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.

Light-dark adaptation of channelrhodopsin C128T mutant.

Channelrhodopsins are microbial type rhodopsins that operate as light-gated ion channels. Largely prolonged lifetimes of the conducting state of channelrhodopsin-2 may be achieved by mutations of crucial single amino acids, i.e. cysteine 128. Such mutants are of great scientific interest in the field of neurophysiology because they allow neurons to be switched on and off on demand (step function rhodopsins). Due to their slow photocycle, structural alterations of these proteins can be studied by vibrational spectroscopy in more detail than possible with wild type. Here, we present spectroscopic evidence that the photocycle of the C128T mutant involves three different dark-adapted states that are populated according to the wavelength and duration of the preceding illumination. Our results suggest an important role of multiphoton reactions and the previously described side reaction for dark state regeneration. Structural changes that cause formation and depletion of the assumed ion conducting state P520 are only small and follow larger changes that occur early and late in the photocycle, respectively. They require only minor structural rearrangements of amino acids near the retinal binding pocket and are triggered by all-trans/13-cis retinal isomerization, although additional isomerizations are also involved in the photocycle. We will discuss an extended photocycle model of this mutant on the basis of spectroscopic and electrophysiological data.

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.

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.

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.