Electron Transport Reactions in Grana Preparations from Spinach Chloroplasts

Fraction 2 (grana-stack) particles prepared with the French press showed absorbance changes, at room temperature and with sodium ascorbate and methyl-viologen, that were produced by the oxidation of cytochrome b-559. This oxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and sensitized by system II of photosynthesis. The oxidation is too slow to account for the rates of the Hill reaction that have been observed with nicotinamide-adenine dinucleotide phosphate (NADP⁺). It appears that this cytochrome is not functioning in the main pathway of electron transport. In the presence of 2,3,5,6-tetramethyl-p-phenylene-diamine (DAD) and ascorbate, light-induced oxidation of cytochrome f took place within 3 msec (or faster) in the grana-stack particles. Treatment with the detergent Triton X-100 disrupted this rapid cytochrome f oxidation as well as the oxidation of cytochrome b-559. Subsequent plastocyanin addition did not restore the rapid oxidation of cytochrome f (nor of cytochrome b-559) but only slow changes of cytochrome f. In view of the fact that these particles contain almost no plastocyanin, it is unlikely that plastocyanin functions in electron transport between cytochrome f and P-700 in the particles derived from the grana-stack regions of the chloroplast.     

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

In the photosynthetic light reactions of plants and cyanobacteria, plastocyanin (Pc) plays a crucial role as an electron carrier and shuttle protein between two membrane protein complexes: cytochrome b₆f (cyt b₆f) and photosystem I (PSI). The rapid turnover of Pc between cyt b₆f and PSI enables the efficient use of light energy. In the Pc-cyt b₆f and Pc-PSI electron transfer complexes, the electron transfer reactions are accomplished within <10⁻⁴ s. However, the mechanisms enabling the rapid association and dissociation of Pc are still unclear because of the lack of an appropriate method to study huge complexes with short lifetimes. Here, using the transferred cross-saturation method, we investigated the residues of spinach (Spinacia oleracea) Pc in close proximity to spinach PSI and cyt b₆f, in both the thylakoid vesicle–embedded and solubilized states. We demonstrated that the hydrophobic patch residues of Pc are in close proximity to PSI and cyt b₆f, whereas the acidic patch residues of Pc do not form stable salt bridges with either PSI or cyt b₆f, in the electron transfer complexes. The transient characteristics of the interactions on the acidic patch facilitate the rapid association and dissociation of Pc.     

Electron transport in chromatophores from Rhodopseudomonas sphaeroides GA fused with liposomes

Chromatophores from Rhodopseudomonas sphaeroides GA were fused with liposomes in order to dilute the components of the cyclic photosynthetic electron-transport chain within the membrane. This dilution led to a decrease in the rate of cytochrome b-561 reduction. The original rates could be restored at potentials around 100 mV (where a large part of the quinone pool is chemically reduced), if ubiquinone was incorporated into the liposomes prior to fusion. Similar dilution effects could be observed in synchronized cultures. The membrane obtained after division contained about twice the amount of phospholipids per reaction center when compared to chromatophores prepared from cells harvested just before division. Chromatophores from synchronized cultures are more uniform with respect to the concentration of the different electron-transport components in the membrane than the membranes from normally grown cells. The kinetic behaviour both of fused chromatophores and of membranes from synchronized cultures are in agreement with a modified Q-cycle model for photosynthetic electron transport in Rps. sphaeroides. The results presented in this paper cannot be explained by postulating the presence of a firmly bound quinone, Qz, in the ubiquinol: cytochrome c2 oxidoreductase, as previously proposed.     

Effect of Anions on Photosystem 1-Mediated Electron Transport in Spinach Chloroplasts

The effect of various anions on photosystem I (PSI)-mediatedelectron transport was studied in control and heat-treated chloroplasts.Results show that heat treatment exposes not only some of thereduced dichlorophenolindophenol binding sites, but also certainanion binding sites. Moreover, the site of action of anionsis at two places in the electron transport chain: one site isbetween the DCMU binding site and the HgCl₂, binding site (onplastocyanin) and the other is on the P700 itself. Key words: Anions, chloroplasts, electron transport, heat-treatment, photosystem I, spinach     

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts

Intact chloroplasts from spinach showed a transient increasein Chl fluorescence after saturating illumination with actiniclight and its yield depended on the duration of illuminationand the intensity of the actinic light (AL). The increase waspartially suppressed when antimycin A was added immediatelyafter termination of the AL. The inhibited fluorescence increase,therefore, reflected the electron flow from the reductant(s)that had accumulated during the actinic illumination to theplastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitiveCyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743].Addition of dihydroxyacetone phosphate (DHAP) to chloroplastscaused the enhancement of the increase in fluorescence afterAL, which was inhibited by antimycin A. Decay of the transientlyraised fluorescence was retarded by 2-heptyl-4-hydroxyquinolineN-oxide and stigmatellin, suggesting that re-oxidation of thereduced PQ pool is coupled with the operation of Q-cycle. Althoughthe activity of the stromal enzyme system that supplies NADPHon addition of DHAP was constant irrespective of light or darkness,the capacity of the intact chloroplasts to show a DHAP-dependentfluorescence increase had a limited lifetime after AL was turnedoff. This result suggests that the antimycin A-sensitive Cytb-559 or ferredoxin-NADP reductase is activated by light anddeactivated in the dark. In ruptured chloroplasts, the additionof NADPH increased the dark fluorescence yield only in the presenceof Fd, which also was inhibited by antimycin A. Thus the photoregulatorymechanism of Cyt b-559 (Fd) in intact chloroplasts appearedto be lost when chloroplasts were ruptured. (Received June 21, 1995; Accepted September 25, 1995)     

Methyl Viologen-Dependent Cyclic Electron Transport in Spinach Chloroplasts in the Absence of Oxygen

The methyl viologen (MV)-dependent, linear electron flow fromPS II to PS I was severely blocked in intact or broken, uncoupledchloroplasts when oxygen was removed from the suspension medium,as revealed by measurements of chlorophyll fluorescence andthe rate of photoreduction of MV. Kinetics of the reductionof pre-oxidized P700 by a saturating light pulse showed thatreduced MV in the absence of oxygen re-reduces P700⁺ via theintersystem electron transport chain. Since the re-reductionof P700⁺ was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone,the MV-mediated cyclic electron flow, in contrast to the phenazinemethosulphate-catalyzed one, involves the plastoquinone pool.However, 2-n-heptyl-4-hydro-xyquinoline-N-oxide, 2-n-nonyl-4-hydroxyquinoline-N-oxideand antimycin A did not inhibit the MV-mediated flow. Thus,the inhibition of the linear electron flow in chloroplasts underanaerobic conditions suggested the overreduction of the plastoquinonepool as a result of the MV-mediated cyclic flow (Received February 13, 1990; Accepted March 31, 1990)     

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.     

Clotrimazole对菠菜叶绿体光合磷酸化和电子传递的抑制作用

10μmol/的clotrimazole不仅抑制光合磷酸化活力,而且抑制各种类型的电子传递,是一个典型的电子传递抑制剂。经过它对叶绿体放氧,荧光和毫秒延迟发光影响的比较研究表明:clotri—mazole在光合电子传递链上的作用部位在Q与PQ之间,即与DGMU的作用部位相同或相近。     

Plasmid distribution and analyses in Rhodopseudomonas sphaeroides

Ten strains of Rhodopseudomonas sphaeroides were analyzed by agarose gel electrophoresis for plasmid DNA content and, by filter-hybridizations, for their molecular relationships. All strains examined contained at least one plasmid. Several strains carried as many as six different plasmid species with sizes ranging from 42 to 140 kilobases (kb). Those larger than 89 kb showed extensive homology with each other; the 42-kb plasmid of R. sphaeroides strain 2.4.1 was homologous to the smaller plasmid DNA of three other strains. A partial map of the 42-kb plasmid derived from R. sphaeroides 2.4.1 was prepared by analysis of restriction endonuclease digests. Cross-hybridization among the large plasmids indicated that those present in any one strain of R. sphaeroides showed homology to one or more of the large plasmids detected in strains L and 2.4.1.     

Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites

The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H₂O→methyl viologen, H₂O→silicomolybdic acid H₂O→ferricyanide, and H₂O→dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H₂O→silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition.     

Nano-Anatase Relieves the Inhibition of Electron Transport Caused by Linolenic Acid in Chloroplasts of Spinach

Linolenic acid is an inhibitor of electron transport in chloroplasts of higher plants. It has obvious effects on the structure and function of chloroplasts. In the present paper, we investigated the nano-anatase relieving the inhibition of photoreduction activity and oxygen evolution caused by linolenic acid in spinach chloroplasts. The results showed that linolenic acid in various concentrations could obviously reduce the whole chain electron transport and the photoreduction activity of two photosystems, especially on the oxidative reside and reduce reside of photosystem II (PS II). After adding nano-anatase to chloroplasts treated by linolenic acid, the whole chain electron transport rate, the photoreduction activity of two photosystems, and the oxygen evolution rate were increased significantly, indicating that nano-anatase could obviously decrease the inhibition of linolenic acid on the electron transport, photoreduction activity, and oxygen evolution of spinach chloroplasts.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

Electron Transfer between Plastocyanin and P700 in Various Types of Photosystem I Complex

Three types of PS I Chl-protein complex, PS I 180, PS I 65,and PS I 30, have been prepared and the kinetic properties ofthe transfer of electrons from plastocyanin to P700 in the PSI complexes with different sized antennae were examined. ThePS I 180 complex, which consists of 180 Chi per P700, showedthe almost same rate constant and effects of cations for thetransfer of electrons from plastocyanin to P700 as those obtainedwith PS I-enriched membrane fragments. The rate constant increasedwith the addition of low concentrations of monovalent and divalentcations, but decreased with high concentrations of cations.However, the rate was severely reduced in the case of the PSI 65 and PS I 30 complexes, and quite different effects of cationswere observed. Given the presence of additional 25- to 28-kDapolypeptides in the PS I 180 complex as compared to the PS I65 and PS I 30 complexes, we discuss a possible function forthese polypeptides in the regulation of the reaction betweenplastocyanin and P700. ¹This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science and Cultureof Japan. (Received May 27, 1988; Accepted November 7, 1988)     

Palisade Tissue Chloroplasts and Spongy Tissue Chloroplasts in Spinach: Biochemical and Ultrastructural Differences

Palisade tissue chloroplasts (P-Chlts) and spongy tissue chloroplasts(S-Chlts) were separately isolated from spinach leaves, andtheir photosynthetic properties were compared. The followingresults were obtained: (1) At saturating light, the activities of overall electrontransport and CO₂ fixation in P-Chlts were respectively 1.6–2.0and 2.5–3.0 times higher than those in S-Chlts on a Chlbasis. (2) The contents of PS I and PS II reaction centers (P700 and47 kDa polypeptide, respectively) were slightly higher in P-Chltsthan in S-Chlts, while the contents of plastoquinone, Cyt f,plastocyanin, ferredoxin, ferredoxin-NADP⁺ reductase, couplingfactor and ribulose-bisphosphate carboxylase were 1.6–2.2times higher in P-Chlts than in S-Chlts on a Chl basis. (3) Electron microscopic examination of chloroplast ultrastructureshowed that S-Chlts have highly stacked grana accompanied byhigher proportion of appressed thylakoids relative to non-appressedthylakoids, while P-Chlts have poorly stacked grana. The volumeratio of thylakoids to stroma was higher in S-Chlts than inP-Chlts. These results indicate that mesophyll chloroplasts adapt tothe light environment within a leaf in a similar way that thesun and shade plant chloroplasts adapt to the light environmentwithin a canopy. (Received July 19, 1984; Accepted October 13, 1984)     

Plasmid transfer and expression in Rhodopseudomonas sphaeroides.

Plasmid RP4 (among others) has been transferred from Escherichia coli to Rhodopseudomonas sphaeroides. Data bearing on the physical presence of the plasmid and its expression of drug resistance determinants in R. sphaeroides are presented. Conditions of transfer between R. sphaeroides strains, between R. sphaeroides and R. capsulata, and between R. sphaeroides and E. coli have been carefully defined.     

Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts

The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO₂ fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO₂ fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5-phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions.     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)