Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus

The oxyhydrogen reaction in the presence and absence of CO₂ was studied in H₂-adapted Scenedesmus obliquus by monitoring the initial rates of H₂, O₂, and ¹⁴CO₂ uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere O₂, the pH₂ was varied from 0 to 1 atmosphere. Whereas the rate of O₂ uptake increased by only 30%, the rate of H₂ uptake increased severalfold over the range of pH₂ values. At 0.1 atmosphere H₂ and 0.02 atmosphere O₂, rates for H₂ and O₂ uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH₂ was changed from 0 to 1 atmosphere, the quotient H₂:O₂ changed from 0 to roughly 2. This change may reflect the competition between H₂ and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H₂ uptake. KCN inhibited equally respiration (O₂ uptake in the absence of H₂) and the oxyhydrogen reaction in the presence and absence of CO₂. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO₂ has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast.     

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.     

Hydrogen uptake by the nitrogen-starved cyanobacterium Anabaena cylindrica

The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H₂ and O₂ in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO₂ in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H₂ uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia.     

Properties of the hydrogenase system in Rhizobium japonicum bacteroids

The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of $\text{Rhizobium japonicum}$ is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K_m for H₂ of 2.8 μM and an apparent K_m for O₂ of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO₂ production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H₂ to O₂ uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H₂.     

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.     

Light and dark reactions of the uptake hydrogenase in anabaena 7120

Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole filaments or isolated heterocysts that contained nitrogenase consumed H₂ in the presence of C₂H₂ or N₂ in a light-dependent reaction. If nitrogenase was inactivated by O₂ shock, filaments catalyzed H₂ uptake to an unidentified endogenous acceptor in the light. Addition of NO₃⁻ or NO₂⁻ enhanced these rates. Isolated heterocysts consumed H₂ in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H₂ uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanide-sensitive oxyhydrogen reaction that was inactivated by O₂ at O₂ concentrations above 2%. Light-dependent H₂ uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of O₂, light-dependent H₂ uptake occurred simultaneously with the oxyhydrogen reaction. The pH optima for both types of H₂ uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena.     

Aerobic hydrogenase activity in Anacystis Nidulans The oxyhydrogen reaction

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction.2. Besides O₂, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O₂ levels <10–15%; aerobically the artificial acceptors were reduced simultaneously with O₂.3. H₂-supported O₂ uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction.4. The kinetic properties of O₂ uptake by isolated membranes were the same in presence of H₂ and of other respiratory substrates.5. Low rates of H₂ evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction.6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H₂ to O₂ through the respiratory chain.     

Maintenance of High Photosynthetic Rates in Mesophyll Cells Isolated from Papaver somniferum

The establishment and maintenance of high rates of photosynthetic CO₂ incorporation in mesophyll cells of Papaver somniferum (opium poppy) depend on a regime of dark and light periods immediately following isolation, as well as carefully adjusted conditions of isolation. Analysis of the incorporation pattern of ¹⁴CO₂ by the isolated cells indicates an initial “stress-response” period of approximately 20 hours characterized by increased respiratory-type metabolism and diminished photosynthesis. Under the favorable regime, this period is followed by rapid recovery and the reinstatement of a metabolic state strikingly similar to that of intact leaves in which the initial rate of CO₂ incorporation is between 110 and 175 μmoles CO₂ fixed per mg chlorophyll per hour. The photosynthetic viability of these cells can be maintained for up to 80 hours.     

Hydrogen metabolism in isolated heterocysts of Anabaena 7120

Isolated heterocysts of Anabaena 7120 evolve H₂ in an ATP-dependent nitrogenase-catalyzed process that is inhibited by N₂ and C₂H₂. Heterocysts have an active uptake hydrogenase that only requires an electron acceptor of positive redox potential, e.g., methylene blue, dichlorophenolindophenol or potassium ferricyanide. O₂ supplied at low partial pressures is a very effective physiological oxidant for H₂ uptake. High concentrations of O₂ are inhibitory to H₂ uptake. The oxyhydrogen reaction in heterocysts appears to be mediated by a cytochrome-cytochrome oxidase system, and it supports ATP synthesis via oxidative phosphorylation. Attempts to demonstrate acetylene reduction in isolated heterocysts employing H₂ as an electron donor were unsuccessful. It is suggested that the uptake hydrogenase functions to conserve reductant that otherwise would be dissipated via nitrogenase-catalyzed H₂ evolution.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

H2-Uptake and evolution in the unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758

The unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758 growing photoautotrophically synthesised a hydrogenase which catalysed an in vivo H₂ uptake in the oxyhydrogen reaction at a significant rate and showed only low level of in vitro MV-dependent H₂ evolution. The in vitro hydrogenase activity was not induced under microaerobic or nitrate-limiting conditions. Some correlation observed between the two activities indicated that the same enzyme may be involved in both H₂ uptake and H₂ evolution. Heterologous Southern hybridisations, using cyanobacterial hup and hox DNA fragments as probes, showed the presence of sequences similar to hox (encoding for a bidirectional hydrogenase) in C. thermalis CALU 758 with no indication for the presence of any sequences corresponding to an uptake hydrogenase. Further molecular experiments, using specific primers directed against different conserved regions of the large subunit (hoxH) of the bidirectional hydrogenase confirmed the presence of corresponding sequences in C. thermalis CALU 758. Low-stringency Southern hybridisations detected only one copy of hoxH within the genome of C. thermalis CALU 758.     

Coupling of Methanothermobacter thermautotrophicus Methane Formation and Growth in Fed-Batch and Continuous Cultures under Different H2 Gassing Regimens

In nature, H₂- and CO₂-utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth, Methanothermobacter thermautotrophicus was cultured in a fed-batch fermentor and in a chemostat under different 80% H₂-20% CO₂ gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (p_H2). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (Y_{CH4 max}), notably approximately 3 and 7 g (dry weight) of methane formed mol⁻¹, for growth under low (p_H2 < 12 kPa)- and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the “specific maintenance” requirements or, more likely, the degree of proton leakage and proton slippage processes. At low p_H2 values, the “specific maintenance” diminished and the specific growth yields approached Y_{CH4 max}, indicating that growth and methanogenesis became fully coupled.     

Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

The H₂-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, ²H¹H exchange from a mixture of ²H₂ and ¹H₂ in presence of ²H₂O and ¹H₂O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H₂ oxidation reaction, there was no significant discrimination between ²H₂ and ¹H₂, indicating that the initial H₂-activation step in the over-all H₂ oxidation reaction is not rate-limiting. By use of improved methods, an apparent K_m for H₂ of 0.05 micromolar was determined. The H₂ oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H₂ oxidation and stimulated O₂ uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H₂ to O₂. Partial pressures of H₂ at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO₂ evolution by low partial pressures of H₂ suggests that H₂ utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H₂ uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO₂ (initial concentration) in solution inhibited H₂ uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H₂ uptake by succinate, acetate, formate, and CO₂ in the metabolism of the H₂-uptake-positive strains of Rhizobium.     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Oxyleghemoglobin was used to supply low concentrations of O₂ to H₂-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H₂ oxidation system of these bacteroids was capable of effectively utilizing O₂ at the low concentrations of O₂ expected to be found in soybean nodules. Apparent K_m values of approximately 10 nanomolar O₂ have been calculated for the oxyhydrogen reaction. These values include the K_m values for both H₂ oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H₂ additions stimulated C₂H₂ reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H₂ oxidation system is capable of recycling all of the H₂ evolved during the N₂ fixation process.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

The Effect of Adenine Nucleotides on Purified Phosphoenolpyruvate Carboxylase from the CAM Plant Crassula argentea

The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the K_m for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the K_m for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the K_a for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg²⁺ was held constant and saturating, these effects cannot be ascribed to Mg²⁺ chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the “R” type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day.     

Regulation of glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum in response to proton fluxes at the plasmalemma

The present experiments were carried out to investigate the effect of increased fluxes of H⁺ across the plasmalemma on glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum L. (1) Increased H⁺ influx was produced by adding glucose, 6-deoxyglucose, 2-deoxyglucose, or sodium fluoride. The net influx decreased to zero after 3 min. This recovery was accompanied by an increase in the rate of O₂ uptake, but not of dark CO₂ fixation. When glucose or fluoride were added, the increase of O₂ uptake occurred without a decrease in the ATP/ADP ratio, and was large enough to provide the ATP that would be needed for compensatory H⁺ extrusion via the plasmalemma H⁺-ATPase. When 2-deoxyglucose was added, the rise of respiration was restricted by sequestration of phosphate and depletion of phosphorylated metabolites, the ATP/ADP ratio declined, and a slow net H⁺ influx started again after 4 min. (2) Alkalinisation of the medium to induce an H⁺ efflux resulted in rapid activation of dark CO₂ fixation, but not of O²-uptake. (3) A stimulation of respiration or dark CO₂ fixation was always accompanied by a decrease of phosphoenolpyruvate. This shows that the primary sites for regulation of glycolysis are pyruvate kinase and phosphoenolpyruvate carboxylase, respectively. (4) There was no consistent relation between glycolytic flux and triose-phosphates or hexose-phosphates. This shows that the reactions involved in carbohydrate mobilisation and the conversion of hexose-phosphates to triose-phosphates only have a secondary role in stimulation of glycolysis. (5) Phosphofructokinase will be stimulated as a consequence of the decrease in phosphoenolpyruvate. (6) The increase in glycolytic flux occurred independently of (in the case of 2-deoxyglucose and fluoride), or before (in the case of glucose), any increase of fructose-2,6-bisphosphate. When fructose-2,6-bisphosphate did increase (after supplying glucose), this was accompanied by an increase of triose-phosphate and fructose-1,6-bisphosphate, which otherwise remained very low. It is argued that fructose-2,6-bisphosphate increases as a consequence of the decrease of glycerate-3-phosphate, a known inhibitor of the synthesis of this regulator metabolite. However, activation of pyrophosphate fructose-6-phosphate phosphotransferase by fructose-2,6-bisphosphate does not play an obligatory role in the stimulation of glycolysis.     

Fermentative Metabolism of Chlamydomonas reinhardii: III. Photoassimilation of Acetate

The anaerobic photodissimilation of acetate by Chlamydomonas reinhardii F-60 adapted to a hydrogen metabolism was studied utilizing manometric and isotopic techniques. The rate of photoanaerobic (N₂) acetate uptake was approximately 20 μmoles per milligram chlorophyll per hour or one-half that of the photoaerobic (air) rate. Under N₂, cells produced 1.7 moles H₂ and 0.8 mole CO₂ per mole of acetate consumed. Gas production and acetate uptake were inhibited by monofluoroacetic acid (MFA), 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and by H₂. Acetate uptake was inhibited about 50% by 5% H₂ (95% N₂). H₂ in the presence of MFA or DCMU stimulated acetate uptake and the result was interpreted to indicate a transition from oxidative to reductive metabolism. Carbon-14 from both [1-¹⁴C]- and [2-¹⁴C]acetate was incorporated under N₂ or H₂ into CO₂, lipids, and carbohydrates. The methyl carbon of acetate accumulated principally (75-80%) in the lipid and carbohydrate fractions, whereas the carboxyl carbon contributed isotope primarily to CO₂ (56%) in N₂. The presence of H₂ caused a decrease in carbon lost from the cell as CO₂ and a greater proportion of the acetate was incorporated into lipid. The results support the occurrence of anaerobic and light-dependent citric acid and glyoxylate cycles which affect the conversion of acetate to CO₂ and H₂ prior to its conversion to cellular material.     

Hydrogenase mediated nitrite reduction in chlorella

The assay of the hydrogenase of glucose-grown cells of Chlorella pyrenoidosa, strain 7-11-05 by means of nitrite reduction with molecular hydrogen is described. The hydrogenase of Chlorella shows maximum activity immediately after equilibration in the hydrogen atmosphere. The hydrogenase mediated reduction of nitrite to ammonia requires the presence of CO₂. However, at pH 6.4. when the reaction proceeds optimally, there is apparently sufficient retention of metabolic CO₂ to support the reaction, which goes to completion, at near maximum rates.

Reduction of nitrite in the hydrogenase system when CO₂ is present results in the uptake of 3 moles of H₂ per mole of nitrite and ammonia is the product. When CO₂ is absent or limiting, ammonia is also formed from nitrite but with the uptake of less than the stoichiometric amount of H₂. It is concluded that CO₂ is essential for the uptake of H₂, and that in the absence of CO₂ internal hydrogen donors support nitrite reduction.

The possibility that CO₂ exerts a catalytic effect in all reductions mediated by hydrogenase in algae is considered, and a further hypothesis, that hydrogenase arises from that portion of the photosynthetic machinery which also shows a catalytic requirement for CO₂, is proposed.