ABSTRACT
Photosystem II (PSII) catalyzes the light-driven charge separation and water oxidation reactions of photosynthesis. Eukaryotic PSII core is usually associated with membrane-embedded light-harvesting antennae, which greatly increase the absorbance cross-section of the core. The peripheral antennae in different phototrophs vary considerably in protein composition and arrangement. Photosynthetic cryptophytes possess chlorophyll a/c binding proteins (CACs) that serve as their antennae. How these CACs assemble with the PSII core remains unclear. Here, we report the 2.57-Å resolution structure of cryptophyte PSII-CAC purified from cells at nitrogen-limited stationary growth phase. We show that each monomer of the PSII homodimer contains a core complex, six chlorophyll a/c binding proteins (CACs) and a previously unseen chlorophyll-binding protein (termed CAL-II). Six CACs are arranged as a double-layered arc-shaped non-parallel belt, and two such belts attach to the dimeric core from opposite sides. The CAL-II simultaneously interacts with a number of core subunits and five CACs. The distinct organization of CACs and the presence of CAL-II may play a critical role in stabilizing the dimeric PSII-CAC complex under stress conditions. Our study provides mechanistic insights into the assembly and function of the PSII-CAC complex as well as the possible adaptation of cryptophytes in response to environmental stresses.
Subject(s)
Cryptophyta , Photosystem II Protein Complex , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Cryptophyta/metabolism , Chlorophyll Binding Proteins/metabolism , Chlorophyll Binding Proteins/chemistry , Photosynthesis , Models, Molecular , Light-Harvesting Protein Complexes/metabolism , Light-Harvesting Protein Complexes/chemistryABSTRACT
This research aimed to develop natural plant systems to serve as biological sentinels for the detection of organophosphate pesticides in the environment. The working hypothesis was that the presence of the pesticide in the environment caused changes in the content of pigments and in the photosynthetic functioning of the plant, which could be evaluated non-destructively through the analysis of reflected light and emitted fluorescence. The objective of the research was to furnish in vivo indicators derived from spectroscopic parameters, serving as early alert signals for the presence of organophosphates in the environment. In this context, the effects of two pesticides, Chlorpyrifos and Dimethoate, on the spectroscopic properties of aquatic plants (Vallisneria nana and Spathyfillum wallisii) were studied. Chlorophyll-a variable fluorescence allowed monitoring both pesticides' presence before any damage was observed at the naked eye, with the analysis of the fast transient (OJIP curve) proving more responsive than Kautsky kinetics, steady-state fluorescence, or reflectance measurements. Pesticides produced a decrease in the maximum quantum yield of PSII photochemistry, in the proportion of PSII photochemical deexcitation relative to PSII non photochemical decay and in the probability that trapped excitons moved electrons into the photosynthetic transport chain beyond QA-. Additionally, an increase in the proportion of absorbed energy being dissipated as heat rather than being utilized in the photosynthetic process, was notorious. The pesticides induced a higher deactivation of chlorophyll excited states by photophysical pathways (including fluorescence) with a decrease in the quantum yields of photosystem II and heat dissipation by non-photochemical quenching. The investigated aquatic plants served as sentinels for the presence of pesticides in the environment, with the alert signal starting within the first milliseconds of electronic transport in the photosynthetic chain. Organophosphates damage animals' central nervous systems similarly to certain compounds found in chemical weapons, thus raising the possibility that sentinel plants could potentially signal the presence of such weapons.
Subject(s)
Chlorophyll , Chlorpyrifos , Chlorophyll/metabolism , Chlorophyll/chemistry , Chlorpyrifos/metabolism , Chlorpyrifos/toxicity , Fluorescence , Pesticides/toxicity , Pesticides/metabolism , Photosynthesis/drug effects , Dimethoate/toxicity , Dimethoate/metabolism , Spectrometry, Fluorescence , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Environmental Monitoring/methods , Chlorophyll A/metabolism , Chlorophyll A/chemistry , Kinetics , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/toxicity , Water Pollutants, Chemical/metabolismABSTRACT
Photosynthesis system II (PS II) is an important target for the development of bioherbicides. In this study, a series of natural naphthoquinone derivatives containing diaryl ether were designed and synthesized based on the binding model of lawsone and PS II D1. Bioassays exhibited that most compounds had more than 80% inhibition of Portulaca oleracea and Echinochloa crusgalli roots at a dose of 100 µg/mL and compounds B4, B5, and C3 exhibited superior herbicidal activities against dicotyledonous and monocotyledon weeds to commercial atrazine. In particular, compound B5 exhibited excellent herbicidal activity at a dosage of 150 g a.i./ha. In addition, compared with atrazine, compound B5 causes less damage to crops. Molecular docking studies revealed that compound B5 effectively interacted with Pisum sativum PS II D1 via diverse interaction models, such as π-π stacking and hydrogen bonds. Molecular dynamics simulation studies and chlorophyll fluorescence measurements revealed that compound B5 acted on PS II. This is the first report of natural naphthoquinone derivatives targeting PS II and compound B5 may be a candidate molecule for the development of new herbicides targeting PS II.
Subject(s)
Drug Design , Echinochloa , Herbicides , Molecular Docking Simulation , Naphthoquinones , Plant Weeds , Herbicides/chemistry , Herbicides/pharmacology , Herbicides/chemical synthesis , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Echinochloa/drug effects , Echinochloa/growth & development , Plant Weeds/drug effects , Plant Weeds/growth & development , Structure-Activity Relationship , Ethers/chemistry , Ethers/pharmacology , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/antagonists & inhibitors , Photosystem II Protein Complex/metabolism , Molecular Structure , Plant Proteins/chemistry , Plant Roots/chemistry , Plant Roots/drug effectsABSTRACT
The structure and mechanism of the water-oxidation chemistry that occurs in photosystem II have been subjects of great interest. The advent of X-ray free electron lasers allowed the determination of structures of the stable intermediate states and of steps in the transitions between these intermediate states, bringing a new perspective to this field. The room-temperature structures collected as the photosynthetic water oxidation reaction proceeds in real time have provided important novel insights into the structural changes and the mechanism of the water oxidation reaction. The time-resolved measurements have also given us a view of how this reaction-which involves multielectron, multiproton processes-is facilitated by the interaction of the ligands and the protein residues in the oxygen-evolving complex. These structures have also provided a picture of the dynamics occurring in the channels within photosystem II that are involved in the transport of the substrate water to the catalytic center and protons to the bulk.
Subject(s)
Lasers , Photosystem II Protein Complex , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/ultrastructure , Photosystem II Protein Complex/metabolism , Electrons , Water/chemistry , Water/metabolism , X-Rays , Oxidation-Reduction , Models, MolecularABSTRACT
As a critical step in advancing the simulation of photosynthetic complexes, we present the Martini 3 coarse-grained (CG) models of key cofactors associated with light harvesting (LHCII) proteins and the photosystem II (PSII) core complex. Our work focuses on the parametrization of beta-carotene, plastoquinone/quinol, violaxanthin, lutein, neoxanthin, chlorophyll A, chlorophyll B, and heme. We derived the CG parameters to match the all-atom reference simulations, while structural and thermodynamic properties of the cofactors were compared to experimental values when available. To further assess the reliability of the parameterization, we tested the behavior of these cofactors within their physiological environments, specifically in a lipid bilayer and bound to photosynthetic complexes. The results demonstrate that our CG models maintain the essential features required for realistic simulations. This work lays the groundwork for detailed simulations of the PSII-LHCII super-complex, providing a robust parameter set for future studies.
Subject(s)
Light-Harvesting Protein Complexes , Molecular Dynamics Simulation , Photosynthesis , Photosystem II Protein Complex , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/metabolism , Chlorophyll/metabolism , Chlorophyll/chemistry , Thermodynamics , beta Carotene/chemistry , beta Carotene/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Heme/chemistry , Heme/metabolism , Chlorophyll A/chemistry , Chlorophyll A/metabolismABSTRACT
Recent resolution advancement of in situ cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) enables us to visualize large enzymes-in-action in atomic detail in their native environments inside living cells, such as photosystem II (PSII) and the ribosome. A variety of crystallographic and cryo-EM structures of PSII have been published for the purified PSII dimeric core complexes by itself, in supercomplexes with photosystem I (PSI) and light-harvesting complexes (LHC), and in megacomplexes with phycobilisome (PBS). In the latter case, two or five copies of asymmetric dimeric PSII molecules are present in highly asymmetric environments that differ from other 2-fold symmetric structures. Previous systematic analysis of X-ray free-electron laser (XFEL) crystal structures of PSII has shown different degrees of composition heterogeneity of metal ion cofactor bound at the oxygen-evolving center (OEC), including between two monomers of the same PSII dimer. This study analyzed the metal ions bound at four OECs in two asymmetric dimeric PSII molecules within in situ cryo-ET structures reported for an asymmetric PBS-PSII-PSI-LHC megacomplex determined in a living organism without purification and shows that composition heterogeneity with reduced metal ion occupancies at the OEC of PSII is a general phenomenon. This finding could have profound implications for spectroscopic interpretations of unpurified PSII samples.
Subject(s)
Oxygen , Photosystem II Protein Complex , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Oxygen/metabolism , Oxygen/chemistry , Cryoelectron Microscopy , Models, Molecular , Thermosynechococcus/metabolism , Metals/metabolism , Metals/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolismABSTRACT
Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a Mn4CaO5 cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the Mn4CaO5 cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.
Subject(s)
Catalytic Domain , Photosystem II Protein Complex , Water , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/genetics , Water/metabolism , Water/chemistry , Oxidation-Reduction , Mutation , Cryoelectron Microscopy , Manganese/metabolism , Manganese/chemistryABSTRACT
The deprotonation of O6 within the S3 state marks the final deprotonation event before the formation of oxygenoxygen bond interactions and eventual production and release of dioxygen. Gaining a thorough understanding of this event, from the proton acceptors involved, to the exfiltration pathways available, is key in determining the nature of the resulting oxygen species, influencing the mechanism through which the first oxygenoxygen bond forms. Computational analysis, using BS-DFT methodologies, showed that proton abstraction by the local Glu189 residue provides consistent evidence against this being a viable mechanistic pathway due to the lack of a stable product structure. In contrast, abstraction via W3 shows an increasingly stable oxo-oxo product state between r[O5O6] = 2.1 Å & 1.9 Å. The resulting oxo-oxo state is stabilised through donation of ß electron character from O6 to Mn1 and α electron character from O6 to O5. This donation from the O6 lone pair is shown to be a key factor in stabilising the oxo-oxo state, in addition to showing the initiation of first O5-O6 bond.
Subject(s)
Oxygen , Photosystem II Protein Complex , Protons , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Oxygen/chemistry , Oxygen/metabolism , Density Functional Theory , Models, MolecularABSTRACT
Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thus sustains life on Earth. It catalyzes two chemical reactions: water oxidation to molecular oxygen and plastoquinone reduction. Coupling of electron and proton transfer is crucial for efficiency; however, the molecular basis of these processes remains speculative owing to uncertain water binding sites and the lack of experimentally determined hydrogen positions. We thus collected high-resolution cryo-electron microscopy data of fully hydrated photosystem II from the thermophilic cyanobacterium Thermosynechococcus vestitus to a final resolution of 1.71 angstroms. The structure reveals several previously undetected partially occupied water binding sites and more than half of the hydrogen and proton positions. This clarifies the pathways of substrate water binding and plastoquinone B protonation.
Subject(s)
Hydrogen , Photosystem II Protein Complex , Protons , Thermosynechococcus , Water , Binding Sites , Cryoelectron Microscopy , Electron Transport , Hydrogen/chemistry , Oxidation-Reduction , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/ultrastructure , Photosystem II Protein Complex/metabolism , Plastoquinone/metabolism , Plastoquinone/chemistry , Thermosynechococcus/enzymology , Water/chemistryABSTRACT
Understanding how water ligands regulate the conformational changes and functionality of the oxygen-evolving complex (OEC) in photosystem II (PSII) throughout the catalytic cycle of oxygen evolution remains a highly intriguing and unresolved challenge. In this study, we investigate the effect of water insertion (WI) on the redox state of the OEC by using the molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) hybrid methods. We find that water binding significantly reduces the free energy change for proton-coupled electron transfer (PCET) from Mn to YZâ¢, underscoring the important regulatory role of water binding, which is essential for enabling the OEC redox-leveling mechanism along the catalytic cycle. We propose a water binding mechanism in which WI is thermodynamically favored by the closed-cubane form of the OEC, with water delivery mediated by Ca2+ ligand exchange. Isomerization from the closed- to open-cubane conformation at three post-WI states highlights the importance of the location of the MnIII center in the OEC and the orientation of its Jahn-Teller axis to conformational changes of the OEC, which might be critical for the formation of the O-O bond. These findings reveal a complex interplay between conformational changes in the OEC and the ligand environment during the activation of the OEC by YZâ¢. Analogous regulatory effects due to water ligand binding are expected to be important for a wide range of catalysts activated by redox state transitions in aqueous environments.
Subject(s)
Oxidation-Reduction , Oxygen , Photosystem II Protein Complex , Water , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Water/chemistry , Ligands , Oxygen/chemistry , Oxygen/metabolism , Molecular Dynamics Simulation , Thermodynamics , Quantum TheoryABSTRACT
Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)-ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII-ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII-ACPII, evolutionary variations in PSII-LHCII, and the origin of red-lineage LHCIIs.
Subject(s)
Cryoelectron Microscopy , Cryptophyta , Light-Harvesting Protein Complexes , Photosystem II Protein Complex , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Light-Harvesting Protein Complexes/metabolism , Light-Harvesting Protein Complexes/chemistry , Cryptophyta/metabolism , Photosynthesis , Models, Molecular , Energy Transfer , Photosystem I Protein Complex/metabolism , Photosystem I Protein Complex/chemistry , Chlorophyll A/metabolism , Chlorophyll A/chemistryABSTRACT
Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.
Subject(s)
Chlamydomonas reinhardtii , Cysteine Proteases , Cysteine Proteases/metabolism , Cysteine Proteases/chemistry , Chlamydomonas reinhardtii/enzymology , Oxidative Stress , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Plant Proteins/metabolism , Plant Proteins/chemistry , Hydrogen Peroxide/metabolism , Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/chemistryABSTRACT
The light-harvesting complexes (LHCs) of diatoms, specifically fucoxanthin-Chl a/c binding proteins (FCPs), exhibit structural and functional diversity, as highlighted by recent structural studies of photosystem II-FCP (PSII-FCPII) supercomplexes from different diatom species. The excitation dynamics of PSII-FCPII supercomplexes isolated from the diatom Thalassiosira pseudonana was explored using time-resolved fluorescence spectroscopy and two-dimensional electronic spectroscopy at room temperature and 77 K. Energy transfer between FCPII and PSII occurred remarkably fast (<5 ps), emphasizing the efficiency of FCPII as a light-harvesting antenna. The presence of long-wavelength chlorophylls may further help concentrate excitations in the core complex and increase the efficiency of light harvesting. Structure-based calculations reveal remarkably strong excitonic couplings between chlorophylls in the FCP antenna and between FCP and the PSII core antenna that are the basis for the rapid energy transfer.
Subject(s)
Diatoms , Energy Transfer , Light-Harvesting Protein Complexes , Photosystem II Protein Complex , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Diatoms/chemistry , Diatoms/metabolism , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/metabolism , Spectrometry, Fluorescence , Chlorophyll/chemistryABSTRACT
Cryptophyte algae are an evolutionarily distinct and ecologically important group of photosynthetic unicellular eukaryotes. Photosystem II (PSII) of cryptophyte algae associates with alloxanthin chlorophyll a/c-binding proteins (ACPs) to act as the peripheral light-harvesting system, whose supramolecular organization is unknown. Here, we purify the PSII-ACPII supercomplex from a cryptophyte alga Chroomonas placoidea (C. placoidea), and analyze its structure at a resolution of 2.47 Å using cryo-electron microscopy. This structure reveals a dimeric organization of PSII-ACPII containing two PSII core monomers flanked by six symmetrically arranged ACPII subunits. The PSII core is conserved whereas the organization of ACPII subunits exhibits a distinct pattern, different from those observed so far in PSII of other algae and higher plants. Furthermore, we find a Chl a-binding antenna subunit, CCPII-S, which mediates interaction of ACPII with the PSII core. These results provide a structural basis for the assembly of antennas within the supercomplex and possible excitation energy transfer pathways in cryptophyte algal PSII, shedding light on the diversity of supramolecular organization of photosynthetic machinery.
Subject(s)
Cryoelectron Microscopy , Cryptophyta , Photosystem II Protein Complex , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Cryptophyta/metabolism , Chlorophyll/metabolism , Chlorophyll Binding Proteins/metabolism , Chlorophyll Binding Proteins/chemistry , Protein Multimerization , Chlorophyll A/metabolism , Chlorophyll A/chemistry , Models, Molecular , Light-Harvesting Protein Complexes/metabolism , Light-Harvesting Protein Complexes/chemistryABSTRACT
Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS for which the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type strain. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions interpreted in terms of the changes of the PSII and PSI fluorescence emission intensity. The results revealed yet-to-be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems.
Subject(s)
Bacterial Proteins , Energy Transfer , Phycobilisomes , Synechocystis , Phycobilisomes/metabolism , Phycobilisomes/chemistry , Synechocystis/metabolism , Synechocystis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Photosystem I Protein Complex/metabolism , Photosystem I Protein Complex/chemistry , Photosystem I Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/genetics , Peptides/metabolism , Peptides/chemistryABSTRACT
The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.
Subject(s)
Hydrophobic and Hydrophilic Interactions , Light-Harvesting Protein Complexes , Photosynthesis , Photosystem II Protein Complex , Thylakoids , Thylakoids/metabolism , Thylakoids/chemistry , Light-Harvesting Protein Complexes/metabolism , Light-Harvesting Protein Complexes/chemistry , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Galactolipids/metabolism , Galactolipids/chemistry , LightABSTRACT
Primary processes of light energy conversion by Photosystem II (PSII) were studied using femtosecond broadband pump-probe absorption difference spectroscopy. Transient absorption changes of core complexes isolated from the cyanobacterium Synechococcus sp. PCC 7335 grown under far-red light (FRL-PSII) were compared with the canonical Chl a containing spinach PSII core complexes upon excitation into the red edge of the Qy band. Absorption changes of FRL-PSII were monitored at 278 K in the 400-800 nm spectral range on a timescale of 0.1-500 ps upon selective excitation at 740 nm of four chlorophyll (Chl) f molecules in the light harvesting antenna, or of one Chl d molecule at the ChlD1 position in the reaction center (RC) upon pumping at 710 nm. Numerical analysis of absorption changes and assessment of the energy levels of the presumed ion-radical states made it possible to identify PD1+ChlD1- as the predominant primary charge-separated radical pair, the formation of which upon selective excitation of Chl d has an apparent time of â¼1.6 ps. Electron transfer to the secondary acceptor pheophytin PheoD1 has an apparent time of â¼7 ps with a variety of excitation wavelengths. The energy redistribution between Chl a and Chl f in the antenna occurs within 1 ps, whereas the energy migration from Chl f to the RC occurs mostly with lifetimes of 60 and 400 ps. Potentiometric analysis suggests that in canonical PSII, PD1+ChlD1- can be partially formed from the excited (PD1ChlD1)* state.
Subject(s)
Chlorophyll , Photosystem II Protein Complex , Synechococcus , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Synechococcus/metabolism , Chlorophyll/metabolism , Chlorophyll/chemistry , Light , Electron Transport , Spinacia oleracea/metabolismABSTRACT
The O2-evolving Mn4CaO5 cluster in photosystem II is ligated by six carboxylate residues. One of these is D170 of the D1 subunit. This carboxylate bridges between one Mn ion (Mn4) and the Ca ion. A second carboxylate ligand is D342 of the D1 subunit. This carboxylate bridges between two Mn ions (Mn1 and Mn2). D170 and D342 are located on opposite sides of the Mn4CaO5 cluster. Recently, it was shown that the D170E mutation perturbs both the intricate networks of H-bonds that surround the Mn4CaO5 cluster and the equilibrium between different conformers of the cluster in two of its lower oxidation states, S1 and S2, while still supporting O2 evolution at approximately 50% the rate of the wild type. In this study, we show that the D342E mutation produces much the same alterations to the cluster's FTIR and EPR spectra as D170E, while still supporting O2 evolution at approximately 20% the rate of the wild type. Furthermore, the double mutation, D170E + D342E, behaves similarly to the two single mutations. We conclude that D342E alters the equilibrium between different conformers of the cluster in its S1 and S2 states in the same manner as D170E and perturbs the H-bond networks in a similar fashion. This is the second identification of a Mn4CaO5 metal ligand whose mutation influences the equilibrium between the different conformers of the S1 and S2 states without eliminating O2 evolution. This finding has implications for our understanding of the mechanism of O2 formation in terms of catalytically active/inactive conformations of the Mn4CaO5 cluster in its lower oxidation states.
Subject(s)
Carboxylic Acids , Mutation , Oxygen , Photosystem II Protein Complex , Calcium/metabolism , Calcium/chemistry , Carboxylic Acids/chemistry , Carboxylic Acids/metabolism , Electron Spin Resonance Spectroscopy , Ligands , Manganese/chemistry , Manganese/metabolism , Models, Molecular , Oxygen/chemistry , Oxygen/metabolism , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/genetics , Spectroscopy, Fourier Transform InfraredABSTRACT
Structural distortion of protein-bound ligands can play a critical role in enzyme function by tuning the electronic and chemical properties of the ligand molecule. However, quantifying these effects is difficult due to the limited resolution of protein structures and the difficulty of generating accurate structural restraints for nonprotein ligands. Here, we seek to quantify these effects through a statistical analysis of ligand distortion in chlorophyll proteins (CP), where ring deformation is thought to play a role in energy and electron transfer. To assess the accuracy of ring-deformation estimates from available structural data, we take advantage of the C2 symmetry of photosystem II (PSII), comparing ring-deformation estimates for equivalent sites both within and between 113 distinct X-ray and cryogenic electron microscopy PSII structures. Significantly, we find that several deformation modes exhibit considerable variability in predictions, even for equivalent monomers, down to a 2 Å resolution, to an extent that probably prevents their utilization in optical calculations. We further find that refinement restraints play a critical role in determining deformation values to resolution as low as 2 Å. However, for those modes that are well-resolved in the structural data, ring deformation in PSII is strongly conserved across all species tested from cyanobacteria to algae. These results highlight both the opportunities and limitations inherent in structure-based analyses of the bioenergetic and optical properties of CPs and other protein-ligand complexes.
Subject(s)
Chlorophyll , Photosynthesis , Ligands , Chlorophyll/chemistry , Photosystem II Protein Complex/chemistry , Proteins/metabolismABSTRACT
At room temperature and neutral pH, the oxygen-evolving center (OEC) of photosystem II (PSII) catalyzes water oxidation. During this process, oxygen is released from the OEC, while substrate waters are delivered to the OEC and protons are passed from the OEC to the lumen through water channels known as the narrow or the O4 channel, broad or the Cl1 channel, and large or the O1 channel. Protein residues lining the surfaces of these channels play a critical role in stabilizing the hydrogen-bonding networks that assist in the process. We carried out an occupancy analysis to better understand the structural and possible substrate water dynamics in full PSII monomer molecular dynamics (MD) trajectories in both the S1 and S2 states. We find that the equilibrated positions of water molecules derived from MD-derived electron density maps largely match the experimentally observed positions in crystallography. Furthermore, the occupancy reduction in MD simulations of some water molecules inside the single-filed narrow channel also correlates well with the crystallographic data during a structural transition when the S1 state of the OEC advances to the S2 state. The overall reduced occupancies of water molecules are the source of their "vacancy-hopping" dynamic nature inside these channels, unlike water molecules inside an ice lattice where all water molecules have a fixed unit occupancy. We propose on the basis of findings in our structural and molecular dynamics analysis that the water molecule occupying a pocket formed by D1-D61, D1-S169, and O4 of the OEC could be the last steppingstone to enter into the OEC and that the broad channel may be favored for proton transfer.