Etude de la photophosphorylation endogene des chloroplastes isoles d'epinardStudy on endogenous photophosphorylation of isolated spinach chloroplasts

Whole spinach chloroplasts were able to perform photophosphorylation under nitrogen without the addition of any redox cofactor. This “endogenous” phosphorylation was totally insensitive to 3-(p-chlorophenyl)-1,1-dimethylurea. After osmotic shock endogenous ATP formation decreased but the addition of 3-(p-chlorophenyl)-1,1-dimethylurea stimulated it.

Under a stream of nitrogen, whole chloroplasts reduced NADP⁺ after an osmotic shock, in the absence of added ferredoxin. The resulting ATP/NADPH ratios were high (approx. 2 or 3). They decreased to 1 in the presence of either exogenous ferredoxin, 3-(p-chlorophenyl)-1,1-dimethylurea or limiting light: i.e. high ATP/NADPH ratios were observed only when the terminal step of NADP⁺ reduction was limiting.

The endogenous anaerobic phosphorylation was inhibited by antimycin A to the same extent as the O₂-dependent endogenous non-cyclic phosphorylation.

A direct inhibition of electron transport by antimycin A has never been observed.     

The site of phosphorylation associated with Photosystem I

1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP⁺ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.

2. 2. Under aerobic conditions a low O₂ uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.

3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O₂ can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.

One-way electron discharge subsequent to the photochemical charge separation in Photosystem I

Subsequent to the photochemical charge separation in Photosystem I, three fates are possible: (a) recombination of the photooxidized P700⁺ and photoreduced P430⁻; (b) a cyclic electron flow involving P700⁺, P430⁻ and another electron carrier present in its oxidized and reduced forms; and (c) a non-cyclic electron flow involving one electron donor reacting with P700⁺ and another electron acceptor reacting with P430⁻. This note deals with a fourth fate which is brought about only when an autooxidizable secondary electron acceptor is present but the secondary electron donor is either absent or blocked. In this case, only P430⁻ reverts to the uncharged state in the dark by discharging its electron; P700⁺ remains oxidized and reverts to the uncharged state only extremely slowly.     

On an Inhibitor of Ferredoxin Catalyzed Cyclic Photophosphorylation in Thylakoid Membranes*

Dibromo- and diiodo-naphthoquinones are shown to be inhibitors of the cytochrome b₆/f complex in isolated thylakoid membranes from spinach chloroplasts. Dibromo-naphthoquinone inhibits ferredoxin catalyzed cyclic photophosphorylation at 0.1 μM concentrations, but non cyclic e-flow only at 10 μM. It does not inhibit cyclic systems with artifical cofactors, nor non-cyclic electron flow from duroquinol through photosystem I via the cytochrome b₆/f complex. Dibromo-naphthoquinone does however, lower the stoichiometry for ATP formation in the duroquinol donor system. This inhibitory pattern is quite different from that of DBMIB, but very similar to that of antimycin. This antimycin-like behaviour of these inhibitors is interpreted to indicate a) the existence of a Q_c site in the cytochrome b₆/f complex and its obligate function in ferredoxin catalyzed cyclic electron flow and b) a non-essential role of the Q_c site in non-cyclic electron flow, but which — when operative — pumps an extra proton across the thylakoid membrane increasing the ATP yield.     

Role of the superoxide free radical ion in photosynthetic ascorbate oxidation and ascorbate-mediated photophosphorylation

The mechanism of ascorbate photooxidation in isolated chloroplasts has been studied. The enzyme superoxide dismutase has been used as a tool to show that ascorbate is oxidized by the superoxide free radical ion, which is formed during the autooxidation of a low-potential electron acceptor.

In the absence of an artificial, low-potential electron acceptor, addition of ascorbate stimulates photophosphorylation in isolated chloroplasts. This effect of ascorbate is abolished by superoxide dismutase, indicating that both the superoxide free radical ion and ascorbate are responsible for the stimulation of photophosphorylation. In this case, the superoxide free radical ion seems to be formed during the autooxidation of an endogenous electron acceptor.

In the presence of ferredoxin and NADP⁺, photophosphorylation in isolated chloroplasts stops as soon as the available NADP⁺ is fully reduced. If ascorbate is present in this system, however, a linear rate of photophosphorylation is maintained in spite of the fact, that NADP⁺ is fully reduced. This ascorbate-mediated photophosphorylation again is abolished by superoxide dismutase.

During the catalysis of this oxygen-dependent photophosphorylation, ascorbate consumption is not observed. These findings support the idea, that in chloroplasts ascorbate together with the superoxide free radical ion may function in providing additional ATP by an oxygen-dependent photophosphorylation.     

Photosynthetic electron transport: Emergence of a concept, 1949–59

Historically, two main concepts guided research into possible mechanisms of light-induced atomic rearrangements in oxygenic photosynthesis: Photodecomposition of CO₂ and photodecomposition of water. Both concepts envisioned photoinduced transfers of cumbersome whole atoms and not, as is currently held, photoinduced electron transfers. Early proposals for light-induced electron transfers were relegated to obscurity because they were speculative ideas, not supported by meaningful experimental findings and tied to hypothetical and ephemeral schemes. The concept of photoinduced rearrangements of whole atoms rather than electrons was so well entrenched that it was even invoked to explain their findings by the discoverers of the Hill reaction and cyclic photophosphorylation. The light-induced electron flow concept gained acceptance in photosynthesis research only with the discovery of non-cyclic photophosphorylation in which ATP formation is coupled with electron transport to ferredoxin/NADP⁺ or to artificial substitutes like ferricyanide.     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Photosynthetic control by isolated pea chloroplasts

Isolated pea chloroplasts undergo both cyclic and non-cyclic electron flow. Both processes are coupled to photophosphorylation. During non-cyclic flow the rate of oxygen production showed ADP-governed ;photosynthetic control' analogous to respiratory control of isolated mitochondria. Measurements of ADP/O and photosynthetic control ratios yielded values of 1-1.3 and 2-5.7 respectively. ;Photosynthetic control' was shown to be dependent on the intactness of the chloroplasts.     

Electron transfer components of wild-type and photosynthetic mutant strains of Scenedesmus obliquus D3

To investigate the possible alteration of various components of the photosynthetic electron transport system of certain mutants of Scenedesmus techniques were developed for their extraction and purification from whole cells of this alga. The components identified in the normal alga were cytochrome c 549, cytochrome b 562, a cytochrome c 551, flavoprotein-ferredoxin reductase, plastocyanin, cytochrome c 552, and ferredoxin. Lamellar-bound cytochrome c 552 and cytochromes b were also detected. Application of the extraction and purification techniques to two photosynthetic mutants revealed that Mutants 26 and 50 lacked cytochrome f in both the bound and soluble forms (Mutant 50) or in only the bound form (Mutant 26). Chloroplasts prepared from either of these mutants lacked Hill reaction activity with a variety of oxidants with water as the electron donor but photoreduced NADP⁺ with 2,6-dichlorophenolindophenol and ascorbate as the electron donor system. No photophosphorylation in vivo was detected with either mutant, but isolated chloroplasts performed a cyclic photophosphorylation with phenazine methosulphate as cofactor. Fluorescence analysis revealed that both mutants possess a measurable Photosystem II activity.

It was concluded that the loss of cytochrome f prevents the normal flow of electrons from Photosystem II to NADP and also to a variety of other Hill reaction oxidants. Furthermore, cytochrome f is not required for the reduction of NADP with electron donor systems other than water nor is it an essential component of the mechanism of cyclic photophosphorylation with phenazine methosulphate as cofactor.     

Inhibition of photo-induced electron transport and related reactions in isolated chloroplasts by phenol

Phenol inhibits the Hill reaction with several Hill oxidants and the accompanying non-cyclic phosphorylation. It inhibits also pseudocyclic and cyclic phosphorylation. Partial reactions which are dependent on cyclic electron flow are inhibited too, but electron flow from ascorbate via dichlorophenolindophenol to TPN is not. The activity of various benzene and phenol derivatives was compared. It is concluded that the inhibition is a result of interfering with an electron carrier which participates in cyclic and non-cyclic electron flow.     

The role of the chloroplast coupling factor in the inactivation of thylakoid membranes at low temperatures

Thylakoids isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) were exposed to variable low temperatures under non-freezing conditions. After incubation, changes in the activities of several photochemical reactions and physical properties of the membranes were measured at room temperature.
Cyclic photophosphorylation was strictly dependent on the temperature and the electrolyte concentration: decrease in temperature and increase in NaCl concentration enhanced membrane damage. Inactivation of photophosphorylation was accompanied by stimulation of non-cyclic electron transport, increase in proton permeability and decrease in δpH. When dicyclohexylcarbodiimide was added, the proton gradient became completely restored. The temperature- and salt-dependent breakdown of photophosporylation was closely related to the release of the chloroplast coupling factor (CF₁) from the membranes. The addition of Mg²⁺, very low concentrations of ATP or ADP, or higher concentrations of low-molecular-weight polyols prior to temperature treatment prevented thylakoid damage.
The data indicate that inactivation of photophosphorylation of thylakoids at low temperatures is determined to a considerable extent by the cold lability of the CF₁. As a consequence, it must be concluded that damage of biomembranes caused by freezing is not due solely to changes resulting from the ice formation but additionally by temperature-dependent alterations of cold-labile proteins. Moreover, the data explain the mechanism of non-colligative cryoprotection of isolated thylakoid membranes.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark.

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of Photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg²⁺. The reaction is inhibited by (i) an antibody to ferredoxin-NADP⁺ reductase (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q.It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Cyclic photophosphorylation reactions catalyzed by ferredoxin,methyl viologen and anthraquinone sulfonate. Use of photochemical reactions to optimize redox poising

The flavin analogue 5-deazariboflavin is a convenient catalyst for the photoreduction of low-potential redox compounds. In an anaerobic medium with Tricine buffer as the electron donor, 5-deazariboflavin is capable of photoreducing both ferredoxin and methyl viologen. We have used this method to conduct a comparative study of the Photosystem I photophosphorylation activities supported by the reduced forms of ferredoxin, methyl viologen and anthraquinone sulfonate. All of these catalysts are capable of generating high rates (200–500 μmol ATP/h per mg chlorophyll) of cyclic photophosphorylation, but only the activity dependent on ferredoxin exhibits sensitivity to antimycin A. This finding suggests that the size of the catalyst and its ability to approach the thylakoid membrane, rather than low-redox potential, governs antimycin A sensitivity. Ferredoxin-catalyzed activity is, however, less sensitive to inhibition by dibromothymoquinone than are the activities supported by methyl viologen and anthraquinone sulfonate. This discrepancy is due to binding of the inhibitor by ferredoxin.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes

1. A reversible light-induced enhancement of the fluorescence of a “hydrophobic fluorophore”, 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1′-ethylene-2,2′-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response.

2. This effect is decreased under phosphorylating conditions (presence of ADP, P_i, Mg²⁺), and completely inhibited by the uncoupler of phosphorylation NH₄Cl (5–10 mM), as well as the ionophores nigericin and gramicidin-D (both at 5 · 10⁻⁸ M). The MgCl₂ concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a “high energy intermediate”. The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the “high energy state” of the thylakoid membrane rather than a terminal phosphorylation step.

3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fluorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H⁺ uptake per se because (a) the pH dependence of H⁺ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H⁺ uptake, reduces anthroyl stearate fluorescence.

Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate or Diquat catalyzed electron flow and is the first “non-amine” fluorescence probe utilized for this purpose in chloroplasts.     

The ATP Levels Obtained in Photophosphorylation, Glycolysis and Oxidative Phosphorylation in Scenedesmus Titrated with Desaspidin

The ATP levels in photophosphorylation, glycolysis and oxidative phosphorylation, in the unicellular green alga Scenedesmus obtusiusculus, were titrated with narrow concentration intervals of desaspidin in the presence of different concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which allows the differentiation between non-cyclic, pseudocyclic and true cyclic photophosphorylation. The data on photophosphorylative ATP levels were compared with earlier data on total binding of phosphate. In the true cyclic process, both parameters are equally sensitive towards desaspidin. Under pseudocyclic conditions and in non-cyclic photophosphorylation, the level of ATP is more sensitive towards desaspidin than is total binding of phosphate. This suggests a structural difference between the cyclic and the two non-cyclic (one of which is also pseudocyclic) sites. The non-cyclic ATP level is more sensitive towards desaspidin than is pseudocyclic. This may be connected with the higher ATP level under pseudocyclic as compared to non-cyclic conditions.     

Localization of photophosphorylation and proton transport activities in various regions of the chloroplast lamellae

Membrane fragments released by French pressure cell treatment of whole chloroplasts and isolated by differential centrifugation have been characterized structurally and with respect to phosophorylating and proton transport activities. In agreement with results of other workers, the heavy fraction released by pressure treatment was found by electron microscopy studies to be made up of mostly intact grana stacks while the light fraction was comprised of vesicles derived from the stromal lamellae. Both fractions were found to carry out rapid rates of cyclic photophosphorylation catalyzed by phenazine methosulfate (PMS). However, only the grana membranes demonstrated active proton accumulation in the presence of PMS. No light induced H⁺ uptake could be detected in the stromal lamellae fraction; and as expected, proton gradient dissipating agents such as NH₄Cl, nigericin in the presence of K⁺, and gramicidin were only slightly inhibitory to phosphorylation at concentrations which were very inhibitory in the grana membrane fraction.

Further evidence that stromal lamellae do not have active proton transport in the intact chloroplast was obtained by comparing various chloroplasts having different amounts of stromal and grana membranes. Comparative studies on young and old chloroplasts from lettuce, mesophyll and bundle sheath cell plastids from sorghum, and greening plastids from etiolated corn seedlings revealed a direct correlation between the extent of grana formation and the amount of proton transport activity. Samples which had larger amounts of stromal lamellae had high rates of ATP formation but a reduced capacity for H⁺ accumulation.     

Inhibition of photosynthesis in Phaseolus vulgaris by treatment with toxic concentrations of zinc: effects on electron transport and photophosphorylation

Dwarf beans ( Phaseolus vulgaris L. cv. Limburgse Vroege) were grown on a nutrient medium containing a toxic non-lethal ZnSO₄ concentration. The electron transport and photophosphorylation activities of chloroplasts, isolated from these beans, and from control plants, grown under standard nutrient conditions, were compared. Electron transport was significantly inhibited by Zn²⁺ treatment. Photosystem 2 activity proved to be more sensitive than photosystem 1 activity.
Inhibition was dependent on electron flow rate. Activity was fully restored with semicarbazide. EDTA-washed thylakoid membranes were strongly manganese-deficient. The results suggest that photolysis of water was primarily inhibited, due to a zinc-induced deficiency in loosely bound manganese at the water-splitting site. Manganese is probably substituted by zinc, since the zinc content of thylakoids increased five-fold. Non-cyclic photophosphorylation capacity was also limited as a result of inhibition of electron transport. Phosphorylation efficiency (ATP/2e ratio) involving both energy conserving sites was hardly affected.     

The relation of electron transport and photophosphorylation to conformational changes in chloroplasts

1. The rate of the Hill reaction and photophosphorylation, and the ratio of ATP produced to the electron flow are shown to be strongly dependent on the solute concentration of the medium.

2. A large part, but not all, of the requirement for MgCl₂ or phosphate in photophosphorylation can be replaced by SrCl₂ or other solutes.

3. In two-stage photophosphorylation, solutes are required during the light-activation stage.

4. The presence of solutes causes marked changes in the packed volume of the chloroplasts, and their light-scattering properties. These changes are essentially complete within 1 min.

5. The effectiveness of solutes in enhancing the rate of electron transport and photophosphorylation parallels their effectiveness in inducing conformational changes in chloroplasts.

6. It is suggested that the solutes act by inducing a conformational change of the chloroplast structure which is more optimal for electron transfer and coupled phosphorylation.