Electron transport routes in whole cells of Synechocystis sp. strain PCC 6803: the role of the cytochrome bd-type oxidase

The plastoquinone pool is the central switching point of both respiratory and photosynthetic electron transport in cyanobacteria. Its redox state can be monitored noninvasively in whole cells using chlorophyll fluorescence induction, avoiding possible artifacts associated with thylakoid membrane preparations. This method was applied to cells of Synechocystis sp. PCC 6803 to study respiratory reactions involving the plastoquinone pool. The role of the respiratory oxidases known from the genomic sequence of Synechocystis sp. PCC 6803 was investigated by a combined strategy using inhibitors and deletion strains that lack one or more of these oxidases. The putative quinol oxidase of the cytochrome bd-type was shown to participate in electron transport in thylakoid membranes. The activity of this enzyme in thylakoids was strongly dependent on culture conditions; it was increased under conditions where the activity of the cytochrome b(6)f complex alone may be insufficient for preventing over-reduction of the PQ pool. In contrast, no indication of quinol oxidase activity in thylakoids was found for a second alternative oxidase encoded by the ctaII genes.     

The plastoquinone pool as possible hydrogen pump in photosynthesis.

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H+ + e-) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H+ uptake and the internal H+ release led to the following conclusions: (1) A stoichiometric coupling of 1 : 1 : 1 between the external H+ uptake, the electron translocation through the plastoquinone pool and the internal H+ release (corrected for H+ release due to H2O oxidation) is valid (pHout = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation. These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H+ + e-) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Structure of the chloroplast NADH dehydrogenase-like complex: nomenclature for nuclear-encoded subunits

The chloroplast NADH dehydrogenase-like complex (NDH) was first discovered based on its similarity to complex I in respiratory electron transport, and is involved in electron transport from photoproduced stromal reductants such as NADPH and ferredoxin to the intersystem plastoqunone pool. However, a recent study suggested that it is a ferredoxin-dependent plastoquinone reductase rather than an NAD(P)H dehydrogenase. Furthermore, recent advances in subunit analysis of NDH have revealed the presence of a novel hydrophilic subcomplex on the stromal side of the thylakoid membrane, as well as an unexpected lumenal subcomplex. This review discusses these new studies on the structure of NDH, and proposes a unified nomenclature for newly discovered NDH subunits.     

Chlororespiration and poising of cyclic electron transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase

Polypeptides encoded by plastid ndh genes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H(2)O(2). By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgare L.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H(2)O(2) in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O(2). When FeSP is a component of cytochrome b(6).f complex or of the same Ndh complex, O(2) may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O(2) concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities.     

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.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Regulation of electron-transport pathways in cells of Chlamydomonas reinhardtii under stress conditions

The kinetics of interactions between electron-transport pathways in the thylakoid membrane was examined. A mathematical model was proposed to describe the kinetics of redox transitions in photosystem II, proton concentration changes in the chloroplast stroma, and the plastoquinone pool reduction due to photosynthetic and chlororespiratory pathways. A kinetic mechanism is considered that redirects electron flows between photosynthetic and chlororespiratory pathways in response to the increased NADPH content under mineral deficiency. According to the simulation model, the electron transport flows via different routes are switched over in a stepwise manner. The results of numerical simulations are qualitatively consistent with experimental data for Chlamydomonas reinhardtii cells subjected to mineral deprivation.     

A model of electron transport in chloroplasts taking into account the Mitchell Q-cycle. Calculation of J(e) and J(H) fluxes in steady state

On the basis of the earlier proposed model of electron transport, which takes into account the Mitchell Q-cycle, stationary values for the fluxes of electrons Je along the electron transport chain and of protons JH across the thylakoid membrane were obtained, which are calculated now as the functions of photoexcitation of reaction centers and the medium acidity inside and outside of the thylakoid, pHin and pHout. In the framework of the model, the stoichiometric ratio JH/Je is determined virtually only by the time of reduction of plastoquinone (tau B) on the B site of photosystem II and restoration of plastoquinone (tau C) on the C site to the b/f complex.     

Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled "Regulation of Electron Transport in Chloroplasts".     

Reprint of: Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Location,expression and orientation of the putative chlororespiratory enzymes,Ndh and IMMUTANS,in higher-plant plastids

The chloroplasts of many plants contain not only the photosynthetic electron transport chain, but also two enzymes, Ndh and IMMUTANS, which might participate in a chloroplast respiratory chain. IMMUTANS encodes a protein with strong similarities to the mitochondrial alternative oxidase and hence is likely to be a plastoquinol oxidase. The Ndh complex is a homologue of complex I of mitochondria and eubacteria and is considered to be a plastoquinone reductase. As yet these components have not been purified to homogeneity and their expression and orientation within the thylakoid remain ill-defined. Here we show that the IMMUTANS protein, like the Ndh complex, is a minor component of the thylakoid membrane and is localised to the stromal lamellae. Protease digestion of intact and broken thylakoids indicates that both Ndh and IMMUTANS are orientated towards the stromal phase of the membrane in Spinacia oleracea L. Such an orientation is consistent with a role for the Ndh complex in the energisation of the plastid membrane. In expression studies we show that IMMUTANS and the Ndh complex are present throughout the development of both Pisum sativum L. cv Progress No. 9 and Arabidopsis thaliana (L.) Heynh. leaves, from early expansion to early senescence. Interestingly, both the Ndh complex and the IMMUTANS protein accumulate within etiolated leaf tissue, lacking the photosystem II complex, consistent with roles outside photosynthetic electron transport.Abbreviations PQ plastoquinone - PSI, PSII photosystem I, II     

Is the 9 kDa thylakoid membrane phosphoprotein functionally and structurally analogous to the 'H' subunit of bacterial reaction centres?

Although the amino acid sequence of the 9 kDa (phospho)protein of chloroplasts has been determined, the function of this thylakoid membrane protein in photosynthetic electron transport and the reason for its physiological control remains unclear. In this paper, I briefly review the evidence which indicates that the phosphorylation of the 9 kDa protein results in a partial inhibition of photosynthetic oxygen evolution by increasing the stability of the semiquinone bound to QA the primary, plastoquinone-binding site of photosystem II (PS II). I propose that in its dephosphorylated state, the 9 kDa thylakoid membrane protein may serve PS II to ensure efficient photochemical charge separation by aiding the transfer of reducing equivalents out of the reaction centre to the attendant plastoquinone pool. This function is analogous to that proposed for the H-subunit of the reaction centre of photosynthetic eubacteria. Whether these two proteins have evolved from a common ancestral reaction centre protein is discussed in the light of a comparison of their amino acid sequences and predicted secondary structures.     

Inorganic-pyrophosphate-dependent phosphorylation of spinach thylakoid proteins.

It has been shown for the first time that several photosystem-II thylakoid proteins and the main chlorophyll-a/b light-harvesting complex can be phosphorylated with inorganic pyrophosphate as phosphate donor. With pyrophosphate, as with ATP, the protein-kinase reaction is dependent on light or a strong reducing agent. The reaction which can be demonstrated in well-washed spinach thylakoids is dependent on electron transport and is controlled by the redox state of the plastoquinone pool. It is suggested that the pyrophosphate-dependent thylakoid protein phosphorylation is mediated by the same kinase which is responsible for the ATP-dependent protein phosphorylation. This pyrophosphate-dependent kinase activity may be derived from an evolutionary precursor from which ATP-dependent protein phosphorylation also developed.     

Analysis of Chlorophyll Fluorescence by Means of Noisy Light

Linearization is an efficient means of detecting individualcomponents in complex fluorescence induction curves. In a previousinvestigation based on sine-waves (Hansen, Kolbowski, and Dau,1987) the components related to the plastoquinone pool, thehigh-energy state of the thylakoid membrane and the state 1-state2 transition controller could be identified. In this paper,binary noise is used as an alternative input signal which canalso allow linearization by mathematical tools. Comparison withexperiments using sine-waves shows that curve-fitting of thenoise experiments yields the same data as the sine-wave analysis.Further, some additional components were revealed as labelledby the related time-constants whose values depended on lightintensity (e.g. 5, 15, 20, 70, 85, 550 s for spinach at 2.5W m^–2). The advantages of noise analysis are a shorter measuring time and the availability ofan on-line criterion which indicates when a sufficient signal-to-noiseratio is reached. Key words: Chlorophyll fluorescence, correlation functions, linearization, noise, spinach, time-constants     

A strain of Synechocystis sp. PCC 6803 without photosynthetic oxygen evolution and respiratory oxygen consumption: implications for the study of cyclic photosynthetic electron transport

Cyclic electron transport around photosystem (PS) I is believed to play a role in generation of ATP required for adaptation to stress in cyanobacteria and plants. However, elucidation of the pathway(s) of cyclic electron flow is difficult because of low rates of this electron flow relative to those of linear photosynthetic and respiratory electron transport. We have constructed a strain of Synechocystis sp. PCC 6803 that lacks both PSII and respiratory oxidases and that, consequently, neither evolves nor consumes oxygen. However, this strain is still capable of cyclic electron flow around PSI. The photoheterotrophic growth rate of this strain increased with light intensity up to an intensity of about 25 mumol photons m-2 s-1, supporting the notion that cyclic electron flow contributes to ATP generation in this strain. Indeed, the ATP-generating ability of PSI is demonstrated by the fact that the PSII-less oxidase-less strain is able to grow at much higher salt concentrations than a strain lacking PSI. A quinone electrode was used to measure the redox state of the plastoquinone pool in vivo in the various strains used in this study. In contrast to what is observed in chloroplasts, the plastoquinone pool was rather reduced in darkness and was oxidized in the light. This is in line with significant electron donation by respiratory pathways (NADPH dehydrogenase and particularly succinate dehydrogenase) in darkness. In the light, the pool becomes oxidized due to the presence of much more PSI than PSII. In the oxidase-less strains, the plastoquinone pool was very much reduced in darkness and was oxidized in the light by PSI. Photosystem II activity did not greatly alter the redox state of the plastoquinone pool. The results suggest that cyclic electron flow around PSI can contribute to generation of ATP, and a strain deficient in linear electron transport pathways provides an excellent model for further investigations of cyclic electron flow.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

Thiol regulation of the thylakoid electron transport chain--a missing link in the regulation of photosynthesis?

Avoidance of over-reduction of the chloroplast ferredoxin pool is of paramount importance for plants in avoiding oxidative stress. The redox state of this pool can be controlled through regulation of the thylakoid electron transport chain. A model is presented for regulation of this chain via a thiol reduction mechanism, possibly involving a thioredoxin. It is shown in isolated thylakoids that electron transport is inhibited by the thiol reducing agent dithiothreitol. The kinetics of this reduction are rapid and readily reversible. The midpoint redox potential is -365 mV at pH 7.7, with a pH dependency of about -90 mV/pH. At physiological pH values, this places the potential of the species titrated between that of ferredoxin and NADPH and thus in the right potential range to be regulating the redox poise of the ferredoxin pool. This is also close to the potential of NADPH-malate dehydrogenase, an enzyme known to be regulated by thioredoxin. Regulation of electron transport by thioredoxin provides a mechanistic link between the regulation of photosynthesis and gene expression by sugars and the redox regulation of gene expression mediated through the plastoquinone pool.