Purification of an acidic plastocyanin from Microcystis aeruginosa

Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria. Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu. In the absence of Cu, an acidic cytochrome c-553 alone was isolated. With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated. A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity. This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae. Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Inhibition of photosynthetic electron transport by amphotericin B

By assaying partial reactions of the photosynthetic electron transport system using thylakoids from spinach as well as from the algae Bumilleriopsis, Dunaliella , and Anabaena , it was demonstrated that the polyene antibiotic amphotericin B has no specific effect on plastocyanin. Pretreating spinach and algal thylakoids with this antibiotic decreased photosystem-II as well as photosystem-I activity regardless of whether the membranes contained plastocyanin or cytochrome c-553. Different sensitivity of cell-free electron transport activity against this antibiotic was observed due to the species used. With Dunaliella , the photosystem-II region was inhibited more strongly than photosystem-I, while Bumilleriopsis chloroplasts – although not containing plastocyanin – exhibited a stronger inhibition of the photosystem-I region. Apparently, amphotericin B mainly solubilizes redox compounds that form connecting pools in the photosynthetic electron transport chain.     

Physiological factors determining formation of plastocyanin and plastidic cytochrome c-553 in Scenedesmus

Physiological conditions necessary for the formation of plastocyanin and the concurrent cessation of cytochrome c-553 formation were studied in cells of copper-deficient Scenedesmus acutus after the addition of copper. Plastocyanin is formed after a lag-phase, leaving constant the content of plastidic cytochrome c-553. Therefore, the concentration of plastocyanin per cell increases and the concentration of cytochrome c-553 decreases during growth. Formation of plastocyanin during the induction period studied is dependent on light intensity. In the dark, there is a 90% inhibition, whereas under light intensities above 50 Wm^-2, a ratio of 1.3 molecules plastocyanin per 1,000 molecules chlorophyll is attained.Plastocyanin formations is inhibited by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), but not by moderate concentrations of 3-[3,4-dichlorophenyl]-1,1-dimethylurea (DCMU), and by keeping the algae under a nitrogen atmosphere without CO₂. Concurrently, the cultures treated with FCCP show a decreased endogenous ATP level. The ATP is necessary for plastocyanin formation.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-[3,4-dichlorophenyl]-1,1-dimethylurea - pcv packed cell volume     

Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6

The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.     

Isolation of photosynthetic catalysts from cyanobacteria.

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.     

Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803.

In certain cyanobacteria and algae, cytochrome c553 or plastocyanin can serve to carry electrons from the cytochrome bf complex to photosystem I. The availability of copper in the growth medium regulates which protein is present. To investigate copper induced control of gene expression we isolated these proteins from the cyanobacterium Synechocystis 6803. Using immunodetection and optical spectroscopy, the steady state levels of cytochrome c553 and plastocyanin were measured in cells grown at different copper concentrations. The results show that in cells grown in 20-30 nM copper, cytochrome c553 was present, whereas plastocyanin was not detected. The opposite behavior was observed in cells grown in the presence of 1 microM copper; plastocyanin was present, whereas cytochrome c553 could not be detected. Both proteins were present in cells grown in 0.3 microM copper. Northern analysis of total RNA, probed with a gene fragment for cytochrome c553 or the plastocyanin gene, showed that cells grown in the presence of 20-30 nM copper have message for cytochrome c553, but not for plastocyanin, whereas cells grown in 1 microM copper have message for plastocyanin, but not for cytochrome c553. These results demonstrate that copper regulates expression of both of the genes encoding cytochrome c553 and plastocyanin prior to translation in Synechocystis 6803.     

Formation of silver plastocyanin in Scenedesmus

Silver ions up to 5 microM do not affect growth of the green microalga Scenedesmus acutus. They induce formation of protein species precipitable by an antibody specific against plastocyanin. The metal is incorporated into a part of the induced protein in competition with copper. Bismuth, lead and molybdenum had no effect. The amount of both silver- and copper-containing plastocyanins so formed apparently regulates concurrently inhibition of soluble plastidic cytochrome c-553. The silver-copper competition for the build-up of blue plastocyanin can be shown with intact cells, not with isolated algal plastocyanin.     

The role of plastidic cytochrome c in algal electron transport and photophosphorylation.

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the P/2e ratios remain unaltered. In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.     

Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum.

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.     

Photosynthetic and respiratory electron transport in Cu2+-deficient Dunaliella

Growth inhibition of the green alga Dunalietla parva Lerche has been observed during cultivation in low Cu²⁺ media. A minimum endogenous Cu concentration for unrestricted growth of 100 to 200 nmol ml⁻¹ packed cell volume was estimated. At lower concentrations, Cu deficiency causes a decrease in photosynthesis and respiration. Assay of photosynthetic electron transport rates as well as the determination of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin. Consequently, inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport. A secondary, indirect effect of Cu deficiency is the reduction of thylakoid formation resulting in an additional decrease of photosynthesis compared to cultures with sufficient Cu²⁺.
The inhibitory influence of low Cu²⁺ on respiration was located at the site of cytochrome oxidase. In contrast to blue-green algae, a strong coordination of the biosynthesis of the cytochrome oxidase complex was evident. During restricted Cu²⁺ supply the formation of cytochiome aa₃ , another component besides Cu, was stalled. The resulting low activities of cytochrome oxidase are responsible for decreased respiratory electron transfer activity from NADPH to oxygen. At Cu²⁺ concentrations which exert only moderate effects on Dunalietla , the cytochrome oxidase reaction was more strongly affected than the photosystem I reaction.     

Components and activity of the photosynthetic electron transport system of intact heterocysts isolated from the blue-green alga Nostoc muscorum

Heterocysts of the blue-green alga Nostoc muscorum have been isolated by prolonged treatment with lysozyme. Quantitative data are presented which show the occurrence of cytochromes c-553, f-557 and b-563 in heterocysts in amounts comparable to vegetative cells. Particularly the content of the water-soluble cytochrome c-553 can be used to evaluate the intactness of a heterocyst preparation. Cytochrome f-557 has been partially purified and found to be a c-type cytochrome corresponding to cytochrome f of higher plants and other algae. Cytochrome b-559 is present in vegetative cells but not in heterocysts. The content of plastoquinone in heterocysts is reduced to 42% of the amount present in vegetative cells. These data suggest a degradation of Photosystem II during heterocyst differentiation. Measurements of photosynthetic electron transport in heterocysts proved the inability of the photosynthetic apparatus to carry out electron transport with electrons donated by water or diphenylcarbazide. In Tris-washed thylakoids from vegetative cells, however, diphenylcarbazide can act as an electron donor to Photosystem II.     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

A Brownian dynamics study of the effects of cytochrome f structure and deletion of its small domain in interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii

The availability of seven different structures of cytochrome f (cyt f) from Chlamydomonas reinhardtii allowed us, using Brownian dynamics simulations, to model interactions between these molecules and their redox partners, plastocyanin (PC) and cytochrome c6 (cyt c6) in the same species to study the effect of cyt f structure on its function. Our results showed that different cyt f structures, which are very similar, produced different reaction rates in interactions with PC and cyt c6. We were able to attribute this to structural differences among these molecules, particularly to a small flexible loop between A-184 and G-191 (which has some of the highest crystallographic temperature factors in all of the cyt f structures) on the cyt f small domain. We also showed that deletion of the cyt f small domain affected cyt c6 more than PC, due to their different binding positions on cyt f. One function of the small domain in cyt f may be to guide PC or cyt c6 to a uniform dock with cyt f, especially due to electrostatic interactions with K-188 and K-189 on this domain. Our results could serve as a good guide for future experimental work on these proteins to understand better the electron transfer process between them. Also, these results demonstrated the sensitivity and the power of the Brownian dynamics simulations in the study of molecular interactions.     

Isolation and characterization of soluble cytochrome c-553 and membrane-bound cytochrome f-553 from thylakoids of the green alga Scenedesmus acutus

Soluble cytochrome c-553 and membrane-bound cytochrome f-553 from the alga Scenedesmus acutus were purified to apparent homogeneity. The properties of cytochrome c-553 are comparable to preparations obtained from other eukaryotic algae, whereas the thylakoid-bound species resembles higher plant cytochrome f. Common characteristics are: 1. An asymmetrical alpha-band at 553 nm. 2. A midpoint redox potential of +38 MV (pH 7.0), with a pH dependency above pH 8.0 of -60mV/pH unit. 3. Formation of a pyridine hemochromogen with a maximum at 550 nm; no adducts with CN- or CO are observed. Distinguishing features are: 1. Cytochrome f-553 has a more complicated beta-band, with maxima at 531.5 and 524 nm, and hence a more complex low-temperature spectrum. Also the positions of the gamma- and delta-bank at 421.5 and 331 nm, respectively, distinguish cytochrome f-553 from cytochrome c-553, with gamma- and delta-bands at 416 and 318 nm. 2. The ferricytochrome c-553 spectrum exhibits a weak band at 692 nm, which is not observed with cytochrome f.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium.

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

The roles of c-type cytochromes in algal photosynthesis. Extraction from algae of a cytochrome similar to higher plant cytochrome f.

A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas. Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with alpha-band maxima near 554 nm, very asymmetrical beta-bands, and gamma-band maxima at 421 nm. The cytochrome from Anacystis had alpha and gamma-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c-552. The roles of the membrane-bound and soluble c-type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane-bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its alpha-band (c-552, c-553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.