Effect of dibromothymoquinone (DBMIB) on reduction rates of Photosystem I donors in intact chloroplasts

Dual effect of dibromothymoquinone ( DBMIB ), inhibitor and reducing agent at the donor side of Photosystem I, was investigated in isolated intact chloroplasts by flash-induced absorbance changes at 820 and 515 nm. We show that in the absence of other electron donors, rereduction of P700+ by DBMIB proceeds at a very low rate (half-time of approximately 10 s) Dual effect of DBMIB explains that the initial rise of electrochromic absorbance change induced by repetitive flashes is usually not diminished while the slow rise is fully inhibited by this compound.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Inhbition of photosystem II-dependent phosphorylation in chloroplasts by mercurials.

Photophosphorylation supported by the coupling site associated with Phostosystem II electron transport (coupling site II) is 50 to 60 times less sensitive to the energy transfer inhibitor HgCl₂ than phosphorylation supported by the coupling site associated with Photosystem I electron transport (coupling site I). Coupling site II phosphorylation is only about 2 times less sensitive to the lipophilic mercurial p-hydroxymercuribenzoate (PHMB), however. Both coupling sites are equally sensitive to CF₁ antiserum. These results suggest that a portion of the energy conserving apparatus associated with coupling site II is in a more hydrophobic environment than the corresponding apparatus associated with coupling site I.     

Differential changes in the electron transport properties of thylakoid membranes during aging of attached and detached leaves, and of isolated chloroplasts

Abstract. Aging of chloroplasts both in vivo and in vitro causes a considerable loss in the 2,6-dichlorophenol indophenol (DCPIP)-Hill reaction with water as electron donor. The loss can be reduced by exogenous electron donors like diphenyl carbazide (DPC). suggestive of aging-induced damage of the oxygen evolving system. Aging also brings about a considerable loss in methylviologen (MV) reduction mediated by Photosystem I (PS I) of chloroplasts with an ascorbate-DCPIP couple as the electron donating system.
The loss in the electron transport ability of the plastids is faster during in vitro compared to in vivo aging of the chloroplasts.
Light protects the photo-electron transport ability of chloroplasts during aging of intact leaves in contrast to its action during aging of the isolated organelles.     

Inhibition of the photosynthetic electron transport of isolated thylakoids by hemolyzed rabbit sera. Evidence for the potential involvement of parallel electron transport in photosystem I Mehler reactions

The inhibition patterns of rabbit sera (RS1 & RS2) from two different rabbits on the photosynthetic electron transport of isolated spinach thylakoids were studied. Fifty l of RSI were required for 100% inhibition of a H₂O MV/O₂ reaction, while only 10 l of a 1:10 dilution of RS2 were needed for 100% inhibition. The RS2 serum was greatly hemolyzed. The -globulin fraction from purified rabbit serum (RS1) did not inhibit photosynthetic electron transport, indicating that the antibody fraction of the rabbit serum does not contain the inhibitor. It appears that the inhibitor is from the hemolyzed red blood cells. Rabbit sera added prior to chloroplast illumination caused no inhibition, while addition of rabbit sera during illumination inhibited a H₂O MV/O₂ reaction within 1–3s. Aminotriazole, a catalase inhibitor, did not affect the efficacy of the rabbit sera indicating that the unknown rabbit serum inhibitor is not catalase. Various Hill reactions were employed to determine the site of inhibition. Rabbit sera inhibited the following reactions: DHQ/DCMU MV/O₂, DAD/Asc/DBMIB MV/O₂, and DCIP/Asc/DBMIB MV/O₂. Rabbit sera did not inhibit a H₂O DAD_ox reaction indicating that inhibition is on the reducing side of PSI. However, a H₂O Fd/NADP⁺ reaction was not inhibited by rabbit sera. NADP did not interfere with the ability of RS2 to inhibit a MV-mediated Mehler reaction. In simultaneously measured assays of Fd-mediated O₂ and NADP⁺ reductions, RS2 serum inhibited the reduction of O₂ by ferredoxin without inhibiting the reduction of NADP⁺. These results indicate the potential involvement of parallel (branched) electron transport of the reducing side of PSI in the reduction of oxygen.Abbreviations RS1 and RS2 Rabbit serum 1 and 2 - MV methylviologen - DCMU 3,4-dichlorophenyl-N,N-dimethylurea - KFeCN potassium ferricyanide - DCIP dichlorophenolindolphenol - DAD 2,3,5,6-tetramethyl-p-phenylenediamine - DHQ tetramethyl-p-hydroquinone (durohydroquinone) - MES [2-(N-morpholino)-esthanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid] - DBMIB dibromothymoquinone - PSI and PSII photosystem I and II - Fd ferredoxin - Chl chlorophyll - Asc ascorbate - SOD superoxide dismutase     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions

Assimilation of atmospheric CO₂ by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.     

Impairment of the photosynthetic apparatus by oxidative stress induced by photosensitization reaction of protoporphyrin IX

Treatment with the herbicide acifluorfen-sodium (AF-Na), an inhibitor of protoporphyrinogen oxidase, caused an accumulation of protoporphyrin IX (Proto IX) , light-induced necrotic spots on the cucumber cotyledon within 12-24 h, and photobleaching after 48-72 h of light exposure. Proto IX-sensitized and singlet oxygen (¹O₂)-mediated oxidative stress caused by AF-Na treatment impaired photosystem I (PSI), photosystem II (PSII) and whole chain electron transport reactions. As compared to controls, the F_v/F_m (variable to maximal chlorophyll a fluorescence) ratio of treated samples was reduced. The PSII electron donor NH₂OH failed to restore the F_v/F_m ratio suggesting that the reduction of F_v/F_m reflects the loss of reaction center functions. This explanation is further supported by the practically near-similar loss of PSI and PSII activities. As revealed from the light saturation curve (rate of oxygen evolution as a function of light intensity), the reduction of PSII activity was both due to the reduction in the quantum yield at limiting light intensities and impairment of light-saturated electron transport. In treated cotyledons both the Q (due to recombination of Q_A⁻ with S₂) and B (due to recombination of Q_B⁻ with S₂/S₃) band of thermoluminescence decreased by 50% suggesting a loss of active PSII reaction centers. In both the control and treated samples, the thermoluminescence yield of B band exhibited a periodicity of 4 suggesting normal functioning of the S states in centers that were still active. The low temperature (77 K) fluorescence emission spectra revealed that the F₆₉₅ band (that originates in CP-47) increased probably due to reduced energy transfer from the CP47 to the reaction center. These demonstrated an overall damage to the PSI and PSII reaction centers by ¹O₂ produced in response to photosensitization reaction of protoporphyrin IX in AF-Na-treated cucumber seedlings.     

Putrescine stimulates chemiosmotic ATP synthesis

Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a ΔpH and (iv) a pool of putrescine.     

Unconventional and effective methods for the regeneration of NAD(P) H in microorganisms or crude extracts of cells

Several yeasts, as well as aerobic and anaerobic bacteria catalyze the reduction of NAD and NADP in the presence of reduced methylviologen. The rates are usually much higher than those of reductions of unsaturated substrates by the organisms in cofermentations with carbohydrates. Since methylviologen can be continuously reduced at the cathode of an electrochemical cell it acts in catalytic amounts as a regenerable electron donor. Such systems may be superior to that with glucose as electron donor, because the NAD(P)H can be used exclusively for the reduction of the unsaturated substrate. The rate of the NAD(P)H formation depends very much on the organism and for the same organism on the growth procedure, the growth medium, the pretreatment of the cells, the pH, the buffer as well as on the ionic strength. Cells of Candida utilis which were frozen and thawed several times were superior to cells freshly harvested. Crude extracts revealed the best activities.Clostridia show the highest activities (up to 15 U per mg protein in the crude extract) and are suitable catalysts for the preparation of [4S-²H]NADH and [4S-²H]NADPH using ²H₂O-buffer in an electrochemical cell.The combinations of Alcaligenes eutrophus or Clostridium kluyveri and Candida utilis extracts in the presence of methylviologen are effective systems to reduce hydroxyacetone with hydrogen gas as electron donor or in an electrochemical cell. In this combination of microorganisms NADH is formed mainly by A. eutrophus or C. kluyveri and consumed for the reduction of hydroxyacetone by a reductase present in Candida utilis. The productivity numbers of such combinations are 10–30 times higher than those of yeasts alone.NAD(P)H regeneration, methylviologen-dependent NAD(P)H formation, deuterated NAD(P)H, Clostridia, yeast, bioreduction     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.