The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA * chemie   metabolismus   MeSH
• flavinmononukleotid * chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce * MeSH
• simulace molekulární dynamiky MeSH
• světlo * MeSH
• Thermosynechococcus metabolismus   MeSH
• transkripční faktory metabolismus   chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA vazebné proteiny MeSH
• DNA * MeSH
• flavinmononukleotid * MeSH
• flaviny MeSH
• transkripční faktory MeSH

Quenching of chlorophyll triplet states by carotenoids is an essential photoprotective process, which prevents formation of reactive singlet oxygen in photosynthetic light-harvesting complexes. The process is usually very efficient in oxygenic organisms under physiological conditions, thus preventing any observable accumulation of chlorophyll triplets. However, it subsequently prevents also the determination of the triplet transfer rate. Here we report results of nanosecond transient absorption spectroscopy on photosystem I core complexes, where a major part of chlorophyll a triplet states (~60 %) accumulates on a nanosecond time scale at ambient temperature. As a consequence, the triplet energy transfer could be resolved and the transfer time was determined to be about 24 ns. A smaller fraction of chlorophyll a triplet states (~40 %) is quenched with a faster rate, which could not be determined. Our analysis indicates that these chlorophylls are in direct contact with carotenoids. The overall chlorophyll triplet yield in the core antenna was estimated to be ~0.3 %, which is a value two orders of magnitude smaller than in most other photosynthetic light-harvesting complexes. This explains why slower quenching of chlorophyll triplet states is sufficient for photoprotection of photosystem I. Nevertheless, the core antenna of photosystem I represents one of only few photosynthetic complexes of oxygenic organisms in which the quenching rate of the majority of chlorophyll triplets can be directly monitored under physiological temperature.

• Klíčová slova
• Carotenoid, Chlorophyll, Photoprotection, Triplet state,
• MeSH
• chlorofyl a MeSH
• chlorofyl chemie   MeSH
• fotosystém I (proteinový komplex) * MeSH
• karotenoidy * chemie   MeSH
• kyslík MeSH
• přenos energie MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Thermosynechococcus MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• fotosystém I (proteinový komplex) * MeSH
• karotenoidy * MeSH
• kyslík MeSH
• světlosběrné proteinové komplexy MeSH

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

• MeSH
• chlorofyl analogy a deriváty   chemie   MeSH
• diuron farmakologie   MeSH
• fluorescence MeSH
• fluorescenční spektrometrie MeSH
• fotosystém II (proteinový komplex) chemie   účinky léků   metabolismus   MeSH
• konformace proteinů MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• Spinacia oleracea chemie   MeSH
• světlo MeSH
• teplota MeSH
• Thermosynechococcus chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• chlorophyll a' MeSH Prohlížeč
• diuron MeSH
• fotosystém II (proteinový komplex) MeSH

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

• Klíčová slova
• EPR, mass spectrometry, photosystem II, reactive oxygen species, tocopherol,
• MeSH
• alfa-tokoferol chemie   metabolismus   MeSH
• aminokyseliny chemie   metabolismus   MeSH
• Arabidopsis enzymologie   genetika   účinky záření   MeSH
• fotosyntéza fyziologie   účinky záření   MeSH
• fotosystém II (proteinový komplex) chemie   genetika   metabolismus   MeSH
• hydroxylový radikál chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• intramolekulární transferasy chemie   genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• kyslík chemie   metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• oxidace-redukce MeSH
• superoxidy chemie   metabolismus   MeSH
• světlo MeSH
• termodynamika MeSH
• Thermosynechococcus enzymologie   genetika   účinky záření   MeSH
• tylakoidy enzymologie   genetika   účinky záření   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• železo chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• alfa-tokoferol MeSH
• aminokyseliny MeSH
• fotosystém II (proteinový komplex) MeSH
• hydroxylový radikál MeSH
• intramolekulární transferasy MeSH
• kyslík MeSH
• superoxidy MeSH
• tocopherol cyclase MeSH Prohlížeč
• železo MeSH

Photosystem II (PSII) is a multi-subunit pigment-protein complex and is one of several protein assemblies that function cooperatively in photosynthesis in plants and cyanobacteria. As more structural data on PSII become available, new questions arise concerning the nature of the charge separation in PSII reaction center (RC). The crystal structure of PSII RC from cyanobacteria Thermosynechococcus vulcanus was selected for the computational study of conformational changes in photosystem II associated to the charge separation process. The parameterization of cofactors and lipids for classical MD simulation with Amber force field was performed. The parametrized complex of PSII was embedded in the lipid membrane for MD simulation with Amber in Gromacs. The conformational behavior of protein and the cofactors directly involved in the charge separation were studied by MD simulations and QM/MM calculations. This study identified the most likely mechanism of the proton-coupled reduction of plastoquinone QB. After the charge separation and the first electron transfer to QB, the system undergoes conformational change allowing the first proton transfer to QB- mediated via Ser264. After the second electron transfer to QBH, the system again adopts conformation allowing the second proton transfer to QBH-. The reduced QBH2 would then leave the binding pocket.

• Klíčová slova
• MD simulations, Photosystem II reaction center, Plastoquinone, Proton-coupled reduction,
• MeSH
• bakteriální proteiny chemie   MeSH
• fotosystém II (proteinový komplex) chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• simulace molekulární dynamiky * MeSH
• sinice enzymologie   MeSH
• Thermosynechococcus MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fotosystém II (proteinový komplex) MeSH
• lipidové dvojvrstvy MeSH