RESUMO
Non-photochemical quenching (NPQ) is a fast acting photoprotective response to high light stress triggered by over excitation of photosystem II. The mechanism for NPQ in the globally important diatom algae has been principally attributed to a xanthophyll cycle, analogous to the well-described qE quenching of higher plants. This study compared the short-term NPQ responses in two pennate, benthic diatom species cultured under identical conditions but which originate from unique light climates. Variable chlorophyll fluorescence was used to monitor photochemical and non-photochemical excitation energy dissipation during high light transitions; whereas whole cell steady state 77 K absorption and emission were used to measure high light elicited changes in the excited state landscapes of the thylakoid. The marine shoreline species Nitzschia curvilineata was found to have an antenna system capable of entering a deeply quenched, yet reversible state in response to high light, with NPQ being highly sensitive to dithiothreitol (a known inhibitor of the xanthophyll cycle). Conversely, the salt flat species Navicula sp. 110-1 exhibited a less robust NPQ that remained largely locked-in after the light stress was removed; however, a lower amplitude, but now highly reversible NPQ persisted in cells treated with dithiothreitol. Furthermore, dithiothreitol inhibition of NPQ had no functional effect on the ability of Navicula cells to balance PSII excitation/de-excitation. These different approaches for non-photochemical excitation energy dissipation are discussed in the context of native light climate.
Assuntos
Diatomáceas/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Clorofila/metabolismo , Clorofila/fisiologia , Clima , Diatomáceas/efeitos da radiação , Transporte de Elétrons , Complexos de Proteínas Captadores de Luz/metabolismo , Complexos de Proteínas Captadores de Luz/fisiologia , Processos Fotoquímicos , Estresse Fisiológico , Luz Solar , Tilacoides/metabolismo , Tilacoides/fisiologiaRESUMO
William (Bill) E. Vidaver (February 2, 1921-August 31, 2017), who did his Ph.D. with Laurence (Larry) R. Blinks at Stanford (1964) and a postdoc with C. Stacy French (1965), taught and did research at Simon Fraser University (SFU) for almost 30 years. Here he published over 80 papers in photosynthesis-related areas co-authored by his graduate students, postdocs, visiting professors and SFU colleagues. He developed a unique high-pressure cuvette for the study of oxygen exchange and studied high-pressure effects in photosynthesis. Ulrich (Uli) Schreiber, as a postdoctoral fellow from Germany, introduced measurements on chlorophyll (Chl) a fluorescence to Bill's lab, leading to the discovery of reversible inhibition of excitation energy transfer between photosynthetic pigments and of a pivotal role of O2 in the oxidation of the electron transport chain between Photosystem II (PS II) and PS I. Bill's and Uli's work led to a patent of a portable chlorophyll fluorometer, the first available commercially, which was later modified to measure whole plantlets. The latter was used in pioneering measurement of the health of forest and crop plants undergoing in vitro clonal micropropagation. With several other researchers (including Doug Bruce, the late Radovan Popovic, and Sarah Swenson), he localized the quenching site of O2 and showed a dampening effect on measurements of the four-step process of O2 production by endogenous oxygen uptake. Bill is remembered as a hard-working but fun-loving person with a keen mind and strong sense of social justice.
Assuntos
Oxigênio/história , Fotossíntese , Plantas , Transporte de Elétrons , Transferência de Energia , Alemanha , História do Século XX , História do Século XXI , Pessoal de Laboratório/história , Oxigênio/metabolismoRESUMO
Photosystem II (PSII) of photosynthesis catalyzes one of the most challenging reactions in nature, the light driven oxidation of water and release of molecular oxygen. PSII couples the sequential four step oxidation of water and two step reduction of plastoquinone to single photon photochemistry with charge accumulation centers on both its electron donor and acceptor sides. Photon capture, excitation energy transfer, and trapping occur on a much faster time scale than the subsequent electron transfer and charge accumulation steps. A balance between excitation of PSII and the use of the absorbed energy to drive electron transport is essential. If the absorption of light energy increases and/or the sink capacity for photosynthetically derived electrons decreases, potentially deleterious side reactions may occur, including the production of reactive oxygen species. In response, a myriad of fast (second to minutes timescale) and reversible photoprotective mechanisms are observed to regulate PSII excitation when the environment changes more quickly than can be acclimated to by gene expression. This review compares the diverse photoprotective mechanisms that are used to dissipate (quench) PSII excitation within the antenna systems of higher land plants, green algae, diatoms, and cyanobacteria. The molecular bases of how PSII excitation pressure is sensed by the antenna system and how the antenna then reconfigures itself from a light harvesting to an energy dissipative mode are discussed.
Assuntos
Meio Ambiente , Fotoquímica , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Transporte de ElétronsRESUMO
The oxygen-evolving complex (OEC) of photosystem II (PSII) is a unique Mn4O5Ca cluster that catalyzes water oxidation via four photoactivated electron transfer steps. As the protein influence on the redox and protonation chemistry of the OEC remains an open question, we present a classical valence model of the OEC that allows the redox state of each Mn and the protonation state of bridging µ-oxos and terminal waters to remain in equilibrium with the PSII protein throughout the redox cycle. We find that the last bridging oxygen loses its proton during the transition from S0 to S1. Two possible S2 states are found depending on the OEC geometry: S2 has Mn4(IV) with a proton lost from a terminal water (W1) trapped by the nearby D1-D61 if O5 is closer to Mn4, or Mn1(IV), with partial deprotonation of D1-H337 and D1-E329 if O5 is closer to Mn1. In S3, the OEC is Mn4(IV) with W2 deprotonated. The estimated OEC Em's range from +0.7 to +1.3 V, enabling oxidation by P680(+), the primary electron donor in PSII. In chloride-depleted PSII, the proton release increases during the S1 to S2 transition, leaving the OEC unable to properly advance through the water-splitting cycle.
Assuntos
Oxigênio/química , Complexo de Proteína do Fotossistema II/química , Prótons , Teoria Quântica , Cálcio/química , Cálcio/metabolismo , Transporte de Elétrons , Elétrons , Manganês/química , Manganês/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismoRESUMO
Retinoic acid, the active metabolite of vitamin A, is important for nervous system development, regeneration, as well as cognitive functions of the adult central nervous system. These central nervous system functions are all highly dependent on neuronal activity. Retinoic acid has previously been shown to induce changes in the firing properties and action potential waveforms of adult molluscan neurons in a dose- and isomer-dependent manner. In this study, we aimed to determine the cellular pathways by which retinoic acid might exert such effects, by testing the involvement of pathways previously shown to be affected by retinoic acid. We demonstrated that the ability of all-trans retinoic acid (atRA) to induce electrophysiological changes in cultured molluscan neurons was not prevented by inhibitors of protein synthesis, protein kinase A or phospholipase C. However, we showed that atRA was capable of rapidly reducing intracellular calcium levels in the same dose- and isomer-dependent manner as shown previously for changes in neuronal firing. Moreover, we also demonstrated that the transmembrane ion flux through voltage-gated calcium channels was rapidly modulated by retinoic acid. In particular, the peak current density was reduced and the inactivation rate was increased in the presence of atRA, over a similar time course as the changes in cell firing and reductions in intracellular calcium. These studies provide further evidence for the ability of atRA to induce rapid effects in mature neurons.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Tretinoína/farmacologia , Potenciais de Ação , Animais , Apamina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Lymnaea , Neurônios/fisiologia , Imagem Óptica , Técnicas de Patch-Clamp , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
When modelling tissue-level cardiac electrophysiology, a continuum approximation to the discrete cell-level equations, known as the bidomain equations, is often used to maintain computational tractability. Analysing the derivation of the bidomain equations allows us to investigate how microstructure, in particular gap junctions that electrically connect cells, affect tissue-level conductivity properties. Using a one-dimensional cable model, we derive a modified form of the bidomain equations that take gap junctions into account, and compare results of simulations using both the discrete and continuum models, finding that the underlying conduction velocity of the action potential ceases to match up between models when gap junctions are introduced at physiologically realistic coupling levels. We show that this effect is magnified by: (i) modelling gap junctions with reduced conductivity; (ii) increasing the conductance of the fast sodium channel; and (iii) an increase in myocyte length. From this, we conclude that the conduction velocity arising from the bidomain equations may not be an accurate representation of the underlying discrete system. In particular, the bidomain equations are less likely to be valid when modelling certain diseased states whose symptoms include a reduction in gap junction coupling or an increase in myocyte length.
Assuntos
Junções Comunicantes/fisiologia , Coração/fisiologia , Modelos Cardiovasculares , Potenciais de Ação , Animais , Simulação por Computador , Fenômenos Eletrofisiológicos , Humanos , Conceitos Matemáticos , Miócitos Cardíacos/fisiologiaRESUMO
Photosystem II (PSII) of photosynthesis has the unique ability to photochemically oxidize water. Recently an engineered bacterioferritin photochemical 'reaction centre' (BFR-RC) using a zinc chlorin pigment (ZnCe6) in place of its native heme has been shown to photo-oxidize bound manganese ions through a tyrosine residue, thus mimicking two of the key reactions on the electron donor side of PSII. To understand the mechanism of tyrosine oxidation in BFR-RCs, and explore the possibility of water oxidation in such a system we have built an atomic-level model of the BFR-RC using ONIOM methodology. We studied the influence of axial ligands and carboxyl groups on the oxidation potential of ZnCe6 using DFT theory, and finally calculated the shift of the redox potential of ZnCe6 in the BFR-RC protein using the multi-conformational molecular mechanics-Poisson-Boltzmann approach. According to our calculations, the redox potential for the first oxidation of ZnCe6 in the BRF-RC protein is only 0.57 V, too low to oxidize tyrosine. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. We discuss the possibilities for modifying the BFR-RC to achieve this goal.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Ferritinas/metabolismo , Metaloporfirinas/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Porfirinas/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Clorofilídeos , Grupo dos Citocromos b/química , Ferritinas/química , Concentração de Íons de Hidrogênio , Ligantes , Metaloporfirinas/química , Modelos Moleculares , Conformação Molecular , Método de Monte Carlo , Oxirredução , Complexo de Proteína do Fotossistema II/química , Porfirinas/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Soluções , Tirosina/química , Tirosina/metabolismoRESUMO
Photosystem II (PSII) catalyzes the oxidation of water in the conversion of light energy into chemical energy in photosynthesis. Water delivery and oxygen removal from the oxygen evolving complex (OEC), buried deep within PSII, are critical requirements to facilitate the reaction and minimize reactive oxygen damage. It has often been assumed that water and oxygen travel through separate channels within PSII, as demonstrated in cytochrome c oxidase. This study describes all-atom molecular dynamics simulations of PSII designed to investigate channels by fully characterizing the distribution and permeation of both water and oxygen. Interestingly, most channels found in PSII were permeable to both oxygen and water, however individual channels exhibited different energetic barriers for the two solutes. Several routes for oxygen diffusion within PSII with low energy permeation barriers were found, ensuring its fast removal from the OEC. In contrast, all routes for water showed significant energy barriers, corresponding to a much slower permeation rate for water through PSII. Two major factors were responsible for this selectivity: (1) hydrogen bonds between water and channel amino acids, and (2) steric restraints. Our results reveal the presence of a shared network of channels in PSII optimized to both facilitate the quick removal of oxygen and effectively restrict the water supply to the OEC to help stabilize and protect it from small water soluble inhibitors.
Assuntos
Metabolismo Energético , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Água/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Permeabilidade , Complexo de Proteína do Fotossistema II/química , Conformação ProteicaRESUMO
The influence of electrostatic interactions on the free energy of proton coupled electron transfer in biomimetic oxomanganese complexes inspired by the oxygen-evolving complex (OEC) of photosystem II (PSII) are investigated. The reported study introduces an enhanced multiconformer continuum electrostatics (MCCE) model, parametrized at the density functional theory (DFT) level with a classical valence model for the oxomanganese core. The calculated pKa's and oxidation midpoint potentials (E(m)'s) match experimental values for eight complexes, indicating that purely electrostatic contributions account for most of the observed couplings between deprotonation and oxidation state transitions. We focus on pKa's of terminal water ligands in [Mn(II/III)(H2O)6](2+/3+) (1), [Mn(III)(P)(H2O)2](3-) (2, P = 5,10,15,20-tetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrinato), [Mn2(IV,IV)(µ-O)2(terpy)2(H2O)2](4+) (3, terpy = 2,2':6',2â³-terpyridine), and [Mn3(IV,IV,IV)(µ-O)4(phen)4(H2O)2](4+) (4, phen = 1,10-phenanthroline) and the pKa's of µ-oxo bridges and Mn E(m)'s in [Mn2(µ-O)2(bpy)4] (5, bpy = 2,2'-bipyridyl), [Mn2(µ-O)2(salpn)2] (6, salpn = N,N'-bis(salicylidene)-1,3-propanediamine), [Mn2(µ-O)2(3,5-di(Cl)-salpn)2] (7), and [Mn2(µ-O)2(3,5-di(NO2)-salpn)2] (8). The analysis of complexes 6-8 highlights the strong coupling between electron and proton transfers, with any Mn oxidation lowering the pKa of an oxo bridge by 10.5 ± 0.9 pH units. The model also accounts for changes in the E(m)'s by ligand substituents, such as found in complexes 6-8, due to the electron withdrawing Cl (7) and NO2 (8). The reported study provides the foundation for analysis of electrostatic effects in other oxomanganese complexes and metalloenzymes, where proton coupled electron transfer plays a fundamental role in redox-leveling mechanisms.
Assuntos
Materiais Biomiméticos/química , Manganês/química , Oxigênio/química , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Prótons , Eletricidade Estática , Transporte de Elétrons , Ligantes , Modelos Moleculares , Conformação Molecular , Compostos Organometálicos/química , Oxirredução , Teoria Quântica , Solventes/química , Termodinâmica , Água/químicaRESUMO
The Mn(4)Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn(4)Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn(4)Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca(2+) and Cl(-) from the active site.
Assuntos
Metabolismo Energético , Simulação de Dinâmica Molecular , Complexo de Proteína do Fotossistema II/metabolismo , Água/química , Sítios de Ligação , Permeabilidade , Fotossíntese , Água/metabolismoRESUMO
The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or "site." Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment-proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment-protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna-Mathews-Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.
Assuntos
Proteínas de Bactérias/fisiologia , Bacterioclorofilas/fisiologia , Complexos de Proteínas Captadores de Luz/fisiologia , Simulação de Dinâmica Molecular , Transferência de Energia , Eletricidade EstáticaRESUMO
Chloride binding in photosystem II (PSII) is essential for photosynthetic water oxidation. However, the functional roles of chloride and possible binding sites, during oxygen evolution, remain controversial. This paper examines the functions of chloride based on its binding site revealed in the X-ray crystal structure of PSII at 1.9 Å resolution. We find that chloride depletion induces formation of a salt bridge between D2-K317 and D1-D61 that could suppress the transfer of protons to the lumen.
Assuntos
Cloretos/química , Cloretos/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Oxigênio/metabolismo , Prótons , Relação Estrutura-AtividadeRESUMO
The prediction of protein side-chain conformation is central for understanding protein functions. Side-chain packing is a sub-problem of protein folding and its computational complexity has been shown to be NP-hard. We investigated the capabilities of a hybrid (genetic algorithm/simulated annealing) technique for side-chain packing and for the generation of an ensemble of low energy side-chain conformations. Our method first relies on obtaining a near-optimal low energy protein conformation by optimizing its amino-acid side-chains. Upon convergence, the genetic algorithm is allowed to undergo forward and "backward" evolution by alternating selection pressures between minimal and higher energy setpoints. We show that this technique is very efficient for obtaining distributions of solutions centered at any desired energy from the minimum. We outline the general concepts of our evolutionary sampling methodology using three different alternating selective pressure schemes. Quality of the method was assessed by using it for protein pK(a) prediction.
Assuntos
Algoritmos , Biologia Computacional/métodos , Modelos Genéticos , Proteínas/química , Sequência de Aminoácidos , Animais , Galinhas/genética , Galinhas/metabolismo , Clara de Ovo/química , Evolução Molecular , Modelos Moleculares , Muramidase/química , Muramidase/genética , Conformação Proteica , Dobramento de Proteína , Proteínas/genéticaRESUMO
The CaMn(4) cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the CaMn(4) cluster by moving through protein. Channels for water entrance, and proton and oxygen exit, have previously been proposed following the analysis of cavities found within X-ray structures of PSII. However, these analyses do not account for the dynamic motion of proteins and cannot track the movement of water within PSII. To study water dynamics in PSII, we performed molecular dynamics simulations and developed a novel approach for the visualization of water diffusion within protein based on a streamline tracing algorithm used in fluid dynamics and diffusion tensor imaging. We identified a system of branching pathways of water diffusion in PSII leading to the OEC that connect to a number of distinct entrance points on the lumenal surface. We observed transient changes in the connections between channels and entrance points that served to moderate both the flow of water near the OEC and the exchange of water inside and outside of the protein. Water flow was significantly altered in simulations lacking the OEC which were characterized by a simpler and wider channel with only two openings, consistent with the creation of an ion channel that allows entry of Mn(2+), Ca(2+), and Cl(-) as required for construction of the CaMn(4) cluster.
Assuntos
Simulação de Dinâmica Molecular , Complexo de Proteína do Fotossistema II/química , Movimentos da Água , Água/química , Algoritmos , Cristalografia por Raios X , Difusão , Oxigênio/química , Complexo de Proteína do Fotossistema II/análise , Proteínas de Plantas/química , Água/análiseRESUMO
Phycobilisomes are the major light-harvesting complexes for cyanobacteria, and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. Phycocyanobilin chromophores are covalently bonded to the phycocyanin beta subunit (CpcB) by specific lyases which have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, we found that mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin when grown under low-light conditions. Absorbance measurements at 10 K revealed the energy states of the beta phycocyanin chromophores to be slightly shifted, and 77 K steady state fluorescence emission spectroscopy showed that excitation energy transfer involving the targeted chromophores was disrupted. This evidence indicates that the position of the phycocyanobilin chromophore within the binding domain of the phycocyanin beta subunit had been modified. We hypothesize that alternate, less specific lyases are able to add chromophores, with varying effectiveness, to the beta binding sites.
Assuntos
Proteínas de Bactérias/metabolismo , Liases/metabolismo , Mutação , Ficobilinas/metabolismo , Ficocianina/metabolismo , Synechococcus/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz , Liases/genética , Ficobilinas/química , Ficobiliproteínas/genética , Ficobiliproteínas/metabolismo , Ficobilissomas/metabolismo , Ficocianina/química , Ligação Proteica/efeitos da radiação , Espectrofotometria/métodos , Synechococcus/enzimologia , Synechococcus/genéticaRESUMO
The photophysical properties of axial-bonding types (terpyridoxy)aluminum(III) porphyrin (Al(PTP)), bis(terpyridoxy)tin(IV) porphyrin (Sn(PTP) 2), and bis(terpyridoxy)phosphorus(V) porphyrin ([P(PTP) 2] (+)) are reported. Compared with their hydroxy analogues, the fluorescence quantum yields and singlet-state lifetimes were found to be lower for Sn(PTP) 2 and [P(PTP) 2] (+), whereas no difference was observed for Al(PTP). At low temperature, all of the compounds show spin-polarized transient electron paramagnetic resonance (TREPR) spectra that are assigned to the lowest excited triplet state of the porphyrin populated by intersystem crossing. In contrast, at room temperature, a triplet radical-pair spectrum that decays to the porphyrin triplet state with a lifetime of 175 ns is observed for [P(PTP) 2] (+), whereas no spin-polarized TREPR spectrum is found for Sn(PTP) 2 and only the porphyrin triplet populated by intersystem crossing is seen for Al(PTP). These results clarify the role of the internal molecular structure and the reduction potential for electron transfer from the terpyridine ligand to the excited porphyrin. It is argued that the efficiency of this process is dependent on the oxidation state of the metal/metalloid present in the porphyrin and the reorganization energy of the solvent.
RESUMO
Conversion of light energy in photosynthesis is extremely fast and efficient, and understanding the nature of this complex photophysical process is challenging. This review describes current progress in understanding molecular mechanisms of light harvesting and charge separation in photosystem II (PSII). Breakthroughs in X-ray crystallography have allowed the development and testing of more detailed kinetic models than have previously been possible. However, due to the complexity of the light conversion processes, satisfactory descriptions remain elusive. Recent advances point out the importance of variations in the photochemical properties of PSII in situ in different thylakoid membrane regions as well as the advantages of combining sophisticated time-resolved spectroscopic experiments with atomic level computational modeling which includes the effects of molecular dynamics.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Radicais Livres/química , Íons/química , Cinética , Modelos MolecularesRESUMO
Lichens, a symbiotic relationship between a fungus (mycobiont) and a photosynthetic green algae or cyanobacteria (photobiont), belong to an elite group of survivalist organisms termed resurrection species. When lichens are desiccated, they are photosynthetically inactive, but upon rehydration they can perform photosynthesis within seconds. Desiccation is correlated with both a loss of variable chlorophyll a fluorescence and a decrease in overall fluorescence yield. The fluorescence quenching likely reflects photoprotection mechanisms that may be based on desiccation-induced changes in lichen structure that limit light exposure to the photobiont (sunshade effect) and/or active quenching of excitation energy absorbed by the photosynthetic apparatus. To separate and quantify these possible mechanisms, we have investigated the origins of fluorescence quenching in desiccated lichens with steady-state, low temperature, and time-resolved chlorophyll fluorescence spectroscopy. We found the most dramatic target of quenching to be photosystem II (PSII), which produces negligible levels of fluorescence in desiccated lichens. We show that fluorescence decay in desiccated lichens was dominated by a short lifetime, long-wavelength component energetically coupled to PSII. Remaining fluorescence was primarily from PSI and although diminished in amplitude, PSI decay kinetics were unaffected by desiccation. The long-wavelength-quenching species was responsible for most (about 80%) of the fluorescence quenching observed in desiccated lichens; the rest of the quenching was attributed to the sunshade effect induced by structural changes in the lichen thallus.
Assuntos
Fluorescência , Líquens/efeitos da radiação , Luz , Água/metabolismo , Líquens/metabolismo , Espectrometria de Fluorescência , TemperaturaRESUMO
A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.
Assuntos
Complexo de Proteína do Fotossistema II/fisiologia , Tilacoides/química , Temperatura Baixa , Cinética , Modelos Biológicos , Complexo de Proteína do Fotossistema I/análise , Espectrometria de Fluorescência , Spinacia oleracea/químicaRESUMO
Exposure to blue light has previously been shown to induce the reversible quenching of fluorescence in cyanobacteria, indicative of a photoprotective mechanism responsible for the down regulation of photosynthesis. We have investigated the molecular mechanism behind fluorescence quenching by characterizing changes in excitation energy transfer through the phycobilin pigments of the phycobilisome to chlorophyll with steady-state and time-resolved fluorescence excitation and emission spectroscopy. Quenching was investigated in both a photosystem II-less mutant, and DCMU-poisoned wild-type Synechocystis sp. PCC 6803. The action spectra for blue-light-induced quenching was identical in both cell types and was dominated by a band in the blue region, peaking at 480 nm. Fluorescence quenching and its dark recovery was inhibited by the protein cross-linking agent glutaraldehyde, which could maintain cells in either the quenched or the unquenched state. We found that high phosphate concentrations that inhibit phycobilisome mobility and the regulation of energy transfer by the light-state transition did not affect blue-light-induced fluorescence quenching. Both room temperature and 77 K fluorescence emission spectra revealed that fluorescence quenching was associated with phycobilin emission. Quenching was characterized by a decrease in the emission of allophycocyanin and long wavelength phycobilisome terminal emitters relative to that of phycocyanin. A global analysis of the room-temperature fluorescence decay kinetics revealed that phycocyanin and photosystem I decay components were unaffected by quenching, whereas the decay components originating from allophycocyanin and phycobilisome terminal emitters were altered. Our data support a regulatory mechanism involving a protein conformational change and/or change in protein-protein interaction which quenches excitation energy at the core of the phycobilisome.