Photoisomerization and photoionization of the photoactive yellow protein chromophore in solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.     

Photoisomerization in Rhodopsin

This article reviews the primary reaction processes in rhodopsin, a photoreceptive pigment for twilight vision. Rhodopsin has an 11-cis retinal as the chromophore, which binds covalently with a lysine residue through a protonated Schiff base linkage. Absorption of a photon by rhodopsin initiates the primary photochemical reaction in the chromophore. Picosecond time-resolved spectroscopy of 11-cis locked rhodopsin analogs revealed that the cis-trans isomerization of the chromophore is the primary reaction in rhodopsin. Then, generation of femtosecond laser pulses in the 1990s made it possible to follow the process of isomerization in real time. Formation of photorhodopsin within 200 fsec was observed by a transient absorption (pump–probe) experiment, which also revealed that the photoisomerization in rhodopsin is a vibrationally coherent process. Femtosecond fluorescence spectroscopy directly captured excited-state dynamics of rhodopsin, so that both coherent reaction process and unreacted excited state were observed. Faster photoreaction of the chromophore in rhodopsin than that in solution implies that the protein environment facilitates the efficient isomerization process. Such contributions of the protein residues have been monitored by infrared spectroscopy of rhodopsin, bathorhodopsin, and isorhodopsin (9-cis rhodopsin) at low temperatures. The crystal structure of bovine rhodopsin recently reported will lead to better understanding of the mechanism in future.     

Excited-state dynamics of all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics

The rhodopsin mimic is a chemically synthetized complex with retinyl Schiff base (RSB) formed between protein and the retinal chromophore that can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the ultrafast transient absorption spectra of three mutants of the cellular retinoic acid-binding protein II-based rhodopsin mimic at acidic environment were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the β-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. The experimental results provide implications on designing a rhodopsin-like protein with significant infrared fluorescence, which can be particularly useful in the applications in biosensing or bioimaging in deeper tissues.     

Ultrafast light-induced response of photoactive yellow protein chromophore analogues.

The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1-10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor-acceptor structure: structures of stronger electron donor-acceptor character lead to faster decays and less photoisomerisation.     

Role of protein in the primary step of the photoreaction of yellow protein

We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination. The driving force of the trans-to-cis isomerization reaction was analyzed by a computational method. The force was separated into two different components: the term due to the protein-chromophore interaction and the intrinsic term of the chromophore itself. As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term. This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments. We then analyzed the relaxation process of the chromophore on the excited-state energy surface and compared the process in the protein environment and that in a vacuum. Based on this analysis, we found that the bond-selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment.     

Fluorescence study of brevin, the Mr 92 000 actin-capping and -fragmenting protein isolated from serum. Effect of Ca2+ on protein conformation

The fluorescence characteristics of brevin and the effects of Ca2+ on the protein conformation were fully investigated. Brevin contains 18 tryptophans and 27 tyrosines. Analysis of the fluorescence spectra and the accessibility to quenching molecules indicate that the emitting tryptophans are located in a hydrophobic environment (lambda max = 324 nm) close to the protein surface. In native brevin, tyrosyl residues do not contribute to the fluorescence emission. Partial quenching of these chromophores has to be attributed to tyrosine----tryptophan resonance energy transfer which is highly efficient. The effect of brevin on actin polymerization has been shown to be Ca2+ sensitive [Harris, D. A., & Schwartz, J. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6798-6802; Thorstensson, R., Utter, G., & Norberg, R. (1982) Eur. J. Biochem. 126, 11-16; Wilkins, J. A., Schwartz, J. H. & Harris, D. A. (1983) Cell Biol. Int. Rep. 7, 1097-1104; Harris, H. E., & Weeds, A. G. (1983) Biochemistry 22, 2728-2741] and brevin binding to hydrophobic matrices to be Ca2+ dependent (Z. Soua, personal communication). Ca2+ binding to brevin decreases the tryptophan fluorescence polarization degree (without affecting the excited-state lifetime), which suggests a higher chromophore mobility. This effect may be partly related to the slight unshielding of the tryptophan residues observed in fluorescence quenching experiments. Moreover, the reactivity of brevin sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) increases in the presence of Ca2+. On the other hand, fluorescence spectra, quantum yields, excited-state lifetimes, and thermostability remain unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early molecular events in the photoactive yellow protein: role of the chromophore photophysics.

We report a comparative study of the isomerization reaction in native and denatured photoactive yellow protein (PYP) and in various chromophore analogues in their trans deprotonated form. The excited-state relaxation dynamics was followed by subpicosecond transient absorption and gain spectroscopy. The free p-hydroxycinnamate (pCA(2-)) and its amide analogue (pCM(-)) are found to display a quite different transient spectroscopy from that of PYP. The excited-state deactivation leads to the formation of the ground-state cis isomer without any detectable intermediate with a mechanism comparable to trans-stilbene photoisomerization. On the contrary, the early stage of the excited-state deactivation of the free thiophenyl-p-hydroxycinnamate (pCT(-)) and of the denatured PYP is similar to that of the native protein. It involves the formation of an intermediate absorbing in the spectral region located between the bleaching and gain bands in less than 2 ps. However, in these two cases, the formation of the cis isomer has not been proved yet. This difference with pCA(-) and pCM(-) might result from the fact that, in the thioester substituted chromophore, simultaneous population of two quasi-degenerate excited states occurs upon excitation. This comparative study highlights the determining role of the chromophore structure and of its intrinsic properties in the primary molecular events in native PYP.     

Green fluorescent protein variants as ratiometric dual emission pH sensors. 2. Excited-state dynamics

In the preceding paper [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S., Xi, L., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488], novel mutants of the green fluorescent protein (GFP) that exhibit dual steady-state emission properties were characterized structurally and discussed as potential intracellular pH probes. In this work, the excited-state dynamics of one of these new dual emission GFP variants, deGFP4 (C48S/S65T/H148C/T203C), is studied by ultrafast fluorescence upconversion spectroscopy. Following excitation of the high-energy absorption band centered at 398 nm and assigned to the neutral form of the chromophore, time-resolved emission was monitored from the excited state of both the neutral and intermediate anionic chromophores at both high and low pH and upon deuteration of exchangeable protons. The time-resolved emission dynamics and isotope effect appear to be very different from those of wild-type GFP [Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8362-8367]; however, due to overlapping emission bands, the apparent difference can be analyzed quantitatively within the same framework used to describe GFP excited-state dynamics. The results indicate that the pH-sensitive steady-state emission characteristics of deGFP4 are a result of a pH-dependent modulation of the rate of excited-state proton transfer. At high pH, a rapid interconversion from the excited state of the higher energy neutral chromophore to the lower energy intermediate anionic chromophore is achieved by proton transfer. At low pH, excited-state proton transfer is slowed to the point where it is no longer rate limiting.     

On the involvement of single-bond rotation in the primary photochemistry of photoactive yellow protein

Prior experimental observations, as well as theoretical considerations, have led to the proposal that C₄-C₇ single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C₄-C₇ single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is ∼60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C₄-C₇ single-bond rotation in PYP is not an alternative to C₇=C₈ double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore.     

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies

Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (相似文献   

Femtosecond kinetics of photoconversion of the higher plant photoreceptor phytochrome carrying native and modified chromophores

The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (P_r) and far-red absorbing phytochrome (P_fr) forms. Native P_r phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I₇₀₀, absorbing at ∼690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional ∼15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I₇₀₀ formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65 kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes P_r to P_fr isomerization primarily via a different configurational pathway, largely circumventing I₇₀₀ as an intermediate. The isomerization process is accompanied by strong coherent oscillations due to wavepacket motion on the excited-state surface for both phytochrome forms. The femto- to (sub-)nanosecond kinetics of the P_fr forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within ∼150 fs, the chromophores return to the first ground-state intermediate in 400-800 fs followed by two additional ground-state intermediates which are formed with 2-3 ps and ∼400 ps lifetimes. We call the first ground-state intermediate in native phytochrome I_fr·750, due to its pronounced absorption at that wavelength. The other intermediates are termed I_fr·675 and pseudo-P_r. The absorption spectrum of the latter already closely resembles the absorption of the P_r chromophore. PCB-65 kDa-phy shows a very similar kinetics, although many of the detailed spectral features in the transients seen in native phy are blurred, presumably due to wider inhomogeneous distribution of the chromophore conformation. Iso-PCB-65 kDa-phy shows similar features to the PCB-65 kDa-phy, with some additional blue-shift of the transient spectra of ∼10 nm. The sub-200 fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124 kDa phytochrome or in PCB-65 kDa-phy. We interpret the data within the framework of two- and three-dimensional potential energy surface diagrams for the photoisomerization processes and the ground-state intermediates involved in the two photoconversions.     

Mechanism of rhodopsin activation as examined with ring-constrained retinal analogs and the crystal structure of the ground state protein

The guanine nucleotide-binding protein (G-protein)-coupled receptor superfamily (GPCR) is comprised of a large group of membrane proteins involved in a wide range of physiological signaling processes. The functional switch from a quiescent to an active conformation is at the heart of GPCR action. The GPCR rhodopsin has been studied extensively because of its key role in scotopic vision. The ground state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Light induces cis-trans isomerization and rhodopsin activation. Here we show that rhodopsin regenerated with a ring-constrained 11-cis-retinal analog undergoes photoisomerization; however, it remains marginally active because isomerization occurs without the chromophore-induced conformational change of the opsin moiety. Modeling the locked chromophore analogs in the active site of rhodopsin suggests that the beta-ionone ring rotates but is largely confined within the binding site of the natural 11-cis-retinal chromophore. This constraint is a result of the geometry of the stable 11-cis-locked configuration of the chromophore analogs. These results suggest that the native chromophore cis-trans isomerization is merely a mechanism for repositioning of the beta-ionone ring which ultimately leads to helix movements and determines receptor activation.     

On mechanisms of fluorescence quenching by water

Mechanisms of fluorescence quenching of aromatic chromophores by water are reviewed. The mechanisms include polarity of chromophore environment, proton or electron transfer between the excited chromophore and water. A hypothesis is proposed that the quenching can be a result of chromophore-solvent hydrogen bond breaking in the excited state.     

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties

In the preceding accompanying paper [Shu, X., et al. (2007) Biochemistry 46, 12005-12013], the 1.5 A resolution crystal structure of green fluorescent protein (GFP) variant S65T/H148D is presented, and the possible consequences of an unusual short hydrogen bond (相似文献   

Charge transfer in green fluorescent protein.

Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene-imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the ramifications of the observation of strong conformational perturbations below the protein dynamical transition at approximately 200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.     

Function and structure of GFP-like proteins in the protein data bank

The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.     

Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein

We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.     

Reversible photocontrol of DNA binding by a designed GCN4-bZIP protein

Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain. As predicted, the trans form of the chromophore destabilizes the helical structure of the coiled-coil region of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis photoisomerization of the chromophore increases helical content and substantially enhances DNA binding. The system is observed to be readily reversible; thermal relaxation of the chromophore to the trans state and concomitant dissociation of the protein-DNA complex occurs with tau(1/2) approximately 10 min at 37 degrees C. It appears that conformational dynamics in the zipper domain make the transition state for isomerization readily available so that retention of reversible switching is observed.     

Characterization of the photoconversion on reaction of the fluorescent protein Kaede on the single-molecule level

Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with lambda = 350-410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (lambda = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at lambda = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s(-1) for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.     

Fluorescence of phytochrome adducts with synthetic locked chromophores

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.