Ferricyanide Photoreduction Research Articles

Reactions of Antisera to Lutein and Plastoquinone with Chloroplast Preparations and their Effects on Photosynthetic Electron Transport

An antiserum to lutein inhibits photosynthetic electron transport between water and potassium ferricyanide in diloroplasts from green Nicotiana tabacum var. John William’s Breadleaf. However, electron transport between diphenylcarbazide and potassium ferricyanide is not impaired. From this it is concluded that the photochemically active carotenoid should feed its electrons into the photosynthetic electron transport chain before the site from which diphenyl-carbazide donates electrons. The inhibition of the ferricyanide Hill reaction in diloroplasts by antibodies to lutein depends on the accessibility of the carotenoid antigen in the thylakoid membrane. In fresh preparations the accessibility is greater in diloroplasts in which photo- synthetic electron transport is coupled to photophosphorylation. Concomitantly the antiserum to lutein agglutinates only such chloroplast preparations in which the Hill reaction is impaired by the antiserum. An antiserum to plastoquinone inhibits ferricyanide photoreduction of diloroplasts regardless whether driven by water or diphenylcarbazide as the electron donors. Typical photosystem-I-reactions are not influenced by the antiserum. In a certain type of chloroplast preparations the antiserum does not inhibit PMS-mediated photophosphorylation inferring that plastoquinone, eventually involved in this reaction, is either not accessible to antibodies, or that this cyclic electron flow does not necessarily pass through plastoquinone.

PH Dependence and Cofactor Requirements of Photochemical Reactions in Maize Chloroplasts

The pH dependence of the photoreduction of ferricyanide and the photoreduction of NADP from water and photosystem I activity have been compared in isolated chloroplasts from mesophyll and bundle sheath cells of Zea mays. The maximum activity of photoreduction of ferricyanide occurs at pH 8.5 in isolated mesophyll chloroplasts. The addition of methylamine does not cause a marked shift in the pH maximum, but brief sonication lowers the pH maximum to 7.0. In contrast, isolated bundle sheath chloroplasts have a pH maximum at 7.0 and the shape of the pH versus activity curve is similar to that of sonicated mesophyll chloroplasts. When photoreduction of ferricyanide by the isolated chloroplasts is measured at their pH maxima, the values for bundle sheath chloroplasts are about half those of methylamine-treated mesophyll chloroplasts on a chlorophyll basis.The pH maxima for the photoreduction of NADP from water and photosystem I activity are similar in both mesophyll and bundle sheath chloroplasts with maximum activity occurring at pH 7.0 in both cases. In the presence of added plastocyanin and ferredoxin NADP-reductase, the photosystem I activities of both sonicated mesophyll and sonicated bundle sheath chloroplasts are significantly higher than those of the unsonicated preparations. On a chlorophyll basis, photosystem I activity of bundle sheath chloroplasts is at least twice that of mesophyll chloroplasts.

Photochemically Active Chlorophyll-Containing Proteins from Chloroplasts and their Localization in the Thylakoid Membrane

After solubilization of stroma-freed chloroplasts with deoxycholate, the lipids and the detergent used are separated from the proteins by gel filtration. In this way not denatured pigment-con-taining protein preparations were obtained. The particles in fraction 1 exhibited a molecular weight of 600 000 and contained an average of 25 chlorophyll molecules. The circular dichroism spectrum showed exciton splitting of the red band. The particles in fraction 2 contained 1 chlorophyll molecule and exhibited a molecular weight of 110 000. The particles in fraction 3 also contained only 1 chlorophyll molecule and had a molecular weight of between 80 000 and 100 000. Pure preparations of fraction 1 only carried out the methylviologen Mehler reaction with the dichlorophenol indophenol/ascorbate couple as electron donor. Fraction 3 only reduced ferricyanide with diphenylcarbazide as an electron donor in the light. Fraction 2 exhibited both the photosystem I reaction and the photosystem II reaction. An antiserum to extracted fraction 1 does not inhibit electron transport in the intact lamellar system. The photoreduction of methylviologen is only inhibited after disruption of the thylakoids. The antiserum to fraction 2 inhibits the photoreduction of methylviologen in the intact lamellar system. Consequently, one inhibition site for this photosystem I reaction must be located on the inner and another on the outer surface of the thylakoid membrane. In addition, antibodies to fraction 1 are specifically adsorbed onto the lamellar system without any effect on electron transport and without a concomitant agglutination. Antibodies to fraction 3 partially inhibit the photoreduction of ferricyanide with diphenylcarbazide as an electron donor in the intact lamellar system. Hence, the inhibition site of this system II reaction is located on the outer surface of the thylakoids. We have reason to believe that the inhibition sites not reacting are located in the partitions, which are not accessible to antibodies.

Studies on Effect of Certain Quinones

The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.CO(2) fixation was inhibited by all the quinones to varying degrees. In chloroplasts treated with 06K-quinone or benzoquinone, CO(2) fixation was inhibited to a greater extent than the photoreduction of ferricyanide or ATP formation, indicating the possibility that the two quinones may also inhibit certain reactions in the carbon reduction cycle. The effect of dichlone and chloranil, but not of 06K-quinone, was overcome by the addition of reduced glutathione. The quinones caused an increase in the proportion of (14)C incorporated into 3-phosphoglyceric acid and a reduction in the amount of glycolic acid.

The relation of the alteration of Photosystem II. Activity to the inhibition of energy transfer and the proton pump in tris-washed chloroplasts

Washing of spinach chloroplasts with high concentrations of Tris 3 3 Abbreviations used in this paper include: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl)amino methane; Tricine, tris (hydroxymethyl) methyl-glycine; DCMU, dichlorophenyl-1,1-dimethyl-urea; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; PSII, Photosystem 2; PMS, N-methylphenazonium methosulfate. induces pH-dependent changes in chloroplast reactions. At high pH (8.4) Tris washing causes the inhibition of Photosystem 2 activity which can be prevented by the maintenance of reducing conditions during washing. Washing at low pH (7.2) causes an enhancement of oxygen evolution and increased rate of ferricyanide photoreduction which is not influenced by the presence of reducing conditions. The increased rate of electron flow is accompanied by the inhibition of light mediated phosphorylating activity, acid-induced ATP synthesis, light-induced proton uptake and light triggered Mg 2+ ATPase activity. Tris treatment at low pH also causes a sensitization of Photosystem 2 activity such that oxygen evolution is inhibited by low concentrations of tris at high pH. This inhibition of the stimulated electron flow is not accompanied by a reconstitution of the photophosphorylation activity. A detailed analysis of the effect of tris treatment on Photosystem II activity and membrane dependent energy conversion shows that the treatment of chloroplasts causes an inhibition of the energy conversion process which is independent of the effect on oxygen evolution. Determination of the presence of coupling factor (as determined by ATPase activity) and membrane osmotic properties reveal normal levels of enzyme activity and osmotic response in treated chloroplasts. The inhibition of the energy conversion process is accompanied by reduced capacity to maintain a proton gradient. Kinetic analysis of the proton uptake reaction reveals that Tris treatment renders the grana membranes more permeable to protons.

Membrane structure and function: II. Alterations in the photo-induced absorption changes after treatment of isolated chloroplasts with large pulses of the ruby laser

Treatment of isolated chloroplasts with high-energy pulses of the ruby laser causes graded structural changes in the chloroplast membranes and is here correlated with the biochemical changes produced. The laser treatment caused decreases in the photoinducible absorption changes of cytochromes b 559, b 563, and P 520 (the carotenoid shift), but smaller decreases in cytochrome f. The decreases correlated with the quantum efficiency alterations produced by the laser treatment. Ferricyanide photoreduction and O 2 evolution was only slightly affected by the laser treatment. The slow phase of the dark recovery kinetics of P 520 was increased maximally by the lowest laser input energies and NADP + photoreduction induced by carbonylcyanide- P-trifluoromethoxyphenylhydrazone (FCCP) was decreased maximally by the lowest energies, suggesting that uncoupling of the chloroplasts was the most sensitive parameter. This was corroborated by our previous observation (5) that chloroplast membrane bound surface particles (coupling factor) was the ultrastructural change most sensitive to the laser pulses. Electron flow from photosystem II to photosystem I was not altered by the laser treatment. The laser treatments did not cause a detectable decrease in total chlorophyll in the chloroplasts, however, approximately 10% of the total chlorophyll was present in the solution phase after the treatment, whereas no detectable cytochromes were present in the solution phase.

The site of ferricyanide photoreduction in the lamellae of isolated spinach chloroplasts: a cytochemical study.

ABSTRACT Ferricyanide was used as a Hill oxidant to localize the site of photoreduction in chloroplast lamellae. The ferrocyanide formed on illumination was complexed with copper ions to form insoluble, electron-dense precipitates of Cu ferrocyanide, which can be easily seen in unstained preparations in the electron microscope. Control experiments showed no precipitates in the dark, at zero time, in the absence of ferricyanide, or on addition of sodium ascorbate. Discrete precipitates of Cu ferrocyanide were seen on both stroma and grana lamellae. It is concluded that both stroma and grana lamellae have photosystem II activity necessary for reduction of ferricyanide.

On the Role of Plastoquinone in Photosynthesis

Dibromothymoquinone and its hydroquinone are inhibitors of non cyclic electron flow from water to NADP, anthraquinone or methylviologen. The inhibition is competetively reversed by plastoquinone. It appears that dibromothymoquinone is an antagonist of plastoquinone and that it prevents the enzymic (by the next endogenous carrier of the chloroplast electron transport chain) but not the chemical (by ferricyanide) reoxidation of reduced plastoquinone. This follows from the result that the photoreduction of ferricyanide and DCPIP * is not inhibited by dibromothymoquinone in sonicated chloroplasts and is inhibited in intact chloroplasts to only 60% or 80% respectively. It is concluded that dibromothymoquinone does not inhibit photoreductions by photosystem II. According to their response to dibromothymoquinone, cyclic photophosphorylations can be subdivided in those requiring plastoquinone and those which do not. Menadione catalyzed cyclic photophosphorylation is inhibited by dibromothymoquinone, whereas the PMS catalyzed system is not. The DAD cyclic system is only partly inhibited by dibromothymoquinone. The PMS catalyzed cyclic photophosphorylation in the presence of dibromothymoquinone is antimycin sensitive, which suggests that the PMS system can switch from a plastoquinone dependent system to a plastoquinone independent, but cytochrome b dependent system, which is now antimycin sensitive. Ferredoxin catalyzed cyclic photophosphorylation is inhibited by dibromothymoquinone as well as by antimycin. The data indicate that non cyclic electron flow through both photosystems is obligatory dependent on plastoquinone, whereas cyclic systems do not necessarily include plastoquinone. The relevance of the results to the possibility of different coupling sites in cyclic and non cyclic electron flow systems is discussed.

Enhancement of photophosphorylation and photoreduction by a chloroplast factor from spinach leaves

A factor from spinach chloroplasts stimulates both photoreduction of ferricyanide and photophosphorylation where assayed using isolated chloroplasts. This factor consists of a low molecular weight chromophore attached by non-covalent bonds to a protein. Spectral and chemical evidence suggests that the factor is a direct link in the photosynthetic electron transport chain and does not act as an uncoupling agent nor in pseudo-cyclic phosphorylation.

Inhibition of the Hill Reaction by Tris and Restoration by Electron Donation to Photosystem II

Experiments in which chloroplasts were washed with tris and tricine buffers at different pH's indicated that the non-protonated (uncharged) form of tris was inhibitory to the Hill reaction while the protonated form of tris and the zwitterionic forms of tricine were non-inhibitory. Buffers analogous to tris and tricine gave similar results. Photoreduction of NADP could be restored to the inhibited chloroplasts by adding the reduced forms of p-hydroquinone, p-aminophenol, p-phenylenediamine, benzidine, semicarbazide, and dihydroxydiphenyl, all of which donated electrons to photosystem II. Photoreduction of ferricyanide was shown with those donor systems (benzidine and semicarbazide) which did not react chemically with ferricyanide. Photophosphorylation was also restored with all of the electron donors except semicarbazide.

A Study of Plastoquinones in Photochemical Reactions in the Chloroplasts of Euglena gracilis Strain Z

Plastoquinone-9 (PQ-9) was isolated from the chloroplasts of Euglena gracilis Strain Z and spinach. The functional involvement and the structural specificity of PQ-9 in photochemical reactions was investigated in the isolated chloroplasts of Euglena gracilis. It was found that PQ-9 was required for both photoreduction of ferricyanide and photosynthetic phosphorylation in Euglena chloroplasts. The structural integrity of PQ-9 was not required to the same degree in the 2 photochemical reactions. Photosynthetic phosphorylation seemed to require the entire molecular structure of PQ-9 for the activity, whereas shortening in isoprenoid chain and modification of quinoid nucleus of PQ-9 do not seem to alter the photoreduction activity significantly. Addition of PQ-9 to the lyophilized Euglena chloroplasts inhibited the photoreduction of ferricyanide significantly, while it stimulated photosynthetic phosphorylation activity.

Photosynthetic Reactions by Lysed Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP(+) or ferricyanide. P/2e(-) ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C(554) and C(549) and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C(554) or plastocyanin, and may be a coupling factor.Analysis of the well-washed membranes by low temperature (77 degrees K) difference spectrophotometry reveals the presence of cytochrome b(6) and a bound form of cytochrome C(554) in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C(554), relative to cytochrome b(6) is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C(554), which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b(6): chlorophyll a was found to be 1:500.Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C(549).

Inhibition of Photosynthetic Electron Transport by Ioxynil

THE herbicide ioxynil (4-hydroxy-3,5-diiodo benzonitrile)1 has been found to inhibit the photoreduction of ferricyanide by chloroplasts from a range of plants2 and also NADP photoreduction, the accompanying photophosphorylation and carbon dioxide fixation by the broad bean (Vicia faba) chloroplasts3. Recently, Paton and Smith4 further reported that ioxynil inhibited the photoreduction of endogenous plastoquinone in broad bean chloroplasts, even in the presence of ascorbate and indophenol. It was previously found5 that although o-phenanthroline and the herbicide DCMU inhibit the photoreduction of endogenous plastoquinone they do not inhibit the enhanced photoreduction in the presence of ascorbate and indophenol. Furthermore, ascorbate and indophenol reduce plastoquinone in darkness6, in a reaction which has been shown to be non-enzymatic7. No other inhibitors previously investigated have inhibited the dark reduction and we therefore decided to investigate the effects of ioxynil on plastoquinone reduction in more detail.

Plastoquinone B

A compound found in spinach and other higher plants previously referred to as R 263 has now been found to be a breakdown product of plastoquinone B. This quinone, PQ B, is found with 8 other quinones in spinach chloroplasts. These 9 quinones are PQ A, PQ B, PQ C, PQ D (7, 8, 15) Vitamin K(1) (10, 12), an unknown naphthoquinone (13) and alpha-, beta- and gamma-tocopherylquinones (7, 12). An improved method for purification of plastoquinone B is described. Previous confusion of this compound with other quinoid material on silica gel is described and corrected R(F) values are given. The activity of PQ B is similar to the activity of PQ C in restoration studies of the photo-reduction of ferricyanide and indophenol.

Photosynthetische Reaktionen in lyophilisierten Zellen der Blaualge Anacystis

Intact cells of the bluegreen algae Anacystis nidulans photoreduce p-benzoquinone under O2-evolution but not ferricyanide. Catalytic amounts of a quinone catalyze the photoreduction of ferricyanide; the more positive its redoxpotential the better is the stimulation. By the simple procedure of lyophilizing Anacystis in 5% sucrose and resuspending in 4·10—2-m. magnesiumchloride a preparation is obtained which exhibits the same photosynthetic activities as known from isolated chloroplast fragments, i. e. they evolve oxygen and photoreduce ferricyanide, quinones and NADP *; these Hill- reactions are coupled to ATP-formation; they catalyze pseudocyclic and cyclic photophosphorylation. Rates compare favorably with those of isolated chloroplasts except for the NADP-system. A number of known inhibitors like DCMU, KCN, dinitrophenol, salicylaldoxime and ammoniumchloride were checked and found to behave in principal as in photosynthetic chloroplast reactions.

FURTHER STUDIES ON PHOTOPHOSPHORYLATION ACCOMPANYING THE HILL REACTION WITH FERRICYANIDE.

Abstract The striking pH dependence of the Hill reaction with ferricyanide was confirmed for reactions carried out in a low anion buffer. The pH dependence of inhibition of the Hill reaction by ADP, of stimulation by associated phosphorylation and of the stoichiometry between ferricyanide photoreduction and phosphate consumption was also examined. It was found that at every pH, the “extra” ferricyanide reduced when phosphate was added to the ADP-inhibited system, was equal, on a molar basis, to the amount of phosphate consumed. The pH dependence of ferricyanide photoreduction stimulated by trichlorophenol indophenol dye and NH4Cl was examined. Taking into consideration the variation in the concentration of NH3 in the presence of NH4Cl at various pH values, it is concluded that the Hill reaction as stimulated by both dye and NH4Cl is relatively independent of pH. However, the degree of stimulation induced by either agent was strongly dependent upon pH, and was greatest at low pH, where the unstimulated reaction (and phosphorylation) is most sluggish. Concentrations of dye which have little effect upon the ferricyanide Hill reaction were found to relieve or prevent the marked inhibition of ferricyanide photoreduction induced by ADP, and to stimulate ferricyanide photoreduction in the presence of ADP and suboptimal concentrations of phosphate. In such concentrations of dye its uncoupling effects are thus seen to resemble those of arsenate. These results are discussed in terms of the possible participation of an activated intermediate which is unstable at alkaline pH. Recent studies on the formation of activated intermediates during photophosphorylation are discussed and the results of such studies are shown to be consistent with a novel scheme for the Hill reaction.

Photophosphorylation accompanying the hill reaction with ferricyanide

A study of the dependence upon pH of ferricyanide photoreduction revealed that the reaction was most rapid but least stable at high pH (above pH 8) and that inadequate buffering could readily lead to differences in rate due to pH changes. Since the occurrence of phosphorylation may be associated with a pH change, precautions are necessary to prevent artifacts due to pH when determining the effect of phosphorylation on Hill reaction rates. Depending upon circumstances one may overestimate or underestimate the effect. For this reason a reappraisal has been made of the relationship between photophosphorylation and the photoreduction of ferricyanide as catalyzed by spinach chloroplasts. A study of the stoichiometry of photophosphorylation in relation to the Hill reaction revealed that the quantity of ATP formed approached a maximum of 1 mole for 2 moles of ferricyanide reduced and for 0.5 mole of O 2 evolved, in agreement with previous reports from other laboratories. The effects of concomitant phosphorylation upon rates of ferricyanide photoreduction were smaller than those reported previously by other workers. They may best be described as an inhibition of ferricyanide photoreduction by added ADP and the relief of this inhibition by added phosphate. When phosphate was added to the ADP-inhibited system the extra ferricyanide reduced was equal to the amount of phosphate consumed. A scheme for the mechanism of the Hill reaction which can accommodate all of the observed stoichiometry is presented and discussed. It is pointed out that the only mechanism for the Hill reaction which can easily be brought into congruence with this scheme appears to be that proposed by Warburg.