ATP-dependent uptake of nitrate in Nostoc muscorum and inhibition by ammonium ions

A high rate of nitrate uptake was observed in Nostoc muscorum when cells were grown on elemental nitrogen as compared to that when they were grown on nitrate or ammonium. The uptake of nitrate was light dependent. However, supplementation with ATP (50 μM) stimulated nitrate uptake both in light and darkness. ADP, under similar conditions had no effect. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-n-heptyl-4-hydroxyquinoline, (HOQNO) and KCN inhibitied nitrate uptake in light which could be partially reversed by adition of ATP. Inhibitiion by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler of photophosphorylation, was complete and could not be restored by the addition of ATP. N,N′-dicyclohexylcarbodiimide (DCCD), a specific inhibitor of ATPase, blocked nitrate uptake in the presence or absence of externally added ATP. Although no nitrate uptake was observed under anaerobic conditions in dark, addition of ATP resulted in uptake of nitrate, which was similar in magnitude to that observed under aerobic condition in the light, and was inhibited by DCCD. Ammonium ions inhibited the uptake of nitrate in the absence of ATP but in its presence there was simultaneous uptake of nitrate and ammonium ions. However, uptake of ammonium ions alone was not affected by presence or absence of ATP in the external medium. It was concluded that nitrate ion uptake was energy dependent and may be linked with a proton gradient which can be formed either by photophosphorylation or ATP hydrolysis.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans. The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C.The Arrhenius plot of the phosphorylation mediated by N-methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C.The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.     

Protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Anacystis nidulans R2

The protein composition of the photosynthetic membrane from the cyanobacterium, Anacystis nidulans R2, was analyzed by acrylamide gel electrophoresis following solubilization with lithium dodecyl sulfate. Autoradiograms of ³⁵S-labelled membranes revealed over 90 bands by this procedure. The effect of solubilization conditions on protein resolution was analyzed by modifying temperature and sulfhydryl concentrations. Labelling cells with ⁵⁹Fe yielded nine iron-containing bands on these gels. Three of these bands, at 33, 19, and 14 kDa, were also heme proteins as determined by tetramethylbenzidine staining, and represent cytochromes f, b₆ and c-552, respectively. The remaining iron proteins are highly sensitive to solubilization conditions, especially the presence of 2-mercaptoethanol, and we suggest that these bands may be Fe-S proteins. Lactoperoxidase-catalyzed iodination of the membranes indicated that at least 41 proteins have surface-exposed domains. Some of the known proteins with external surfaces include cytochrome c-552 and the chlorophyll-binding proteins of Photosystems I and II. Neither cytochrome f nor b₆ appear to be accessible to external labelling. When this structural information was combined with the isolation of functional submembrane complexes, we constructed a topological model of the membrane. Using this model we have discussed the protein architecture of the cyanobacterial membrane.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophyenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.     

Factors affecting energy transfer from phycobilisomes to thylakoids in Anacystis nidulans

Short illumination with white light of dark-maintained Anacystis nidulans prior to immersion in liquid nitrogen resulted in a marked change of fluorescence emission characteristics at 77 K. The fluorescence of Photosystem II-associated membrane bound pigments increases, while the emission due to phycobilins decreases. This effect seems to be due to a light-dependent alteration in the extent of contact between phycobilisomes and thylakoids, since the effect is reversible in the dark and is abolished by short glutaraldehyde fixation. The preillumination effect is not inhibited by DCMU. Emission spectra obtained with actively growing and CO₂-starved cells indicate that the light-dependent increase in energy transfer from phycobilins to chlorophyll depends upon the physiological state of the cells.     

Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae

The long-wave chlorophyll a forms in Phaeodactylum tricornutum (688 and 703 nm) change into a short-wave form, 670 nm, as a result of incubation with 55% glycerol, freeze-thawing, short ultraviolet irradiation and, probably, chloroplast preparation. This short-wave form is non-fluorescent. Fluorescence polarisation measurements indicate that the long-wave chlorophyll a molecules are oriented parallel to each other. Although “labile” long-wave chlorophyll a receives energy from Photosystem II pigments at room temperatures and follows the induction phenomena of fluorescence, it is indicated by afterglow experiments that it probably does not participate in Photosystem II.Long-wave chlorophyll forms in Fucus are stable and probably are related to Photosystem I.     

Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421

The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus.     

Photosystem I-initiated postillumination CO2 burst in a cyanobacterium,Anabaena variabilis

In Anabaena variabilis, a postillumination CO₂ burst originating from a pool of HCO₃^? is described here. This burst is insensitive to the electron-transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and N,N′-dicyclohexylcarbodiimide (inhibitors of photophosphorylation). The action spectrum for the burst shows that only Photosystem I is involved.     

Isolation of a fast decay in submillisecond Chlorella luminescence

Using a simple He-Ne (632.8-nm) laser phosphoroscope steady-state luminescence from Chlorella pyrenoidosa was studied from 50 μs to 1.1 ms between 1 ms long exciting flashes. The following results were obtained: (1) prior freezing or ultraviolet irradiation changed the time course of the luminescence to a rapid decay with a half-time of about 110 μs; (2) 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed the 110-μs luminescence; (3) spectrally, all observed luminescence was, within possible error, identical to fluorescence; (4) no effect on the luminescence intensity from pulsed magnetic fields up to 30 kgauss was observed; (5) the relative fluorescence yield, measured simultaneously with luminescence, was found to be constant.Our principal conclusions, supported mainly by experiments with DCMU, are: (1) the 110-μs decay is a distinct component of the total steady-state luminescence; (2) prior freezing or ultraviolet irradiation isolates this component of the luminescence by suppressing all other components; (3) the half-time and intensity of this component are temperature independent in the interval 0–22 °C.     

Kinetics of luminescence in the 10−6–10−4-s range in Chlorella

The decay of luminescence in the 6–600-μs range following a microsecond flash has been studied in Chlorella. The decay is highly polyphasic; three kinetic components are outlined, in confirmation of the results of K. L. Zankel (1971, Biochim. Biophys. Acta 245, 373–385).Extrapolation of the decay to zero dark time suggests that a unique metastable species C^?₊, resulting from photochemical charge separation in the System II reaction center, is the substrate of the recombination reaction which gives rise to luminescence.The fast (5–10 μs) and medium (50–70 μs) phases of the decay denote different stabilization steps, preceding relaxation of the centers by electron and proton transduction to the photosynthetic chain.NH₂OH specifically inhibits the fast phase and enhances the medium phase. This effect is explained by assuming that the fast phase results from electron transfer from the water splitting system Z to the oxidized primary donor Y.3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), in the presence of NH₂OH elicits another fast phase. It is believed that DCMU affords a parasitic stabilization of C^?₊ by forming a complex with Q^?.     

Regulatory interaction of photosynthetic nitrate utilization and carbon dioxide fixation in the cyanobacterium Anacystis nidulans

The rate of photosynthetic nitrate utilization in Anacystis nidulans is strongly influenced by the availability of carbon dioxide. This dependence can be relieved by inhibiting the metabolism of the ammonium derived from nitrate reduction. Nitrate uptake seems to be modulated through a sensitive regulatory system integrating the photosynthetic metabolism of carbon and nitrogen, with CO₂ fixation products antagonizing the inhibitory effect of ammonium derivatives.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Vanadium in photosynthesis of Chlorella fusca and higher plants

The influence of vanadium compounds (vanadate, vanadyl citrate) on photosynthesis in Chlorella fusca and in algal and spinach chloroplasts has been investigated. It was found that: 1. At moderately high concentrations (at least 0.1 mM) both vanadate and vanadyl citrate enhance photosynthetic O₂ production in intact C. fusca cells. At lower V concentration (about 2 μM) only vanadate stimulates photosynthesis. The increase is dependent on culture conditions and on light intensity. 2. Up to 1 mM V, neither vanadium compound influences PS II activity, either in intact cells or in algal or spinach chloroplasts. 3. The PS I reaction in algal and spinach chloroplasts is maximally enhanced (3-fold) in presence of vanadium (20 μM). The increase is independent of light intensity. 4. Cr(VI), Mo(VI), and W(VI) (1 mM) stimulate photosynthesis in intact C. fusca cells, but do not influence the photosystems of isolated chloroplasts. Vanadium is suggested to act as a redox catalyst in the electron transport from PS II to PS I.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. I. Formation of System II photosynthetic units during greening under optimal light intensity

The relationships between light-harvesting chlorophyll and reaction centers in Photosystem II were analyzed during the chloroplast development of dark-grown, non-dividing Euglena gracilis Z. Comparative measurements included light saturation of photosynthesis, oxygen evolution under flashing-light and fluorescence induction. The results obtained can be summarized as follows: (1) Photosystem II photocenters are formed in parallel with chlorophyll synthesis, but after a longer lag phase. (2) As a consequence, the chlorophyll: reaction center ratio (Emerson's type photosynthetic unit) decreases during greening. (3) This decrease is accompanied by considerable changes in the energy transfer and trapping properties of Photosystem II. Most of the initially synthesized chlorophylls are inactive in the transfer of excitations to active photochemical centers and are shared among newly formed Photosystem II photocenters; as a consequence, the number of chlorophylls functionally connected to each Photosystem II photocenter decreases and cooperativity between these centers appears. Results are discussed in terms of chlorophyll organization in developing photosynthetic membranes with reference to the lake or puddle models of photosynthetic unit organization.