Rhodopsins constitute a broad class of retinal-binding photoreceptors. Microbial rhodopsins are canonically activated through an all-trans to 13-cis photoisomerization, whereas animal rhodopsins are mostly activated through an 11-cis to all-trans isomerization. Bestrhodopsins constitute a special microbial rhodopsin subfamily, with bistable rhodopsin domains that can be photoswitched between a far red-absorbing state D661 and a green-absorbing state P540. Its photochemistry involves a peculiar all-trans to 11-cis isomerization for the D661 to P540 photoreaction and vice versa. Here, we present the P. antarctica bestrhodopsin 11-cis to all-trans photoreaction as determined by femtosecond-to-submillisecond transient absorption, femtosecond stimulated Raman and flash-photolysis spectroscopy. The primary photoreaction involves ultrafast isomerizations in 240 fs from the 11-cis reactant to a mixture of highly distorted all-trans and 13-cis photoproducts. The 13-cis fraction then thermally isomerizes to a distorted all-trans RSB in 120 ps. We propose bicycle pedal models for the branched photoisomerizations with corotation of the C11═C12 and C13═C14 double bonds. One reactant fraction undergoes bicycle pedal motion aborted at the C13═C14 double bond, resulting in all-trans retinal. The other fraction undergoes a full bicycle pedal motion of both C11═C12 and C13═C14, resulting in 13-cis retinal. The primary products are trapped high up the ground-state potential energy surface with a low energetic barrier that facilitates thermal isomerization from 13-cis to all-trans retinal in 120 ps. All-trans retinal then structurally and energetically relaxes with subsequent time constants of 0.7 and 62 μs and 4.4 ms, along with counterion protonation, completing the P540 to D661 photoreaction.

• MeSH
• fotochemické procesy MeSH
• isomerie MeSH
• retinaldehyd * chemie   MeSH
• rhodopsiny mikrobiální * chemie   MeSH
• stereoizomerie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• retinaldehyd * MeSH
• rhodopsiny mikrobiální * MeSH

Neorhodopsin (NeoR) is a newly discovered fungal bistable rhodopsin that reversibly photoswitches between UV- and near-IR absorbing states denoted NeoR367 and NeoR690, respectively. NeoR367 represents a deprotonated retinal Schiff base (RSB), while NeoR690 represents a protonated RSB. Cryo-EM studies indicate that NeoR forms homodimers with 29 Å center-to-center distance between the retinal chromophores. UV excitation of NeoR367 takes place to an optically allowed S3 state of 1Bu+ symmetry, which rapidly converts to a low-lying optically forbidden S1 state of 2Ag- symmetry in 39 fs, followed by a multiexponential decay to the ground state on the 1-100 ps time scale. A theoretically predicted nπ* (S2) state does not get populated in any appreciable transient concentration during the excited-state relaxation cascade. We observe an intradimer retinal to retinal excitation energy transfer (EET) process from the NeoR367 S1 state to NeoR690, in competition with photoproduct formation. To quantitatively assess the EET mechanism and rate, we experimentally addressed and modeled the EET process under varying NeoR367-NeoR690 photoequilibrium conditions and determined the EET rate at (200 ps)-1. The NeoR367 S1 state shows a weak stimulated emission band in the near-IR around 700 nm, which may result from mixing with an intramolecular charge-transfer (ICT) state, enhancing the transition dipole moment of the S1-S0 transition and possibly facilitating the EET process. We suggest that EET may bear general relevance to the function of bistable multiwavelength rhodopsin oligomers.

• MeSH
• dimerizace MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• přenos energie MeSH
• retinaldehyd * chemie   MeSH
• rhodopsiny mikrobiální * chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• retinaldehyd * MeSH
• rhodopsiny mikrobiální * MeSH

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

• Klíčová slova
• conical intersection, far-red absorbing rhodopsin, femtosecond stimulated Raman spectroscopy, femtosecond to millisecond spectroscopy, multiple retinal isomerization,
• MeSH
• diterpeny * MeSH
• isomerie MeSH
• konformace proteinů MeSH
• retinaldehyd * chemie   MeSH
• rodopsin * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 13-cis-retinal MeSH Prohlížeč
• diterpeny * MeSH
• retinaldehyd * MeSH
• rodopsin * MeSH

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

• MeSH
• bakteriální proteiny chemie   MeSH
• flavinadenindinukleotid chemie   MeSH
• fotoreceptory mikroorganismů * chemie   MeSH
• glutamin * chemie   MeSH
• organické látky MeSH
• protony MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• světlo MeSH
• tyrosin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• flavinadenindinukleotid MeSH
• fotoreceptory mikroorganismů * MeSH
• glutamin * MeSH
• organické látky MeSH
• protony MeSH
• tyrosin MeSH

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

• Klíčová slova
• CP47, Chlorophyll, High-light-inducible proteins, Photosystem II, Synechocystis,
• MeSH
• bakteriální proteiny metabolismus   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• protein TNFSF14 metabolismus   MeSH
• světlosběrné proteinové komplexy * metabolismus   MeSH
• Synechocystis * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fotosystém II (proteinový komplex) MeSH
• protein TNFSF14 MeSH
• světlosběrné proteinové komplexy * MeSH

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.

• MeSH
• chlorofyl metabolismus   MeSH
• karotenoidy metabolismus   MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• přenos energie MeSH
• proteiny chloroplastové chemie   genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• sbalování proteinů MeSH
• Synechocystis genetika   metabolismus   MeSH
• vazba proteinů MeSH
• xanthofyly metabolismus   MeSH
• zeaxanthiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• ELIP2 protein, Arabidopsis MeSH Prohlížeč
• karotenoidy MeSH
• light-harvesting-like protein 3, Arabidopsis MeSH Prohlížeč
• proteiny chloroplastové MeSH
• proteiny huseníčku MeSH
• violaxanthin MeSH Prohlížeč
• xanthofyly MeSH
• zeaxanthiny MeSH

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

• Publikační typ
• časopisecké články MeSH

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

• Publikační typ
• časopisecké články MeSH

The major light-harvesting complex of photosystem II (LHCII) is the main contributor to sunlight energy harvesting in plants. The flexible design of LHCII underlies a photoprotective mechanism whereby this complex switches to a dissipative state in response to high light stress, allowing the rapid dissipation of excess excitation energy (non-photochemical quenching, NPQ). In this work, we locked single LHCII trimers in a quenched conformation after immobilization of the complexes in polyacrylamide gels to impede protein interactions. A comparison of their pigment excited-state dynamics with quenched LHCII aggregates in buffer revealed the presence of a new spectral band at 515 nm arising after chlorophyll excitation. This is suggested to be the signature of a carotenoid excited state, linked to the quenching of chlorophyll singlet excited states. Our data highlight the marked sensitivity of pigment excited-state dynamics in LHCII to structural changes induced by the environment.

• Klíčová slova
• Molecular Structure, Optical Property, Physical Optics,
• Publikační typ
• časopisecké články MeSH

Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.

• Klíčová slova
• Synechocystis, carotenoid, chlorophyll, chloroplast, ferrochelatase, heme, light harvesting complex (LHC)-like proteins, membrane protein, photosynthesis, photosynthetic pigment, pigment binding, plant biochemistry,
• MeSH
• chlorofyl a metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• dimerizace MeSH
• ferrochelatasa chemie   metabolismus   MeSH
• fylogeneze MeSH
• karotenoidy metabolismus   MeSH
• konformace proteinů MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Synechocystis enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• chlorophyll b MeSH Prohlížeč
• ferrochelatasa MeSH
• karotenoidy MeSH
• světlosběrné proteinové komplexy MeSH