Two forms of pea leaf ferredoxin]

Some causative factors resulting in the appearance of two components of ferredoxin (Fd) isolated from the pea leaves preparations have been studied by 15% PAG electrophoresis. It has been shown that the composition of the buffer systems (tris-glycine buffer, pH 8,3; veronal buffer, pH 7.0) does not cause Fd separation into two components. Study of the effects of the modifying agents on the active centre and apoprotein of Fd. Showed that the destruction of the active centre does not affect Fd separation and modification of the Fd protein part results in greater amount of the bands on the electrophoregrams. It has been concluded that heterogeneity of Fd is possibly of the genetic nature. The following facts can be regarded as an evidence for existence of both forms of Fd in pea leaves: occurrence of two forms of Fd in chloroplasts isolated in organic media, differing in Fd content during pea ontogenesis and upon varying illumination conditions during their growth.     

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP⁺. The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 μM in the absence of NADP⁺ increased the proportion of oxygen reduction by Fd from 25–35 to 40–60% in different experiments. This proportion decreased with increasing light intensity. When NADP⁺ was added in the presence of 15 μM Fd, which was optimal for the NADP⁺ reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP⁺ and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.     

Participation of ferredoxin in oxygen reduction by the photosynthetic electron transport chain

Oxygen reduction by isolated pea thylakoids was studied in the presence of ferredoxin (Fd), Fd + NADP, and cytochrome c. At Fd concentrations optimal for NADP reduction, it contributed 30–50% of the reducing equivalents (as deduced by comparing the rates of oxygen reduction and light oxidation of reduced Fd). The oxygen reduction rate in the presence of Fd + NADP was 3–4 times lower than with Fd alone, and comparable to that with cyt c. It is supposed that the process involves a photosystem I component whose reaction with oxygen depends on the rate of electron efflux from the PS I terminal acceptors, and that this component is phylloquinone.     

Sites of electron donation by alkylhydroquinones in the electron transport chain of spinach chloroplasts

Alkyl derivatives of p-hydroquinones were examined as electrondonors for the electron transport chain in spinach chloroplasts.Hydrophobic hydroquinones with long side chains donate relativelymore electrons to photosystem 1, while hydrophilic hydroquinoneand methylhydroquinone donate electrons specifically to photosystem2. ¹On leave; Research Laboratories, Fuji Photo Film Co., Ltd.,Asaka, Saitama. (Received March 2, 1973; )     

Changes in the electron transport chain of pea leaf mitochondria metabolizing malate

Pea leaf mitochondria showed complex kinetics for malate metabolism. O₂ uptake increased as malate concentration increased from 0 to 10 mm, reached a plateau between 10 and 20 mm malate, and then increased again up to 40 mm malate. Analysis of the products of malate oxidation by high-performance liquid chromatography revealed that the first phase of O₂ uptake coincided with the synthesis of both pyruvate and oxalacetate (OAA) while the second phase of O₂ uptake at higher malate levels usually occurred with a large increase in OAA formation. The biphasic response in O₂ uptake and the changing ratios of pyruvate and OAA synthesis did not appear to be the direct result of the differing K_m values of malate dehydrogenase and malic enzyme. Rather, they resulted from thermodynamic properties of these two malate oxidases and the kinetics of the two NADH dehydrogenases found in plant mitochondria. At low malate concentrations the rotenone-sensitive NADH dehydrogenase was active and could accept electrons from both malate oxidases. This NADH dehydrogenase became saturated at about 10 mm malate. At higher malate concentrations the rotenone-insensitive NADH dehydrogenase was increasingly important and its increased electron transport capacity was best exploited by malate dehydrogenase. At the higher malate concentrations an increasing portion of the electrons from malate reduce O₂ through the alternative oxidase. Although this coincided with the second phase of malate-dependent O₂ uptake it was not required for this phase to be seen.     

Light-induced changes of b-type cytochromes in the electron transport chain of Euglena chloroplasts

Light-induced changes of b-type cytochromes in Euglena chloroplastswere studied spectrophotometrically.

1. In the dark and at pH 6.5, most of the cytochrome 558 in chloroplastswas in the reduced state, and most of the cytochrome 563, inthe oxidized state. Illumination of chloroplasts at pH 6.5 induceda rapid, but slight oxidation of cytochrome 552 and cytochrome558. The magnitude of photooxidation of cytochrome 558 was greatlyenhanced by the addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea(DCMU). The rate of photooxidation in the presence of DCMU wasstimulated by the addition of 0.15 µM Euglena cytochrome552, or 100 µM methyl viologen.
2. Euglena chloroplasts,incubated at 55°C for 5 min showedno significant absorbancechanges for about 10 min after theonset of illumination. However,greater photooxidation of cytochrome558 was observed afterprolonged illumination, or in the presenceof DCMU or ethylenediaminetetraaceticacid (EDTA). Similar resultswere obtained with chloroplastspre-treated at pH 9.0–10.0for 5 min.
3. At pH 9.5, andin the dark, both cytochrome 563 and cytochrome558 were inan almost reduced state. On illumination at thispH, both cytochromeswere photooxidized, with a complicatedkinetics, showing aninitial rapid and small absorbance decrease,followed by a stagnantphase of temporary retarded reaction.In the presence of DCMUor EDTA, photooxidation proceeded rapidlywithout a stagnantphase.
4. At pH 6.5 cytochrome 558, on cessation of illumination,wasquickly reduced to the initial level. At pH 9.5, there wasalsoappreciable re-reduction of cytochrome 558 and 563 whenthelight was turned off at an early stage of illumination.Theamounts of re-reduction of the cytochromes in the subsequentdark period, however, decreased as photooxidation of cytochromesproceeded. This decrease was accelerated by the presence ofDCMU.
5. At pH 9.5 ascorbate and manganese served as electrondonorsfor die DCMU-sensitive photooxidation of cytochromes558 and563.
6. Experimental results are discussed with specialreference tothe occurrence of two pools of electron carriers,one at thereducing side and the other at the oxidizing sideof photosystem2. The role of manganese in the latter pool ofelectron carriersis also discussed.

(Received March 11, 1970; )     

Two types of ammonium uncoupling in pea chloroplasts

The effect of ammonium on ATP synthesis, electron transfer, and light-induced uptake of hydrogen ions in pea chloroplasts was studied. It is shown that the dependence of these reactions on ammonium concentration could be due to effects of two different uncoupling processes. The first process is induced by low ammonium concentrations (<0.2 mM); the second one is observed in the NH₄Cl concentration interval of 0.5–5.0 mM. The first type of uncoupling is stimulated by palmitic acid or by N,N′-dicyclohexylcarbodiimide, while the second is stimulated by chloroplast thylakoid swelling caused by energy-dependent osmotic gradients. In the presence of the fluorescent dye sulforhodamine B, which does not penetrate through the cell membrane, this swelling causes the dye to enter the lumens. It is supposed that ammonium activates two different routes of cation leakage from the lumen. The first route involves channel proteins, while the second is a mechanosensitive pore that opens in response to osmotic gradients.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Targeting of ricin A chain into pea chloroplasts

A chimaeric gene was constructed encoding the pre-sequence of the 33 kDa oxygen-evolving complex protein from wheat (a thylakoid lumen protein) linked to ricin A chain. The fusion protein is efficiently imported by isolated pea chloroplasts and localised partly in the stroma, with the remainder bound to the stromal surface of the thylakoids. The imported protein is fully processed by both the stromal and thylakoidal processing peptidases, indicating that partial or complete translocation across the thylakoid membrane has taken place.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Tetracaine inhibition of electron transport in pea chloroplasts is coupled to calcium displacement by the local anesthetic

Chloroplasts isolated from pea seadlings grown on water containing 45Ca2+ were treated with local anesthetic tetracaine. Addition of tetracaine inactivated the electron transport activity of donor side photosystem II. This inhibition was accompanied by 45Ca2+ release from the chloroplast membranes as the whole and destroyed by osmotic shock. No such effect was observed when Tris or hydroxylamine were used to inhibit the donor side photosystem II. Upon thermal inactivation of chloroplasts 45Ca2+ release occurred but at temperatures above 80 degrees. The functional role of Ca2+ in photosystem II is discussed.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.     

Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide

In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO₂ reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of ¹⁸O₂ during reduction of CO₂ was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO₂ assimilation resulted in the formation of H₂O₂. This was indicated by a large stimulation of CO₂ reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H₂O₂ was shown to be at the level of phosphoglycerate formation. Evidence for electron transport to oxygen was also obtained from ¹⁴CO₂ experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO₂ reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO₂ and oxygen during photosynthesis.     

Auxiliary electron transport pathways in chloroplasts of microalgae

Microalgae are photosynthetic organisms which cover an extraordinary phylogenic diversity and have colonized extremely diverse habitats. Adaptation to contrasted environments in terms of light and nutrient’s availabilities has been possible through a high flexibility of the photosynthetic machinery. Indeed, optimal functioning of photosynthesis in changing environments requires a fine tuning between the conversion of light energy by photosystems and its use by metabolic reaction, a particularly important parameter being the balance between phosphorylating (ATP) and reducing (NADPH) power supplies. In addition to the main route of electrons operating during oxygenic photosynthesis, called linear electron flow or Z scheme, auxiliary routes of electron transfer in interaction with the main pathway have been described. These reactions which include non-photochemical reduction of intersystem electron carriers, cyclic electron flow around PSI, oxidation by molecular O₂ of the PQ pool or of the PSI electron acceptors, participate in the flexibility of photosynthesis by avoiding over-reduction of electron carriers and modulating the NADPH/ATP ratio depending on the metabolic demand. Forward or reverse genetic approaches performed in model organisms such as Arabidopsis thaliana for higher plants, Chlamydomonas reinhardtii for green algae and Synechocystis for cyanobacteria allowed identifying molecular components involved in these auxiliary electron transport pathways, including Ndh-1, Ndh-2, PGR5, PGRL1, PTOX and flavodiiron proteins. In this article, we discuss the diversity of auxiliary routes of electron transport in microalgae, with particular focus in the presence of these components in the microalgal genomes recently sequenced. We discuss how these auxiliary mechanisms of electron transport may have contributed to the adaptation of microalgal photosynthesis to diverse and changing environments.     

Stoichiometries of electron transport complexes in spinach chloroplasts

The stoichiometric relationship among photosystem II complexes, photosystem I complexes, cytochrome b/f complexes, high-potential cytochrome b-559, and chlorophyll in spinach chloroplasts has been determined. Two features of this data stand out, in contrast to currently proposed stoichiometries in which the ratio of photosystem II to photosystem I is reported to be 2:1 and the chlorophyll to reaction center ratio to be as low as 260:1. Using a variety of techniques it was found that the stoichiometry of photosystem II:photosystem I:cytochrome b/f complex was 1:1:1, within 10%, and that the ratio of total chlorophyll to these components was 600:1, also within 10%. A ratio of two high-potential cytochrome b-559 molecules per 640 chlorophyll, or two molecules per photosystem II reaction center, was found. These ratios were remarkably constant regardless of the time of year or the source of the spinach. The concentration of photosystem II complexes was determined using a pH electrode to measure the flash-induced proton release resulting from water oxidation. The photosystem I reaction center concentration was measured by two different techniques that compared favorably. In the first method a pH electrode was used to measure the amount of flash-induced proton consumption associated with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive oxidation of N,N,N',N'- tetramethylphenylenediamine , resulting in the production of hydrogen peroxide. In the second method the amount of P700 oxidized by far-red light was determined using dual-wavelength spectroscopy. The concentration of the cytochrome b/f complex was determined assuming 1 mol of cytochrome f per complex. The concentration of cytochrome f was measured spectroscopically by its light-induced turnover and by chemical difference spectra. The concentration of high-potential cytochrome b-559 was determined by chemical difference spectra. In addition to these studies, the light-induced absorbance change exhibiting a peak at 323 nm that has been attributed to the reduction of the primary quinone acceptor of photosystem II has been investigated. This measurement frequently has been used to quantitate the photosystem II to chlorophyll ratio. However, in view of these results it is argued that this technique significantly overestimates the photosystem II concentration.     

Porton uptake by isolated chloroplasts during cyclic and non-cyclic electron transport catalyzed by photosystem I in the presence of ferredoxin]

Proton uptake by isolated chloroplasts during cyclic electron transport in the presence of ferredoxin and under NADP+ reduction from the ascorbate--TMPD donor pair under anaerobic conditions was studied. It was found that during cyclic transport the proton uptake is less intensive than under NADP+ reduction. In the presence of ATP the proton uptake is increased in the first case and is decreased in the second one. During cyclic transport in the presence of gramicidin D the proton uptake is completely suppressed and under NADP+ reduction is decreased down to 0,08--0,09 mk equiv H+ per mg of chlorophyll, irrespective of ferredoxin or NADP+ concentrations. The role of ferredoxin NADP+ reductase in a proton uptake by thylakoids is discussed.