Regulation of Electron Transport Composition in Cyanobacterial Photosynthetic System: Stoichiometry among Photosystem I and II Complexes and Their Light-Harvesting Antennae and Cytochrome b6/fComplex

This study was done to confirm our previous observation withthe pattern of changes in electron transport composition inducedby an imbalance of the electron transport state. Contents ofphotosystem (PS) I and II complexes and their antennae and Cytb₆/f complex were determined for systems of cyanobacterium SynechocystisPCC 6714 of different PS I/PS II ratios. The results indicatedthat (1) the observed changes in the PS I/PS II ratio are not-dueto regulation of the activities of the respective PS's but tochanges in their contents, (2) the molar ratio between PS IIand Cyt b₆/f complexes was fairly constant when marked changesoccurred in the PS I content, and (3) the PS II and Cyt b₆/fcontents per cell remained fairly constant while the PS I contentchanged markedly. These findings agree with our previous observationwith autotrophic cells of Anacystis nidulans Tx 20 and supportour argument that in cyanobacterial and red algal electron transportsystems, the content of the terminalcomponent(s), such as PSI complex, is regulated in order to maintain a balance betweenthe electron influx by PS II action to the system and the effluxby PS I action from it. (Received June 3, 1987; Accepted September 20, 1987)     

Photosystem I and photophosphorylation-dependent leucine incorporation in the blue-green alga Anacystis nidulans

L-Leucine uptake and incorporation in the blue-green alga Anacystisnidulans were measured during illumination with monochromaticlight of 630 and 717 nm. With near as well as far red light,an enhanced uptake of ¹⁴C-L-leucine was observed. In far redlight, the leucine uptake depended on light intensity and pHvalue. After the first few minutes, the uptake remained constantfor more than one hour. The rate of uptake in light was thesame in air as in nitrogen. The incorporation of ¹⁴C-leucinein the soluble fraction decreased in the presence of chloramphenicolwhich prevents protein synthesis. In far red light, its incorporationwas insensitive to DCMU (5 ? 10^–6 M) but was depressedby uncouplers like CCCP or desaspidin. These effects are takenas evidence that leucine incorporation under the conditionsused is dependent on photosystem I reactions and cyclic photophosphorylation.DBMIB and KCN in high concentrations decrease the leucine incorporationin far red light and indicate that plastoquinone and plastocyaninare members of the cyclic electron flow also in intact cellsof Anacystis. Antimycin A has no inhibitory effect. The inhibitionby other less specific inhibitors like salicylaldoxime, desaspidinand DSPD is discussed. (Received August 19, 1978; )     

膜脂对菠菜细胞色素b6f复合体电子传递活性的影响

利用从菠菜(Spinacia oleracea L.)叶绿体分离、纯化出的缺失膜脂的细胞色素b6f蛋白复合体(Cyt b6f)制剂与从菠菜类囊体分离、纯化的膜脂进行体外重组,检测了不同膜脂对Cyt b6f催化电子传递活性的影响.结果表明:被检测的5种膜脂,即单半乳糖基甘油二酯(MGDG)、双半乳糖基甘油二酯(DGDG)、磷脂酰胆碱(PC)、磷脂酰甘油(PG)和硫代异鼠李糖基甘油二酯(SQDG)对Cyt b6f催化电子传递的活性均有明显的促进作用,但促进的程度各不相同,这可能与这些膜脂分子的带电性质密切相关.不带电荷的MGDG和DGDG及分子整体呈电中性的PC对促进Cyt b6f催化电子传递的活性非常有效,可分别使其活性提高89%、75%和77%;而带负电荷的PG和SQDG对活性的促进作用则相对较弱,仅可使其活性分别提高43%和26%.     

Preparation and Identification of Antidecapeptides of Subunit IV of Cytochrome b6-f Complex from Chloroplasts

Three synthetic decapeptides of subunit Ⅳ of cytochrome b6-f complex were coulpled respectively with ovalumine and purified using High-Performance Liquid Chromatography. They were served as antigens and injected into rabbits. Five weeks later, three kinds of monoclonal antibodies were obtained. Western blotting showed that there were immunoaffinity reactions between anti 17 kD-1, anti 17 kD-2, anti 17 kD-3 and 17kD band of cytochrome b6-f complex from spinach chloroplasts. Enzyme-linked immunosorbent assay showed that there were immunoaffinity reactions between anti 17 kD-1, anti 17 kD-2, anti 17 kD-3 and thylakoid membranes from spinach chloroplasts. Therefore, these antibodies can be used as probes to survey the location and orientation of 17 kD on the two sides of thylakoid membranes.     

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)     

Chromatic Regulation of Photosystem Composition in the Cyanobacterial Photosynthetic System: Kinetic Relationship between Change of Photosystem Composition and Cell Proliferation

Kinetics of the change of photosystem (PS) composition in cyanobacteriainduced by chromatic light were studied in relation to cellproliferation. The study was made for two unicellular strains,Synechococcus NIBB 1059 and Synechocystis (Aphanocapsa) PCC6714. We found that (1) the change to a higher or lower PS I/IIratio was due to acceleration or suppression of apparent PSI formation, and (2) it progressed on a similar time scale tothat of the cell proliferation. The apparent rate constant ofthe change in the PS I/II ratio was proportional to that ofcell proliferation, µ, when this was low, but at highvalues of µ the increase in the rate constant of the changein the PS I/II ratio became smaller, causing a deviation fromthe linear relationship. Results indicate that under autotrophicconditions, the photoregulated composition change occurs asa result of thylakoid development, which accompanies cell proliferation. (Received June 23, 1986; Accepted December 5, 1986)     

Chromatic Regulation of Photosystem Composition in the Photosynthetic System of Red and Blue-Green Algae

A chromatic adaptation in the photosynthetic quantum yield forthe light mainly absorbed by chlorophyll a (Chl a light) firstfound by Yocum (1951) was studied with one red and three blue-greenalgal strains. When the cells were grown under a weak Chl alight, the quantum yield in all the strains increased. Comparisonof photosystem (PS) compositions, including phycobilin (PBP)and Chl a antennae, reaction centers I and II, in the cellsgrown under the light mainly absorbed by PBP and Chl a revealedthat changes in quantum yield could be attributed to changesin the ratio of PS I/II; PS I/II becomes larger than 1 underPBP light but decreases to 1 in most cases under Chl a light.The change in the PS I/II ratio is due solely to the changesin the PS I population in the cell; PS II remains constant.These results are similar to the intensity-dependent responsein PS composition. A common hypothesis for both the chromatic and intensity-inducedregulation of PS composition was proposed based on the ideaof balance between the electron flow from H₂O to NADP drivenby PS I and II and the cyclic one driven by PS I. (Received May 16, 1985; Accepted September 4, 1985)     

Bidirectional Electron Transfer in the Reaction Centre of Photosystem I

In the past decade light-induced electron transfer reactions in photosystem I have been the subject of intensive investigations that have led to the elucidation of some unique characteristics, the most striking of which is the existence of two parallel, functional, redox active cofactors chains. This process is generally referred to as bidirectional electron transfer. Here we present a review of the principal evidences that have led to the uncovering of bidirectionality in the reaction centre of photosystem I. A special focus is dedicated to the results obtained combining time-resolved spectroscopic techniques, either difference absorption or electron paramagnetic resonance, with molecular genetics, which allows, through modification of the binding of redox active cofactors with the reaction centre subunits, an effect on their physical-chemical properties.     

膜脂对菠菜细胞色素b6f复合体电子传递活性的影响

利用从菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）叶绿体分离、纯化出的缺失膜脂的细胞色素ｂ６ｆ蛋白复合体（Ｃｙｔ ｂ６ｆ）制剂与从菠菜类囊体分离、纯化的膜脂进行体外重组,检测了不同膜脂对Ｃｙｔ ｂ６ｆ催化电子传递活性的影响。结果表明：被检测的５种膜脂,即单半乳糖基甘油二酯（ＭＧＤＧ）、双半乳糖基甘油二酯（ＤＧＤＧ）、磷脂酰胆碱（ＰＣ）、磷脂酰甘油（ＰＧ）和硫代异鼠李糖基甘油二酯（ＳＱＤＧ）对Ｃ     

Regulation of Photosystem Composition in Cyanobacterial Photosynthetic System: Evidence Indicating that Photosystem I Formation is Controlled in Response to the Electron Transport State

The quantitative composition of thylakoid components such asthe photosystem (PS) I/PS II ratio in the cyanobacterial photosyntheticsystem is regulated in response to the light regime. The regulationoccurs as changes in PS I content due to control of either PSI formation or decomposition. In order to determine which ofthese two is controlled in this regulation, experiments wereperformed to determine the light-induced PS I decrease in cellsof Synechocystis PCC 6714 under conditions where protein synthesiswas suppressed, i.e. the incubation without a nitrogen sourcefor cell growth or with chloramphenicol. The results revealedthat light-induced PS I decrease did not occur when synthesisof the thylakoid system was suppressed by incubation withouta nitrogen source or by the addition of chloramphenicol, indicatingthat (1) the thylakoid composition is regulted in the processof thylakoid formation and (2) the regulation is achieved bythe control of PS I formation. (Received November 6, 1987; Accepted March 2, 1988)     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.     

Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach

The effect of photosystem II core phosphorylation on the secondary quinone acceptor of photosystem II (Q_B) domain environment was analyzed by comparative herbicide-binding studies with photosystem II preparations from spinach (Spinacia oleracea L.). It was found that phosphorylation reduces the binding affinity for most photosynthetic herbicides. The binding of synthetic quinones and of the electron acceptor 2,6-dichlorophenolindophenol is also reduced by photosystem II phosphorylation. Four photosystem II core populations isolated from membranes showed different extents of phosphorylation as well as different degrees of affinity for photosynthetic herbicides. These findings support the idea that heterogeneity of photosystem II observed in vivo could be, in part, due to phosphorylation.     

Chlorophyll Composition in an Isolated Chlorophyll a-Protein Complex of Photosystem I

A P700-chlorophyll a-protein complex, solubilized by the detergent Triton X-100, has been isolated by hydroxyl apatite column chromatography. The chlorophyll composition was determined by thin-layer chromatography and spectrofluorimetric analysis. This photosystem I reaction centre complex, prepared at pH 7, contained pheophytin a and P700 in a ratio of 2/1, high enough to account for a composition similar to that in the reaction centre of photosynthetic bacteria. Prepared at pH 9, the same ratio was 0.2/1, which excludes pheophytin a from having the same function as that of bacterio-pheophytin in the photosynthetic bacteria.     

Cytochrome b6-f complex : The carburettor of exciton distribution in oxygenic photosynthesis

Efficient oxygenic photosynthesis not only requires synchronous turover and operation of photosystem I (PS I) and photosystem II (PS II) but also the preferential turnover of PS I for cyclic photophosphorylation to maintain required ATP and NADPH ratio during carbon dioxide reduction. Ohe initial higher rate of turnover of PS IIin viva is accounted by the fact that (i) PS I contains only about one-third of total chlorophylls, (ii) about 90% of light harvesting a/b protein (LAC) which accounts for about 50% of the total chlorophylls, remains associated with PS II as PS II-LHC II complexes (PS II_α and (iii) the ratio of PS II/PS I is always greater than unity, in the range of 1–2 : 1 under different environmental regimes. Ohe initial preferential feeding of PS II, due to its larger antenna, is bound to result in faster rate of turn over of PS II than PS I, leading to higher rate of reduction of an intersystem carrier than the rate of its oxidation by PS I. Ohe light dependent phosphorylation of a ‘mobile’ and small pool (−20%) of LHC II of PS II_α (possibly located at the edge of appressed regions of the membranes) increases the repulsive forces of LHC II resulting in its migration to non-appressed region associating itself with PS 1. Ohe phosphorylation itself is controlled by the redox state of an intermediate of electron transport. Several experimental approaches have provided evidence which suggest that (i) phosphorylation of LAC II involves interaction of cyt b5-f complex with LAC II kinase and the interaction of QA with cyt b₅-f complex and (ii) different kinases may be involved in phosphorylation of LHC IIversus PS II polypeptides. Ohe major purpose of light dependent LAC II phosphorylation and its consequent migration close to PS I appears to balance the rate of cyclicversus non-cyclic photophosphorylation. Ohe mechanism by which cyt b₅-f complex controls the activation of LAC II is not known. Ohe role of membrane bound ealmodulin, electron transfer through cyt b₆-f complex in activation of LAC II kinase should be explored.     

Transfer of the Excitation Energy in Anacystis nidulans Grown to Obtain Different Pigment Ratios

The blue-green alga, Anacystis nidulans, was grown in lights of different colors and intensities, and its absorption and fluorescence properties were studied. Strong orange light, absorbed mainly by phycocyanin, causes reduction in the ratio of phycocyanin to chlorophyll a; strong red light, absorbed mainly by chlorophyll, causes an increase in this ratio. This confirms the earlier findings of Brody and Emerson (12) on Porphyridum, and of Jones and Myers (8) on Anacystis. Anacystis cultures grown in light of low intensity show, upon excitation of phycocyanin, emission peaks at 600 mmu and 680 mmu, due to the fluorescence of phycocyanin and chlorophyll a, respectively. Changes in the efficiency of energy transfer from phycocyanin to chlorophyll a are revealed by changes in the ratios of these two bands. A decrease in efficiency of energy transfer from phycocyanin to chlorophyll a seems to occur whenever the ratio of chlorophyll a to phycocyanin deviates from the normal. Algae grown in light of high intensity show, upon excitation of phycocyanin, only a fluorescence band at 660 mmu and no band at 680 mmu. This suggests reduced efficiency of energy transfer from phycocyanin to the strongly fluorescent form of chlorophyll a (chlorophyll a(2)) and perhaps increased transfer to the weakly fluorescent form of chlorophyll a (chlorophyll a(1)).     

Nanodomains of Cytochrome b6f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b₆f (cytb₆f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb₆f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb₆f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb₆f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb₆f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb₆f complexes. We suggest that the close proximity between PSII and cytb₆f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

CO2 fixation and its regulation in Anacystis nidulans (Synechococcus)

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30 degrees C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occured at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow. CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15-20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purfied GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.     

Light-Induced Changes in Pigment Composition of Photosynthetic Lamellae and Cell-Free Extracts from the Blue-Green Alga Anacystis nidulans

Anacystis nidulans was grown at two different levels of white light, 7 and50 W^.m^?2. The cells were disrupted through French press treatment, and phycocyanin-free photosynthetic lamellae were obtained from the homogenate by fractionated centrifugation. Comparative absorption studies of the lamellae revealed that high intensity gave an increased carotenoid content relative to chlorophyll a. The spectral characteristics of the cell-free supernatants were also analysed. The high light intensity gave increased contents of both pteridines (410 nm) and allophycocyanin (655 nm) compared with the contents in algae grown at the low light level.     

Entrapment of active ion-permeable cyanobacteria (Anacystis nidulans) in calcium alginate

Summary Cells of the unicellular cyanobacterium Anacystis nidulans were made permeable to ions by treating them with lysozyme and EDTA in a way that leaves the photosynthetic water-splitting function, the photoreduction of exogenous oxidants and the peptidoglycan exoskeleton of the cell virtually intact. The permeabilized cells (permeaplasts) were subsequently immobilized by entrapment in calcium alginate beads. The immobilized preparation exhibits remarkable stability both on storage and in action. On prolonged storage at room temperature in darkness, its photosynthetic activity deteriorates one-third as fast as the activity of immobilized intact cells. Illumination accelerates deactivation. Tested in prolonged runs, however, performed in an illuminated open reactor, alginate-immobilized Anacystis permeaplasts were capable of photoreducing ionic oxidants (ferricyanide) and of exporting ionic reductants (ferrocyanide) to the suspension medium continuously for more than 5 h before being totally inactivated. It is also shown that the major impediment to the photoreduction performance of immobilized permeaplasts arises from diffusion limitations, while the photonic limitation due to light reflection and scattering is approx. 7%.Abbreviations Chl chlorophyll - CSTR continuously stirred tank reactor - EDTA ethylenediaminetetraacetate - FeCN potassium ferricyanide - pBQ p-benzoquinone - PD p-phenylenediamine - PD_ox p-phenylenediamine in the presence of excess potassium ferricyanide - Hepes N-2-hydroxyethylpiperazine-N-2 ethane-sulphonic acid