James A. Bassham

The Path of Carbon in Photosynthesis. XXI. The Cyclic Regenerationof Carbon Dioxide Acceptor

IJCEL-2369 Unclassified Chemistry D i s t r i b u t i o n UNIVERSITY O CALIFORML4 F Radiation Laboratory Contract No. M-7405- eng THE P T O CARBON I N PHdGSYNTHESIS .BI. A H F T E CYCLIC REGENERATION O CARBON DIOXIDE ACCEPTOR H F J. A , Bassham, &Ae A. Benson, Lorel D. K r ~ r ~ Anne Z. Harris, A. T. Wilson and M. Calvin October, 1953 Berkeley, California

Free energy changes and metabolic regulation in steady-state photosynthetic carbon reduction

Abstract The standard physiological free energy changes of reactions of glycolysis, the reductive pentose phosphate cycle (photosynthetic carbon reduction cycle) and the oxidative pentose phosphate cycle have been calculated from available data. The concentrations of metabolites of the photosynthetic carbon reduction cycle, measured during steady-state photosynthesis in Chlorella pyrenoidosa in the presence of radioactive tracers, and the concentrations of some intermediates of the oxidative pentose phosphate cycle measured during a subsequent dark period, have been employed to calculate the free energy changes of each reaction of the reductive cycle and of some of the reactions of the oxidative cycle during steady-state light and dark conditions. With respect to the magnitude of the negative free energy change, at steady state, such reactions have been found to be of two types. Those with high negative free energy changes (−6 to −11 kcal) are in each case reactions from which there exists independent evidence of a role in metabolic regulation. Those with small negative free energy changes (o to −2 kcal) are not regulated reactions and are highly reversible. Thus most of the negative free energy change occurring under steady-state conditions in this metabolic system is dissipated for purposes of control. By the criterion of negative free energy change, ribulosediphosphate carboxylase, and fructose- and heptosediphosphatases are regulated enzymes. The activities of these enzymes are known to be high in the light and low in the dark. Phosphoribulokinase, which mediates the one reaction with an intermediate negative free energy change (−3.82 kcal) also may be a regulated enzyme with greater activity in the light than in the dark. In the oxidative cycle, the reaction mediated by glucose-6-phosphate dehydrogenase has a very high negative free energy change and appears to be active in the dark and inactive in the light. One function of these controls is thought to be the exclusive operation of the reductive cycle in the light and the oxidative cycle in the dark.

Photosynthetic and dark carbon metabolism in unicellular blue-green algae

Summary0946 091.The kinetics of 14CO2 incorporation into cellular intermediates was used to determine the primary pathway of carbon fixation by four genetically diverse unicellular blue-green algae. In each case label was first detected in 3-phosphoglycerate and then in compounds of the reductive pentose cycle.2.A light to dark transition evoked the same response in all four strains: Immediate cessation of biosynthesis, rapid increase in the concentration of 6-phosphogluconate and changes in the concentrations of sugar mono- and diphosphates. On the other hand, after the first few seconds of dark incubation little or comparatively slow change was noted in the concentrations of 3-phosphoglycerate, phosphoenolpyruvate, citrate, aspartate, and glutamate.3.For one strain (aphanocapsa 6308) an experiment using both 32P and 14CO2 as tracers revealed comparatively rapid turnover of metabolites common to the oxidative pentose cycle during dark incubation. Much slower turnover of labeled carbon was found in other metabolites of glycolysis and the biosynthetic portions of the tricarboxylic acid cycle.4.During dark incubation of Aphanocapsa 6308 the concentration of adenosine triphosphate decreased to approximately 50% of the photosynthetic value within the first 50 sec. In the same period, adenosine diphosphate nearly doubled in concentration. By 4 min after the beginning of the dark period, the steady-state levels of the two adenylates had been restored to photosynthetic levels.

Photosynthesis by isolated chloroplasts I. Diffusion of labeled photosynthetic intermediates between isolated chloroplasts and suspending medium

Abstract The diffusion of 14 C- and 32 P-labeled photosynthetic intermediate compounds from isolated chloroplasts has been investigated. Those intermediate compounds of the photosynthetic carbon reduction cycle lying between the carboxylation reaction and the diphosphatase reaction were found to diffuse rapidly from the chloroplast to the suspending medium. In contrast, those intermediates lying between the diphosphatase reaction and the carboxylation reaction, with the exception of the pentose monophosphates, tend to be retained by the chloroplasts during the 15 min or more when the photosynthesis of CO 2 by the isolated chloroplasts is most active. An experiment in which changes in the level of ATP were observed on addition of CO 2 shows that ATP and ADP, which had diffused from the choroplasts, can apparently re-enter the chloroplasts and be used metabolically. It is proposed that the diphosphatase reaction plays a general role in metabolic regulation in biosynthesis as well as in the light-dark transition.

REGULATORY EFFECTS OF AMMONIA ON CARBON METABOLISM IN PHOTOSYNTHESIZING CHLORELLA PYRENOIDOSA

Abstract Addition of ammonia to Chlorella pyrenoidosa , photosynthesizing under steady-state conditions, causes changes in the metabolism which are due not only to the increased availability of NH 4 + for reductive amination but also to regulation of controlled enzymes. One such effect is an increased rate of the reaction which converts phosphoenolpyruvate to pyruvate in vivo . This regulatory effect was revealed by kinetic tracer studies with 14 CO 2 , paper chromatography and radioautographic analysis, which showed that upon addition of NH 4 + (1) the levels of both 3-phosphoglycerate and phosphoenolpyruvate drop, with the ratio of 3-phosphoglycerate/phosphoenolpyruvate increasing, (2) the level of labeled pyruvic acid increases and the rate of formation of alanine increases rapidly, while the rate of formation of serine is unaffected, (3) the rate of flow of carbon into the tricarboxylic acid cycle acids, malate and citrate, increases along with the increased rates of formation of glutamate, glutamine and aspartate and (4) the rate of labeling of lipids increases. The increased flow of carbon into amino acids is mostly at the expense of sucrose synthesis; starch synthesis decreases only slightly. The interruption of sucrose synthesis apparently is due to stopping the reaction between UDP-glucose and fructose 6-phosphate. The rate of conversion of fructose 1,6-diphosphate to fructose 6-phosphate is also decreased upon NH 4 + addition.

GROWTH AND BRANCHED HYDROCARBON PRODUCTION IN A STRAIN OF BOTRYOCOCCUS BRAUNII (CHLOROPHYTA)1

A strain Botryococcus braunii Kütz. that produces high levels of branched hydrocarbons (botryococcenes) was grown under different environmental conditions to investigate the relationship between growth and hydrocarbon production. Carbon dioxide concentration had the most significant influence on growth; 0.3% CO2‐enriched cultures demonstrated a minimum mass doubling time of ca. 40 h, compared to ca. 6 days for ambient air cultures grown on the same buffered growth medium. The botryococcene fraction, which consisted of 10 identified compounds (CnH2n‐10; n = 30–34), usually constituted ca. 25–40% of the culture dry weight under various growth regimes, including nitrogen‐ and/or phosphate‐deficiencies. CO2 enrichment initially favored the production of the lower botryococcenes (C30–C32), whereas relatively slow‐growing ambient air cultures accumulated C33 and C34 compounds.

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.

DYNAMICS OF THE PHOTOSYNTHESIS OF CARBON COMPOUNDS II. AMINO ACID SYNTHESIS

Further kinetic studies have been made of the rates of appearance of 14C in individual compounds formed by Chlorella pyrenoidosa during steady state photosynthesis with 14CO2. Total and “active” pools of several amino acids have been determined. The effects of adding unlabeled acetate and of turning off the light have been studied in this system. From these experiments it is concluded that synthesis and utilization of alanine, serine, aspartic acid, glutamic acid and several other amino acids are most active within the chloroplasts during photosynthesis and that these amino acids are formed rather directly from intermediates of the carbon reduction cycle. The major portion of the carbon utilized for amino acid synthesis is accounted for in the synthesis of these compounds. Evidence for the presence of at least two separated pools of these amino acids is given, and the effect of light and dark and of the addition of unlabeled acetate upon the synthesis of these amino acids is discussed.

Regulatory effects of ammonia on carbon metabolism in Chlorella pyrenoidosa during photosynthesis and respiration.

Abstract Addition of ammonia to Chlorella pyrenoidosa , respiring in the dark following a period of photosynthesis, causes a stimulation of the flow of carbon into the synthesis of amino acids similar to that observed upon addition of ammonia during photosynthesis. In both cases, this stimulation is due not only to the increased availability of NH 4 + for reductive amination of α-ketoglutarate to glutamate but is also due to stimulation of the rate of conversion of phosphoenolpyruvate to pyruvate. Addition of NH 4 + in the dark causes a large increase in the formation of 6-phosphogluconate, beyond the increase in 6-phosphogluconate already seen when the light is turned off. When the light is turned off, the level of starch begins to decrease, and the rate of this decrease is not changed by the subsequent addition of ammonia. In contrast, the level of sucrose becomes nearly constant when the light is turned off, but begins immediately to decline when ammonia is added. As observed before, the level of ATP drops temporarily when the light is turned off and then rises to a steady state similar to that seen in the light. Upon the addition of ammonia, a similar transient drop and re-establishment in the level of ATP is seen. These and other reported results are discussed with respect to sites and mechanisms of light-dark metabolic regulation leading to increased flow of carbon from carbohydrate reserves into mitochondrial metabolism in the dark, and the sites and mechanisms by which ammonia affects the rate of this flow.

Dynamics of the photosynthesis of carbon compounds I. Carboxylation reactions

Abstract Kinetic studies have been made of the rates of appearance of 14 C in individual compounds formed by Chlorella pyrenoidosa during steady state photosynthesis with 14 CO 2 . These rates have been compared with rates of CO 2 and 14 C disappearance from the gas phase during the same experiments. 1. The following results were obtained: 2. 1. After the first few seconds, the rate of appearance of 14 C in compounds stable to drying on planchets at room temperature is 95 to 100% of the rate of uptake of carbon from the gas phase. 3. 2. After the first few seconds, the rate of appearance of carbon in compounds isolable by usual methods of paper chromatography constitutes at least 73 to 88% of the rate of uptake of carbon from the gas phase. Compounds formed from the carbon reduction cycle via the carboxylation of ribulose diphosphate account for a least 70 to 85% of the uptake, while carboxylation of phosphoenolpyruvic acid appears to account for at least another 3%. 4. 3. The induction period in the appearance of 14 C in stable compounds may be due to a reservoir of intracellular CO 2 and HCO 3 − or to some other volatile or unstable compound. If so, this reservoir contains no more than 1.5 μmoles of carbon, corresponding to about 7 sec carbon fixation in the experiment in which it was measured. 5. 4. No other carboxylation reactions, such as the carboxylation of γ-aminobutyric acid, could be observed. The rate of labeling of glutamic acid after 5 min of exposure of the algae to 14 CO 2 reached a maximum rate of about 5% of the total uptake rate, but this labeling appears to be due to conversion of labeled intermediates formed from the carbon reduction cycle or phosphoenolpyruvic acid carboxylation. 6. 5. The in vivo carboxylation of ribulose diphosphate in the light appears to be followed by conversion of the product to one molecule of phosphoglyceric acid, containing the newly incorporated 14 CO 2 and one molecule of some other (kinetically distinguishable) three carbon compound. This reaction would be different from the one reported for the isolated enzyme system and the in vivo reaction in the dark, which produces two molecules of 3-phosphoglyceric acid.