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1.
J Chem Phys ; 155(11): 114113, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34551543

RESUMO

We present a methodology that provides a complete parametric description of the time evolution of the electronically and vibrationally excited states as detected by ultrafast transient absorption (TA). Differently from previous approaches, which started fitting the data after ≈100 fs, no data are left out in our methodology, and the "coherent artifact" and the instrument response function are fully taken into account. In case studies, the method is applied to solvents, the dye Nile blue, and all-trans ß-carotene in cyclohexane solution. The estimated Damped Oscillation Associated Spectra (DOAS) and phases express the most important vibrational frequencies present in the molecular system. By global fit alone of the experimental data, it is difficult to interpret in detail the underlying dynamics. Since it is unfeasible to directly fit the data by a theoretical simulation, our enhanced DOAS methodology thus provides a useful "middle ground" where the theoretical description and the fit of the experimental data can meet. ß-carotene in cyclohexane was complementarily studied with femtosecond stimulated Raman spectroscopy (FSRS). The fs-ps dynamics of ß-carotene in cyclohexane in TA and FSRS experiments can be described by a sequential scheme S2 → hot S1 → S1' → S1 → S0 with lifetimes of 167 fs (fixed), 0.35, 1.1, and 9.6 ps. The correspondence of DOAS decaying concomitantly with hot S1 and the Species Associated Difference Spectra of hot S1 in TA and FSRS suggest that we observe here features of the vibrational relaxation and nuclear reorganization responsible for the hot S1 to S1 transition.

2.
J Chem Phys ; 153(22): 224101, 2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33317279

RESUMO

Broad-band pump-probe spectroscopy combined with global and target analysis is employed to study the vibronic and excitonic dynamics of two dimers and a tetramer of perylenediimides. A simultaneous analysis is developed for two systems that have been measured in the same conditions. This enhances the resolvability of the vibronic and excitonic dynamics of the systems, and the solvent contributions that are common in the experiments. We resolve two oscillations of 1399 cm-1 or 311 cm-1 damped with ≈30/ps involved in vibrational relaxation and two more oscillations of 537 cm-1 or 136 cm-1 damped with ≈3/ps. A relaxation process with a rate of 2.1/ps-3.2/ps that is positively correlated with the excitonic coupling was discovered in all three model systems, attributed to annihilation of the one but lowest exciton state.

3.
J Am Chem Soc ; 142(41): 17346-17355, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32878439

RESUMO

Photosynthesis in plants starts with the capture of photons by light-harvesting complexes (LHCs). Structural biology and spectroscopy approaches have led to a map of the architecture and energy transfer pathways between LHC pigments. Still, controversies remain regarding the role of specific carotenoids in light-harvesting and photoprotection, obligating the need for high-resolution techniques capable of identifying excited-state signatures and molecular identities of the various pigments in photosynthetic systems. Here we demonstrate the successful application of femtosecond stimulated Raman spectroscopy (FSRS) to a multichromophoric biological complex, trimers of LHCII. We demonstrate the application of global and target analysis (GTA) to FSRS data and utilize it to quantify excitation migration in LHCII trimers. This powerful combination of techniques allows us to obtain valuable insights into structural, electronic, and dynamic information from the carotenoids of LHCII trimers. We report spectral and dynamical information on ground- and excited-state vibrational modes of the different pigments, resolving the vibrational relaxation of the carotenoids and the pathways of energy transfer to chlorophylls. The lifetimes and spectral characteristics obtained for the S1 state confirm that lutein 2 has a distorted conformation in LHCII and that the lutein 2 S1 state does not transfer to chlorophylls, while lutein 1 is the only carotenoid whose S1 state plays a significant energy-harvesting role. No appreciable energy transfer takes place from lutein 1 to lutein 2, contradicting recent proposals regarding the functions of the various carotenoids (Son et al. Chem. 2019, 5 (3), 575-584). Also, our results demonstrate that FSRS can be used in combination with GTA to simultaneously study the electronic and vibrational landscapes in LHCs and pave the way for in-depth studies of photoprotective conformations in photosynthetic systems.

4.
Nat Commun ; 11(1): 4248, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843623

RESUMO

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/efeitos da radiação , Cinética , Luz , Estrutura Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/efeitos da radiação , Conformação Proteica , Análise Espectral
5.
Biochim Biophys Acta Bioenerg ; 1861(8): 148206, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305412

RESUMO

The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.


Assuntos
Clorofila/análogos & derivados , Luz , Complexo de Proteína do Fotossistema I/metabolismo , Synechococcus/enzimologia , Clorofila/metabolismo , Transferência de Energia , Ligação Proteica
6.
Photosynth Res ; 144(2): 247-259, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32076913

RESUMO

Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been studied by picosecond time-resolved fluorescence spectroscopy at room temperature. Global analysis of the time-resolved fluorescence kinetics revealed two lifetimes of spectral equilibration in the isolated PBS, 30-35 ps and 110-130 ps, assigned primarily to energy transfer within the rods and between the rods and the allophycocyanin core, respectively. An additional intrinsic kinetic component with a lifetime of 500-700 ps was found, representing non-radiative decay or energy transfer in the core. Isolated tetrameric PSI complexes exhibited biexponential fluorescence decay kinetics with lifetimes of about 10 ps and 40 ps, representing equilibration between the bulk antenna chlorophylls with low-energy "red" states and trapping of the equilibrated excitations, respectively. The cascade of EET in the PBS and in PSI could be resolved in intact filaments as well. Virtually all energy absorbed by the PBS was transferred to the photosystems on a timescale of 180-190 ps.


Assuntos
Anabaena/química , Anabaena/metabolismo , Complexo de Proteína do Fotossistema I/química , Ficobilissomas/química , Transferência de Energia , Fluorescência , Cinética , Complexo de Proteína do Fotossistema I/metabolismo , Ficobilissomas/metabolismo , Espectrometria de Fluorescência , Análise Espectral/métodos
7.
Photosynth Res ; 144(2): 261-272, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32076914

RESUMO

The phycobilisome (PBS) serves as the major light-harvesting system, funnelling excitation energy to both photosystems (PS) in cyanobacteria and red algae. The picosecond kinetics involving the excitation energy transfer has been studied within the isolated systems and intact filaments of the cyanobacterium Anabaena variabilis PCC 7120. A target model is proposed which resolves the dynamics of the different chromophore groups. The energy transfer rate of 8.5 ± 1.0/ns from the rod to the core is the rate-limiting step, both in vivo and in vitro. The PBS-PSI-PSII supercomplex reveals efficient excitation energy migration from the low-energy allophycocyanin, which is the terminal emitter, in the PBS core to the chlorophyll a in the photosystems. The terminal emitter of the phycobilisome transfers energy to both PSI and PSII with a rate of 50 ± 10/ns, equally distributing the solar energy to both photosystems. Finally, the excitation energy is trapped by charge separation in the photosystems with trapping rates estimated to be 56 ± 6/ns in PSI and 14 ± 2/ns in PSII.


Assuntos
Anabaena variabilis/química , Anabaena variabilis/metabolismo , Complexo de Proteína do Fotossistema I/química , Ficobilissomas/química , Clorofila A/química , Clorofila A/metabolismo , Transferência de Energia , Modelos Teóricos , Complexo de Proteína do Fotossistema I/isolamento & purificação , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/isolamento & purificação , Ficobilissomas/metabolismo , Espectrometria de Fluorescência , Análise Espectral/métodos , Tilacoides/química
8.
Chemistry ; 26(1): 336-343, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31750960

RESUMO

Photoinduced processes in thiouracil derivatives have lately attracted considerable attention due to their suitability for innovative biological and pharmacological applications. Here, sub-20 fs broadband transient absorption spectroscopy in the near-UV are combined with CASPT2/MM decay path calculations to unravel the excited-state decay channels of water solvated 2-thio and 2,4-dithiouracil. These molecules feature linear absorption spectra with overlapping ππ* bands, leading to parallel decay routes which we systematically track for the first time. The results reveal that different processes lead to the triplet states population, both directly from the ππ* absorbing state and via the intermediate nπ* dark state. Moreover, the 2,4-dithiouracil decay pathways is shown to be strongly correlated either to those of 2- or 4-thiouracil, depending on the sulfur atom on which the electronic transition localizes.

9.
Proc Natl Acad Sci U S A ; 116(17): 8320-8325, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30962362

RESUMO

Sunlight drives photosynthesis but can also cause photodamage. To protect themselves, photosynthetic organisms dissipate the excess absorbed energy as heat, in a process known as nonphotochemical quenching (NPQ). In green algae, diatoms, and mosses, NPQ depends on the light-harvesting complex stress-related (LHCSR) proteins. Here we investigated NPQ in Chlamydomonas reinhardtii using an approach that maintains the cells in a stable quenched state. We show that in the presence of LHCSR3, all of the photosystem (PS) II complexes are quenched and the LHCs are the site of quenching, which occurs at a rate of ∼150 ps-1 and is not induced by LHCII aggregation. The effective light-harvesting capacity of PSII decreases upon NPQ, and the NPQ rate is independent of the redox state of the reaction center. Finally, we could measure the pH dependence of NPQ, showing that the luminal pH is always above 5.5 in vivo and highlighting the role of LHCSR3 as an ultrasensitive pH sensor.


Assuntos
Proteínas de Algas/fisiologia , Chlamydomonas , Concentração de Íons de Hidrogênio , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Proteínas de Algas/metabolismo , Chlamydomonas/fisiologia , Chlamydomonas/efeitos da radiação , Cinética , Complexo de Proteína do Fotossistema II/metabolismo , Espectrometria de Fluorescência , Temperatura
10.
J Am Chem Soc ; 141(1): 520-530, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30511841

RESUMO

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid ß1-ring in picoseconds occurs at a low yield of <1%, whereby the ß1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 µs, which allow the carotenoid to translocate into the N-terminal domain in 10 µs. We identified infrared signatures of helical elements that dock on the C-terminal domain ß-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Luz , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína
11.
J Phys Chem B ; 122(24): 6328-6340, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29847127

RESUMO

Determining the key features of high-efficiency photosynthetic energy transfer remains an ongoing task. Recently, there has been evidence for the role of vibronic coherence in linking donor and acceptor states to redistribute oscillator strength for enhanced energy transfer. To gain further insights into the interplay between vibronic wavepackets and energy-transfer dynamics, we systematically compare four structurally related phycobiliproteins from cryptophyte algae by broad-band pump-probe spectroscopy and extend a parametric model based on global analysis to include vibrational wavepacket characterization. The four phycobiliproteins isolated from cryptophyte algae are two "open" structures and two "closed" structures. The closed structures exhibit strong exciton coupling in the central dimer. The dominant energy-transfer pathway occurs on the subpicosecond timescale across the largest energy gap in each of the proteins, from central to peripheral chromophores. All proteins exhibit a strong 1585 cm-1 coherent oscillation whose relative amplitude, a measure of vibronic intensity borrowing from resonance between donor and acceptor states, scales with both energy-transfer rates and damping rates. Central exciton splitting may aid in bringing the vibronically linked donor and acceptor states into better resonance resulting in the observed doubled rate in the closed structures. Several excited-state vibrational wavepackets persist on timescales relevant to energy transfer, highlighting the importance of further investigation of the interplay between electronic coupling and nuclear degrees of freedom in studies on high-efficiency photosynthesis.


Assuntos
Transferência de Energia , Complexos de Proteínas Captadores de Luz/química , Criptófitas/metabolismo , Cristalografia por Raios X , Dimerização , Cinética , Complexos de Proteínas Captadores de Luz/metabolismo , Ficobiliproteínas/química , Ficobiliproteínas/metabolismo , Estrutura Terciária de Proteína , Espectrofotometria
12.
Photosynth Res ; 137(2): 307-320, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29600442

RESUMO

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB-PSII complexes with closed (saturated) RCs, a quenched or open PB-PSII complex, and a PB-PSII 'not fully closed.' For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB-PSII-PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.


Assuntos
Transporte de Elétrons/fisiologia , Transferência de Energia/fisiologia , Luz , Fotossíntese/fisiologia , Synechocystis/fisiologia , Regulação Bacteriana da Expressão Gênica , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Espectrometria de Fluorescência , Tilacoides/metabolismo
13.
Photosynth Res ; 135(1-3): 87-102, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28721458

RESUMO

In the light-harvesting antenna of the Synechocystis PCC 6803 phycobilisome (PB), the core consists of three cylinders, each composed of four disks, whereas each of the six rods consists of up to three hexamers (Arteni et al., Biochim Biophys Acta 1787(4):272-279, 2009). The rods and core contain phycocyanin and allophycocyanin pigments, respectively. Together these pigments absorb light between 400 and 650 nm. Time-resolved difference absorption spectra from wild-type PB and rod mutants have been measured in different quenching and annihilation conditions. Based upon a global analysis of these data and of published time-resolved emission spectra, a functional compartmental model of the phycobilisome is proposed. The model describes all experiments with a common set of parameters. Three annihilation time constants are estimated, 3, 25, and 147 ps, which represent, respectively, intradisk, interdisk/intracylinder, and intercylinder annihilation. The species-associated difference absorption and emission spectra of two phycocyanin and two allophycocyanin pigments are consistently estimated, as well as all the excitation energy transfer rates. Thus, the wild-type PB containing 396 pigments can be described by a functional compartmental model of 22 compartments. When the interhexamer equilibration within a rod is not taken into account, this can be further simplified to ten compartments, which is the minimal model. In this model, the slowest excitation energy transfer rates are between the core cylinders (time constants 115-145 ps), and between the rods and the core (time constants 68-115 ps).


Assuntos
Modelos Biológicos , Ficobilissomas/metabolismo , Synechocystis/metabolismo , Simulação por Computador , Transferência de Energia , Ficobilissomas/química , Espectrometria de Fluorescência , Fatores de Tempo
14.
Photosynth Res ; 135(1-3): 115-124, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29030777

RESUMO

Excitation energy transfer (EET) and trapping in Synechococcus WH 7803 whole cells and isolated photosystem I (PSI) complexes have been studied by time-resolved emission spectroscopy at room temperature (RT) and at 77 K. With the help of global and target analysis, the pathways of EET and the charge separation dynamics have been identified. Energy absorbed in the phycobilisome (PB) rods by the abundant phycoerythrin (PE) is funneled to phycocyanin (PC645) and from there to the core that contains allophycocyanin (APC660 and APC680). Intra-PB EET rates have been estimated to range from 11 to 68/ns. It was estimated that at RT, the terminal emitter of the phycobilisome, APC680, transfers its energy at a rate of 90/ns to PSI and at a rate of 50/ns to PSII. At 77 K, the redshifted Chl a states in the PSI core were heterogeneous, with maximum emission at 697 and 707 nm. In 72% of the PSI complexes, the bulk Chl a in equilibrium with F697 decayed with a main trapping lifetime of 39 ps.


Assuntos
Transferência de Energia , Synechococcus/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Especificidade da Espécie , Espectrometria de Fluorescência , Synechococcus/citologia , Temperatura
16.
Photosynth Res ; 135(1-3): 285-298, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29151177

RESUMO

Some mosses stay green and survive long even under desiccation. Dissipation mechanisms of excess excitation energy were studied in two drought-tolerant moss species adapted to contrasting niches: shade-adapted Rhytidiadelphus squarrosus and sun-adapted Rhytidium rugosum in the same family. (1) Under wet conditions, a light-induced nonphotochemical quenching (NPQ) mechanism decreased the yield of photosystem II (PSII) fluorescence in both species. The NPQ extent saturated at a lower illumination intensity in R. squarrosus, suggesting a larger PSII antenna size. (2) Desiccation reduced the fluorescence intensities giving significantly lower F 0 levels and shortened the overall fluorescence lifetimes in both R. squarrosus and R. rugosum, at room temperature. (3) At 77 K, desiccation strongly reduced the PSII fluorescence intensity. This reduction was smaller in R. squarrosus than in R. rugosum. (4) Global and target analysis indicated two different mechanisms of energy dissipation in PSII under desiccation: the energy dissipation to a desiccation-formed strong fluorescence quencher in the PSII core in sun-adapted R. rugosum (type-A quenching) and (5) the moderate energy dissipation in the light-harvesting complex/PSII in shade-adapted R. squarrosus (type-B quenching). The two mechanisms are consistent with the different ecological niches of the two mosses.


Assuntos
Adaptação Fisiológica , Briófitas/fisiologia , Briófitas/efeitos da radiação , Secas , Luz Solar , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/efeitos da radiação , Briófitas/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Desidratação , Dessecação , Cinética , Espectrometria de Fluorescência , Temperatura , Fatores de Tempo
17.
Biochim Biophys Acta Bioenerg ; 1859(2): 57-68, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29137991

RESUMO

Cyanobacterial thylakoid membranes are known to host photosynthetic and respiratory complexes. This hampers a straight forward interpretation of the highly dynamic fluorescence originating from photosynthetic units. The present study focuses on dark-to-light transitions in whole cells of a PSI-deficient mutant of the cyanobacterium Synechocystis sp. PCC 6803. The time-dependent cellular fluorescence spectrum has been measured, while having previously exposed the cells to different conditions that affect respiratory activity. The analysis method used allows the detected signal to be decomposed in a few components that are then assigned to functional emitting species. Additionally, we have worked out a minimal mathematical model consisting of sensible postulated species to interpret the recorded data. We conclude that the following two functional complexes play a major role: a phycobilisome antenna complex coupled to a PSII dimer with either two or no closed reaction centers. Crucially, we present evidence for an additional species capable of strongly quenching fluorescence, whose formation requires the presence of oxygen.


Assuntos
Proteínas de Bactérias/química , Complexo de Proteína do Fotossistema I/química , Synechocystis/enzimologia , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Espectrometria de Fluorescência
18.
Nat Commun ; 8(1): 1994, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29222488

RESUMO

To avoid photodamage plants regulate the amount of excitation energy in the membrane at the level of the light-harvesting complexes (LHCs). It has been proposed that the energy absorbed in excess is dissipated via protein conformational changes of individual LHCs. However, the exact quenching mechanism remains unclear. Here we study the mechanism of quenching in LHCs that bind a single carotenoid species and are constitutively in a dissipative conformation. Via femtosecond spectroscopy we resolve a number of carotenoid dark states, demonstrating that the carotenoid is bound to the complex in different conformations. Some of those states act as excitation energy donors for the chlorophylls, whereas others act as quenchers. Via in silico analysis we show that structural changes of carotenoids are expected in the LHC protein domains exposed to the chloroplast lumen, where acidification triggers photoprotection in vivo. We propose that structural changes of LHCs control the conformation of the carotenoids, thus permitting access to different dark states responsible for either light harvesting or photoprotection.


Assuntos
Carotenoides/metabolismo , Clorofila/metabolismo , Transferência de Energia/fisiologia , Complexos de Proteínas Captadores de Luz/metabolismo , Tabaco/fisiologia , Carotenoides/química , Cloroplastos/fisiologia , Simulação por Computador , Luz/efeitos adversos , Complexos de Proteínas Captadores de Luz/química , Simulação de Dinâmica Molecular , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Ligação Proteica/fisiologia , Conformação Proteica , Análise Espectral/métodos
19.
Biochim Biophys Acta Bioenerg ; 1858(10): 854-864, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28801049

RESUMO

The phenomenon of non-photochemical quenching (NPQ) was studied in spinach chloroplasts using pulse amplitude modulated (PAM) fluorometry. We present a new analysis method which describes the observed fluorescence quantum yield as the sum of the product of four different states of PSII and their corresponding quantum yields. These four distinct states are PSII in the quenched or unquenched state, and with its reaction center either open or closed depending upon the reduction of the QA site. With this method we can describe the dynamics of the NPQ induction and recovery as well as quantify the percentage of photoinactivated RC throughout the measurement. We show that after one cycle of quenching followed by a period of recovery, approximately 8-9% of the RC are photoinactivated, after two cycles of illumination this number becomes 15-17%. The recovery from the quenching appeared with rates of (50s)-1 and (1h)-1. The new analysis method presented here is flexible, allowing it to be applied to any type of PAM fluorometry protocol. The method allows to quantitatively compare qualitatively different PAM curves on the basis of statistically relevant fitting parameters and to quantify quenching dynamics and photoinactivation. Moreover, the results presented here demonstrate that the analysis of a single PAM fluorometry quenching experiment can already provide information on the relative quantum yield of the four different states of PSII for the intact chloroplasts - something no other form of spectroscopy could provide in a single measurement.


Assuntos
Complexo de Proteína do Fotossistema II/metabolismo , Percepção de Quorum/fisiologia , Cloroplastos/metabolismo , Fluorescência , Fluorometria/métodos , Cinética , Luz , Spinacia oleracea/metabolismo
20.
Sci Rep ; 7(1): 7217, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28775289

RESUMO

Channelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S1) state through two conical intersections CI1 and CI2, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via CI1 and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via CI2, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection CI2 rationalizes the relatively low quantum yield of photoisomerization (30 ± 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2.


Assuntos
Channelrhodopsins/química , Aminoácidos , Sítios de Ligação , Channelrhodopsins/metabolismo , Isomerismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Teoria Quântica , Análise Espectral , Relação Estrutura-Atividade
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