the NTS. This uncoupling enables structural asymmetry of the overall homodimeric assembly and allows signal transduction to the covalently linked physiological diguanylate cyclase output module in which asymmetry might play a role in the enzyme-catalyzed reaction. The functional differences to other phytochrome systems identified here highlight opportunities for using additional red-light sensors in artificial sensor-effector systems.

Light- and temperature-dependent dynamics of chromophore and protein structural changes in bathy phytochrome Agp2.

Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from Agrobacterium fabrum. In both cases, this intermediate represents a branching point of the phototransformation, since it opens an unproductive reaction channel back to the initial state and a productive pathway to the final active state, including the functional protein structural changes. It is shown that the functional quantum yield, i.e. the events of tongue refolding per absorbed photons, is lower by a factor of ca. two than the quantum yield of the primary photochemical process. However, the kinetic data derived from the spectroscopic experiments imply an increased formation of the final active state upon increasing photon flux or elevated temperature under photostationary conditions. Accordingly, the branching mechanism does not only account for the phytochrome's function as a light intensity sensor but may also modulate its temperature sensitivity.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/ß-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.

Role of the Propionic Side Chains for the Photoconversion of Bacterial Phytochromes.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum. Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain B (PsB) or C (PsC), but in each case, only one monoester adduct is reactive. In the reactive Agp2-BVM-B complex (esterified at ring B), the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated PsC. As in native Agp2, PsC is deprotonated in the final step of the Pfr phototransformation. However, the concomitant α-helix/ß-sheet secondary structure change of the tongue is blocked at the stage of unfolding of the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM toward the enol form are attributed to the drastic changes in the electrostatic potential. The calculations further suggest that deprotonation of PsC and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinarily slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.

The Photoconversion of Phytochrome Includes an Unproductive Shunt Reaction Pathway.

Phytochromes are modular bimodal photoswitches that control gene expression for morphogenetic processes in plants. These functions are triggered by photoinduced conversions between the inactive and active states of the photosensory module, denoted as Pr and Pfr, respectively. In the present time-resolved resonance Raman spectroscopic study of bacterial representatives of this photoreceptor family, we demonstrate that these phototransformations do not represent linear processes but include a branching reaction back to the initial state, prior to (de)activation of the output module. Thus, only a fraction of the photoreceptors undergoing the phototransformations can initiate the downstream signaling process, consistent with phytochrome's function as a sensor for more durable changes of light conditions.

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers.

Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion.

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.

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.

Ultrafast proton-coupled isomerization in the phototransformation of phytochrome.

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2. Transient deprotonation of the chromophore's pyrrole ring D or ring C into a hydrogen-bonded water cluster, revealed by a broad continuum infrared band, is triggered by electronic excitation, coherent oscillations and the sudden electric-field change in the excited state. Subsequently, a dominant fraction of the excited population relaxes back to the Pfr state, while ~35% follows the forward reaction to the photoproduct. A combination of quantum mechanics/molecular mechanics calculations and ultrafast visible and infrared spectroscopies demonstrates how proton-coupled dynamics in the excited state of Pfr leads to a restructured hydrogen-bond environment of early Lumi-F, which is interpreted as a trigger for downstream protein structural changes.

Light-induced activation of bacterial phytochrome Agp1 monitored by static and time-resolved FTIR spectroscopy.

Phytochromes, which regulate many biological processes in plants, bacteria, and fungi, can exist in two stable states, Pr and Pfr, that can be interconverted by light, via a number of intermediates such as meta-Rc. Herein we employ FTIR spectroscopy to study the Pr-to-Pfr conversion of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens. Static FTIR Pfr/Pr and meta-Rc/Pr difference spectra are disentangled in terms of cofactor and protein structural changes. Guided by DFT calculations on cofactor models, the chromophore conformational changes can be grouped into structural adjustments of the cofactor-protein interactions localized in the C-D dipyrrole moiety, that is, the photoisomerisation site, and in the A-B dipyrrole moiety including the protein attachment site. Whereas changes at the C and D rings appear to be largely completed in the meta-Rc state, the structural changes in the A-B unit occur during the transition from meta-Rc to Pfr, concomitant with the main protein structural changes, as demonstrated by static and time-resolved FTIR difference spectroscopy. We employ this technique to monitor, for the first time, the dynamics of the photocycle of phytochrome on the millisecond timescale. By extending the studies to genetically engineered protein variants of Agp1, we further demonstrate that H250 and D197 as well as the PHY domain are essential for formation of the Pfr state. Based on the IR spectroscopic and available crystallographic data we discuss the role of critical amino acid residues for the protein-cofactor interactions during the photoinduced reaction cycle.

Light-induced conformational changes of the chromophore and the protein in phytochromes: bacterial phytochromes as model systems.

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

The Lumi-R Intermediates of Prototypical Phytochromes.

Phytochromes are photoreceptors that upon light absorption initiate a physiological reaction cascade. The starting point is the photoisomerization of the tetrapyrrole cofactor in the parent Pr state, followed by thermal relaxation steps culminating in activation of the physiological signal. Here we have employed resonance Raman (RR) spectroscopy to study the chromophore structure in the primary photoproduct Lumi-R, trapped between 130 and 200 K. The investigations covered phytochromes from plants (phyA) and prokaryotes (Cph1, Agp1, CphB, and RpBphP2) including phytochromobilin (PΦB), phycocyanobilin (PCB), and biliverdin (BV). In PΦB- and PCB-binding phyA and Cph1, two Lumi-R states (Lumi-R1, Lumi-R2) were identified and discussed in terms of sequential and parallel reaction models. In Lumi-R1, the chromophore structural changes are restricted to the C-D methine bridge isomerization site but extended throughout the chromophore in Lumi-R2. Formation and decay kinetics as well as photochemical activity depend on the specific protein-chromophore interactions and thus account for the different distribution between Lumi-R1 and Lumi-R2 in the photostationary mixtures of the various PΦB(PCB)-binding phytochromes. For BV-binding bacteriophytochromes, only a single Lumi-R(BV) state was found. In this state, which is similar for Agp1, CphB, and RpBphP2, the chromophore structural changes comprise major torsions of the C-D methine bridge but also perturbations at the A-B methine bridge remote from the isomerization site. The different structures of the photoproducts in PΦB(PCB)-binding phytochromes and BV-binding bacteriophytochromes are attributed to the different disposition of ring D upon isomerization, which leads to distinct protein-chromophore interactions in the Lumi-R states of these two classes of phytochromes.

Subpicosecond midinfrared spectroscopy of the Pfr reaction of phytochrome Agp1 from Agrobacterium tumefaciens.

Phytochromes are light-sensing pigments found in plants and bacteria. For the first time, the P(fr) photoreaction of a phytochrome has been subject to ultrafast infrared vibrational spectroscopy. Three time constants of 0.3 ps, 1.3 ps, and 4.0 ps were derived from the kinetics of structurally specific marker bands of the biliverdin chromophore of Agp1-BV from Agrobacterium tumefaciens after excitation at 765 nm. VIS-pump-VIS-probe experiments yield time constants of 0.44 ps and 3.3 ps for the underlying electronic-state dynamics. A reaction scheme is proposed including two kinetic steps on the S(1) excited-state surface and the cooling of a vibrationally hot P(fr) ground state. It is concluded that the upper limit of the E-Z isomerization of the C(15) = C(16) methine bridge is given by the intermediate time constant of 1.3 ps. The reaction scheme is reminiscent of that of the corresponding P(r) reaction of Agp1-BV as published earlier.

Spectral properties of phytochrome Agp2 from Agrobacterium tumefaciens are specifically modified by a compound of the cell extract.

Phytochromes are widely distributed photoreceptors that are converted by light between the red absorbing Pr and the far-red absorbing Pfr form. The soil bacterium Agrobacterium tumefaciens contains two phytochromes, Agp1 and Agp2, which act as light-regulated histidine kinases. Whereas most phytochromes are stable in the Pr form, Agp2 and few other phytochromes convert into Pfr in darkness. We have shown in a previous publication that the spectral properties of recombinant Agp2 are modified by compounds of the cell extract from an Agrobacterium agp1(-)/agp2(-) double knockout mutant. In the present work we performed concentration series which show that the interaction is specific and that the modifying factor has a concentration of ca. 0.2 microM. We have also performed a series of mixing experiments with the truncated protein Agp2-M2, which consists of the N-terminal chromophore module (501 amino acids). The cell extract inhibited the photoconversion of Agp2-M2 in an unspecific way. In concentration series, this negative effect was less pronounced when lower concentrations of Agp2-M2 were used. In the presence of excess Agp2-M2 apoprotein, the cell extract did no longer modify the spectral properties of Agp2. The data suggest that the factor of the cell extract interacts specifically with the N-terminal moiety of Agp2.

Long-Range Modulations of Electric Fields in Proteins.

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ Eads due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ Eint, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ Eads and Δ Eint, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ Eads) and attached to membranes (Δ Eads + Δ Eint).

Structural snapshot of a bacterial phytochrome in its functional intermediate state.

Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors.

Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1.

The bacteriophytochrome Agp1 was reconstituted with a locked 5Zs-biliverdin in which the C(4)=C(5) and C(5)-C(6) bonds of the methine bridge between rings A and B are fixed in the Z and syn configuration/conformation, respectively. In Agp1-5Zs the photoconversion proceeds via the Lumi-R intermediate to Meta-R(A), but the following millisecond-transition to Meta-R(C) is blocked. Consistently, no transient proton release was detected. The photoconversion of Agp1-5Zs is apparently arrested in a Meta-R(A)-like intermediate, since the subsequent syn to anti rotation around the C(5)-C(6) bond is prevented by the lock. The Meta-R(A)-like photoproduct was characterized by its distinctive CD spectrum suggesting a reorientation of ring D.