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The plastid terminal oxidase PTOX catalyzes the oxidation of plastoquinol (PQH 2 ) coupled with the reduction of oxygen to water. In vivo PTOX is attached to the thylakoid membrane. PTOX is important for plastid development and carotenoid biosynthesis, and its role in photosynthesis is controversially discussed. To analyze PTOX activity in photosynthetic electron transport recombinant purified PTOX fused to the maltose-binding protein was added to photosystem II-enriched membrane fragments. These membrane fragments contain the plastoquinone (PQ) pool as verified by thermoluminescence. Experimental evidence for PTOX oxidizing PQH 2 is demonstrated by following chlorophyll fluorescence induction. Addition of PTOX to photosystem II-enriched membrane fragments led to a slower rise, a lower level of the maximal fluorescence and an acceleration of the fluorescence decay. This effect was only observed at low light intensities indicating that PTOX cannot compete efficiently with the reduction of the PQ pool by photosystem II at higher light intensities. PTOX attached tightly to the membranes since it was only partly removable by membrane washings. Divalent cations enhanced the effect of PTOX on chlorophyll fluorescence compared to NaCl most likely because they increase connectivity between photosystem II centers and the size of the PQ pool. Using single turnover flashes, it was shown that the level of reactive oxygen species, generated by PTOX in a side reaction, increased when the spacing between subsequent double flashes was enlarged. This shows that PTOX generates reactive oxygen species under limited substrate availability. [ABSTRACT FROM AUTHOR]

Abstract: The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOX). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration and setting the redox poise for cyclic electron transport. We have investigated a homogenously pure MBP fusion of PTOX. The protein forms a homo-tetrameric complex containing 2 Fe per monomer and is very specific for the plastoquinone head-group. The reaction kinetics were investigated in a soluble monophasic system using chemically reduced decyl-plastoquinone (DPQ) as the model substrate and, in addition, in a biphasic (liposomal) system in which DPQ was reduced with DT-diaphorase. While PTOX did not detectably produce reactive oxygen species in the monophasic system, their formation was observed by room temperature EPR in the biphasic system in a [DPQH2] and pH-dependent manner. This is probably the result of the higher concentration of DPQ achieved within the partial volume of the lipid bilayer and a higher Km observed with PTOX-membrane associates which is ≈47mM compared to the monophasic system where a Km of ≈74μM was determined. With liposomes and at the basic stromal pH of photosynthetically active chloroplasts, PTOX was antioxidant at low [DPQH2] gaining prooxidant properties with increasing quinol concentrations. It is concluded that in vivo, PTOX can act as a safety valve when the steady state [PQH2] is low while a certain amount of ROS is formed at high light intensities. [Copyright &y& Elsevier]

The liverwort Marchantia polymorpha contains two isoforms of the plastid terminal oxidase (PTOX), an enzyme that catalyzes the reduction of oxygen to water using plastoquinol as substrate. Phylogenetic analyses showed that one isoform, here called MpPTOXa, is closely related to isoforms occurring in plants and some algae, while the other isoform, here called MpPTOXb, is closely related to the two isoforms occurring in Chlamydomonas reinhardtii. Mutants of each isoform were created in Marchantia polymorpha using CRISPR/Cas9 technology. While no obvious phenotype was found for these mutants, chlorophyll fluorescence analyses demonstrated that the plastoquinone pool was in a higher reduction state in both mutants. This was visible at the level of fluorescence measured in dark-adapted material and by post illumination fluorescence rise. These results suggest that both isoforms have a redundant function. However, when P700 oxidation and re-reduction was studied, differences between these two isoforms were observed. Furthermore, the mutant affected in MpPTOXb showed a slight alteration in the pigment composition, a higher non-photochemical quenching and a slightly lower electron transport rate through photosystem II. These differences may be explained either by differences in the enzymatic activities or by different activities attributed to preferential involvement of the two PTOX isoforms to either linear or cyclic electron flow. • MpPTOX isoforms belong to two different classes. • MpPTOX isoforms have a redundant function keeping the plastoquinone pool oxidized. • Activity of the two isoforms differs depending on illumination conditions. [ABSTRACT FROM AUTHOR]

Abstract: The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool. [Copyright &y& Elsevier]

Abstract: Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between QA and QB, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F 0 ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O2 in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes. [Copyright &y& Elsevier]

Abstract: The physiological role of the plastid terminal oxidase (PTOX) involved in plastoquinol oxidation in chloroplasts has been investigated in vivo in tomato leaves. Enzyme activity was assessed by non-invasive methods based on the analysis of the kinetics of chlorophyll fluorescence changes. In the dark, the maximum PTOX rate was smaller than 1 electron per second per PSII. This value was further decreased upon light acclimation, and became almost negligible upon inhibition of the photosynthetic performances by reducing the CO2 availability. In contrast, prolonged exposure to high light resulted in an increase of the overall PTOX activity, which was paralleled by an increased protein accumulation. Under all the conditions tested the enzyme activity always remained about two orders of magnitude lower than that of electron flux through the linear photosynthetic electron pathway. Therefore, PTOX cannot have a role of a safety valve for photogenerated electrons, while it could be involved in acclimation to high light. Moreover, by playing a major role in the control of the stromal redox poise, PTOX is also capable of modulating the balance between linear and cyclic electron flow around PSI during the deactivation phase of carbon assimilation that follows a light to dark transition. [Copyright &y& Elsevier]

Abstract: An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2–2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100–1800 μmol photons m−2 s−1). Simultaneous measurements of FI and 820-nm transmission kinetics (I820) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2–0.4 s) is accompanied by a decrease of I820, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4–2 s) of the dip is not paralleled by the change in I820. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I–P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I820 kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin–NADP+–oxidoreductase or Mehler–peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed. [Copyright &y& Elsevier]

The present work reports reactions of plastoquinol (PQH 2 -9) and plastoquinone (PQ-9) in organic solvents and summarizes the literature to understand similar reactions in thylakoids. In thylakoids, PQH 2 -9 is oxidized by the cytochrome b 6 /f complex (Cyt b 6 /f ) but some PQH 2 -9 is also oxidized by reactions in which oxygen acts as an electron acceptor and is converted to reactive oxygen species (ROS). Furthermore, PQH 2 -9 reacts with ROS. Light enhances oxygen-dependent oxidation of PQH 2 -9. We examined the oxidation of PQH 2 -9 via dismutation of PQH 2 -9 and PQ-9 and scavenging of the superoxide anion radical (O 2 − ) and hydrogen peroxide (H 2 O 2 ) by PQH 2 -9. Oxidation of PQH 2 -9 via dismutation to semiquinone was slow and independent of pH in organic solvents and in solvent/buffer systems, suggesting that intramembraneous oxidation of PQH 2 -9 in darkness mainly proceeds via reactions catalyzed by the plastid terminal oxidase and cytochrome b 559 . In the light, oxidation of PQH 2 -9 by singlet oxygen and by O 2 − formed in PSI contribute significantly. In addition, Cyt b 6/ f forms H 2 O 2 with a PQH 2 -9 dependent mechanism. Measurements of the reaction of O 2 − with PQH 2 -9 and PQ-9 in acetonitrile showed that O 2 − oxidizes PQH 2 -9, forming PQ-9 and several PQ-9-derived products. The rate constant of the reaction between PQH 2 -9 and O 2 − was found to be 10 4  M −1  s −1 . H 2 O 2 was found to oxidize PQH 2 -9 to PQ-9, but failed to oxidize all PQH 2 -9, suggesting that the oxidation of PQH 2 -9 by H 2 O 2 proceeds via deprotonation mechanisms producing PQH − -9, PQ 2− -9 and the protonated hydrogen peroxide cation, H 3 O 2 + . [ABSTRACT FROM AUTHOR]

Eleven genes encoding chloroplast NADH dehydrogenase-like (NDH) complex have been discovered in plastid genomes on the basis of their homology with genes encoding respiratory complex I. Despite this structural similarity, chloroplast NDH and its evolutionary origin NDH-1 in cyanobacteria accept electrons from ferredoxin (Fd), indicating that chloroplast NDH is an Fd-dependent plastoquinone (PQ) reductase rather than an NAD(P)H dehydrogenase. In Arabidopsis thaliana , chloroplast NDH interacts with photosystem I (PSI); this interaction is needed to stabilize NDH, especially under high light. On the basis of these distinct characters of chloroplast and cyanobacterial NDH, it can be distinguished as a photosynthetic NDH from respiratory complex I. In fact, chloroplast NDH forms part of the machinery of photosynthesis by mediating the minor pathway of PSI cyclic electron transport. Along with the antimycin A-sensitive main pathway of PSI cyclic electron transport, chloroplast NDH compensates the ATP/NADPH production ratio in the light reactions of photosynthesis. In this review, I revisit the original concept of chloroplast NDH on the basis of its similarity to respiratory complex I and thus introduce current progress in the field to researchers focusing on respiratory complex I. I summarize recent progress on the basis of structure and function. Finally, I introduce the results of our examination of the process of assembly of chloroplast NDH. Although the process requires many plant-specific non-subunit factors, the core processes of assembly are conserved between chloroplast NDH and respiratory complex I. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. [ABSTRACT FROM AUTHOR]

Cyanobacteria have evolved elaborate electron transport pathways to carry out photosynthesis and respiration, and to dissipate excess energy in order to limit cellular damage. Our understanding of the complexity of these systems and their role in allowing cyanobacteria to cope with varying environmental conditions is rapidly improving, but many questions remain. We summarize current knowledge of cyanobacterial electron transport pathways, including the possible roles of alternative pathways in photoprotection. We describe extracellular electron transport, which is as yet poorly understood. Biological photovoltaic devices, which measure electron output from cells, and which have been proposed as possible means of renewable energy generation, may be valuable tools in understanding cyanobacterial electron transfer pathways, and enhanced understanding of electron transfer may allow improvements in the efficiency of power output. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux. [ABSTRACT FROM AUTHOR]

In sunflower leaves linear electron flow LEF = 4 O 2 evolution rate was measured at 20 ppm O 2 in N 2 . PSII charge separation rate CSR II = a II ∙ PAD∙( F m − F ) / F m , where a II is excitation partitioning to PSII, PAD is photon absorption density, F m and F are maximum and actual fluorescence yields. Under 630 nm LED + 720 nm far-red light (FRL), LEF was equal to CSR II with a II = 0.51 to 0.58. After FRL was turned off, plastoquinol (PQH 2 ) accumulated, but LEF decreased more than accountable by F increase, indicating PQH 2 -oxidizing cyclic electron flow in PSII (CEF II ). CEF II was faster under conditions requiring more ATP, consistent with CEF II being coupled with proton translocation. We propose that PQH 2 bound to the Q C site is oxidized, one e − moving to P680 + , the other e − to Cyt b 559 . From Cyt b 559 the e − reduces Q B − at the Q B site, forming PQH 2 . About 10–15% electrons may cycle, causing misses in the period-4 flash O 2 evolution and lower quantum yield of photosynthesis under stress. We also measured concentration dependence of PQH 2 oxidation by dioxygen, as indicated by post-illumination decrease of Chl fluorescence yield. After light was turned off, F rapidly decreased from F m to 0.2 F v , but further decrease to F 0 was slow and O 2 concentration dependent. The rate constant of PQH 2 oxidation, determined from this slow phase, was 0.054 s − 1 at 270 μM (21%) O 2 , decreasing with K m (O 2 ) of 60 μM (4.6%) O 2 . This eliminates the interference of O 2 in the measurements of CEF II . [ABSTRACT FROM AUTHOR]

Abstract: The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the ΔpsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase (katG) and iron superoxide dismutase (sodB) was upregulated in the ΔpsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in ΔpsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the ΔpsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the ΔpsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species. [Copyright &y& Elsevier]