True oxygen reduction capacity during photosynthetic electron transfer in thylakoids and intact leaves

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O₂) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O₂ reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H₂O₂) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H₂O₂ accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H₂O₂ from PETC has a potential of increasing mitochondrial respiration and CO₂ release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H₂O₂ accumulation. The putative production of H₂O₂ in any PETC location other than PSI is rapidly quenched and therefore cannot function in H₂O₂ translocation to another cellular location or in signaling.

Photosynthetically derived H₂O₂ only accumulates at Photosystem I and may trigger cooperation with mitochondria during stress.     

Oxygenic photoreduction of ferredoxin independently of the membrane-bound iron-sulfur centers of photosystem I

An investigation of the photoreduction of soluble ferredoxin and membrane-bound Fe-S centers of chloroplasts yielded results that are incompatible with some basic postulates of the now prevalent concept of photosynthetic electron transport (the “Z scheme”). In the Z scheme, plastquinone serves as an essential link in a linear electron transport chain from water via photosystem II to photosystem I and thence to the bound Fe-S centers, soluble ferredoxin and NADP⁺. In this formulation the oxygenic photoreduction of ferredoxin and of the Fe-S centers should have the same sensitivity to the plastoquinone inhibitors, dibromothymoquinone (DBMIB) and dinitrophenol ether of iodonitrothymol (DNP-INT). We found that the photoreduction of ferredoxin and the Fe-S centers exhibited differential sensitivity to these inhibitors. Ferredoxin was fully photoreduced by water at inhibitor concentrations that abolished the photoreduction of the Fe-S centers. These findings suggest that the oxygenic photoreduction of ferredoxin does not involve the participation of the Fe-S centers or other components of photosystem I. Only when an artificial, direct donor to photosystem I is used is the reduction of ferredoxin invariably preceded by the reduction of the Fe-S centers.     

Effects of severe CO2 starvation on the photosynthetic electron transport chain in tobacco plants

Tobacco plants (Nicotiana tabacum) were kept in CO₂ free air for several days to investigate the effect of lack of electron acceptors on the photosynthetic electron transport chain. CO₂ starvation resulted in a dramatic decrease in photosynthetic activity. Measurements of the electron transport activity in thylakoid membranes showed that a loss of Photosystem II activity was mainly responsible for the observed decrease in photosynthetic activity. In the absence of CO₂ the plastoquinone pool and the acceptor side of Photosystem I were highly reduced in the dark as shown by far-red light effects on chlorophyll fluorescence and P700 absorption measurements. Reduction of the oxygen content of the CO₂ free air retarded photoinhibitory loss of photosynthetic activity and pigment degradation. Electron flow to oxygen seemed not to be able to counteract the stress induced by severe CO₂ starvation. The data are discussed in terms of a donation of reducing equivalents from mitochondria to chloroplasts and a reduction of the plastoquinone pool via the NAD(P)H-plastoquinone oxidoreductase during CO₂ starvation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plastoquinone as a common link between photosynthesis and respiration in a blue-green alga

The role of plastoquinone in a thermophilic blue-green alga, Shynechococcus sp., was studied by measuring reduction kinetics of cytochrome 553 which was oxidized with red flash preferentially exciting photosystem I. Sensitivity of the cytochrome reduction to DBMIB indicates that cytochrome 553 accepts electrons from reduced plastoquinone. Plastoquinone is in turn reduced in cells without electrons from photosystem II, since DCMU, which inhibited methyl viologen photoreduction more strongly than DBMIB, failed to affect the cytochrome reduction. Participation of cyclic electron transport around photosystem I in cytochrome reduction in the presence of DCMU was excluded, because methyl viologen and antimycin A had no effect on the cytochrome kinetics. On the other hand, electron donation from endogenous substrates to plastoquinone was suggested from decreases in rate of the cytochrome reduction by dark starvation of cells and also from restoration of fast reduction kinetics by the addition of exogenous substrates to or by reillumination of starved cells.KCN, which completely suppressed respiratory O₂-uptake, induced a marked acceleration of the cytochrome reduction in starved cells. The poison was less or not effective in stimulating the cytochrome reduction in more extensively starved or reilluminated cells.Results indicate that plastoquinone is functioning not only in the photosynthetic but also in the respiratory electron transport chain, thereby forming a common link between the two energy conservation systems of the blue-green alga.

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The effect of low osmotic potential on nitrite reduction in intact spinach chloroplasts

The effect of water stress (reduced osmotic potential) on photosynthetic nitrite reduction was investigated using intact, isolated spinach (Spinacia oleracea) chloroplasts. Nitrite-dependent O₂ evolution was inhibited 39% at −29.5 bars osmotic potential, relative to a control at −11 bars. In the presence of an uncoupler of photophosphorylation this inhibition was not seen. Reduced osmotic potential did not inhibit either methyl viologen reduction or photosynthetic O₂ reduction. These results indicate that an inhibition of electron transport to ferredoxin cannot account for the observed inhibition of nitrite-dependent O₂ evolution. In vitro assay of nitrite reductase activity showed that the interaction of the enzyme with nitrite was not affected by changes in the concentrations of ions or molecules that might be caused by water stress conditions. These results indicate that the most likely site for the effect of water stress on chloroplastic nitrite reduction is the interaction of ferredoxin with nitrite reductase.     

Inhibition of chlororespiration by myxothiazol and antimycin A in Chlamydomonas reinhardtii

Myxothiazol and antimycin A are shown to suppress the oxygen transient previously attributed to the flash-induced inhibition of chlororespiration in Chlamydomonas reinhardtii (Peltier et al. 1987, Biochim Biophys Acta 893: 83–90). However, these two compounds do not affect the photosynthetic electron transport chain as inferred by the insensitivity of the CO₂-dependent photosynthetic O₂ evolution and of the flash-induced electrochromic effect. Chlorophyll fluorescence induction measurements carried out in dark-adapted cells of a mutant of Chlamydomonas lacking photosystem 1, show that myxothiazol and antimycin A significantly increase the redox state of the photosystem 2 acceptors. We conclude from these results that chlororespiration is inhibited by myxothiazol and antimycin A and that the site of inhibition is located on the dark oxidation pathway of the plastoquinone pool. This inhibition is interpreted through the involvement of a myxothiazol and antimycin A sensitive cytochrome in the chlororespiratory chain.Abbreviations cyt cytochrome - PQ plastoquinone - PS photosystem     

Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity

Absorbance changes of ferredoxin measured at 463 nm in isolated thylakoids were shown to arise from the activity of the enzyme ferredoxin-plastoquinone reductase (FQR) in cyclic electron transport. Under anaerobic conditions in the presence of DCMU and an appropriate concentration of reduced ferredoxin, a light-induced absorbance decrease due to further reduction of Fd was assigned to the oxidation of the other components in the cyclic pathway, primarily plastoquinone. When the light was turned off, Fd was reoxidised and this gave a direct quantitative measurement of the rate of cyclic electron transport due to the activity of FQR. This activity was sensitive to the classical inhibitor of cyclic electron transport, antimycin, and also to J820 and DBMIB. Antimycin had no effect on Fd reduction although this was inhibited by stigmatellin. This provides further evidence that there is a quinone reduction site outside the cytochrome bf complex. The effect of inhibitors of ferredoxin-NADP⁺ reductase and experiments involving the modification of ferredoxin suggest that there may be some role for the reductase as a component of FQR. Contrary to expectations, NADPH₂ inhibited FQR activity; ATP and ADP had no effect.Abbreviations AQS 9,10-anthraquinone-2-sulphonate - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - dimaleimide N,N-p-phenylenedimaleimide - EDC N-(dimethylaminopropyl)-N-ethylcarbodiimide - Fd ferredoxin - FNR Fd-NADP⁺ oxidoreductase - FQR Fd-PQ reductase - GME glycine methyl ester - J820 tetrabromo-4-hydroxypyridine - PC plastocyanin - PMS N-methylphenazinium methyl sulphate - PS Photosystems I and II - PQ plastoquinone - Q quinone - Q_r and Q_o sites of quinone reduction and oxidation, respectively - sulpho-DSPD disulphodisalicylidenepropane-1,2-diamine     

Alternative photosynthetic electron flow to oxygen in marine Synechococcus

Cyanobacteria dominate the world's oceans where iron is often barely detectable. One manifestation of low iron adaptation in the oligotrophic marine environment is a decrease in levels of iron-rich photosynthetic components, including the reaction center of photosystem I and the cytochrome b₆f complex [R.F. Strzepek and P.J. Harrison, Photosynthetic architecture differs in coastal and oceanic diatoms, Nature 431 (2004) 689-692.]. These thylakoid membrane components have well characterised roles in linear and cyclic photosynthetic electron transport and their low abundance creates potential impediments to photosynthetic function. Here we show that the marine cyanobacterium Synechococcus WH8102 exhibits significant alternative electron flow to O₂, a potential adaptation to the low iron environment in oligotrophic oceans. This alternative electron flow appears to extract electrons from the intersystem electron transport chain, prior to photosystem I. Inhibitor studies demonstrate that a propyl gallate-sensitive oxidase mediates this flow of electrons to oxygen, which in turn alleviates excessive photosystem II excitation pressure that can often occur even at relatively low irradiance. These findings are also discussed in the context of satisfying the energetic requirements of the cell when photosystem I abundance is low.     

Changes in the mode of electron flow to photosystem I following chilling-induced photoinhibition in a C3 plant, Cucumis sativus L.

This study provides evidence for enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700⁺ via the cytochrome b₆/f complex (Cyt b₆/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation–reduction kinetics studied in the absence of linear electron transport from water to NADP⁺, cyclic electron transfer mediated through the Q-cycle of Cyt b₆/f and charge recombination in photosystem I (PSI). The segregation of these pathways for P700⁺ rereduction were achieved by the use of a 50-ms multiple turnover white flash or a strong pulse of white or far-red illumination together with inhibitors. In cucumber leaves, chilling-induced photoinhibition resulted in ∼20% loss of photo-oxidizible P700. The measurement of P700⁺ was greatly limited by the turnover of cyclic processes in the absence of the linear mode of electron transport as electrons were rapidly transferred to the smaller pool of P700⁺. The above is explained by integrating the recent model of the cyclic electron flow in C₃ plants based on the Cyt b₆/f structural data [Joliot and Joliot (2006) Biochim Biophys Acta 1757:362–368] and a photoprotective function elicited by a low NADP⁺/NAD(P)H ratio [Rajagopal et al. (2003) Biochemistry 42:11839–11845]. Over-reduction of the photosynthetic apparatus results in the accumulation of NAD(P)H in vivo to prevent NADP⁺-induced reversible conformational changes in PSI and its extensive damage. As the ferredoxin:NADP reductase is fully reduced under these conditions, even in the absence of PSII electron transport, the reduced ferredoxin generated during illumination binds at the stromal openings in the Cyt b₆/f complex and activates cyclic electron flow. On the other hand, the excess electrons from the NAD(P)H pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow.     

Salicylhydroxamic acid (SHAM) inhibits O<Subscript>2</Subscript> photoreduction which protects nitrogenase activity in the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. RF-1

Synechococcus sp. RF-1, a unicellular N₂-fixing cyanobacterium, can grow photosynthetically and diazotrophically in continuous light. How the organism protects its nitrogenase from damage by oxygen is unclear. In cyanobacerial cells, electron transport carriers associated with photosynthesis and respiration are all on the thylakoid membranes and share some common components, including plastoquinone pool and cytochrome b ₆ f complex, and the pathways are interacting with each other. In this work, a pulse amplitude modulation (PAM) fluorometer (PAM-101) and an O₂ electrode are used simultaneously to study the chlorophyll a fluorescence and to monitor O₂ exchanges in Synechococcus sp. RF-1 cells. At the CO₂ compensation point, the photochemical quenching activity remained high unless the O₂ was exhausted by the glucose oxidase system (GOS). It indicates that in addition to CO₂, O₂ can also act as electron acceptor to receive electrons derived from Q_A. Studies with various inhibitors of the electron transport chain demonstrated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and salicylhydroxamic acid (SHAM) inhibited the photoreduction of O₂, while glycolaldehyde, disalicylidenepropanediamine (DSPD), methyl viologen (MV) and KCN did not. These results imply that a KCN-resistant and SHAM-sensitive oxidase transfers electrons generated from Photosystem II to O₂ between cytochrome b ₆ f complex and ferredoxin. When SHAM blocked this alternative electron transport pathway, the dinitrogen-fixing activity decreased significantly. The results indicate that a novel oxidase may function as an intracellular O₂-scavenger in Synechococcus sp. RF-1 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential effects of ambient or diminished CO2 and O2 levels on thylakoid membrane structure in light‐stressed plants

Over‐reduction of the photosynthetic electron transport chain may severely damage the photosynthetic apparatus as well as other constituents of the chloroplast and the cell. Here, we exposed Arabidopsis leaves to saturating light either under normal atmospheric conditions or under CO₂‐ and O₂‐limiting conditions, which greatly increase excitation and electron pressures by draining terminal electron acceptors. The two treatments were found to have very different, often opposing, effects on the structure of the thylakoid membranes, including the width of the granal lumenal compartment. Modulation of the latter is proposed to be related to movements of ions across the thylakoid membrane, which alter the relative osmolarity of the lumen and stroma and affect the partitioning of the proton motive force into its electrical and osmotic components. The resulting changes in thylakoid organization and lumenal width should facilitate the repair of photodamaged photosystem II complexes in response to light stress under ambient conditions, but are expected to inhibit the repair cycle when the light stress occurs concurrently with CO₂ and O₂ depletion. Under the latter conditions, the changes in thylakoid structure are predicted to complement other processes that restrict the flow of electrons into the high‐potential chain, thus moderating the production of deleterious reactive oxygen species at photosystem I.     

Photosystem I is not solely responsible for oxygen reduction in isolated thylakoids

It was found that the contribution of segments of photosynthetic electron transport chain (PETC) besides Photosystem I (PSI) to oxygen reduction increased with increase in light intensity, and at high intensities achieved 50% at pH 5.0, and was higher than 60% at pH 6.5 and pH 7.8. The data are explained as the result of O2 reduction in plastoquinone (PQ) pool as well as in PSI followed by reduction of superoxide radicals generated in both processes by plastohydroquinone.     

Participation of ferredoxin in oxygen reduction in a photosynthetic electron-transport chain

Oxygen reduction in a photosynthetic electron-transport chain (PETC) was studied in isolated pea thylakoids in the presence of either ferredoxin, or ferredoxin + NADP+, or cytochrome c. The contribution of the electron flow through ferredoxin to the total oxygen reduction was evaluated by comparing the rate of oxygen reduction and the rate of oxidation of reduced ferredoxin in the light. It was found that at ferredoxin concentrations optimal for NADP+ reduction, 30-50% of electrons transferred to oxygen went through ferredoxin both in the absence and presence of NADP+. However, the absolute rate of oxygen reduction by membrane components of PETC in the presence of NADP+ was 3-4 times less than that in the presence of ferredoxin alone and close to the rate of oxygen reduction in the presence of cytochrome c. It was assumed that a Photosystem I component, whose role in this process depends on the rate of electron outflow from terminal acceptors of this photosystem, participates in oxygen reduction, and this component is phylloquinone.     

Kinetics of NADPH-Induced Non-Photochemical Reduction of the Plastoquinone Pool in Spinach Chloroplasts

Kinetics of non-photochemical reduction of the photosynthetic intersystem electron transport chain by exogenous NADPH was examined in osmotically lysed spinach chloroplasts by chlorophyll (Chl) fluorescence measurements under anaerobic condition. Upon the addition of NADPH, the apparent F₀ increased sigmoidally, and the value of the maximal slope was calculated to give the reduction rate of plastoquinone (PQ) pool. Application of 5 µM antimycin A lowered significantly both the ceiling and the rate of the NADPH-induced Chl fluorescence increase, while the suppressive effect of 10 µM rotenone was slighter. This indicated that dark reduction of the PQ pool by NADPH in spinach chloroplasts under O₂-limitation condition could be attributed mainly to the pathway catalysed sequentially by ferredoxin-NADP⁺ oxidoreductase (FNR) and ferredoxin-plastoquinone reductase (FQR), rather than that mediated by NAD(P)H dehydro- genase (NDH).     

Divergent pathways of photosynthetic electron transfer: The autonomous oxygenic and anoxygenic photosystems

The aim of this article is to assemble and integrate, from a personal perspective of a research participant, seldom examined evidence that is incompatible with some basic tenets of photosynthetic electron transport, the cornerstone of which is the Z scheme. The nonconforming evidence pertaining to the mode of ferredoxin reduction and the role of the copper redox protein, plastocyanin, indicates that contrary to the Z scheme ferredoxin is reduced in two experimentally distinguishable ways: oxygenically by PS II (renamed the oxygenic photosystem), without the participation of PS I, and anoxygenically by PS I (renamed the anoxygenic photosystem). It also indicates that plastocyanin is not only, as the Z scheme asserts, the electron donor to the reaction center chlorophyll of PS I (P700) but also to the reaction center chlorophyll of PS II (P680). Other unconventional findings include evidence that the fully functional oxygenic photosystem, when operating separately from the anoxygenic photosystem, reduces plastoquinone to plastoquinol and subsequently oxidizes plastoquinol by two pathways acting in concert: one being the universally recognized DBMIB-sensitive pathway via the Rieske iron-sulfur center of the cytochrome bf complex and the other, a hitherto unrecognized, DBMIB-insensitive electron transport pathway around P680 that centers on cytochrome b-559. These nonconforming findings form the basis of an alternate hypothesis of photosynthetic electron transport that modifies and complements the Z scheme.Abbreviations PS photosystem - PQ oxidized plastoquinone - PQH₂ reduced plastoquinone (plastoquinol) - Q_A and Q_B specialized membrane-bound forms of PQ - PC plastocyanin - Fd ferredoxin - BISC F_AF_B, membrane-bound iron-sulfur centers of PS I - DBM1B 2,5-dibromo-3-methyl-6-isopropyl-n-benzoquinone (dibromothymoquinone) - DNP-INT dinitrophenol ether of iodonitrothymol - NADP⁺ NADPH, oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate - FCCP carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone - CCCP carbonyl cyanide-3-chlorophenylhydrazone - SF 6847 2,6,-di-(t-butyl)-4-(2,2-dicyanovinyl) phenol - diuron (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - DCIP 2,6-dichloro-phenolindophenol - UHDBT 5-(n-undecyl)-6-hydroxy-4-7-dioxobenzothiazole; cytochrome b-559_HP-cytochrome b-559_LP, high- and low potential states of cytochrome b-559 - oxygenic reductions reductions in which water is the electron donor - BBY PS II preparation made according to Berthold et al. (1981) Dedicated to Professor Achim Trebst on his 65th birthday.Based in part on lecture in Advanced Course on Trends in Photosynthesis Research, Palma de Mallorca, Spain, September 18, 1990.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in Gonyaulax polyedra

Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O₂ uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O₂ evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as ¹⁴C fixation or O₂ evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax.     

Oxygen reduction in a plastoquinone pool of isolated pea thylakoids

Oxygen uptake in isolated pea thylakoids in the presence of an inhibitor of plastoquinol oxidation by b ₆/f-complex dinitrophenylether of 2-iodo-4-nitrothymol (DNP-INT) was studied. The rate of oxygen uptake in the absence of DNP-INT had a distinct maximum at pH 5.0 followed by a decline to pH 6.5 and posterior slow rise, while in the presence of an inhibitor it increased at an increasing pH from 4.5 to 6.5 and then kept close to the rate in its absence up to pH 8.5. Gramicidin D substantially affected the oxygen uptake rate in the absence of DNP-INT, and only slightly in its presence. Such differences pointed to the presence of special oxygen reduction site(s) in photosynthetic electron transport chain `before' cytochrome complex. Oxygen uptake in membrane fragments of Photosystem II (BBY-particles) was low and did not depend on pH. This did not support the participation of Q_B in oxygen reduction in DNP-INT-treated thylakoids. Oxygen uptake in thylakoids in the presence of DNP-INT was inhibited by DCMU as well as by catalase in whole pH range. The catalase effect indicated that oxygen uptake was the result of dioxygen reduction by electrons derived from water, and that H₂O₂ was a final product of this reduction. Photoreduction of Cyt c in the presence of DNP-INT was partly inhibited by superoxide dismutase (SOD), and this pointed to superoxide formation. The latter was confirmed by a rise of the oxygen uptake rate in the presence of ascorbate and by suppression of this rise by SOD. Both tests showed that the detectable superoxide radicals averaged 20–25% of potentially formed superoxide radicals the quantity of which was calculated from the oxygen uptake rate. The obtained data implies that the oxygen reduction takes place in a plastoquinone pool and occurs mainly inside the membrane, where superoxide can be consumed in concomitant reactions. A scheme for oxygen reduction in a plastoquinone pool in thylakoid membranes is proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of Sulfur Re-Addition on H<Subscript>2</Subscript> Photoproduction by Sulfur-Deprived Green Algae

Sulfur deprivation of algal cultures selectively and partially inactivates photosystem II (PSII)-catalyzed O₂ evolution, induces anaerobiosis and hydrogenase expression, and results in sustained H₂ photoproduction for several days. We show that re-addition of limiting amounts of sulfate (1–10 μM final concentration) to the cultures during the H₂-production phase temporarily reactivates PSII photochemical and O₂-evolution activity and re-establishes higher rates of electron transport through the photosynthetic electron transport chain. The reactivation of PSII occurs by de novo D1 protein synthesis, but does not result in the re-establishment of aerobic conditions in the reactor, detectable by dissolved-O₂ sensors. However, concomitant H₂ photoproduction is inhibited, possibly due to excessive intra-cellular levels of photosynthetically-evolved O₂. The partial recovery of electron transport rates correlates with the re-oxidation of the plastoquinone (PQ) pool, as observed by pulse-amplitude modulated (PAM) and fluorescence-induction measurements. These results show that the presence of a more oxidized PQ pool releases some of the down-regulation of electron transport caused by the anaerobic conditions.     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.