QB Binding Research Articles

Phe265 of the D1 protein is required to stabilize plastoquinone binding in the QB-binding site of photosystem II in Synechocystis sp. PCC 6803

In Photosystem II electrons from water splitting pass through a primary quinone electron acceptor (QA) to the secondary plastoquinone (QB). The D2 protein forms the QA-binding site and the D1 protein forms the QB-binding site. A non-heme iron sits between QA and QB resulting in a quinone-Fe-acceptor complex that must be activated before assembly of the oxygen-evolving complex can occur. An extended loop (residues 223–266) between the fourth (helix D) and fifth (helix E) helices of the D1 protein activates forward electron transfer via a conformational change that stabilizes a bidentate bicarbonate ligand to the non-heme iron while simultaneously stabilizing the binding of QB. We show that positioning of D1:Phe265 to provide a hydrogen bond to the distal oxygen of QB is required for forward electron transfer. In addition, mutations targeting D1:Phe265, resulted in a 50 mV decrease in the QB/QB– midpoint potential.

Effect of the insecticide clothianidin on the photosynthetic electron transport chain in pea.

Clothianidin (CL) is a neonicotinoid insecticide widely used in crop protection against insect pests. However, its effects on photosynthesis remain largely unknown. Here, by investigating the influence of CL at the concentrations of 22 and 110 μg/L on the primary processes of photosynthesis, membrane fluidity and structural changes of pea chloroplasts, we located several primary binding sites of this pesticide. Similar dynamics were observed for both concentrations. However, statistically significant differences were only found at 110 μg/L for all methods used. The light saturated rate of linear electron flow decreased mainly due to the disturbance of electron flow on the acceptor side of photosystem II (PSII) associated with the appearance of QB-nonreducing centers and empty QB binding sites of PSII. The functioning of the donor side of PSII, the activity of photosystem I (PSI) and the maximum quantum yield of PSII photochemistry (Fv/Fm) were not found to be significantly altered. Increased membrane fluidity and structural alterations of the thylakoid membrane led to a decrease in the development of the proton gradient ΔрН and membrane energization processes.

Arg24 and 26 of the D2 protein are important for photosystem II assembly and plastoquinol exchange in Synechocystis sp. PCC 6803

Photosystem II (PS II) assembly is a stepwise process involving preassembly complexes or modules focused around four core PS II proteins. The current model of PS II assembly in cyanobacteria is derived from studies involving the deletion of one or more of these core subunits. Such deletions may destabilize other PS II assembly intermediates, making constructing a clear picture of the intermediate events difficult. Information on plastoquinone exchange pathways operating within PS II is also unclear and relies heavily on computer-aided simulations. Deletion of PsbX in [S. Biswas, J.J. Eaton-Rye, Biochim. Biophys. Acta - Bioenerg. 1863 (2022) 148519] suggested modified QB binding in PS II lacking this subunit. This study has indicated the phenotype of the ∆PsbX mutant arose by disrupting a conserved hydrogen bond between PsbX and the D2 (PsbD) protein. We mutated two conserved arginine residues (D2:Arg24 and D2:Arg26) to further understand the observations made with the ∆PsbX mutant. Mutating Arg24 disrupted the interaction between PsbX and D2, replicating the high-light sensitivity and altered fluorescence decay kinetics observed in the ∆PsbX strain. The Arg26 residue, on the other hand, was more important for either PS II assembly or for stabilizing the fully assembled complex. The effects of mutating both arginine residues to alanine or aspartate were severe enough to render the corresponding double mutants non-photoautotrophic. Our study furthers our knowledge of the amino-acid interactions stabilizing plastoquinone-exchange pathways while providing a platform to study PS II assembly and repair without the actual deletion of any proteins.

Physiological and molecular mechanisms of the inhibitory effects of artemisinin on Microcystis aeruginosa and Chlorella pyrenoidosa

Artemisinin, a novel plant allelochemical, has attracted attention for its potential selective inhibitory effects on algae, yet to be fully explored. This study compares the sensitivity and action targets of Microcystis aeruginosa (M. aeruginosa) and Chlorella pyrenoidosa (C. pyrenoidosa) to artemisinin algaecide (AMA), highlighting their differences. Results indicate that at high concentrations, AMA displaces the natural PQ at the QB binding site within M. aeruginosa photosynthetic system, impairing the D1 protein repair function. Furthermore, AMA disrupts electron transfer from reduced ferredoxin (Fd) to NADP+ by interfering with the iron-sulfur clusters in the ferredoxin-NADP+ reductases (FNR) domain of Fd. Moreover, significant reactive oxygen species (ROS) accumulation triggers oxidative stress and interrupts the tricarboxylic acid cycle, hindering energy acquisition. Notably, AMA suppresses arginine synthesis in M. aeruginosa, leading to reduced microcystins (MCs) release. Conversely, C. pyrenoidosa counters ROS accumulation via photosynthesis protection, antioxidant defenses, and by regulating intracellular osmotic pressure, accelerating damaged protein degradation, and effectively repairing DNA for cellular detoxification. Additionally, AMA stimulates the expression of DNA replication-related genes, facilitating cell proliferation. Our finding offer a unique approach for selectively eradicating cyanobacteria while preserving beneficial algae, and shed new light on employing eco-friendly algicides with high specificity.

2-Hydroxychalcone as a Novel Natural Photosynthesis Inhibitor against Bloom-Forming Cyanobacteria.

The control of harmful cyanobacterial blooms has been becoming a global challenge. The development of eco-friendly algicides with strong specificity is urgently needed. The photosynthetic apparatus is a promising target site for algicides to minimize the possible harmful effects on animals and humans. In this study, biologically derived 2-hydroxychalcone efficiently inhibited the growth of bloom-forming M.aeruginosa by selectively interfering with photosynthesis. 2-Hydroxychalcone targeting Photosystem II (PSII) inhibited electron transfer between the primary and secondary electron acceptors (QA and QB) and the binding of plastoquinone (PQ) molecules to the QB binding pocket at the acceptor side of PSII, as revealed by polyphasic chlorophyll (Chl) a fluorescence induction and QA- reoxidation kinetics. Molecular docking for 2-hydroxychalcone to D1 protein and the proteomic responses of M.aeruginosa suggested that 2-hydroxychalcone formed a stable monodentate ligand with the nonheme iron in D1 protein, provoking significant modulation of PSII proteins. The unique binding mode of 2-hydroxychalcone with PSII differentiated it from classical PSII inhibitors. Furthermore, 2-hydroxychalcone down-regulated the expression of microcystin (MC) synthesis-related genes to restrain MC synthesis and release. These results indicated the potential application of 2-hydroxychalcone as an algicide or a template scaffold for designing novel derivatives with superior algicidal activity.

Conformational Changes and H-Bond Rearrangements during Quinone Release in Photosystem II.

In photosystem II (PSII) and photosynthetic reaction centers from purple bacteria (PbRC), the electron released from the electronically excited chlorophyll is transferred to the terminal electron acceptor quinone, QB. QB accepts two electrons and two protons before leaving the protein. We investigated the molecular mechanism of quinone exchange in PSII, conducting molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations. MD simulations suggest that the release of QB leads to the transformation of the short helix (D1-Phe260 to D1-Ser264), which is adjacent to the stromal helix de (D1-Asn247 to D1-Ile259), into a loop and to the formation of a water-intake channel. Water molecules enter the QB binding pocket via the channel and form an H-bond network. QM/MM calculations indicate that the H-bond network serves as a proton-transfer pathway for the reprotonation of D1-His215, the proton donor during QBH-/QBH2 conversion. Together with the absence of the corresponding short helix but the presence of Glu-L212 in PbRC, it seems likely that the two type-II reaction centers undergo quinone exchange via different mechanisms.

PH-Dependent Regulation of Electron Flow in Photosystem II by a Histidine Residue at the Stromal Surface

In photosystem II (PSII), the secondary plastoquinone electron acceptor QB functions as a substrate that converts into plastoquinol upon its double reduction by electrons abstracted from water. It has been suggested that a histidine residue, D1-H252, which is located at the stromal surface near QB, is involved in the pH-dependent regulation of electron flow and proton transfer to QB. However, definitive evidence for the involvement of D1-H252 in the QB reactions has not been obtained yet. Here, we studied the roles of D1-H252 in PSII using a cyanobacterial mutant, in which D1-H252 was replaced with Ala. Delayed luminescence (DL) measurement upon a single flash showed a faster QB- decay at higher pH in the thylakoids from the wild-type strain due to the downshift of the redox potential of QB [Em(QB-/QB)]. This pH dependence of the QB- decay was lost in the D1-H252A mutant. The experimental Em(QB-/QB) changes were well reproduced by the density functional theory calculations for models with different protonation states of D1-H252 and with Ala replaced for H252. It was further shown that the period-four oscillation of the DL intensity by successive flashes was significantly diminished in the D1-H252A mutant, suggesting the inhibition of plastoquinone exchange at the QB pocket in this mutant. It is thus concluded that D1-H252 is a key amino acid residue that regulates electron flow in PSII by sensing pH in the stroma and stabilizes the QB binding site to facilitate the quinone exchange reaction.

6-Ethoxy-4- N-(2-morpholin-4-ylethyl) -2-N-propan-2-yl-1,3, 5-triazine-2, 4-diamineendows herbicidal activity against Phalaris minora weed of wheat crop field: An in -silico and experimental approaches of herbicide discovery.

Phalaris minor is a major weed of wheat crop which has evolved resistance against herbicides. Isoproturon is the most accepted herbicide developedresistance in 1992. Later, introduced herbicides also developed resistance and cross-resistance to their respective binding sites. Isoproturon binds at the QB binding site of the D1 protein of photosystem-II (PS-II), which blocks the electron transfer in photosynthesis. In this work, we have carried out a series of computational studies to prioritize the promising herbicides against D1 protein of P. minor. Through the computational studies, twenty-four lead molecules are prioritized which have shown a higher binding affinity and inhibition constant than the reference ligand molecule. The binding and conformational stability of docked complexes was evaluated by molecular dynamics simulations and binding free energy calculations i.e., MM/PBSA. A list of amino acids such as Ala225, Ser226, Phe227, and Asn229 present in the binding site of protein is obtained to be playing an important role in the stability of the protein-lead complex via hydrogen bond and π-π interactions. Binding free energy calculation revealed that the selected lead molecule binding is energetically favorable and driven by electrostatic interactions. Among 24 leads, computational results have uncovered eight promising compounds as potential herbicides which have shown comparable physiochemical profile, better docking scores, system stability, H-bond occupancy, and binding free energy than terbutryn, a reference molecule. These prioritized molecules were custom synthesized and evaluated for their herbicidal activity and specificity through whole plant assay under laboratory-controlled conditions. The lead molecule ELC5 (6-ethoxy-4-N-(2-morpholin-4-ylethyl)-2-N-propan-2-yl-1,3,5-triazine-2,4-diamine) has shown comparable activity to the reference herbicide(isoproturon) against P. minor.

Properties of Photosystem II lacking the PsbJ subunit.

Photosystem II (PSII), the oxygen-evolving enzyme, consists of 17 trans-membrane and 3 extrinsic membrane proteins. Other subunits bind to PSII during assembly, like Psb27, Psb28, and Tsl0063. The presence of Psb27 has been proposed (Zabret et al. in Nat Plants 7:524-538, 2021; Huang et al. Proc Natl Acad Sci USA 118:e2018053118, 2021; Xiao et al. in Nat Plants 7:1132-1142, 2021) to prevent the binding of PsbJ, a single transmembrane α-helix close to the quinone QB binding site. Consequently, a PSII rid of Psb27, Psb28, and Tsl0034 prior to the binding of PsbJ would logically correspond to an assembly intermediate. The present work describes experiments aiming at further characterizing such a ∆PsbJ-PSII, purified from the thermophilic Thermosynechococcus elongatus, by means of MALDI-TOF spectroscopy, thermoluminescence, EPR spectroscopy, and UV-visible time-resolved spectroscopy. In the purified ∆PsbJ-PSII, an active Mn4CaO5 cluster is present in 60-70% of the centers. In these centers, although the forward electron transfer seems not affected, the Em of the QB/QB- couple increases by ≥ 120mV , thus disfavoring the electron coming back on QA. The increase of the energy gap between QA/QA- and QB/QB- could contribute in a protection against the charge recombination between the donor side and QB-, identified at the origin of photoinhibition under low light (Keren et al. in Proc Natl Acad Sci USA 94:1579-1584, 1997), and possibly during the slow photoactivation process.

Mechanism of the formation of proton transfer pathways in photosynthetic reaction centers

In photosynthetic reaction centers from purple bacteria (PbRCs) from Rhodobacter sphaeroides, the secondary quinone QB accepts two electrons and two protons via electron-coupled proton transfer (PT). Here, we identify PT pathways that proceed toward the QB binding site, using a quantum mechanical/molecular mechanical approach. As the first electron is transferred to QB, the formation of the Grotthuss-like pre-PT H-bond network is observed along Asp-L213, Ser-L223, and the distal QB carbonyl O site. As the second electron is transferred, the formation of a low-barrier H-bond is observed between His-L190 at Fe and the proximal QB carbonyl O site, which facilitates the second PT. As QBH2 leaves PbRC, a chain of water molecules connects protonated Glu-L212 and deprotonated His-L190 forms, which serves as a pathway for the His-L190 reprotonation. The findings of the second pathway, which does not involve Glu-L212, and the third pathway, which proceeds from Glu-L212 to His-L190, provide a mechanism for PT commonly used among PbRCs.

Binding Properties of Photosynthetic Herbicides with the QB Site of the D1 Protein in Plant Photosystem II: A Combined Functional and Molecular Docking Study.

Photosystem II (PSII) is a multi-subunit enzymatic complex embedded in the thylakoid membranes responsible for the primary photosynthetic reactions vital for plants. Many herbicides used for weed control inhibit PSII by interfering with the photosynthetic electron transport at the level of the D1 protein, through competition with the native plastoquinone for the QB site. Molecular details of the interaction of these herbicides in the D1 QB site remain to be elucidated in plants. Here, we investigated the inhibitory effect on plant PSII of the PSII-inhibiting herbicides diuron, metobromuron, bentazon, terbuthylazine and metribuzin. We combined analysis of OJIP chlorophyll fluorescence kinetics and PSII activity assays performed on thylakoid membranes isolated from pea plants with molecular docking using the high-resolution PSII structure recently solved from the same plant. Both approaches showed for terbuthylazine, metribuzin and diuron the highest affinity for the D1 QB site, with the latter two molecules forming hydrogen bonds with His215. Conversely, they revealed for bentazon the lowest PSII inhibitory effect accompanied by a general lack of specificity for the QB site and for metobromuron an intermediate behavior. These results represent valuable information for future design of more selective herbicides with enhanced QB binding affinities to be effective in reduced amounts.

Novel Action Targets of Natural Product Gliotoxin in Photosynthetic Apparatus.

Gliotoxin (GT) is a fungal secondary metabolite that has attracted great interest due to its high biological activity since it was discovered by the 1930s. It exhibits a unique structure that contains a N-C = O group as the characteristics of the classical PSII inhibitor. However, GT’s phytotoxicity, herbicidal activity and primary action targets in plants remain hidden. Here, it is found that GT can cause brown or white leaf spot of various monocotyledonous and dicotyledonous plants, being regarded as a potential herbicidal agent. The multiple sites of GT action are located in two photosystems. GT decreases the rate of oxygen evolution of PSII with an I 50 value of 60 µM. Chlorophyll fluorescence data from Chlamydomonas reinhardtii cells and spinach thylakoids implicate that GT affects both PSII electron transport at the acceptor side and the reduction rate of PSI end electron acceptors’ pool. The major direct action target of GT is the plastoquinone QB-site of the D1 protein in PSII, where GT inserts in the QB binding niche by replacing native plastoquinone (PQ) and then interrupts electron flow beyond plastoquinone QA. This leads to severe inactivation of PSII RCs and a significant decrease of PSII overall photosynthetic activity. Based on the simulated modeling of GT docking to the D1 protein of spinach, it is proposed that GT binds to the-QB-site through two hydrogen bonds between GT and D1-Ser264 and D1-His252. A hydrogen bond is formed between the aromatic hydroxyl oxygen of GT and the residue Ser264 in the D1 protein. The 4-carbonyl group of GT provides another hydrogen bond to the residue D1-His252. So, it is concluded that GT is a novel natural PSII inhibitor. In the future, GT may have the potential for development into a bioherbicide or being utilized as a lead compound to design more new derivatives.

D1:Glu244 and D1:Tyr246 of the bicarbonate-binding environment of Photosystem II moderate high light susceptibility and electron transfer through the quinone-Fe-acceptor complex

In cyanobacteria, Glu-244 and Tyr-246 of the Photosystem II (PS II) D1 protein are hydrogen bonded to two water molecules that are part of a hydrogen-bond network between the bicarbonate ligand to a non-heme iron and the cytosol. Ala substitutions were introduced in Synechocystis sp. PCC 6803 to investigate the roles of these residues and the hydrogen-bond network on electron transfer between the primary plastoquinone acceptor, QA, and the secondary plastoquinone acceptor, QB, of the quinone-Fe-acceptor complex. All mutants assembled PS II; however, an increase in the PS II to PS I ratio was apparent, particularly in the E244A:Y246A double mutant. The mutants also showed impaired oxygen evolution and retarded chlorophyll a fluorescence decays following single turnover actinic flashes, which appeared to be primarily due to reduced QB binding in the E244A strain and an enhanced back reaction with the S2 state of the oxygen-evolving complex in the Y246A mutant. Impaired PS II in the Y246A and E244A:Y246A mutants resulted in inactivation of the psbA gene encoding D1. The Y246A and E244A:Y246A mutants also showed high light sensitivity whereas the E244A mutant showed enhanced resilience towards photodamage. Unlike the control strain, all of the mutants were insensitive to the addition of formate or bicarbonate in assays following chlorophyll decay kinetics that reflect electron transfer between QA and QB, suggesting the bicarbonate binding environment was perturbed. Our data also indicate that waters W582 and W622 (PDB: 4UB6) have essential roles in maintaining the architecture of the acceptor side of PS II.

Unravelling electron transfer processes at photosystem 2 embedded in an Os-complex modified redox polymer

In the development of semi-artificial biophotovoltaic assemblies, deeper understanding of electrochemical processes is required to achieve functional and efficient devices. Evaluation of photosystem 2 embedded in an Os-complex modified redox polymer using scanning photoelectrochemical microscopy (SPECM) provides insight into the intricate electrochemical processes of the immobilized protein complex and its electrical communication pathways with the redox tethers of the polymer matrix. The use of local irradiation during an SPECM array scan prevents sample inactivation prior to analysis. Moreover, the simultaneously possible collection of partially reduced oxygen species in the form of hydrogen peroxide confirms the presence of competing charge transfer pathways involved in the reduction of oxygen at the chlorophyll pigments upon irradiation of the sample. In addition, evaluation of photocurrent in the presence of an inhibitor that blocks the terminal plastoquinone QB binding site of the photosystem reveals electrochemical communication between the intermediate plastoquinone QA and the redox polymer. The obtained information proves to be relevant for further design and optimization of devices for technological applications.

Control of the maximal chlorophyll fluorescence yield by the Q<sub>B</sub> binding site

Differences in maximal yields of chlorophyll variable fluorescence (Fm) induced by single turnover (ST) and multiple turnover (MT) excitation are as great as 40%. Using mutants of Chlamydomonas reinhardtii we investigated potential mechanisms controlling Fm above and beyond the QA redox level. Fm was low when the QB binding site was occupied by PQ and high when the QB binding site was empty or occupied by a PSII herbicide. Furthermore, in mutants with impaired rates of plastoquinol reoxidation, Fm was reached rapidly during MT excitation. In PSII particles with no mobile PQ pool, Fm was virtually identical to that obtained in the presence of PSII herbicides. We have developed a model to account for the variations in maximal fluorescence yields based on the occupancy of the QB binding site. The model predicts that the variations in maximal fluorescence yields are caused by the capacity of secondary electron acceptors to reoxidize QA-.

Mutual relationships between structural and functional changes in a PsbM-deletion mutant of photosystem II.

Photosystem II (PSII) is a membrane protein complex that performs light-induced electron transfer and oxygen evolution from water. PSII consists of 19 or 20 subunits in its crystal form and binds various cofactors such as chlorophyll a, plastoquinone, carotenoid, and lipids. After initial light excitation, the charge separation produces an electron, which is transferred to a plastoquinone molecule (QA) and then to another plastoquinone (QB). PsbM is a low-molecular-weight subunit with one transmembrane helix, and is located in the monomer-monomer interface of the PSII dimer. The function of PsbM has been reported to be stabilization of the PSII dimer and maintenance of electron transfer efficiency of PSII based on previous X-ray crystal structure analysis at a resolution of 4.2 Å. In order to elucidate the structure-function relationships of PsbM in detail, we improved the quality of PSII crystals from a PsbM-deleted mutant (ΔPsbM-PSII) of Thermosynechococcus elongatus, and succeeded in improving the diffraction quality to a resolution of 2.2 Å. X-ray crystal structure analysis of ΔPsbM-PSII showed that electron densities for the PsbM subunit and neighboring carotenoid and detergent molecules were absent in the monomer-monomer interface. The overall structure of ΔPsbM-PSII was similar to wild-type PSII, but the arrangement of the hydrophobic transmembrane subunits was significantly changed by the deletion of PsbM, resulting in a slight widening of the lipid hole involving QB. The lipid hole-widening further induced structural changes of the bicarbonate ion coordinated to the non-heme Fe(ii) atom and destabilized the polypeptide chains around the QB binding site located far from the position of PsbM. The fluorescence decay measurement indicated that the electron transfer rate from QA to QB was decreased in ΔPsbM-PSII compared with wild-type PSII. The functional change in electron transfer efficiency was fully interpreted based on structural changes caused by the deletion of the PsbM subunit.

If Photoinhibition of Soybean Photosystem II Enhances the Hypersensitive Response, It is Not Solely Due to Blockage of Electron Transfer Flow at D1

Previous studies have suggested that photoinhibition, through inactivation of photosystem II (PSII), could be beneficial to plants during defense to pathogens through enhanced reactive oxygen (ROS), especially during the hypersensitive response (HR). In this study, we addressed this question by focusing on a possible role of turnover and inhibition of the PSII subunit D1, in defense to the compatible and incompatible strains of the bacterial pathogen Pseudomonas syringae in soybean leaves. Expression of the D1 encoding gene, psbA, as well as 14 other chloroplast encoded genes, was down regulated in response to P. syringae. This down regulation is consistent with reduced production of PSII components leading to increased photoinhibition of existing photocenters, and is also consistent with multiple studies showing a concerted down regulation of nuclear-encoded chloroplast genes during pathogen attack. However, although expression of the psbA transcript was reduced in response to pathogen within 8 hours of inoculation, the level of the psbA product, the D1 protein, showed no significant changes via western blots, and did not show any signs of degradation. Additionally, infiltrating leaves with the D1 inhibiting herbicide bentazon (competitive inhibitor of QB binding to D1, stopping photosynthesis by blocking electron transfer from QA to QB) together with P. syringae inoculation, showed that D1 inhibition did not enhance defense as expected (if photoinhibition enhanced defense), but actually rendered the host slightly more susceptible. The results reflect two possibilities. One is that PSII inhibition through blockage of electron flow through D1 of PSII, does not enhance resistance to P. syringae. The second possibility supported by the data is that the mechanism of photoinhibition during pathogen defense is not due solely to the blockage of electron flow, but through another means of stimulating photoinhibition, such as an inefficient degradation, removal, and replacement of damaged D1 from the PSII complex.

Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

Long-range interaction between the Mn4CaO5 cluster and the non-heme iron center in photosystem II as revealed by FTIR spectroelectrochemistry.

It is known that inactivation of the Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), induces a positive shift of the redox potential (Em) of the primary quinone electron acceptor QA by ∼+150 mV, resulting in suppression of the electron transfer from QA to the secondary quinone acceptor QB. Although the relevance of this Em(QA(-)/QA) shift to the photoprotection of PSII has been debated, its molecular mechanism is still enigmatic from a structural viewpoint because QA is ∼40 Å from the Mn4CaO5 cluster. In this work, we have investigated the influence of Mn depletion on the Em of the non-heme iron, which is located between QA and QB, and its surrounding structure. Electrochemical measurements in combination with Fourier transform infrared (FTIR) spectroscopy revealed that Mn depletion shifts Em(Fe(2+)/Fe(3+)) by +18 mV, which is ∼8 times smaller than the shift of Em(QA(-)/QA). Comparison of the Fe(2+)/Fe(3+) FTIR difference spectra between intact and Mn-depleted PSII samples showed that Mn depletion altered the pKa's of a His ligand to the non-heme iron, most probably the D1-His215 interacting QB, and a carboxylate group, possibly D1-Glu244, coupled with the non-heme iron. It was further shown that Mn depletion influences the C≡N vibration of bromoxynil bound to the QB site, indicative of the modification of the QB binding site. On the basis of these results, we discuss the mechanism of a long-range interaction between the donor and acceptor sides of PSII.

Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of Chlamydomonas reinhardtii under Extreme Environment in Space

Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, Chlamydomonas reinhardtii, which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the QB binding pocket and in the environment surrounding Tyr-161 (YZ) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the QA − state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.