Photoproduction of hydrogen by photosystem I of Scenedesmus

Summary Anaerobically adapted and illuminated Scenedesmus evolves molecular hydrogen from endogenous organic compounds. This photoproduction of H₂ does not require photosystem II, since 5x10^-6 M DCMU, which inhibited normal photosynthesis almost completely, did not significantly inhibit the photoevolution of H₂. The relative efficiencies in far-red light of photosynthesis, photoreduction and H₂ production were determined. Photohydrogen evolution was comparatively the most efficient of these three processes. Three mutants of Scenedesmus (isolated and characterized by Dr. N. I. Bishop) were also tested. Mutant PS-50, which lacks cytochrome 552, did not photoproduce H₂. Mutant No. 11, blocked in photosystem II, showed rates of H₂ production comparable to those of the wild type. Cl-CCP, an uncoupler of photophosphorylation, caused an apparent stimulation of H₂ production by mutant No. 11 and wild-type cells. Mutant No. 8, which is partially blocked in photosystem I, showed a diminished photohydrogen production which was inhibited by Cl-CCP. These results suggest that photoproduction of hydrogen by photosystem I is due either to cyclic photophosphorylation, which supplies energy needed for a dark, H₂-yielding reaction, or to a more direct photooxidation of organic compounds by the photosynthetic electron transfer chain.The following abbreviations were used: Cl-CCP=carbonyl cyanide m-chlorophenylhydrazone; DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

The mechanism of hydrogen photoproduction by several algae

Summary The contribution of PS II to H₂ photoproduction by several unicellular green algae was measured both when O₂ evolution and photophosphorylation were unimpaired and also when these processes had been eliminated by Cl-CCP. As judged by the effects of DCMU, a PS II contribution was found under both sets of experimental conditions for several strains of Chlorella, Ankistrodesmus and Scenedesmus. However, H₂ photoproduction by Chlamydomonas moewusii was insensitive to DCMU and thus was entirely due to PS I. With cells treated with Cl-CCP, the relative amount of PS II contribution varied from zero in autotrophically grown Chlamydomonas reinhardii, to 20% in photoheterotrophically grown and 50% in autotrophically grown cells of Ankistrodesmus braunii, Chlorella fusca, Chlorella vulgaris and Scenedesmus obliquus. The dehydrogenation of reduced H-donors by PS II of Scenedesmus treated with Cl-CCP showed the same biphasic kinetics previously described for H₂ photoproduction by PS I of this alga.Abbreviations Cl-CCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ICC Indiana Culture Collection - PS photosystem - SAL salicylaldoxime - SIO Marine Botany Culture Collection, Scripps Institution of Oceanography These studies were supported by contract No. AT-(40-1)-2687 from the U.S. Atomic Energy Commission.     

Luminescence decay kinetics in relation to quenching and stimulation of dark fluorescence from high and low CO2 adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii

Two green algal species, Chlamydomonas reinhardtii and Scenedesmus obliquus, exhibited a relative maximum during the decay of luminescence, when adapted to low CO₂ conditions that was not observed in high CO₂ adapted cells.From the kinetics of transient changes in the level of dark fluorescence, after illumination and parallel to the luminescence maxima, it was concluded that the maximum in Scenedesmus was mainly related to a decrease in nonphotochemical quenching, whereas in Chlamydomonas the maximum was mainly related to a dark reduction of the primary PS II acceptor Q_A.ATP/ADP ratios from low CO₂ adapted Scenedesmus showed transient high levels after a dark/light transition that was not observed in high CO₂ adapted cells. After 30 s of illumination the ATP/ADP ratios however stabilized at the same steady state level as in high CO₂ adapted cells.Dark addition of HCO₃ ^- to low CO₂ adapted cells of Chlamydomonas resulted in a rapid transient quenching of luminescence that was not observed in low CO₂ adapted cells of neither species.It is concluded that the luminescence maxima present in both low CO₂ adapted Scenedesmus and Chlamydomonas reflect adaptation of the cells to low CO₂ conditions. It is further suggested that the difference in mechanistic origin of luminescence maxima in the two species reflects differences in adaptation.Abbreviations ADP adenosine-diphosphate - ATP adenosine-triphosphate - C_i inorganic carbon - F_D dark fluorescence recorded under dark adapted conditions - F₀ fluorescence with all reaction centers open - FV variable fluorescence - PS I photosystem I - PS II photosystem II - Q_A the first quinone acceptor of PS II     

The kinetics of hydrogen photoproduction by adapted Scenedesmus

Summary In our earlier work we have shown that hydrogen photoproduction by photosystem I of Scenedesmus does not require O₂ evolution or cyclic photophosphorylation but must be due to non-cyclic electron flow from organic substrate(s) through photosystem I to hydrogenase, where molecular H₂ is released. The kinetics of this reaction are rather complex, in that H₂ photoproduction by Scenedesmus evidently occurs in two phases: a rapid initial phase which depends upon the dehydrogenation of a pool of H donors, and a later and slower second phase which is limited by the flow of electrons from fermentation. When adapted cells were incubated in the dark with an inhibitor (Cl-CCP or salicylaldoxime), the pool utilized by photosystem I gradually disappeared. However, the pool gave a rapid rate of hydrogen photoproduction when the adapted cells were illuminated immediately after adding the inhibitor. The rate at which the pool was utilized depended upon the light intensity and was not light-saturated at the highest intensity tested (3.4×10³ W cm^-2).With light of at least medium intensity (1.67×10³ W cm^-2), the pool was rapidly exhausted and the reaction became dependent upon the leak of electrons from fermentation. The size of the leak was found to depend upon the level of reduced organic compounds in the cell, since this process was depressed by starving the cells and was much enhanced by adding glucose or by growing the cells heterotrophically. A quantitative relationship was found between the amount of glucose added and the resulting stimulation of H₂ photoproduction, in that one mole of glucose gave about 0.5 mole of H₂ gas.The following abbreviations were used: Cl-CCP=carbonyl cyanide m-chlorophenylhydrazone; DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP=dichlorophenol-indophenol; PS=photosystem.These studies were supported by contract No. AT-(40-1)-2687 from the U. S. Atomic Energy Commission.     

Hydrogen production by photosystem I of Scenedesmus: Effect of heat and salicylaldoxime on electron transport and photophosphorylation

Summary Photosynthesis, photoreduction, the p-benzoquinone Hill reaction, and glucose uptake by whole cells, as well as cyclic photophosphorylation (with PMS) by chloroplast particles were strongly inhibited by 10^-2 M salicylaldoxime or by heating whole cells for 1–2 min at 55°. In contrast, H₂ photoproduction by whole cells of mutant No. 11 and wild type Scenedesmus and PS I-mediated MR reduction by chloroplast particles were either stimulated or not significantly inhibited by these agents. H₂ production by mutant No. 8 was slightly depressed by salicylaldoxime. DCMU inhibited H₂ photoproduction with 10^-2 M salicylaldoxime approximately 20%, indicating some contribution of electrons by endogenous organic compounds to photosystem II between the O₂-evolving mechanism and the DCMU-sensitive site. We conclude that photohydrogen production by PS I of Scenedesmus does not require cyclic photophosphorylation but is due to non-cyclic electron flow from organic substrate(s) through PS I to hydrogenase where molecular H₂ is released.The following abbreviations were used CI-CCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenol-indophenol - MR methyl red - PMS phenazine methosulfate - PS photosystem This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

Regulation by Ammonium of Variation in Heterotrophic CO2 Fixation by Euglena gracilis During Growth Cycles*

SYNOPSIS Heterotrophic (dark) CO₂ fixation by Euglena gracilis strain Z varies with phase of batch culture growth and mode of nutrition. Increases in the fixation during growth cycles correlate closely with the depletion of exogenous NH₄* from the medium during growth. It is demonstrated that exogenous NH₄⁺ regulates a component of heterotrophic CO₂ fixation and that another component is independent of NH₄⁺. This is true for cells grown heterotrophically (glucose, dark), autotrophically (CO₂, light) and for a permanently bleached strain (E. gracilis SB3). Some kinetics of the NH₄⁺ regulation are presented.     

Untersuchungen über die Aufnahme von Glykolat bei Chlorogonium

Summary The alga Chlorogonium was cultured either heterotrophically or autotrophically under different partial pressures of CO₂ by aerating with pure air of air enriched with 2% CO₂. Cells were harvested in the logarithmic phase, transferred to phosphate buffer containing 0.01 M 1C¹⁴-glycolate and incubated with shaking in the dark. Under these conditions the rate of glycolate uptake was higher when the cells had been grown in the light. Cells grown in the light at the lower CO₂-concentration took up more glycolate than those grown with 2% CO₂. Approximately 90% of the radioactivity taken up with the glycolate was released as CO₂. The radioactivity remaining in the algae was somewhat higher in those cells which had been cultured heterotrophically or autotrophically under air than in cells grown autotrophically under air enriched with 2% CO₂.Addition of glycolate increased the uptake of oxygen by the cells. The consumption of the oxygen was quantitatively correlated to the uptake of glycolate.     

Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus

Scenedesmus cells grown on high CO₂, when adapted to air levels of CO₂ for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO₂ from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O₂ evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO₂ pump with K_0.5 for O₂ evolution of less than 5 micromolar CO₂ was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO₃⁻ concentrating process between pH 8 to 10 lowered the K_0.5 (DIC) from 7300 micromolar HCO₃⁻ in high CO₂ grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO₃⁻ pump was inhibited by vanadate (K_i of 150 micromolar), as if it involved an ATPase linked HCO₃⁻ transporter. The CO₂ pump was formed on low CO₂ by high-CO₂ grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO₃⁻ pump was partially activated on low CO₂ within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO₃⁻ pump at pH 9 had requirements similar to the activation of the CO₂ pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO₂-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO₂ pump and in some algae an additional vanadate-sensitive plasmalemma HCO₃⁻ transporter for a HCO₃⁻ pump.     

Association of the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardii with Photoreduction and the Oxyhydrogen Reaction

The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO₂ assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chlamydomonas reinhardii 11-32/6. Photoreduction but not photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H-plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO₂ fixation was inhibited more than 95% by 10 micromolar 2,5 - dibromo - 3 - methyl - 6 - isopropyl - p - benzoquinone (DBMIB), a concentration which did not affect respiratory activity. In cells, both photoreduction and the oxyhydrogen reaction exhibited a similar sensitivity to salicylhydroxamic acid (SHAM) showing approximately 90% inhibition by 7 millimolar concentration. Photosynthesis was inhibited only 30% by the same concentration of SHAM. Antimycin A (18 micromolar, 10 micrograms per milliliter) inhibited both photoreduction (80%) and the oxyhydrogen reaction (92%) in cells with the oxyhydrogen reaction being approximately 10 times more sensitive to lower concentrations of the inhibitor. Antimycin A at 18 micromolar concentration did not inhibit photosynthetic CO₂ fixation unless the cells were adapted to an atmosphere of N₂ and the reaction conducted anaerobically. Photosynthesis, photoreduction, and the oxyhydrogen reaction coupled to CO₂ fixation were all inhibited greater than 90% by 10 micromolar carbonylcyanide-p-trifluoromethoxyphenylhydrazone. ATP added to chloroplasts adapted to an atmosphere of H₂ could support CO₂ uptake in the dark. These results are interpreted as evidence that photoreduction and the oxyhydrogen reaction involve some common components of thylakoidal electron transport pathways in Chlamydomonas including NAD(P)H-plastoquinone oxidoreductase and the plastoquinone pool. An O₂-consuming thylakoidal or mitochondrial reaction is an additional component of the oxyhydrogen reaction.     

ATP-dependent carbon transport in perfused Chara cells

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO₂ and HCO₃^- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO₂ and HCO₃^- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H⁺_-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H⁺_-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied ¹⁴C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO₂ and HCO₃^- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ^-3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m^-3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m^-3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation.     

Photoproduction of hydrogen from water in hydrogenase-containing algae.

Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO₂ production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.     

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana

 Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO₂ fluxes in cell suspensions using mass spectrometry. Addition of H¹³CO₃ ⁻ to cell suspensions in the dark caused a transient increase in the CO₂ concentration in the medium far in excess of the equilibrium CO₂ concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO₂ efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO₂ by active transport. Photosynthetic O₂ evolution and the release CO₂ in the dark depend on HCO₃ ⁻ uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO₂ efflux in the dark, indicating that CO₂ efflux was dependent upon the intracellular dehydration of HCO₃ ⁻. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO₂ and HCO₃ ⁻ and thus causes a subsequent release of CO₂ to the external medium. Received: 20 September 1999 / Accepted: 25 October 1999     

Induced Protein Synthesis during the Adaptation to H2 Production in Chlamydomonas moewusii

Gas production by Chlamydomonas moewusii in the light has been followed by manometric techniques during the adaptation to anaerobiosis. The only detectable gases produced are CO₂ and H₂ CO₂ is produced at a rather constant rate whereas H₂ evolution increase with time. This increase of H₂ evolution during the adaptation period can be inhibited by cycloheximide and by chloral hydrate, two inhibitors of protein synthesis. If the inhibitors are added to already adapted cells there is no effect on H₂ evolution. Adapted cell suspensions are sensitive to oxygen. Incubation under O₂ for 10 min inhibits the H₂ evolution to 100%. After removal of oxygen the capability to evolve H₂ can be restored only by a new adaptation period. This second adaptation to H₂ evolution can also be inhibited by cycloheximide.     

Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii

Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H₂ demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H₂ while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H₂. Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO₂ was not detected.     

Regulation of ribulose diphosphate formation in vivo by light

Light-dependent formation of ribulose-1,5 diphosphate is completely inhibited by low concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which do not severely affect cyclic photophosphorylation. Also in Scenedesmus mutant number 11, capable of cyclic photophosphorylation, cellular ribulose-1,5 diphosphate-levels do not increase upon illumination. When mutant cells are H₂ adapted, however, a light-dependent formation of ribulose-1,5 diphosphate is observed in the presence of H₂. From these results it has been concluded that at least part of the Calvin cycle does not operate in the dark, since a reductant is lacking which is generated in the light.     

Respiration of blue-green algae in the light

The CO₂ evolution in the light of Anabaena as well as several other blue-green algae is below 10% of the dark control. Addition of DCMU restores CO₂ evolution in the light almost to the dark level. Furthermore, by adding unlabeled NaHCO₃, a ¹⁴CO₂ release is observed with prelabeled algal cells attaining 15 to 100% of dark control. Analysis by double-reciprocal plots exhibits a competitive relationship between added and endogenously released carbon dioxide. We conclude that CO₂ evolved by respiration is immediately refixed in the light without being liberated.The degree of ¹⁴CO₂ release induced by unlabeled bicarbonate in the light allows to determine true photoinhibition of respiration. Anabaena variabilis Kütz. exhibits almost no inhibition while in eight other species respiration is light-inhibited between 50 and 85% of the dark control.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TCA trichloroacetic acid     

The Gas Exchange of Hydrogen-adapted Algae as Followed by Mass Spectrometry

A mass spectrometer inlet and an oxygen electrode in the same vessel allowed the continuous recording of the gases exchanged (H₂, CO₂, O₂) by hydrogenase-containing anaerobically adapted Scenedesmus obliquus strain D₃ (Gaffron) and Chlorella fusca Shihira et Krauss (= pyrenoidosa) 211-15. A light intensity which produces more photosynthetic oxygen than the cells can re-reduce to water leads to de-adaptation and the substitution of normal photosynthesis for photoreduction. The sequence of these metabolic events was recorded in a matter of a few minutes. Upon exposure of these adapted algae to light, an evolution of hydrogen lasting up to 60 seconds preceded any other light-dependent gas exchange. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, this initial hydrogen production was inhibited approximately 50%, pointing to a contribution of electrons by photosystem II. At very low hydrogen tensions (0.1 microliter per milliliter), a balance between light-induced production and absorption of hydrogen was observed in normal, unpoisoned algae. Addition of either glucose or inhibitors of phosphorylation increased the release of hydrogen in the light very considerably. When the light was turned off the algae consumed the remaining amount of hydrogen, only to release it again upon illumination. This reversible hydrogen exchange persisted even when any concomitant carbon dioxide exchange had been abolished.     

Molecular weight and quaternary structure of ribulose bisphosphate carboxylase from the cyanobacterium,Synechococcus sp.

Inhibition of photosynthetic growth of Rhodopseudomonas capsulata by metronidazole was dependent on the nitrogen supply in culture solutions. Cultures fixing dinitrogen were more susceptible to inhibition by low concentrations than those supplied with NH ₄ ⁺ . Light-dependent C₂H₂ reduction and H₂ production by washed cells were inhibited by 80% and 60% respectively by 1 mM metronidazole. When this compound was first reduced with H₂-palladised asbestos prior to assay, it only partially restricted C₂H₂ reduction in washed cells (33%) compared with unreduced inhibitor (68%). Metronidazole was without effect on other metabolic functions. Thus, even at 40 mM it did not inhibit either (a) dark or light respiration in cells grown under photo- and chemo-heterotrophic conditions; (b) H₂-dependent photoreduction of ¹⁴CO₂; (c) -glutamyltransferase activity of glutamine synthetase in cell-free extracts (25 mM inhibitor).Metronidazole (1 mM) completely inhibited C₂H₂ reduction by washed cells of Azotobacter vinelandii. The dithionite-dependent C₂H₂ reduction of a partially purified nitrogenase was only partially inhibited (30%) by 1 mM metronidazole.     

Reduced sulfur and nitrogen compounds and molecular hydrogen as electron donors for anaerobic CO2 photoreduction in Anacystis nidulans

Photosynthesis by Anacystis nidulans was studied in presence of reduced sulfur or nitrogen compounds, or of hydrogen. O₂ evolution and CO₂ fixation were depressed by sulfide, sulfite, cysteine, thioglycollate, hydroxylamine and hydrazine. Sulfite, cysteine and hydrazine inhibited O₂ evolution much more strongly than CO₂ fixation, indicating ability to supply electrons for CO₂ photoreduction; DCMU suppressed these photoreductions. In contrast, some anoxygenic photosynthetic CO₂ fixation insensitive to DCMU was found with sulfide, thiosulfate and hydrogen. Emerson enhancement studies confirmed that sulfite, cysteine and hydrazine acted on photosystem II, while photoreduction supported by sulfide, thiosulfate and hydrogen needed photosystem I only.Sulfite was photooxidized to sulfate, sulfide to elemental sulfur, and thiosulfate to sulfate plus elemental sulfur; the sulfur accumulated inside the cells. Results on the stoichiometries of the photoreductions were consistent with the photooxidation products determined. Inhibitor studies suggested photosynthetic CO₂ fixation through the Calvin cycle.While photoreduction by all reductants used was found to be constitutive in Anacystis, the process was stimulated by anaerobic preincubation with the reductants only in the cases of hydrogen and thiosulfate; this adaptation was prevented by chloramphenicol and by O₂. Anaerobic photoautotrophic growth of Anacystis was, however, not observed; the increase in dry weight with H₂ and thiosulfate was not accompanied by cell multiplication or by an increase in chlorophyll content. Parallel short-term experiments with Chlorella did not reveal any constitutive photoreduction in this eukaryotic alga.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone - DCMU dichlorophenyl dimethyl urea - DSPD disalicylidenepropane diamine-(1,3) - EDAC 1-ethyl-3(3-dimethylaminopropyl-) carbodiimide     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume