ABSTRACT
Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.
Subject(s)
Bacterial Proteins , Carotenoids , Cyanobacteria , Phycobilisomes , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carotenoids/chemistry , Carotenoids/metabolism , Cyanobacteria/metabolism , Cyanobacteria/chemistry , Phycobilisomes/chemistry , Phycobilisomes/metabolism , Bile Pigments/chemistry , Bile Pigments/metabolism , Photochemical ProcessesABSTRACT
The antenna complexes of Photosystem I present low-lying states visible as red-shifted and broadened absorption and fluorescence bands. Among these, Lhca4 has the most evident features of these "red" states, with a fluorescence band shifted by more than 25 nm from typical LHC emission. This signal arises from a mixing of exciton and charge-transfer (CT) states within the excitonically coupled a603-a609 chlorophyll (Chl) dimer. Here we combine molecular dynamics, multiscale quantum chemical calculations, and spectral simulations to uncover the molecular mechanism for the formation and tuning of exciton-CT interactions in Lhca4. We show that the coupling between exciton and CT states is extremely sensitive to tiny variations in the Chl dimer arrangement, explaining both the red-shifted bands and the switch between conformations with blue and red emission observed in single-molecule spectroscopy. Finally, we show that mutating the axial ligand of a603 diminishes the exciton-CT coupling, removing any red-state fingerprint.
ABSTRACT
The photosynthetic light-harvesting complexes (LHCs) are responsible for light absorption due to their pigment-binding properties. These pigments are primarily Chlorophyll (Chl) molecules of type a and b, which ensure an excellent coverage of the visible light spectrum. To date, it is unclear which factors drive the selective binding of different Chl types in the LHC binding pockets. To gain insights into this, we employed molecular dynamics simulations on LHCII binding different Chl types. From the resulting trajectories, we have calculated the binding affinities per each Chl-binding pocket using the Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) model. To further examine the importance of the nature of the axial ligand in tuning the Chl selectivity of the binding sites, we used Density Functional Theory (DFT) calculations. The results indicate that some binding pockets have a clear Chl selectivity, and the factors governing these selectivities are identified. Other binding pockets are promiscuous, which is consistent with previous in vitro reconstitution studies. DFT calculations show that the nature of the axial ligand is not a major factor in determining the Chl binding pocket selectivity, which is instead probably controlled by the folding process.
Subject(s)
Chlorophyll , Light-Harvesting Protein Complexes , Chlorophyll/chemistry , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/metabolism , Ligands , Binding SitesABSTRACT
The first step of photosynthesis in plants is performed by the light-harvesting complexes (LHC), a large family of pigment-binding proteins embedded in the photosynthetic membranes. These complexes are conserved across species, suggesting that each has a distinct role. However, they display a high degree of sequence homology and their static structures are almost identical. What are then the structural features that determine their different properties? In this work, we compared the two best-characterized LHCs of plants: LHCII and CP29. Using molecular dynamics simulations, we could rationalize the difference between them in terms of pigment-binding properties. The data also show that while the loops between the helices are very flexible, the structure of the transmembrane regions remains very similar in the crystal and the membranes. However, the small structural differences significantly affect the excitonic coupling between some pigment pairs. Finally, we analyzed in detail the structure of the long N-terminus of CP29, showing that it is structurally stable and it remains on top of the membrane even in the absence of other proteins. Although the structural changes upon phosphorylation are minor, they can explain the differences in the absorption properties of the pigments observed experimentally.
Subject(s)
Light-Harvesting Protein Complexes , Photosystem II Protein Complex , Light-Harvesting Protein Complexes/chemistry , Photosystem II Protein Complex/metabolism , Thylakoids/metabolism , Photosynthesis , Plant Proteins/chemistry , Plants/metabolism , Chlorophyll/metabolismABSTRACT
Increasing the absorption cross section of plants by introducing far-red absorbing chlorophylls (Chls) has been proposed as a strategy to boost crop yields. To make this strategy effective, these Chls should bind to the photosynthetic complexes without altering their functional architecture. To investigate if plant-specific antenna complexes can provide the protein scaffold to accommodate these Chls, we have reconstituted the main light-harvesting complex (LHC) of plants LHCII in vitro and in silico, with Chl d. The results demonstrate that LHCII can bind Chl d in a number of binding sites, shifting the maximum absorption â¼25 nm toward the red with respect to the wild-type complex (LHCII with Chl a and b) while maintaining the native LHC architecture. Ultrafast spectroscopic measurements show that the complex is functional in light harvesting and excitation energy transfer. Overall, we here demonstrate that it is possible to obtain plant LHCs with enhanced far-red absorption and intact functional properties.
Subject(s)
Light-Harvesting Protein Complexes , Photosynthetic Reaction Center Complex Proteins , Plants/metabolism , Chlorophyll , Energy Transfer , Photosynthetic Reaction Center Complex Proteins/metabolismABSTRACT
Carotenoids are essential constituents of plant light-harvesting complexes (LHCs), being involved in protein stability, light harvesting, and photoprotection. Unlike chlorophylls, whose binding to LHCs is known to require coordination of the central magnesium, carotenoid binding relies on weaker intermolecular interactions (such as hydrogen bonds and van der Waals forces), whose character is far more elusive. Here we addressed the key interactions responsible for carotenoid binding to LHCs by combining molecular dynamics simulations and polarizable quantum mechanics/molecular mechanics calculations on the major LHC, LHCII. We found that carotenoid binding is mainly stabilized by van der Waals interactions with the surrounding chlorophyll macrocycles rather than by hydrogen bonds to the protein, the latter being more labile than predicted from structural data. Furthermore, the interaction network in the binding pockets is relatively insensitive to the chemical structure of the embedded carotenoid. Our results are consistent with a number of experimental data and challenge the role played by specific interactions in the assembly of pigment-protein complexes.
ABSTRACT
Light-harvesting complex II (LHCII) is a pigment-protein complex present in higher plants and green algae. LHCII represents the main site of light absorption, and its role is to transfer the excitation energy toward the photosynthetic reaction centers, where primary energy conversion reactions take place. The optical properties of LHCII are known to depend on protein conformation. However, the relation between the structural and spectroscopic properties of the pigments is not fully understood yet. In this respect, previous classical molecular dynamics simulations of LHCII in a model membrane [Sci. Rep. 2015, 5, 1-10] have shown that the configuration and excitonic coupling of a chlorophyll (Chl) dimer functioning as the main terminal emitter of the complex are particularly sensitive to conformational changes. Here, we use quantum chemistry calculations to investigate in greater detail the effect of pigment-pigment interactions on the excited-state landscape. While most previous studies have used a local picture in which electrons are localized on single pigments, here we achieve a more accurate description of the Chl dimer by adopting a supramolecular picture where time-dependent density functional theory is applied to the whole system at once. Our results show that specific dimer configurations characterized by shorter inter-pigment distances can result in a sizable intensity decrease (up to 36%) of the Chl absorption bands in the visible spectral region. Such a decrease can be predicted only when accounting for Chl-Chl charge-transfer excitations, which is possible using the above-mentioned supramolecular approach. The charge-transfer character of the excitations is quantified by two types of analyses: one focusing on the composition of the excitations and the other directly on the observable total absorption intensities.
Subject(s)
Light-Harvesting Protein Complexes/chemistry , Chlorophyll/chemistry , Chlorophyll/radiation effects , Density Functional Theory , Light , Light-Harvesting Protein Complexes/radiation effects , Models, Chemical , SpectrophotometryABSTRACT
Photosynthesis is regulated by a dynamic interplay between proteins, enzymes, pigments, lipids, and cofactors that takes place on a large spatio-temporal scale. Molecular dynamics (MD) simulations provide a powerful toolkit to investigate dynamical processes in (bio)molecular ensembles from the (sub)picosecond to the (sub)millisecond regime and from the Å to hundreds of nm length scale. Therefore, MD is well suited to address a variety of questions arising in the field of photosynthesis research. In this review, we provide an introduction to the basic concepts of MD simulations, at atomistic and coarse-grained level of resolution. Furthermore, we discuss applications of MD simulations to model photosynthetic systems of different sizes and complexity and their connection to experimental observables. Finally, we provide a brief glance on which methods provide opportunities to capture phenomena beyond the applicability of classical MD.
Subject(s)
Molecular Dynamics Simulation , Photosynthesis/physiology , Thylakoids/chemistry , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/metabolism , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Quantum Theory , Thylakoids/metabolism , WorkflowABSTRACT
In plants and green algae, light-harvesting complexes (LHCs) are a large family of chlorophyll binding proteins functioning as antennae, collecting solar photons and transferring the absorbed energy to the photosynthetic reaction centers, where light to chemical energy conversion begins. Although LHCs are all highly homologous in their structure and display a variety of common features, each complex finds a specific location and task in the energy transport. One example is CP29, which occupies a pivotal position in Photosystem II, bridging the peripheral antennae to the core. The design principles behind this specificity, however, are still unclear. Here, a synergetic approach combining steady-state and ultrafast spectroscopy, mutational analysis and structure-based exciton modeling allows uncovering the energy landscape of the chlorophylls bound to this complex. We found that, although displaying an overall highly conserved exciton structure very similar to that of other LHCs, CP29 possesses an additional terminal emitter domain. The simultaneous presence of two low energy sites facing the peripheral antennae and the core, allows CP29 to efficiently work as a conduit in the energy flux. Our results show that the LHCs share a common solid architecture but have finely tuned their structure to carry out specific functions.
Subject(s)
Light-Harvesting Protein Complexes/metabolism , Photosystem II Protein Complex/metabolism , Plants/metabolism , Plants/radiation effects , Sunlight , Chlorophyll/metabolism , Energy Transfer , Models, Molecular , Mutation/genetics , ThermodynamicsABSTRACT
Under strong sunlight, plants avoid photooxidation by quenching the excess absorbed energy. Quenching is triggered by PsbS, a membrane protein that is activated and deactivated by the light-dependent pH changes in the thylakoid lumen. The mechanism of action of this protein is unknown, but it was suggested that several glutamates act as pH sensors. However, the p Ka of glutamate is several pH units below the physiological values in the lumen. Thus, how can PsbS sense the pH of the lumen, and how does it respond to it? By applying a nonstandard molecular dynamics method that treats pH explicitly, we show that the lumen-exposed glutamates of PsbS have strongly shifted p Ka values and that such shifts are crucial for the pH sensitivity in physiological conditions. We also demonstrate that protonation drives a systematic unfolding of a region key for protein-protein interactions, indicating that PsbS response to pH is a functional conformational switch.
ABSTRACT
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.
Subject(s)
Carotenoids/metabolism , Chlorophyll/metabolism , Energy Transfer/physiology , Light-Harvesting Protein Complexes/metabolism , Nicotiana/physiology , Carotenoids/chemistry , Chloroplasts/physiology , Computer Simulation , Light/adverse effects , Light-Harvesting Protein Complexes/chemistry , Molecular Dynamics Simulation , Photosynthesis/physiology , Plants, Genetically Modified/physiology , Protein Binding/physiology , Protein Conformation , Spectrum Analysis/methodsABSTRACT
The light harvesting complex II (LHCII), is a pigment-protein complex responsible for most of the light harvesting in plants. LHCII harvests sunlight and transfers excitation energy to the reaction centre of the photo-system, where the water oxidation process takes place. The energetics of LHCII can be modulated by means of conformational changes allowing a switch from a harvesting to a quenched state. In this state, the excitation energy is no longer transferred but converted into thermal energy to prevent photooxidation. Based on molecular dynamics simulations at the microsecond time scale, we have recently proposed that the switch between different fluorescent states can be probed by correlating shifts in the chromophore-chromophore Coulomb interactions to particular protein movements. However, these findings are based upon calculations in the ideal point dipole approximation (IDA) where the Coulomb couplings are simplified as first order dipole-dipole interactions, also assuming that the chromophore transition dipole moments lay in particular directions of space with constant moduli (FIX-IDA). In this work, we challenge this approximation using the time-dependent density functional theory (TDDFT) combined with the frozen density embedding (FDE) approach. Our aim is to establish up to which limit FIX-IDA can be applied and which chromophore types are better described under this approximation. For that purpose, we use the classical trajectories of solubilised light harvesting complex II (LHCII) we have recently reported [Liguori et al., Sci. Rep., 2015, 5, 15661] and selected three pairs of chromophores containing chlorophyll and carotenoids (Chl and Car): Chla611-Chla612, Chlb606-Chlb607 and Chla612-Lut620. Using the FDE in the Tamm-Dancoff approximation (FDEc-TDA), we show that IDA is accurate enough for predicting Chl-Chl Coulomb couplings. However, the FIX-IDA largely overestimates Chl-Car interactions mainly because the transition dipole for the Cars is not trivially oriented on the polyene chain.
Subject(s)
Light-Harvesting Protein Complexes/chemistry , Carotenoids/chemistry , Chlorophyll/chemistry , Light-Harvesting Protein Complexes/metabolism , Molecular Dynamics Simulation , Protein Structure, Tertiary , Solubility , ThermodynamicsABSTRACT
Under excess light, photosynthetic organisms employ feedback mechanisms to avoid photodamage. Photoprotection is triggered by acidification of the lumen of the photosynthetic membrane following saturation of the metabolic activity. A low pH triggers thermal dissipation of excess absorbed energy by the light-harvesting complexes (LHCs). LHCs are not able to sense pH variations, and their switch to a dissipative mode depends on stress-related proteins and allosteric cofactors. In green algae the trigger is the pigment-protein complex LHCSR3. Its C-terminus is responsible for a pH-driven conformational change from a light-harvesting to a quenched state. Here, we show that by replacing the C-terminus of the main LHC of plants with that of LHCSR3, it is possible to regulate its excited-state lifetime solely via protonation, demonstrating that the protein template of LHCs can be modified to activate reversible quenching mechanisms independent of external cofactors and triggers.
Subject(s)
Arabidopsis/genetics , Chlamydomonas reinhardtii/genetics , Genetic Engineering/methods , Light-Harvesting Protein Complexes/chemistry , Photosynthesis/physiology , Pigments, Biological/chemistry , Amino Acid Sequence , Arabidopsis/metabolism , Arabidopsis/radiation effects , Binding Sites , Chlamydomonas reinhardtii/metabolism , Chlamydomonas reinhardtii/radiation effects , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Light , Light-Harvesting Protein Complexes/genetics , Light-Harvesting Protein Complexes/metabolism , Models, Molecular , Pigments, Biological/genetics , Pigments, Biological/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Thermodynamics , Thylakoids/physiology , Thylakoids/radiation effects , TransgenesABSTRACT
LHCSR3 is a member of the Light-Harvesting Complexes (LHC) family, which is mainly composed of pigment-protein complexes responsible for collecting photons during the first steps of photosynthesis. Unlike related LHCs, LHCSR3 is expressed in stress conditions and has been shown to be essential for the fast component of photoprotection, non-photochemical quenching (NPQ), in the green alga Chlamydomonas reinhardtii. In plants, which do not possess LHCSR homologs, NPQ is triggered by the PSBS protein. Both PSBS and LHCSR3 possess the ability to sense pH changes but, unlike PSBS, LHCSR3 binds multiple pigments. In this work we have analyzed the properties of the pigments bound to LHCSR3 and their excited state dynamics. The data show efficient excitation energy transfer between pigments with rates similar to those observed for the other LHCs. Application of an exciton model based on a template of LHCII, the most abundant LHC, satisfactorily explains the collected steady state and time-resolved spectroscopic data, indicating that LHCSR3 has a LHC-like molecular architecture, although it probably binds less pigments. The model suggests that most of the chlorophylls have similar energy and interactions as in LHCII. The most striking difference is the localization of the lowest energy state, which is not on the Chlorophyll a (Chl a) 610-611-612 triplet as in all the LHCB antennas, but on Chl a613, which is located close to the lumen and to the pH-sensing region of the protein.
Subject(s)
Chlamydomonas reinhardtii/chemistry , Light-Harvesting Protein Complexes/chemistry , Chlorophyll/chemistry , Chlorophyll A , Photosystem II Protein Complex/chemistryABSTRACT
Light-Harvesting Complex II (LHCII) is largely responsible for light absorption and excitation energy transfer in plants in light-limiting conditions, while in high-light it participates in photoprotection. It is generally believed that LHCII can change its function by switching between different conformations. However, the underlying molecular picture has not been elucidated yet. The available crystal structures represent the quenched form of the complex, while solubilized LHCII has the properties of the unquenched state. To determine the structural changes involved in the switch and to identify potential quenching sites, we have explored the structural dynamics of LHCII, by performing a series of microsecond Molecular Dynamics simulations. We show that LHCII in the membrane differs substantially from the crystal and has the signatures that were experimentally associated with the light-harvesting state. Local conformational changes at the N-terminus and at the xanthophyll neoxanthin are found to strongly correlate with changes in the interactions energies of two putative quenching sites. In particular conformational disorder is observed at the terminal emitter resulting in large variations of the excitonic coupling strength of this chlorophyll pair. Our results strongly support the hypothesis that light-harvesting regulation in LHCII is coupled with structural changes.
Subject(s)
Light-Harvesting Protein Complexes/chemistry , Light , Plant Physiological Phenomena , Models, MolecularABSTRACT
Electron transfers within and between protein complexes are core processes of the electron transport chains occurring in thylakoid (chloroplast), mitochondrial, and bacterial membranes. These electron transfers involve a number of cofactors. Here we describe the derivation of molecular mechanics parameters for the cofactors associated with the function of the photosystem II core complex: plastoquinone, plastoquinol, heme b, chlorophyll A, pheophytin, and ß-carotene. Parameters were also obtained for ubiquinol and ubiquinone, related cofactors involved in the respiratory chain. Parameters were derived at both atomistic and coarse grain (CG) resolutions, compatible with the building blocks of the GROMOS united-atom and Martini CG force fields, respectively. Structural and thermodynamic properties of the cofactors were compared to experimental values when available. The topologies were further tested in molecular dynamics simulations of the cofactors in their physiological environment, e.g., either in a lipid membrane environment or in complex with the heme binding protein bacterioferritin.
Subject(s)
Photosystem II Protein Complex/metabolism , Chlorophyll/metabolism , Chlorophyll A , Heme/metabolism , Lipid Bilayers/metabolism , Molecular Dynamics Simulation , Molecular Structure , Octanols/chemistry , Pheophytins/metabolism , Plastoquinone/analogs & derivatives , Plastoquinone/metabolism , Protein Conformation , Thermodynamics , Ubiquinone/analogs & derivatives , Ubiquinone/metabolism , Water/chemistry , beta Carotene/metabolismABSTRACT
Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.
Subject(s)
Chlamydomonas reinhardtii/metabolism , Light-Harvesting Protein Complexes/metabolismABSTRACT
Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-ß in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aß peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer's disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.