Higher plant-like fluorescence induction and thermoluminescence characteristics in cyanobacterium, Spirulina mutant defective in PQH2 oxidation by cytb6/f complex

Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers.     

Redox-sensing is the basis of photophobic responses in cyanobacteria

Abstract Cyanobacteria lack specialized photoreceptors for photophobic responses (PPR), the action spectrum for photosynthesis coinciding with that for PPR. In the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethyl urea) (DCMU), which blocks electron transfer prior to plastoquinone (PQ), turning on the light increased the membrane potential, but simultaneously evoked an unusual phobic response, presumably due to oxidation of PQ · H₂ via photosystem I (PS I). White light and the PQ reductant, (DQ · H₂), acted as attractants and produced methylation of a 40 kDa membrane peptide. Repellents, an uncoupler, carbonyl cyanide m -chlorophenylhydrazone (CCCP), and an oxidant, tetramethyl- p -hydroquinone(duroquinon) (DQ), caused demethylation. The results are interpreted in the framework of Häder's 'electron pool hypothesis', according to which PQ · H₂ level governs PPR. It is further concluded that the same system is the mechanism of pmf-sensing:      

Cyclic electron flow around photosystem I in C(3) plants. In vivo control by the redox state of chloroplasts and involvement of the NADH-dehydrogenase complex

Cyclic electron flow around photosystem (PS) I has been widely described in vitro in chloroplasts or thylakoids isolated from C(3) plant leaves, but its occurrence in vivo is still a matter of debate. Photoacoustic spectroscopy and kinetic spectrophotometry were used to analyze cyclic PS I activity in tobacco (Nicotiana tabacum cv Petit Havana) leaf discs illuminated with far-red light. Only a very weak activity was measured in air with both techniques. When leaf discs were placed in anaerobiosis, a high and rapid cyclic PS I activity was measured. The maximal energy storage in far-red light increased to 30% to 50%, and the half-time of the P(700) re-reduction in the dark decreased to around 400 ms; these values are comparable with those measured in cyanobacteria and C(4) plant leaves in aerobiosis. The stimulatory effect of anaerobiosis was mimicked by infiltrating leaves with inhibitors of mitochondrial respiration or of the chlororespiratory oxidase, therefore, showing that changes in the redox state of intersystem electron carriers tightly control the rate of PS I-driven cyclic electron flow in vivo. Measurements of energy storage at different modulation frequencies of far-red light showed that anaerobiosis-induced cyclic PS I activity in leaves of a tobacco mutant deficient in the plastid Ndh complex was kinetically different from that of the wild type, the cycle being slower in the former leaves. We conclude that the Ndh complex is required for rapid electron cycling around PS I.     

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c(6)), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c(6) through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.     

Electrogenic reactions in the photosystem I

The processes of electron transfer in cyanobacterial photosystem I (PS I) and photoelectric methods of the studies were reviewed. Particular emphasis was placed on structural and kinetic characteristics of the electron transport chain. The electrogenicity in PS I complex and its interaction with natural donors (plastocyanin, cytochrome c6), natural acceptors (ferredoxin, flavodoxin), and artificial acceptors and donors (methyl viologen and other redox dyes) were studied. On the basis of photoelectric measurements and the X-ray structural data, the operating dielectric constants in the vicinity of charge carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c6 through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and the electron transfer rate constant in the corresponding segment of the chain were discussed.     

Function and organization of Photosystem I polypeptides

Photosystem I functions as a plastocyanin:ferredoxin oxidoreductase in the thylakoid membranes of chloroplasts and cyanobacteria. The PS I complex contains the photosynthetic pigments, the reaction center P700, and five electron transfer centers (A₀, A₁, F_X, F_A, and F_B) that are bound to the PsaA, PsaB, and PsaC proteins. In addition, PS I complex contains at least eight other polypeptides that are accessory in their functions. Recent use of cyanobacterial molecular genetics has revealed functions of the accessory subunits of PS I. Site-directed mutagenesis is now being used to explore structure-function relations in PS I. The overall architecture of PSI complex has been revealed by X-ray crystallography, electron microscopy, and biochemical methods. The information obtained by different techniques can be used to propose a model for the organization of PS I. Spectroscopic and molecular genetic techniques have deciphered interaction of PS I proteins with the soluble electron transfer partners. This review focuses on the recent structural, biochemical and molecular genetic studies that decipher topology and functions of PS I proteins, and their interactions with soluble electron carriers.Abbreviation NHS N-hydroxysuccinamide This review is dedicated to Prof. J. Philip Thornber, in whose laboratory PRC was introduced to the green world of chlorophyllproteins.     

Double reduction of plastoquinone to plastoquinol in photosystem 1

In Photosystem 1 (PS1), phylloquinone (PhQ) acts as a secondary electron acceptor from chlorophyll ec(3) and also as an electron donor to the iron-sulfur cluster F(X). PS1 possesses two virtually equivalent branches of electron transfer (ET) cofactors from P(700) to F(X), and the lifetime of the semiquinone intermediate displays biphasic kinetics, reflecting ET along the two different branches. PhQ in PS1 serves only as an intermediate in ET and is not normally fully reduced to the quinol form. This is in contrast to PS2, in which plastoquinone (PQ) is doubly reduced to plastoquinol (PQH(2)) as the terminal electron acceptor. We purified PS1 particles from the menD1 mutant of Chlamydomonas reinhardtii that cannot synthesize PhQ, resulting in replacement of PhQ by PQ in the quinone-binding pocket. The magnitude of the stable flash-induced P(700)(+) signal of menD1 PS1, but not wild-type PS1, decreased during a train of laser flashes, as it was replaced by a ~30 ns back-reaction from the preceding radical pair (P(700)(+)A(0)(-)). We show that this process of photoinactivation is due to double reduction of PQ in the menD1 PS1 and have characterized the process. It is accelerated at lower pH, consistent with a rate-limiting protonation step. Moreover, a point mutation (PsaA-L722T) in the PhQ(A) site that accelerates ET to F(X) ~2-fold, likely by weakening the sole H-bond to PhQ(A), also accelerates the photoinactivation process. The addition of exogenous PhQ can restore activity to photoinactivated PS1 and confer resistance to further photoinactivation. This process also occurs with PS1 purified from the menB PhQ biosynthesis mutant of Synechocystis PCC 6803, demonstrating that it is a general phenomenon in both prokaryotic and eukaryotic PS1.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.     

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

Senescence-induced alterations in the photosystem II functions of Cucumis sativus cotyledons: probing of senescence driven alterations of photosystem II by chlorophyll a fluorescence induction O-J-I-P transients

Senescence-induced alterations in photosystem II (PS II) structure and photofunctions were probed in cucumber (Cucumis sativus) cotyledons, using fast O-J-I-P Chlorophyll a (Chl a) fluorescence transients. Analysis of measured and derived parameters of the fast fluorescence O-J-I-P transient revealed senescence-induced alterations in (i), PS II acceptor side electron transfer equilibrium between QA and QB, the primary stable and secondary acceptors of PS II; (ii), intersystem PQ pool size and (iii), affected electron transfer from PS II to PS I. Also, senescence of cotyledons triggered conversion of QA-reducing (fully active) to non- QA-reducing PS II (heat sink) centres. Further, some of the remaining active PS II centres showed a high apparent trapping efficiency due to clustering and energetic connectivity (grouping) between the antennae of active and inactive centers. The overall density of active PS II reaction centers showed a temporal decrease due to the onset of foliar senescence. Thus, the fast Chl a fluorescence transients, with a time resolution of at least 50 mircosec and use of the equations of JIP-test, provide a valuable, non-invasive rapid biophysical probe to study the ageing in plants in terms of detecting photosynthetic activities and the heterogeneity of different types of photosynthetic units. Further, these results were found to be in agreement with the earlier in vitro studies using thylakoids isolated from senescing cotyledons where it was shown that senescence induced heterogeneity in PS II centers affected acceptor side QA<-->QB equilibrium.     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: III. KINETICS OF CHLOROPHYLL FLUORESCENCE EMISSION FROM INTACT LEAVES

The kinetics of 685 nm chlorophyll fluorescence emission weremeasured at 20 °C following illumination of primary leavesof P. vulgaris. During foliar senescence, a large reductionwas observed in the maximal level of fluorescence emission (P)of the induction curve, normalized with respect to the minimallevel (O), and in the time taken to reach P. This suggests thatfewer plastoquinone (PQ) molecules were able to accept electronsfrom each photosystem two (PS II) reaction centre in older leaves.Measurements of fluorescence emission at 77 °K indicatedthat the primary photochemical quantum yield of the PS II reactioncentres remained constant during senescence. The redox stateof the PQ pool was estimated throughout the induction curveat 20 °C. In both mature and senescent leaves PQ was highlyreduced at P. There followed a reoxidation of PQ in the matureleaves, but in the old leaves the PQ pool remained reduced.This indicates that the rate of electron flow from PQ to photosystemone (PS I) decreased considerably during senescence. Fluorescencewas quenched from P to a steady state level (T) in leaves ofall ages, and this was associated with a redistribution of excitationin favour of PS I. Since, in senescent leaves, changes in theredox state of PQ were absent, it is suggested that quenchingresulted from the generation of proton and ion gradients acrossthe thylakoid membranes, and the synthesis of ATP.     

Physiological roles of plastid terminal oxidase in plant stress responses

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase localized in the plastids of plants. It is able to transfer electrons from plastoquinone (PQ) to molecular oxygen with the formation of water. Recent studies have suggested that PTOX is beneficial for plants under environmental stresses, since it is involved in the synthesis of photoprotective carotenoids and chlororespiration, which could potentially protect the chloroplast electron transport chain (ETC) from over-reduction. The absence of PTOX in plants usually results in photo-bleached variegated leaves and impaired adaptation to environment alteration. Although PTOX level and activity has been found to increase under a wide range of stress conditions, the functions of plant PTOX in stress responses are still disputed now. In this paper, the possible physiological roles of PTOX in plant stress responses are discussed based on the recent progress.     

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A_1A/A_1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters F_X, F_A, and F_B have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.     

温州蜜柑叶片光系统反应中心光能分配的变化

为深入了解果树光化学反应中心光能分配的状况,以柑橘为试材,采用调制荧光法对叶片光系统在高光强和低光强下的状态转换进行了研究．结果表明,光系统在100μmol·m^-2·s^-1的低光强下,由于QA的还原使PQ库处于还原状态,导致光能由PSⅡ转向PSⅠ分配,光系统处于状态2;在1000μmol·m^-2·s^-1的高光强下,PQ库无法得到电子而处于氧化状态,导致光能分配由PSⅠ转向PSⅡ,光系统处于状态1,叶片经磷酸酯酶抑制剂NaF处理后,光系统从高光强下状态2到状态1的转换受到抑制,高光强下过多的光能由PSⅠ向PSⅡ分配是导致PSⅡ光破坏的重要原因．     

Alternative Photosystem I-Driven Electron Transport Routes: Mechanisms and Functions

In addition to the linear electron transport, several alternative Photosystem I-driven (PS I) electron pathways recycle the electrons to the intersystem electron carriers mediated by either ferredoxin:NADPH reductase, NAD(P)H dehydrogenase, or putative ferredoxin:plastoquinone reductase. The following functions have been proposed for these pathways: adjustment of ATP/NADPH ratio required for CO(2) fixation, generation of the proton gradient for the down-regulation of Photosystem II (PS II), and ATP supply the active transport of inorganic carbon in algal cells. Unlike ferredoxin-dependent cyclic electron transport, the pathways supported by NAD(P)H can function in the dark and are likely involved in chlororespiratory-dependent energization of the thylakoid membrane. This energization may support carotenoid biosynthesis and/or maintain thylakoid ATPase in active state. Active operation of ferredoxin-dependent cyclic electron transport requires moderate reduction of both the intersystem electron carriers and the acceptor side of PS I, whereas the rate of NAD(P)H-dependent pathways under light depends largely on NAD(P)H accumulation in the stroma. Environmental stresses such as photoinhibition, high temperatures, drought, or high salinity stimulated the activity of alternative PS I-driven electron transport pathways. Thus, the energetic and regulatory functions of PS I-driven pathways must be an integral part of photosynthetic organisms and provides additional flexibility to environmental stress.     

A kinetic assessment of the sequence of electron transfer from F(X) to F(A) and further to F(B) in photosystem I: the value of the equilibrium constant between F(X) and F(A)

The x-ray structure analysis of photosystem I (PS I) crystals at 4-A resolution (Schubert et al., 1997, J. Mol. Biol. 272:741-769) has revealed the distances between the three iron-sulfur clusters, labeled F(X), F(1), and F(2), which function on the acceptor side of PS I. There is a general consensus concerning the assignment of the F(X) cluster, which is bound to the PsaA and PsaB polypeptides that constitute the PS I core heterodimer. However, the correspondence between the acceptors labeled F(1) and F(2) on the electron density map and the F(A) and F(B) clusters defined by electron paramagnetic resonance (EPR) spectroscopy remains controversial. Two recent studies (Diaz-Quintana et al., 1998, Biochemistry. 37:3429-3439;, Vassiliev et al., 1998, Biophys. J. 74:2029-2035) provided evidence that F(A) is the cluster proximal to F(X), and F(B) is the cluster that donates electrons to ferredoxin. In this work, we provide a kinetic argument to support this assignment by estimating the rates of electron transfer between the iron-sulfur clusters F(X), F(A), and F(B). The experimentally determined kinetics of P700(+) dark relaxation in PS I complexes (both F(A) and F(B) are present), HgCl(2)-treated PS I complexes (devoid of F(B)), and P700-F(X) cores (devoid of both F(A) and F(B)) from Synechococcus sp. PCC 6301 are compared with the expected dependencies on the rate of electron transfer, based on the x-ray distances between the cofactors. The analysis, which takes into consideration the asymmetrical position of iron-sulfur clusters F(1) and F(2) relative to F(X), supports the F(X) --> F(A) --> F(B) --> Fd sequence of electron transfer on the acceptor side of PS I. Based on this sequence of electron transfer and on the observed kinetics of P700(+) reduction and F(X)(-) oxidation, we estimate the equilibrium constant of electron transfer between F(X) and F(A) at room temperature to be approximately 47. The value of this equilibrium constant is discussed in the context of the midpoint potentials of F(X) and F(A), as determined by low-temperature EPR spectroscopy.     

Mechanisms for regulating electron transfer in multi-centre redox proteins.

Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase.     

Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco

Chlororespiration has been defined as a respiratory electron transport chain in interaction with photosynthetic electron transport involving both non-photochemical reduction and oxidation of plastoquinones. Different enzymatic activities, including a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the non-photochemical reduction of plastoquinones. However, the enzyme responsible for plasquinol oxidation has not yet been clearly identified. In order to determine whether the newly discovered plastid oxidase (PTOX) involved in carotenoid biosynthesis acts as a plastoquinol oxidase in higher plant chloroplasts, the Arabidopsis thaliana PTOX gene (At-PTOX) was expressed in tobacco under the control of a strong constitutive promoter. We showed that At-PTOX is functional in tobacco chloroplasts and strongly accelerates the non-photochemical reoxidation of plastoquinols; this effect was inhibited by propyl gallate, a known inhibitor of PTOX. During the dark to light induction phase of photosynthesis at low irradiances, At-PTOX drives significant electron flow to O(2), thus avoiding over-reduction of plastoquinones, when photo- synthetic CO(2) assimilation was not fully induced. We proposed that PTOX, by modulating the redox state of intersystem electron carriers, may participate in the regulation of cyclic electron flow around photosystem I.