Leo P. Vernon

Ion and water transport processes related to the light-dependent shrinkage of spinach chloroplasts☆

Abstract When spinach chloroplasts are illuminated in the presence of an appropriate electron acceptor, they undergo rapid, partially reversible increases in the light-scattering level which have been related, by indirect evidence, to chloroplast shrinkage. This paper presents experimental evidence that a volume decrease does occur (measured by the Coulter counter and packed-cell volume techniques), and it is associated with an efflux of Mg ++ and K + as well as an H + consumption. All reactions have kinetics similar to those of the light-scattering change. Under the conditions used, the light-induced water, Mg ++ , K + , and H + fluxes follow the same pattern of reversal in an ensuing dark period. Mg ++ and K + fluxes follow the same pattern as the water flux (as measured by light scattering) during quinacrine treatment. Kinetic analysis indicates that the relative order of speeds for the reaction is: H + > K + = Mg ++ > H 2 O. The simplest model which accomodates these observations involves protonation of fixed negative sites within the chloroplast as the primary event, followed consequentially by an efflux of the cations Mg ++ and K + (and in some cases Na + ) to maintain electric neutrality, with a final water efflux occurring to reestablish osmotic equilibrium. The cation fluxes occur under conditions which suggest that they are a manifestation of the operation of the chloroplast energy conservation mechanism (ATP formation). The idea is put forward that such rapid and large scale fluxes of Mg ++ and K + in and out of the chloroplast could possibly have an important bearing on the activities of various Mg ++ - and K + -dependent enzymes which occur in the cytoplasm and chloroplasts of spinach leaf cells.

STRUCTURE OF PHOTOSYSTEM I AND PHOTOSYSTEM II OF PLANT CHLOROPLASTS

Abstract— The action of Triton X‐100 upon photosynthetic membranes which are devoid of carotenoids produces a small Photosystem I particle (HP700 particle) which is active in N ADP photoreduction and has a [Chl]/[P700] ratio of 30. The properties of the HP700 particle indicate that it is a reaction center complex which is served by an accessory complex containing the additional light‐harvesting chlorophyll of Photosystem I as well as the cytochromes and plastoquinone. When Photosystem II particles obtained by the action of Triton X‐100 are further washed with a solution 0.5 M in sucrose and 0.05 M in Tris buffer (pH 8.0), chlorophyll‐containing material is released. After centrifugation, the supernatant contains about 1 per cent of the chlorophyll and contains three types of particles which can be separated by sucrose density gradient centrifugation. One of these particles, designated TSF‐2b, has the same pigment composition as the original Photosystem II fragment, contains cytochrome 559, and shows Photosystem II activity (DCMU‐sensitive diphenylcarbazide‐supported photoreduction of 2,6‐dichlorophenolindophenol). The other two particles (TSF‐2a and TSF‐2a′) have a [Chl a]/[Chl b] ratio of 8, have a low concentration of xanthophylls, and show a [Chl]/[Cyt 5591 ratio of about 20. Only the TSF‐2a particle is active in the Photosystem II reaction described above. On the basis of these data, it is proposed that the Photosystem II unit consists of a reaction center complex which contains Chl a, Cyt 559, and an acceptor for the photochemical reaction. The reaction center complex would be served by an accessory complex which contains the light‐harvesting pigments, Chl a. Chi b, and xanthophyils.

Oxidation of 1,5-diphenylcarbazide as a measure of photosystem 2 activity in subchloroplast fragments☆

Abstract The use of 1,5-diphenylcarbazide as an electron donor coupled with DPIP 2 reduction allows the detection of photosystem 2 in subchloroplast fragments which have lost the ability to evolve oxygen. Various subchloroplast fragment obtained by detergent treatment of spinach chloroplasts have been assayed in this manner. Photosystem 1 activity was measured as NADP reduction in the presence of ascorbate and DPIP. The best separation of the two photosystems was obtained with Triton X-100 at a concentration of 40 mg Chl/gram detergent.

Increased content of cytochromes 554 and 562 in Anabaena variabilis cells grown in the presence of diphenylamine

Abstract Of the four major cytochromes (cytochromes 550, 554, 557 and 562) present in Anabaena variabilis cells, two c type cytochromes (cytochromes 550 and 554) were removed from the membrane when cells were sonicated and cytochromes 557 and 562 (which are probably b type cytochromes) remained tightly bound to the membrane. Cytochrome 554 was obtained in the reduced state while the others were in the oxidized form. Cytochromes 554 and 562 were present in markedly higher amounts in Anabaena cells grown in the presence of diphenylamine, whereas cytochromes 550 and 557 were present at about the same concentrations in both types of cells. The contents of P700 and chlorophyll a in normal cells were twice as high as those in the diphenylamine-grown cells (expressing concentrations on the basis of cell weight of freezedried cells). The data on cytochrome and P700 composition of the membrane of the two cell types indicate that at least the cytochromes 554 and 562 are inserted into the membrane system independently of the chlorophyll and other cytochromes. Photooxidation of cytochrome 557 was observed with membrane fragments from diphenylamine-grown cells. The kinetics of the absorbance changes observed in the blue region were biphasic, and relatively slow changes were observed following the initial fast P700 photooxidation. The slow changes may be related to an unidentified component of the membrane fragments.

Photoreduction of NAD and NADP by chlorophyllin a in the presence of ascorbate: Requirement for NADP reductase

Abstract Chlorophyllin a catalyzes the photoreduction of NADP (and NAD to a lesser extent) in the presence of ascorbate as electron donor and NADP reductase as the coupling enzyme. Photosynthetic pyridine nucleotide reductase is inactive in the reaction and does not stimulate the reaction observed with NADP reductase. The presence of deoxycholate (0.6%) and methanol (30%) serves to stimulate markedly the NADP reaction, with rates as high as 615 μmoles reduced per hour per μmole chlorophyllin being observed in their presence. Removal of magnesium somewhat increases the activity of chlorophyllin a for this reaction. An aggregated form of chlorophyllin a, which is retained by a dialysis membrane, is the most active form for the photocatalyst. The reaction does not saturate at light intensities up to 20,000 foot candles, proceeds best under anaerobic conditions, and is inhibited by the addition of phenazine methosulfate, 2,6-dichlorophenolindophenol or reduced trimethyl benzoquinone. The reaction is a Krasnovsky-type reaction in which the activated chlorophyll couples with the enzyme NADP reductase to transfer electrons to NADP from ascorbate.

Nicotinamide-adenine dinucleotide photoreduction in Rhodospirillum rubrum chromatophores☆

Abstract 1. 1. The rates of succinate and ascorbate- N , N , N ′, N ′-tetramethyl- p -phenylenediamine (TMPD)-supported NAD + photoreduction by Rhodospirillum rubrum chromatophores were approximately one-half and one-quarter, respectively, of the photophosphorylation rates. 2. 2. All four reactions showed a very similar sensitivity to the uncoupler m -chlorocarbonyl cyanide phenylhydrazone. 3. 3. NAD + photoreduction was strongly inhibited by the presence of an active phosphorylation system, whereas photophosphorylation showed no net decrease in activity in the presence of an active NAD + photoreduction system. 4. 4. When both NAD + photoreduction and photophosphorylation were allowed to occur simultaneously, there appeared to be an increased utilization of high-energy intermediate. 5. 5. At concentrations of antimycin A which strongly inhibited succinate and ascorbate-TMPD-associated photophosphorylation, succinate-supported NAD + photoreduction was also strongly inhibited, whereas NAD + photoreduction from ascorbate-TMPD was not affected. 6. 6. These results are discussed in relation to pathways of electron transfer to NAD + and to the interaction of a high-energy intermediate with several energy-dependent reactions in R. rubrum chromatophores.

Light-induced absorption changes in Chromatium subchromatophore particles exhaustively extracted with non-polar solvents

Abstract Chromatium subchromatophore particles completely freed of ubiquinone were prepared by Triton treatment preceeded by an exhaustive extraction with non-polar solvents at different stages of their preparation. Reconstituted particles were prepared by recondensing appropriate amounts of the quinone extract onto the extracted particles. The following observations are made and conclusions drawn from the light-induced absorption changes. 1. 1. In the extracted particles, where cytochrome-422 is present in the oxidized state, light causes charge separation, which is followed by charge recombination in the dark with a half time of 20 ms; the light- minus -dark difference spectrum between 240 and 950 nm is essentially the same as that of the unextracted subchromatophore particles. 2. 2. In the reconstituted particles, presumably because of the presence of ubiquinone as the secondary electron acceptor, charge recombination is prevented. After addition of 1 mM o -phenanthroline, which blocks electron transfer from the primary reductant to the secondary acceptor, charge recombination is re-established, as indicated by the return of the decay kinetics to the 20-ms half time. 3. 3. The room-temperature light- minus -dark difference spectrum of the extracted particles indicates that the majority of the absorption decrease at 280 nm may be attributed to P890 photooxidation and not ubiquinone reduction. This is further confirmed by the fact that the light- minus -dark difference spectrum at 77 °K is essentially the same for extracted and unextracted particles. 4. 4. If the oxidized cytochrome-422 in the extracted or reconstituted particles is reduced chemically, the reduced cytochrome can couple to the photooxidized P890 + , leading to its own oxidation and the reduction of P890 + in the dark. 5. 5. With the reconstituted particles, a steady, slow absorption decrease associated with the reduction of ubiquinone can be observed in the ultraviolet region when an electron donor such as N , N , N ′ N ′,-tetramethylphenylenediamine is present. No such reaction occurs in the extracted particles. 6. 6. Studies with the extracted particles have provided evidence that endogenous ubiquinone does not play the role of a primary electron acceptor in bacterial photosynthesis. Recent trends in the studies of the primary electron acceptor and possible approaches to the problem are briefly commented upon.

Photochemistry of Chlorophyll in Vivo

Publisher Summary This chapter focuses on the photochemistry of chlorophyll in vivo technique. Chlorophyll functions in photosynthesis by virtue of its ability to produce and maintain a charge separation in the highly ordered lamellar structure of the chloroplast. This charge separation initially involves chlorophyll and some other molecule complexed to it but is subsequently evidenced in discrete biochemical entities other than chlorophyll, which become either reduced or oxidized after reaction with photoexcited chlorophyll. The major chlorophylls are Chl a and Chl b. Some algae do not contain Chl b, but all photosynthetic plant cells contain Chl a, which sets it apart as the essential photoactive pigment in plant photosynthesis. Complexing to different proteins and lipids would suffice to alter the absorption and photochemical properties of Chl a. However, it is reported that the different forms observed in the intact chloroplasts are because of chemically different chlorophyll molecules, which can be physically separated in the purified state. Light energy absorbed by any chlorophyll molecule in the unit is transferred with high efficiency to the reactive chlorophyll, which initiates the photochemistry. As extraction of chloroplasts with organic solvents yields only one Chl a, the reaction-center chlorophyll must differ only in its environment in the chloroplast, being more available to adjoining redox agents.

Increased rate of cyclic photophophorylation in preparations from Anabaena variabilis cells grown in the presence of diphenylamine

Cell free homogenates and membrane fractions prepared from Anabaena variabilis cells grown in the presence of diphenylamine have markedly higher activities for cyclic phosphorylation than similar preparations from normal cells. The preparations from diphenylamine‐grown cells are also more active in system I mediated electron transport from reduced dichloroindophenol to oxygen or methyl viologen. The light intensity required to saturate phenazine methosulphate‐supported cyclic phosphorylation, in such preparations, is higher than for preparations for normal cells.

Light-induced electron transfer reactions and adenosine triphosphate formation by Rhodospirillum rubrum chromatophores☆

Abstract The photooxidation of reduced phenazine methosulfate (PMS) by chromatophores from Rhodospirillum rubrum in a terminal reaction with added ubiquinone as an electron acceptor is primarily a nonphosphorylating reaction. In the presence of reduced PMS (ubiquinone absent), chromatophores can also cycle electrons through a nonphosphorylating as well as a phosphorylating system. The photooxidation of reduced PMS is a monophasic, first-order reaction while the subsequent dark reduction of oxidized PMS by endogenous components of the chromatophores is biphasic and has kinetics consistent with two first-order reactions of different rate constants. Comparison of the rates of PMS turnover and ATP formation show a decreasing efficiency for PMS-stimulated photophosphorylation with increasing light intensity, indicating that more electrons cycle via the nonphosphorylating pathway at higher intensities. Changing the percentage of reduced PMS present in reaction mixtures from less than 10 to about 80 had little effect on the rate of PMS-stimulated cyclic photophosphorylation. The photooxidation of horse heart ferrocytochrome c by chromatophores has been coupled to the reduction of various quinones, including ubiquinones, plastoquinone, α-tocopheryl quinone, vitamine K 3 , and trimethyl- p -benzoquinone. Under anaerobic conditions these reactions are completely reversible in the dark. Oxidized ubiquinone-2, but not ubiquinone-6, was found to be a potent inhibitor of photophosphorylation. In the reduced form both ubiquinones behaved similarly, and show some inhibition with fresh preparations and stimulation with aged chromatophore fragments. An active reduced ubiquinone-cytochrome c reductase activity was demonstrated. This reaction was sensitive to antimycin A, but no associated ATP forming capacity was observed. ATP formation was not associated with the rapid photooxication of ferrocytochrome c with either oxygen or ubiquinone acting as the electron acceptor. Previously observed increases in ATP formation under these conditions can be explained by the role of reduced cytochrome c in stimulating cyclic photophosphorylation.