Secondary Quinone Electron Acceptor Research Articles

Transcriptome analysis of the bloom-forming dinoflagellate Prorocentrum donghaiense exposed to Ginkgo biloba leaf extract, with an emphasis on photosynthesis.

Ginkgo biloba leaf extract (GBE) can effectively treat bloom-forming freshwater algae. However, there is limited information about the underlying suppression mechanism of the marine bloom-forming Prorocentrum donghaiense-the most dominant algal bloom species in the East China Sea. We investigated the effect of GBE on P. donghaiense in terms of its response to photosynthesis at the molecular/omic level. In total, 93,743 unigenes were annotated using six functional databases. Furthermore, 67,203 differentially expressed genes (DEGs) were identified in algae treated with 1.8 g∙L-1 GBE. Among these DEGs, we identified the genes involved in photosynthesis. PsbA, PsbB and PsbD in photosystem II, PsaA in photosystem I, and PetB and PetD in the cytochrome b6/f complex were downregulated. Other related genes, such as PsaC, PsaE, and PsaF in photosystem I; PetA in the cytochrome b6/f complex; and atpA, atpD, atpH, atpG, and atpE in the F-type H+-ATPase were upregulated. These results suggest that the structure and activity of the complexes were destroyed by GBE, thereby inhibiting the electron flow between the primary and secondary quinone electron acceptors, primary quinone electron acceptor, and oxygen-evolving complex in the PSII complex, and interrupting the electron flow between PSII and PSI, ultimately leading to a decline in algal cell photosynthesis. These findings provide a basis for understanding the molecular mechanisms underlying P. donghaiense exposure to GBE and a theoretical basis for the prevention and control of harmful algal blooms.

Photoinduced electron transfer from spinach chloroplasts to carbon paste electrodes through an exogenous electron acceptor

The photocurrent generated from chloroplasts of spinach is investigated using a carbon paste electrode. Chloroplasts extracted from spinach are shown to interact with a carbon paste electrode through five types of redox mediators. Among these mediators, 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ) is found to be the best electron-transfer mediator, generating the highest photocurrent of approximately 310 nA cm−2 per μg chlorophyll. In addition, the photocurrent density is investigated as a function of the photon flux density, concentration of 2,5-DCBQ, and the amount of chloroplasts deposited onto the surface of the electrode. Photoreduction of 2,5-DCBQ exhibited Michaelis–Menten-like kinetics, and the reduction rates were dependent on the amount of 2,5-DCBQ and the light intensity. Even 10 days after the chloroplasts were extracted, the electrodes with the extracted spinach chloroplasts exhibited ∼90% of the photoinduced current that they exhibited immediately after the chloroplast extraction. The source of photocurrent was confirmed in experiments using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron transfer from primary quinone electron acceptor QA to secondary quinone electron acceptor QB in photosystem II, and KCN, an inhibitor of electron transfer from cytochrome b6f complex to photosystem I.

Effects of Mycotoxin Fumagillin, Mevastatin, Radicicol, and Wortmannin on Photosynthesis of Chlamydomonas reinhardtii.

Mycotoxins are one of the most important sources for the discovery of new pesticides and drugs because of their chemical structural diversity and fascinating bioactivity as well as unique novel targets. Here, the effects of four mycotoxins, fumagillin, mevastatin, radicicol, and wortmannin, on photosynthesis were investigated to identify their precise sites of action on the photosynthetic apparatus of Chlamydomonas reinhardtii. Our results showed that these four mycotoxins have multiple targets, acting mainly on photosystem II (PSII). Their mode of action is similar to that of diuron, inhibiting electron flow beyond the primary quinone electron acceptor (QA) by binding to the secondary quinone electron acceptor (QB) site of the D1 protein, thereby affecting photosynthesis. The results of PSII oxygen evolution rate and chlorophyll (Chl) a fluorescence imaging suggested that fumagillin strongly inhibited overall PSII activity; the other three toxins also exhibited a negative influence at the high concentration. Chl a fluorescence kinetics and the JIP test showed that the inhibition of electron transport beyond QA was the most significant feature of the four mycotoxins. Fumagillin decreased the rate of O2 evolution by interrupting electron transfer on the PSII acceptor side, and had multiple negative effects on the primary photochemical reaction and PSII antenna size. Mevastatin caused a decrease in photosynthetic activity, mainly due to the inhibition of electron transport. Both radicicol and wortmannin decreased photosynthetic efficiency, mainly by inhibiting the electron transport efficiency of the PSII acceptor side and the activity of the PSII reaction centers. In addition, radicicol reduced the primary photochemical reaction efficiency and antenna size. The simulated molecular model of the four mycotoxins' binding to C. reinhardtii D1 protein indicated that the residue D1-Phe265 is their common site at the QB site. This is a novel target site different from those of commercial PSII herbicides. Thus, the interesting effects of the four mycotoxins on PSII suggested that they provide new ideas for the design of novel and efficient herbicide molecules.

Cold priming improves chilling resistance in wheat seedlings: Changing of photosystem II imprints during recovery from priming

Chilling stress oriented from frequent extreme events has seriously depressed growth and productivity of wheat. Cold priming can effectively improve cold tolerance of crops. However, it is still unclear about the changes of stress memory imprints during recovery from priming and the potential function of the imprints on improved cold tolerance. Here we subjected wheat plants to cold priming (10 °C/6 °C, 1 d) at the trifoliate stage, and then to chilling stresses (2 °C/0 °C, 1 d) at 3, 6, 9, 12, 15 and 18 days of recovery from priming, respectively. Compared with non-primed plants (NL), primed plants (PL) showed the most obvious resistance phenotype at 9 days after priming. Its terminal spreading leaves exhibit temporally specific physiological memory and persistent a high photochemical quenching capacity of photosystem II after priming. In addition, it appeared faster photosynthetic performance in the post-stress rewarming phase during 3–12 days after priming. Further analysis of different leaf positions revealed that priming increased light energy absorption by the augmented antennal pigments content and synthesis of light-harvesting proteins, and a higher percentage of primary and secondary quinone electron acceptors (QAQB) active reaction centers (RCs) which enhanced energy capture and transfer performance in primed control plants (PC). Meanwhile, the increased antenna pigments in PC indirectly affected PL. Compared with NL, priming increased electron flux into the inter-system by alleviating the degradation of antenna pigments and increased Photosystem II Subunit S (PsbS) gene expression to promote the occurrence of non-photochemical quenching (NPQ), lowered the excitation pressure per RC, and increased the percentage of QA-RCs, it finally improved the light energy absorption and utilization capacity in PL. In summary, priming-induced memory is mainly present in primed leaves (3rd and 4th leaves during R6-R15) and diminishes with spatial and temporal changes, it exists even no obvious difference in phenotype. This study contributes to a deeper understanding of the long-term effects of fluctuating temperatures on the plant.

Insights into melatonin-induced photosynthetic electron transport under low-temperature stress in cucumber.

In this study, the differences in chlorophyll fluorescence transient (OJIP) and modulated 820 nm reflection (MR820) of cucumber leaves were probed to demonstrate an insight into the precise influence of melatonin (MT) on cucumber photosystems under low temperature stress. We pre-treated cucumber seedlings with different levels of MT (0, 25, 50, 100, 200, and 400 μmol · L-1) before imposing low temperature stress (10 °C/6 °C). The results indicated that moderate concentrations of MT had a positive effect on the growth of low temperature-stressed cucumber seedlings. Under low temperature stress conditions, 100 μmol · L-1 (MT 100) improved the performance of the active photosystem II (PSII) reaction centers (PIabs), the oxygen evolving complex activity (OEC centers) and electron transport between PSII and PSI, mainly by decreasing the L-band, K-band, and G-band, but showed differences with different duration of low temperature stress. In addition, these indicators related to quantum yield and energy flux of PSII regulated by MT indicated that MT (MT 100) effectively protected the electron transport and energy distribution in the photosystem. According to the results of WO-I ≥ 1 and MR820 signals, MT also affected PSI activity. MT 100 decreased the minimal value of MR/MRO and the oxidation rate of plastocyanin (PC) and PSI reaction center (P700) (Vox ), while increased △MRslow/MRO and deoxidation rates of PC+ and P700 + (Vred ). The loss of the slow phase of MT 200 and MT 400-treated plants in the MR820 kinetics was due to the complete prevention of electron movement from PSII to re-reduce the PC+ and P700 +. These results suggest that appropriate MT concentration (100 μmol · L-1) can improve the photosynthetic performance of PS II and electron transport from primary quinone electron acceptor (QA) to secondary quinone electron acceptor (QB), promote the balance of energy distribution, strengthen the connectivity of PSI and PSII, improve the electron flow of PSII via QA to PC+ and P700 + from reaching PSI by regulating multiple sites of electron transport chain in photosynthesis, and increase the pool size and reduction rates of PSI in low temperature-stressed cucumber plants, All these modifications by MT 100 treatment promoted the photosynthetic electron transfer smoothly, and further restored the cucumber plant growth under low temperature stress. Therefore, we conclude that spraying MT at an appropriate concentration is beneficial for protecting the photosynthetic electron transport chain, while spraying high concentrations of MT has a negative effect on regulating the low temperature tolerance in cucumber.

PSII photochemistry responses to drought stress in autochthonous and modern sweet cherry cultivars

In this study, the JIP test was used to assess the drought tolerance of two sweet cherry cultivars (Prunus avium L.) (modern and autochthonous). Plants were exposed to progressive drought by withholding water and their fast (< 1 s) chlorophyll fluorescence kinetics was evaluated. JIP test analysis showed that drought stress caused a greater decrease in performance indices (PIABS and PItotal) in a modern cultivar, as compared to an autochthonous one. Our results suggest that limited reoxidation of primary quinone electron acceptor (QA), higher amount of secondary quinone electron acceptor (QB-) nonreducing reaction centres, or inhibition of the electron transport between QA and QB, decreased more seriously the photosynthetic performance of the modern cultivar. Further, higher positive L- and K-bands observed for the modern cultivar also suggest lower energetic connectivity between PSII units and increased inhibition of oxygen-evolving complex over autochthonous cultivar. Our results suggest that the autochthonous cultivar Crveni hruĹĄt had better photosynthetic performance under drought conditions, compared to the modern cultivar New Star.

The Mechanisms of Electrogenic Reactions in Bacterial Photosynthetic Reaction Centers: Studies in Collaboration with Alexander Konstantinov.

In this review, we discuss our studies conducted in 1985-1988 in collaboration with A. A. Konstantinov, one of the top scientists in the field of membrane bioenergetics. Studying fast kinetics of membrane potential generation in photosynthetic reaction centers (RCs) of purple bacteria in response to a laser flash has made it possible to examine in detail the mechanisms of electrogenic reactions at the donor and acceptor sides of RCs. Electrogenesis associated with the intraprotein electron transfer from the exogenous secondary donors, redox dyes, and soluble cytochrome (cyt) c to the photooxidized dimer of bacteriochlorophyll P870 was studied using proteoliposomes containing RCs from the non-sulfur purple bacterium Rhodospirillum rubrum. It was found that reduction of the secondary quinone electron acceptor QB accompanied by its protonation in the chromatophores from R. rubrum in response to every second light flash was electrogenic. Spectral characteristics and redox potentials of the four hemes in the tightly bound cyt c in the RC of Blastochloris viridis and electrogenic reactions associated with the electron transfer within the RC complex were identified. For the first time, relative amplitudes of the membrane potential generated in the course of individual electrogenic reactions were compared with the distances between the redox cofactors determined based on the three-dimensional structure of the Bl. viridis RC.

The D1:Ser268 residue of Photosystem II contributes to an alternative pathway for QB protonation in the absence of bound bicarbonate.

Photosystem II catalyses the splitting of water and the reduction in plastoquinone in thylakoid membranes of all oxygenic photosynthetic organisms. The final step in quinol formation is protonation of the reduced secondary quinone electron acceptor to give QB H2 . The proton for this step is hypothesized to be provided by a hydrogen-bond network incorporating amino acids from the Photosystem II D1 and D2 reaction center proteins, together with several bound waters and a bicarbonate ion ligated to a non-heme iron found between the primary plastoquinone electron acceptor QA and QB . The aim of this study was to investigate the role of bicarbonate and the D1:Ser268 residue in the formation of QB H2 . Using targeted mutagenesis of the D1 protein in the cyanobacterium Synechocystis sp. PCC 6803, we have created two mutants, S268A and S268T. Our D1:Ser268 mutants exhibited increased sensitivity to formate-induced inhibition of electron transfer between QA and QB and indicate that D1:Ser268 and bicarbonate support the second protonation in the formation of QB H2 via two different pathways that both lead to the protonation of by D1:His215.

PH-Dependent Regulation of the Relaxation Rate of the Radical Anion of the Secondary Quinone Electron Acceptor QB in Photosystem II As Revealed by Fourier Transform Infrared Spectroscopy.

Photosystem II (PSII) is a protein complex that performs water oxidation using light energy during photosynthesis. In PSII, electrons abstracted from water are eventually transferred to the secondary quinone electron acceptor, QB, and upon double reduction, QB is converted to quinol by binding two protons. Thus, excess electron transfer in PSII increases the pH of the stroma. In this study, to investigate the pH-dependent regulation of the electron flow in PSII, we have estimated the relaxation rate of the QB- radical anion in the pH region between 5 and 8 by direct monitoring of its population using light-induced Fourier transform infrared difference spectroscopy. The decay of QB- by charge recombination with the S2 state of the water oxidation center in PSII membranes was shown to be accelerated at higher pH, whereas that of QA- examined in the presence of a herbicide was virtually unaffected at pH ≤7.5 and slightly slowed at pH 8. These observations were consistent with the previous studies that included rather indirect monitoring of the QB- and QA- decays using fluorescence detection. The accelerated relaxation of QB- was explained by the shift of a redox equilibrium between QA- and QB- to the QA- side due to the decrease in the redox potential of QB at higher pH, which is induced by deprotonation of a single amino acid residue near QB. It is proposed that this pH-dependent QB- relaxation is one of the mechanisms of electron flow regulation in PSII for its photoprotection.

Effects of modified clay on the physiological and photosynthetic activities of Amphidinium carterae Hulburt

Among the strategies for treating harmful algal blooms, flocculation using modified clay (MC) has been widely applied in the field. This paper studied the mitigation of MC on Amphidinium carterae Hulburt, finding that MC could not only effectively remove A. carterae, but also affect the physiological activities of the residual algae and inhibit their normal growth. The superoxide dismutase (SOD) activity, catalase (CAT) activity and malondialdehyde (MDA) content of the residual algae significantly increased compared with the control, indicating that MC stimulated the accumulation of reactive oxygen species (ROS) in algal cells. In addition, the cell density was significantly correlated with the SOD activity, CAT activity and MDA content in the experiment groups, suggesting that intracellular ROS might be the main internal factor inhibiting cell growth. To reveal the mechanism of ROS generation, this paper further evaluated the effect of MC on photosynthesis in the residual microalgae, and found that compared with the control the absorption flux per photosystem II (PSII) reaction center (ABS/RC), the trapping flux per RC (TR0/RC) and the electron transport flux per RC (ET0/RC) increased, while the TR0/ABS and ET0/ABS decreased after adding 0.10g/L and 0.25g/L MC. These findings indicate that the MC led to an imbalance between photosynthetic light absorption and energy utilization and that the partial RCs became non-primary quinone electron acceptor (QA)-RCs, further inducing the over-excitation of the active RCs. And MC caused the suppression of the electron transport chain (ETC): the ETC from the QA to the secondary quinone electron acceptor (QB) was blocked and the size of plastoquinone pool decreased, which could induce the over-reduction of PSII. The over-excitation of PSII and the damaged ETC likely induce the generation of ROS during photosynthesis. Thus, MC likely induced the accumulation of intracellular ROS due to photosynthesis inhibition, consequently hindering the growth of the residual algae.

Redox potential of the terminal quinone electron acceptor QB in photosystem II reveals the mechanism of electron transfer regulation

Photosystem II (PSII) extracts electrons from water at a Mn4CaO5 cluster using light energy and then transfers them to two plastoquinones, the primary quinone electron acceptor QA and the secondary quinone electron acceptor QB. This forward electron transfer is an essential process in light energy conversion. Meanwhile, backward electron transfer is also significant in photoprotection of PSII proteins. Modulation of the redox potential (Em) gap of QA and QB mainly regulates the forward and backward electron transfers in PSII. However, the full scheme of electron transfer regulation remains unresolved due to the unknown Em value of QB. Here, for the first time (to our knowledge), the Em value of QB reduction was measured directly using spectroelectrochemistry in combination with light-induced Fourier transform infrared difference spectroscopy. The Em(QB (-)/QB) was determined to be approximately +90 mV and was virtually unaffected by depletion of the Mn4CaO5 cluster. This insensitivity of Em(QB (-)/QB), in combination with the known large upshift of Em(QA (-)/QA), explains the mechanism of PSII photoprotection with an impaired Mn4CaO5 cluster, in which a large decrease in the Em gap between QA and QB promotes rapid charge recombination via QA (-).

1P258 光化学系IIにおける第二キノン電子受容体Q_Bの電子・プロトン移動機構(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

1P258 光化学系IIにおける第二キノン電子受容体Q_Bの電子・プロトン移動機構(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

Effects of hydrogen bonding interactions on the redox potential and molecular vibrations of plastoquinone as studied using density functional theory calculations

The effects of H-bonding on the redox potential and molecular vibrations of plastoquinone (PQ) that functions as a primary and a secondary quinone electron acceptor (QA and QB, respectively) in photosystem II (PSII) in plants and cyanobacteria were investigated using density functional theory calculations. Calculations were performed on the neutral and semiquinone anion forms of PQ and its H-bonded complexes, which form H-bonds with water molecules, or using amino acid models mimicking the interactions of QA and QB. The calculated redox potential (E(o)) of PQ showed a linear relationship with the number of H-bonds, and the E(o) increased by +100-200 mV with the addition of one H-bond. Vibrational analysis of the model PQ complexes showed that the CO stretching vibrations of neutral PQ are sensitive to the number and symmetry of H-bonding interactions, providing criteria to determine the H-bonding structure. Although no specific trend in the H-bonding dependency was found for anionic PQ, complex spectral features in the CO stretching region due to significant couplings with other PQ vibrations and the vibrations of H-bonding amino acids are useful monitors of the change in the H-bonding structure of anionic PQ in proteins. The calculated E(o) values and infrared spectra of the QA and QB models are consistent with the view that one additional H-bond to QB from D1-Ser264 largely contributes to the redox potential gap between QA and QB in PSII.

PHOTOPHYSIOLOGICAL RESPONSES OF FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) TO NITROGEN DEPLETION AT TWO TEMPERATURES(1).

The photosynthetic efficiency and photoprotective capacity of the sea-ice diatom, Fragilariopsis cylindrus (Grunow) W. Krieg., grown in a matrix of nitrogen repletion and depletion at two different temperatures (-1°C and +6°C) was investigated. Temperature showed no significant effect on photosynthetic efficiency or photoprotection in F. cylindrus. Cultures under nitrogen depletion showed enhanced photoprotective capacity with an increase in nonphotochemical quenching (NPQ) when compared with nitrogen-replete cultures. This phenomenon was achieved at no apparent cost to the photosynthetic efficiency of PSII (FV /FM ). Nitrogen depletion yielded a partially reduced electron transport chain in which maximum fluorescence (FM ) could only be obtained by adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). reoxidation curves showed the presence of QB nonreducing PSII centers under nitrogen depletion. Fast induction curves (FICs) and electron transport rates (ETRs) revealed slowing of the electrons transferred from the primary (QA ) to the secondary (QB ) quinone electron acceptors of PSII. The data presented show that nitrogen depletion in F. cylindrus leads to the formation of QB nonreducing PSII centers within the photosystem. On a physiological level, the formation of QB nonreducing PSII centers in F. cylindrus provides the cell with protection against photoinhibition by facilitating the rapid induction of NPQ. This strategy provides an important ecological advantage, especially during the Antarctic spring, maintaining photosynthetic efficiency under high light and nutrient-limiting conditions.

Characterization of photosystem II photochemistry in transgenic tobacco plants with lowered Rubisco activase content

Rubisco activase plays an important role in the regulation of CO 2 assimilation. However, it is unknown how activase regulates photosystem II (PSII) photochemistry. To investigate the effects of Rubisco activase on PSII photochemistry, we obtained transgenic tobacco ( Nicotiana tabacum) plants with 50% (i7), 25% (i28), and 5% (i46) activase levels as compared to wild type plants by using a gene encoding tobacco activase for the RNAi construct. Both CO 2 assimilation and PSII activity were significantly reduced only in transgenic i28 and i46 plants, suggesting that activase deficiency led to decreased PSII activity. Flash-induced fluorescence kinetics indicated that activase deficiency resulted in a slow electron transfer between Q A (primary quinine electron acceptor of PSII) and Q B (secondary quinone electron acceptor of PSII). Thermoluminescence measurements revealed that activase deficiency induced a shift of S 2Q A − and S 2Q B − recombinations to higher temperatures in parallel, and a decrease in the intensities of the thermoluminescence emissions. Activase deficiency also dampened the period-four oscillation of the thermoluminescence B-band. Protein gel blot analysis showed that activase deficiency resulted in a significant decrease in the content of D1, D2, CP43, CP47, and PsbO proteins. Transmission electron microscopy analysis demonstrated that activase deficiency induced a significant decrease in the number of grana stacks per chloroplast and discs per grana stack. Our results suggest that activase plays an important role in maintaining PSII function and chloroplast development.

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Different enhancement of senescence induced by metabolic products of Alternaria alternata in tobacco leaves of different ages

The purpose of this study was to explore the mechanisms by which Alternaria alternata damages tobacco (Nicotiana tabacum) leaves. Treatment with A. alternata metabolic products enhanced senescence in leaves of different ages, as indicated by the significant decrease in chlorophyll, soluble protein, photosynthetic O(2) evolution and catalase (CAT, EC 1.11.1.6) activity as well as an increase in H(2)O(2) content. The induction of senescence by A. alternata metabolic products increased as the age of the leaves increased. A. alternata metabolic products greatly influenced the behavior of photosystem II (PSII) in the leaves: oxygen evolving complex (OEC) activity and electron transport from primary quinone electron acceptor of PS II (Q(A)) to secondary quinone electron acceptor of PS II (Q(B)) were both significantly inhibited. This inhibition also became more pronounced in older leaves. In vitro experiments revealed that, without the influence of natural senescence, the A. alternata metabolic products directly inhibited the activity of a commercial CAT solution and inhibited photosynthetic O(2) evolution, which resulted in excess PSII excitation pressure and an overaccumulation of H(2)O(2) in leaf segments. These results suggest that the significant declines in photosynthesis and CAT activity induced by the metabolic products of A. alternata were important contributors to the overaccumulation of reactive oxygen species (ROS), which accelerated senescence in tobacco leaves. The fact that the enhancement of senescence was getting more pronounced with the age of tobacco leaves might be related to the fact that older leaves already had higher H(2)O(2) levels and less antioxidant activity as reflected in lower CAT activity.

Evidence for Delocalized Anticooperative Flash Induced Proton Binding as Revealed by Mutants at the M266His Iron Ligand in Bacterial Reaction Centers

Bacterial reaction centers (RCs) convert light energy into chemical free energy via the double reduction and protonation of the secondary quinone electron acceptor, QB, to the dihydroquinone QBH2. Two RC mutants (M266His --> Leu and M266His --> Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, QA, and QB. However, in the M266HL mutant a destabilization of approximately 40 meV of the free energy level of QA- was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the QA region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of QA/QA- in M266HL caused accelerated recombination of the charges in the P+ QA- state of the RCs where the native QA was replaced by a low potential anthraquinone (AQA). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P+ AQA- --> P AQA charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon QA- and QB- formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either QA- or QB-. We surmise that the presence of an extended protonated water H-bond network providing protons to QB is responsible for these effects.

Proton Uptake in the Reaction Center Mutant L210DN from Rhodobacter sphaeroides via Protonated Water Molecules,

The reaction center (RC) of Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, QB (the secondary quinone electron acceptor), to form quinol, QBH2. Asp210 in the L-subunit has been shown to be a catalytic residue in this process. Mutation of Asp210 to Asn leads to a deceleration of reoxidation of QA- in the QA-QB --> QAQB- transition. Here we determined the structure of the Asp210 to Asn mutant to 2.5 A and show that there are no major structural differences as compared to the wild-type protein. We found QB in the distal position and a chain of water molecules between Asn210 and QB. Using time-resolved Fourier transform infrared (trFTIR) spectroscopy, we characterized the molecular reaction mechanism of this mutant. We found that QB- formation precedes QA- oxidation even more pronounced than in the wild-type reaction center. Continuum absorbance changes indicate deprotonation of a protonated water cluster, most likely of the water chain between Asn210 and QB. A detailed analysis of wild-type structures revealed a highly conserved water chain between Asp210 or Glu210 and QB in Rb. sphaeroides and Rhodopseudomonas viridis, respectively.

A Protein Dynamics Study of Photosystem II: The Effects of Protein Conformation on Reaction Center Function

Molecular dynamics simulations have been performed to study photosystem II structure and function. Structural information obtained from simulations was combined with ab initio computations of chromophore excited states. In contrast to calculations based on the x-ray structure, the molecular-dynamics-based calculations accurately predicted the experimental absorbance spectrum. In addition, our calculations correctly assigned the energy levels of reaction-center (RC) chromophores, as well as the lowest-energy antenna chlorophyll. The primary and secondary quinone electron acceptors, Q A and Q B, exhibited independent changes in position over the duration of the simulation. Q B fluctuated between two binding sites similar to the proximal and distal sites previously observed in light- and dark-adapted RC from purple bacteria. Kinetic models were used to characterize the relative influence of chromophore geometry, site energies, and electron transport rates on RC efficiency. The fluctuating energy levels of antenna chromophores had a larger impact on quantum yield than did their relative positions. Variations in electron transport rates had the most significant effect and were sufficient to explain the experimentally observed multi-component decay of excitation in photosystem II. The implications of our results are discussed in the context of competing evolutionary selection pressures for RC structure and function.