Samuel Ruben's Contributions to Research on Photosynthesis and Bacterial Metabolism with Radioactive Carbon

The earliest experiments on the pathways of carbon in photosynthetic and heterotrophic metabolism using radioactive carbon, (11)C, as a tracer were performed by Samuel (Sam) Ruben, Martin Kamen, and their colleagues. The short half-life of (11)C (20 min), however, posed severe limitations on identification of metabolic intermediates, and this was a major stimulus to search for a radioactive carbon isotope of longer half-life. (14)C was discovered by Ruben and Kamen in 1940, but circumstances prevented continuation of their research using the long-lived isotope. Because of the untimely accidental death of Ruben in 1943, there are very few published accounts on the life and work of this extraordinary scientist. This paper summarizes highlights of Ruben's outstanding accomplishments.     

Photosynthesis: 1900-1930.

During the second half of the 19th century Julius von Sachs established the main principles of the photosynthetic production of sugars. From then, a growing number of biochemists and physiologists attended to the process, that appeared like a "black box", in order to detect what came in and what went out of it. The English group of Frederick Blackman gave a remarkable contribution in individuating the close connection between temperature, light and CO2 concentration. Later, the great importance of light was stressed by Otto Warburg, who evaluated the radiant energy necessary to the process in terms of quantum theory. The biochemical mechanism of photosynthesis was interpreted by the main European schools on the basis of Adolf Baeyer's suggestion which posed formaldehyde as the core of the process. Formaldehyde's theory hold engaged the biochemists for about fifty years although some voices rose up against it. However, nobody could put forward more coherent theories until the 1940s, when Sam Ruben and Martin Kamen individuated the cyclic pattern of the process. Ultimately, the first thirty years of the 20th century must be seen as a preliminary stage studded with light and shade even if, in spite of controversial trends, several findings of remarkable interest became to disclose that "black box" as we know today chlorophyll photosynthesis.     

Physiological studies on nitrogen fixation in the blue-green alga anabaena cylindrica

Summary Short-term manometric experiments with bacteria-free cultures of Anabaena cylindrica showed that the close dependency of nitrogen fixation upon photosynthesis could be temporarily eliminated in nitrogen-starved cells. Initial rates of nitrogen uptake by these cells in the absence of carbon dioxide were equally rapid in the light and dark, decreasing and finally ceasing after two hours. Continued steady nitrogen uptake was only maintained for long periods in the presence of carbon dioxide in the light. In the dark, nitrogen uptake was accompanied by carbon dioxide evolution.More oxygen was evolved in the light by cells fixing nitrogen than by those incubated under argon. This additional oxygen evolution could be accounted for by extra carbon dioxide fixation in the presence of nitrogen.Of a number of organic compounds tested, only sodium pyruvate stimulated nitrogen fixation. This stimulation was achieved both in the light and dark and in the presence and absence of carbon dioxide, showing that the role of pyruvate was other than acting as a carbon skeleton.Three metabolic inhibitors, cyanide and chlorpromazine (chiefly respiratory) and phenylurethane (photosynthetic) differentially inhibited photosynthesis and nitrogen fixation. The latter inhibitor had a more marked effect on photosynthesis while the two chiefly respiratory inhibitors had a stronger effect on nitrogen fixation.     

THE CHLOROPHYLL-CARBON DIOXIDE RATIO DURING PHOTOSYNTHESIS

Using a rapid spectrographic method of carbon dioxide measurement previously described by McAlister (1937) further studies on the time course of photosynthesis in the higher plant, wheat, variety Marquis, are herein reported. Of major importance in this work is the discovery of a pick-up of carbon dioxide in darkness immediately following a high rate of photosynthesis (see Figs. 3 and 4). This pick-up is believed to be due to the action of a carbon dioxide-combining intermediate; i.e., the "acceptor molecule" for carbon dioxide in photosynthesis. The conditions under which this phenomenon has so far been observed indicate that the intermediate is formed in relatively large quantities during the actual process of photosynthesis and not before. That the intermediate is chlorophyllous in nature is suggested by a simple stoichiometry of the order of unity that is found to exist between the number of carbon dioxide molecules taken up and the total number of chlorophyll molecules present in the plant. This is in opposition to the idea of a large photosynthetic unit of some 2000 chlorophyll molecules operating together in the reduction of 1 carbon dioxide molecule. Further studies of the induction phase under various conditions of previous dark rest and of carbon dioxide and light limitation are herein described. Employing the simple hypothesis that the number of carbon dioxide molecules not reduced during the induction period (induction loss) gives a measure of the number of elementary photosynthetic cycles unoperative or compensated for during induction together with the experimental fact that this induction loss is of the order of the total number of chlorophyll molecules present, these latter studies also indicate, in a less direct manner, that chlorophyll participates in photosynthesis as an individual molecule and not as part of a very large multimolecular chlorophyll unit. The fast dark reaction lasting about 1 minute (Fig. 7) required to reproduce both (a) the phenomena of induction in carbon dioxide assimilation and (b) the recovery of fluorescence of chlorophyll in leaves in darkness as observed by Franck and Wood (1936), demonstrates a close relationship between the fluorescence of chlorophyll and induction in photosynthesis. The rate of respiration (carbon dioxide production) of the higher plant, wheat, was measured under intense illumination and in the absence of carbon dioxide (to suppress assimilation). This value was found to be identical with the dark respirational rate measured before and after the light period, indicating very positively the absence of any direct effect of light on respiration.     

Evolution of the Z-scheme of photosynthesis: a perspective

The concept of the Z-scheme of oxygenic photosynthesis is in all the textbooks. However, its evolution is not. We focus here mainly on some of the history of its biophysical aspects. We have arbitrarily divided here the 1941–2016 period into three sub-periods: (a) Origin of the concept of two light reactions: first hinted at, in 1941, by James Franck and Karl Herzfeld; described and explained, in 1945, by Eugene Rabinowitch; and a clear hypothesis, given in 1956 by Rabinowitch, of the then available cytochrome experiments: one light oxidizing it and another reducing it; (b) Experimental discovery of the two light reactions and two pigment systems and the Z-scheme of photosynthesis: Robert Emerson’s discovery, in 1957, of enhancement in photosynthesis when two light beams (one in the far-red region, and the other of shorter wavelengths) are given together than when given separately; and the 1960 scheme of Robin Hill & Fay Bendall; and (c) Evolution of the many versions of the Z-Scheme: Louis Duysens and Jan Amesz’s 1961 experiments on oxidation and reduction of cytochrome f by two different wavelengths of light, followed by the work of many others for more than 50 years.     

Evidence for the participation of the reductive pentose phosphate cycle in photoreduction and the oxyhydrogen reaction

The assimilation of ¹⁴C-sodium bicarbonate has been measured in Scenedesmus obliquus as 1) photosynthesis, 2) photoreduction (light dependent incorporation of carbon dioxide by hydrogen adapted cells under conditions where photosynthesis is inoperative), and 3) the oxyhydrogen reaction (dark assimilation of carbon dioxide by hydrogen adapted cells in an atmosphere of hydrogen and 1% oxygen). Degradation of the glucose formed in each of these reactions using the Leuconostoc technique establishes the participation of the reductive pentose phosphate cycle.     

Following the path of carbon in photosynthesis: a personal story

Chronological recognition of the intermediates and mechanisms involved in photosynthetic carbon dioxide fixation is delineated. Sam Ruben and Martin Kamen's development of application of radioactive carbon for the study of carbon dioxide fixation provided impetus and techniques for following the path of carbon in photosynthesis. Discovery The identity of the primary carboxylation enzyme and its identity with the major protein of photosynthetic tissues (`Fraction 1' protein of Sam Wildman) is reviewed. Memories are dimmed by sixty years of exciting discoveries exploration in newer fields [see Benson 2002 (Annu Rev Plant Biol 53: 1–25), for research and perspectives beyond the early Berkeley days]. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glycolate Stimulation of Oxygen Evolution During Photosynthesis

Glycolate and glyoxylate stimulated 100% to 300% the rate of oxygen evolution by Scenedesmus in the light in the absence of added carbon dioxide. This stimulation occurred either aerobically or anaerobically, and was sensitive to CMU. Aerobic dark respiration was stimulated 25% to 100% by glycolate. This phenomenon was best demonstrated with synchronized Scenedesmus at the stage of cell division. For glycolate stimulation of oxygen evolution, a dark preincubation of 1 minute or less was necessary. In comparative test with other compounds of metabolism and photosynthesis, the stimulation of oxygen evolution was greatest by glycolate and glyoxylate. In a proposed scheme glyoxylate serves as a terminal hydrogen acceptor from NADPH produced by photosynthesis, and it thereby stimulates oxygen evolution when carbon dioxide is not available. Transformation of glycolate to glyoxylate in these cells would have to occur in the absence of oxygen.     

Light-Dependent CO2 Retrieval in Immature Barley Caryopses

Immature detached caryopses from barley (Hordeum vulgare L.var. distichum cv. Midas) were shown to be capable of light-dependentretrieval of internally-produced CO₂. In the first set of experiments,caryopses were radioactively labelled by supplying (U-14C)-sucroseto detached ears in liquid culture. Caryopses were then removedfrom the ear and given a 12 h chase of non-radioactive sucrosein either the light or dark. More ¹⁴C was recovered in the caryopsesafter the chase in the light than in the dark but the differenceswere not significant. In the second set of experiments, ¹⁴C-labelledcaryopses obtained by a 15 min light incubation in ¹⁴CO₂ weremaintained in either the light or dark for 3 h and any redistributionof label between the tissues recorded. The results show thatunder these conditions, photosynthesis in the Chl-containinggreen layer of the pericarp can prevent losses of internally-producedCO₂, since 3 times as much radiocarbon remained in the caryopsesincubated in the light as in the dark. These differences weresignificant at P=0.001. Experiments with the mutant barley Albinolemma, which has no Chi in the pericarp, showed that there waslittle difference between light and dark treatments. This confirmsthe suggestion that photosynthesis in the pericarp of the normalcultivar Midas may be concerned in the refixation of CO₂. Key words: Barley, pericarp, photosynthesis, carbon dioxide     

On the requirement of minimum number of four versus eight quanta of light for the evolution of one molecule of oxygen in photosynthesis: A historical note

A bitter controversy had existed as to the minimum number of quanta required for the evolution of one molecule of oxygen in photosynthesis: Otto Warburg had insisted since 1923 that this value was 3–4, whereas Robert Emerson and others continued to obtain a value of 8–12 since the 1940s. It is shown in this letter that the 1931 Nobel-laureate of Physiology & Medicine Otto Warburg published, in his last and final paper, just before his death in 1970, a measured minimum quantum requirement of oxygen evolution of 12 at the lowest intensities of light he used. Although using his theory on photolyte, Warburg calculated a value of 3–4 for the quantum requirement, this is the first confirmation by Warburg of the higher measured quantum requirement. However, it has remained unknown to most investigators. It is expected that this information will be of general interest not only to those interested in the history and research on photosynthesis, but to the entire sci entific community, especially the writers of text books in biology, biochemistry and biophysics.     

Light-Dark Transients in Levels of Intermediate Compounds during Photosynthesis in Air-Adapted Chlorella

The labeling of intermediate compounds and photosynthetic cofactors during photosynthesis and periods of darkness by Chlorella pyrenoidosa in the presence of ³²P-labeled phosphate and ¹⁴CO₂ have heen investigated. Algae adapted to photosynthesis in air were used, and the level of carbon dioxide was maintained at approximately 0.04 % and at constant specific radioactivity during the course of the experiments. The transient changes which occur in the levels of labeled fructose-1,6-diphosphate and in sedoheptulose-1,7-diphosphate, and in the corresponding monophosphates when the light is turned off suggest a light activation of the diphosphatase enzymes which decays after about 2 minutes of darkness. It is suggested that a light-dark switch in enzymic activities permits photosynthesis and glycolysis to occur in light and dark respectively with the same enzymic apparatus. The greatly diminished rate of disappearanec of the carboxylation substrate, ribulose-1,5-diphosphate, after about 2 minutes suggests that there is also a light activation of the carboxylation reaction in vivo. Large transient changes in the level of pyrophosphate between light and dark indicate that there may be an unstable cofactor which decomposes to give pyrophosphate during or alter killing of the algal cells. The possibility that this cofactor is involved in an activation of Carbon dioxide for the carboxylation reaction in vivo is suggested. Light-dark transient changes in labeling of other compounds of the photosynthetic carbon reduction cycle and related compounds were determined, and possible significance of these changes is discussed.(PDF DAMAGED)     

From Nuclear Science to Bacterial Cytochromes: the Work of Martin D. Kamen

Structural Bases for Function in Cytochromes c. An Interpretation of Comparative X-ray and Biochemical Data (Salemme, F. R., Kraut, J., and Kamen, M. D. (1973) J. Biol. Chem. 248, 7701–7716) Martin David Kamen (1913–2002) was born in Toronto, Canada, but grew up in Chicago. He enrolled at the University of Chicago in 1930, intending to study English. However, the Great Depression took a toll on his family''s finances, and his father suggested he switch his major to chemistry in order to make a living after graduating. By his junior year, Kamen was hooked on chemistry. He graduated in 1933 with honors in physical chemistry, working with William Draper Harkins to determine ammonia gas emission spectra excited by electrodeless discharge. He remained in Harkins'' lab for graduate school, earning his doctorate in physical chemistry in 1936 for an article on neutron scattering (1) that was accepted as his dissertation.Open in a separate windowMartin D. KamenBecause economic conditions were still bleak, Kamen followed the suggestion of one of his mentors, David Gans, and applied for a research post with Nobel laureate Ernest O. Lawrence, who had developed the cyclotron at the radiation laboratory in Berkeley, California. Kamen used his savings to move to Berkeley and worked at the laboratory without pay for 6 months before Lawrence offered him a staff position as a chemist. In addition to troubleshooting the cyclotrons and preparing samples of radioisotopes, Kamen performed numerous photosynthetic studies with Samuel Ruben, using carbon-11. Because carbon-11 had a half-life of only 21 min, Lawrence assigned Kamen and Ruben the task of finding carbon-14. The pair succeeded by bombarding graphite in the cyclotron, producing carbon-14, which had a 5730-year half-life (2).Kamen and Ruben planned to use their discovery to create a company that would construct and operate several cyclotrons dedicated to carbon-14 production and expand on the laboratory''s radioisotope program. However, the war intervened, and all non-war-related research at Berkeley was halted. Kamen was assigned to head a program studying the separation of uranium isotopes for the Manhattan Project. But, unexpectedly in 1944, he was declared a security risk and dismissed from the lab. A few years later, Kamen was called before the House Un-American Activities Committee, being wrongly linked to an espionage ring working for the USSR. Subsequently, the State Department refused to issue Kamen a passport, and the Chicago Tribune named him as a suspected spy. During the next decade, he fought recurring rumors and accusations that he had leaked atomic bomb secrets. Eventually, he won a libel suit against the Chicago Tribune, and the State Department reinstated his passport.In 1945, Kamen moved to the Mallinckrodt Institute of Radiology at the Washington University School of Medicine where he supervised cyclotron production of radioisotopes for medical research. His own research interests gradually shifted away from nuclear physics and radiochemistry to biochemistry, and he began several collaborations involving the use of radioisotopic tracers in biological and biomedical research.Kamen also initiated a series of experiments using carbon-14 to study photosynthesis in bacteria. This resulted in a number of important discoveries, including hydrogen photoevolution (3) and nitrogen fixation (4) in Rhodospirillum rubrum. While working with the bacteria, Kamen and Leo Vernon discovered that R. rubrum contained a c-type cytochrome (5), which they later named “cytochrome c₂.”Twenty years after it was discovered, the structure of cytochrome c₂ was solved (6). By comparing this structure with the recently solved structure of eukaryotic mitochondrial cytochrome c (7), Kamen and his colleagues were able to deduce information about the structural, functional, and evolutionary relationships in the cytochromes c. This is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here.Despite the fact that both eukaryotic cytochrome c and cytochrome c₂ serve analogous functions in their respective physiological electron transport chains, i.e. they both transport electrons to the terminal and most oxidizing electron carrier of each system, Kamen was able to find several differences between the molecules. For example, he noted that cytochrome c₂ has a more positive electrochemical potential and does not exhibit the large oxidation state-dependent conformational change characteristic of mitochondrial cytochrome c. Open in a separate windowKamen continued to study other bacterial cytochromes, showing that at least 12 subgroups of the cytochromes c exist. This resulted in new perspectives on potential variations in structure and function of the heme group in relation to protein.In 1957 Kamen moved to Brandeis University to help establish the graduate department of biochemistry, and in 1961 he joined the University of California, San Diego chemistry department to help found their new campus. He remained there until 1975, when he became director of the Chemical-Biological Development Laboratory at the University of Southern California. Kamen continued to teach into his eighties, being one of six faculty members of the Oregon Institute of Science and Medicine.Kamen received numerous awards and honors for his contributions to science, including the American Chemical Society''s Award for Applications of Nuclear Chemistry (1963), the American Society of Plant Physiologists'' Charles F. Kettering Research Award (1968), the American Society of Biological Chemists'' Merck Award (1982), the John Scott Medal of the City of Philadelphia (1988), the World Cultural Council''s Einstein Award (1990), and the U.S. Department of Energy''s Enrico Fermi Award (1996). He was a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and the American Philosophical Society. ¹     

The ferredoxin/thioredoxin system: a key element in the regulatory function of light in photosynthesis

In addition to its well-established function in supplying the energy for carbon dioxide assimilation, light plays a regulatory role in photosynthesis. The ferredoxin/thioredoxin system is a major mechanism whereby light functions in this capacity. Here, light absorbed by chlorophyll is converted via ferredoxin into a reductant messenger, reduced thioredoxin, that interacts with key target enzymes, thereby changing their catalytic activities. In this way, the green plant achieves maximum efficiency of its photosynthetic (light) and heterotrophic (dark) capabilities.     

Studies on the Biochemistry and Fine Structure of Silica Shell Formation in Diatoms. Photosynthesis and Respiration in Silicon-Starvation Synchrony of Navicula pelliculosa

Rates of photosynthesis, measured by oxygen electrode or by ¹⁴CO₂ fixation, dark respiration and ³²P-phosphate incorporation are reported for the silicon-starvation synchrony of the fresh water diatom Navicula pelliculosa. During late exponential growth the rates were consistent with increase in carbon mass. During silicon starvation, rates of carbon dioxide fixation, oxygen evolution and ³²P incorporation fell, and the saturating light intensity decreased from 27,000 lux to 5000 lux. Reintroduction of silicon led to immediate transients in all parameters studied, followed by a prolonged increase in rate of dark respiration and a gradual increase in apparent photosynthesis. During release of daughter cells, the rates of dark respiration decreased as photosynthesis and ³²P incorporation increased. These results are discussed in relation to effects of silicon on the energy metabolism of the diatom.     

Compensation Points and Carbon Dioxide Enrichment for Lettuce Grown Under Glass in Winter

Measurements of , the minimum intercellular-space carbon dioxideconcentration or carbon dioxide compensation point, made attwo temperatures and at light intensities from 15 to 1,500 f.c.,were used to study the relation between the compensation pointsfor light and carbon dioxide. This was found to be hyperbolicand consistent with the Warburg–Maskell model for photosynthesis.The apparently beneficial effects of carbon dioxide enrichmentof the atmosphere at increased temperature, found in practicewith lettuce or other crops grown under glass at light intensitiesthat might be expected to be severely limiting, must be in partdue to the reduction of the light compensation point at highcarbon dioxide concentration.     

Effect of Potassium Deficiency upon Translocation of C in Detached Blades of Sugarcane

The translocation of radioactive photosynthate was studied in blades detached from plants which had been grown in nutrient solution with and without potassium. Basipetal translocation decreased in the blades of plants deprived of potassium, even when deficiency symptoms were not visible. In such slightly potassium-deficient leaves, translocation was decreased in the light but not in the dark. More severe potassium deficiency decreased translocation both in darkness and in light. Potassium deficiency decreased translocation at light intensities giving no difference in rate of photosynthesis between plus and minus potassium leaves and even at light intensities at which there was no net fixation of carbon dioxide. At all levels of deficiency, the relative decrease in translocation was greater than the relative decrease in photosynthesis. Translocation was affected at potassium deficiency levels which had no effect upon photosynthesis.     

Influence of Carbon Dioxide Concentration on Growth, Carbohydrate Content, Translocation and Photosynthesis of White Clover

White clover ramets were grown at various carbon dioxide concentrations(200, 350 and 1000 µl 1^–1), defoliated and regrownat the same concentrations. Morphological characteristics, dryweights and non-structural carbohydrate contents of plant organs,diurnal variation of sugar and starch content of leaves, translocationof assimilates and photosynthesis were determined. Carbon dioxide concentration influenced the dry weights, butnot the number and size of the plant organs. However, defoliationof plants at low carbon dioxide concentration resulted in decreasedleaf size and stolon length. Carbon dioxide concentration influencedthe content and diurnal variation of starch and sugar in theleaves. Starch was accumulated at medium carbon dioxide concentrationand sugar at a higher concentration when the storage capacityfor starch seemed to be exceeded. Starch was preferentiallyaccumulated in the first and sugar in the second half of thelight period. Translocation was decreased during the periodsof accumulation. Sugar accumulation in the leaves seemed tobe a consequence of the imbalance between sink and source, whereasstarch accumulation seemed to follow an in-built diurnal pattern.Accumulation of both starch and sugar during the photoperiodwas followed by degradation and export during the dark period.Decreased dark export occurred at low carbon dioxide concentrationwhen neither starch nor sugar was accumulated during the photoperiod. Carbon dioxide, white clover, Trifolium repens L., growth, carbohydrates, starch, sugar, translocation, photosynthesis     

Photosynthetic metabolism and cell-wall polysaccharide accumulation inGelidium sesquipedale (Clem.) Born. et Thur. under different light qualities

The influence of different light qualities on the photosynthetic rate, dark respiration, intracellular carbon and nitrogen content, and accumulation of photosynthetic pigments and cell-wall polysaccharides during short-term incubation (5 h) of the red algaGelidium sesquipedale was investigated. The same photon irradiance of 50mol m^–2 s^–2 below the light saturation point of photosynthesis was applied in each case. Blue light stimulated photosynthesis, dark respiration and the accumulation of chlorophyll and biliproteins, phycoerythrin in particular. The accumulation of internal carbon and nitrogen was greater under blue light than under the other light qualities. In contrast, the percentage of cell-wall polysaccharides was higher in red light. The content of cell-wall polysaccharides decreased during the time of incubation in all light treatments except in red light. The action of a non-photosynthetic photoreceptor in the control of cell-wall polysaccharide synthesis is suggested because the accumulation of cell-wall polysaccharides was not correlated with net photosynthesis in contrast to what occurred with carbon, chlorophyll and phycoerythrin accumulation.     

A Dynamic Model for Photosynthesis

A dynamic mathematical model of the effect of radiant flux densityand CO₂ concentration on the rate of photosynthesis is proposed.An appropriate dynamic experimental method for ecological studiesof this subject is described. The methodology permits the analysisof numerous problems, including the effect of changes in CO₂concentration on photosynthesis and the effectiveness of energyconversion by a leaf of a plant in different environmental conditions. The dynamic model for photosynthesis is composed of two separateinteracting non-linear parts; one describes the dynamics ofthe complex set of light reactions, and the other describesthe dark reactions. The model explains the dynamics of leafphotosynthesis in a closed circuit flow system, and also explainsthe expressions for the equilibrium states of photosyntheticrate widely used in the literature. photosynthesis, mathematical model, carbon dioxide fixation, light reactions     

Changing concepts about the distribution of Photosystems I and II between grana-appressed and stroma-exposed thylakoid membranes

Thylakoid membranes of higher plants and some green algae, which house the light-harvesting and energy transducing functions of the chloroplast, are structurally unique. The concept of the photosynthetic unit of the 1930s (Robert Emerson, William Arnold and Hans Gaffron), needing one reaction center per hundreds of antenna molecules, was modified by the discovery of the Enhancement effect in oxygen evolution in two different wavelengths of light (Robert Emerson and his coworkers) in the late 1950s, followed by the 1960 Z scheme of Robin Hill and Fay Bendall. It was realized that two light reactions and two pigment systems were needed for oxygenic photosynthesis. Changing ideas about the distribution of Photosystem II (PS II) and PS I between the green-appressed and stroma-exposed thylakoid membrane domains, which led to the concept of lateral heterogeneity, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.