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The History of Photosynthesis

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The History of Photosynthesis

The History of Photosynthesis

384-322 (BC) Aristotle compared the soil (earth) to the stomach and concluded that the earth is like the stomach of plants as they gain their nutrients directly from earth and water without having a proper digestive system. 1648 Johan Baptist van Helmont (from Brussels) considered water to be the source of life and the basic nutrient for plants. Therefore he devised an experiment by which he showed that small potted willows can thrive on soil and water alone while they gain their substance (weight) solely from the water as the weight of the soil in the pots did not decrease significantly. He published his conclusion in his book Ortus medicinae wherein he also used the term gas for the first time. 1675 Marcellus Malphigi (from Bologna) was the first to study the anatomy of plants (and insects) concisely by making use of the microscope and he claimed that plants take up nutrients which are dissolved in water via their roots. 1679 In a letter to a friend which was later published in 1717 Edmé Mariotte (from Dijon) summarized the current knowledge about the composition and nutrition of plants and thus provided the first theory of metabolism by stating that all plants are made up of certain basic substances (principes: like sulphur,salt, oil, ammonia and nitre) which must be contained in the soil, where from they are consequently taken up by the plants and transformed into their metabolites. As plentiful different plants can grow from the same soil and water he concluded that each plant must provide its own specific metabolism in order to create its substances and form. 1684 In this year the Irishman Robert Boyle who was a prominent proponent of the Corpuscular Philosophy published his Memoirs for the Natural History of Human Blood in which he documented that the changing colour of blood during its passage through the capillaries of the lung (from dark to light red) was due to a certain ingredient of air which was consumed by fire or breathing. 1703 The German G.E. Stahl (later the physician of Friedrich Wilhelm I. in Berlin) put forward his Phlogiston Theory according to which an element called phlogiston was given off by combustible materials when they burned. Air in which things had been burned became less able to support combustion because, it was thought, it was saturated with phlogiston which, according to the current belief, could then also mix with other substances. It was assumed that phlogiston ought to have a negative weight because substances which had been burned were lighter than before. 1727 Stephan Hales (an English clergyman) described the leaves as organs of transpiration and he postulated that plants exchange gases with their surrounding air. Furthermore, he was the first to point out a possible role of light in plant nutrition by quoting Newton: Könnte nicht eine beiderseitige ineinander wirckende Verwandlung (transformation reciproque) zwischen dicken Körpern und Lichte vorgehen ? und die Körper einen grossen Theil ihrer Activität von denen Lichtparticuln haben, die in ihren Zusammensatz mit kommen. Daß Körper in Licht und Licht in Körper verwandelt werde, ist doch der Natur Lauf gar gemäß, welche am liebsten mit Verwandlungen (transformations) zu schaffen hat. 1772 Joseph Priestley (from Yorkshire) published his Experiments and Observations on different kinds of air and was the first to prove the different qualities of the gases released by plants and the one?s exhaled by animals (mice). He discovered that, although a candle burned out in a closed container, when he added a living sprig of mint to the container, the candle would continue to burn. At the time, Priestley did not know of O, but he correctly concluded that the mint sprig restored the air that 2the burning candle (or mice which he used in a similar set of experiments) had depleted. As he still believed in the Phlogiston Theory he called the gas which was given off by plants dephlogisticated air because a candle would burn brightly in it. 1779 Jan Ingenhousz (from Breda, Netherlands): Experiments upon vegetables, discovering their great power of purifying the common air in the sunshine and of injuring it in the shade and at night, to which is joined a new method of examining the accurate degree of the atmosphere systematically investigated the release of dephlogisticated air from green parts of plants during day time especially from the lower side of leaves, while he also discovered that this depends on the effects of light as plants give off noxious air during night time. 1783 Priestley divulged his discovery personally to the eminent French chemist Antoin-Laurent Lavoiser who immediately understood the theoretical implications of it, as Priestly did not. Lavoiser had already announced, in 1772, that he was destined to bring about a revolution in physics and chemistry. Unlike the older scientists he realized that atmospheric air was not an element but a compound of gases, and he identified Priestley?s discovery as the active component of air for which he had been searching. He called it oxygen (Greek: acid former), in the belief that all acids contained it. 1796 After Ingenhousz learned about the findings of Lavoiser he elaborated on his on investigations and stated that green plants absorb carbon dioxide and release oxygen during day time. 1793-98 Hedewig, Schrank and Humboldt discuss the function of stomata. 1804 N. Theodore de Saussure (Switzerland):Recherche chimiques sur la végétation. He verified Ingenhousz?s hypothesis that plants assimilate carbon dioxide from the air while nitrogen and other nutrients are derived from the soil. Furthermore he was the first to distinguish systematically between the principles of assimilation and dissimilation. 1817 Pelletier and Caventou isolated the green substance in leaves and named it chlorophyll. 1840 Liebig?s findings help to establish the use of fertilizers in agriculture. 1845 Robert Mayer: Plants transform energy of sunlight into chemical energy. Energy can be transformed but not created or destroyed (first law of thermodynamics or energy conservation). See also: Hermann v. Helmholtz, “Über die Erhaltung der Kraft, 1847. 1845 Mohl: Discovery of starch associated with chlorophyll. 1851 L. Garreau: again delineated the differences between dissimilation and assimilation and emphasizes that dissimilation continuously takes place in all parts of a plant. 1864 Bossingault made the first accurate measurements of the gas exchange and found that the volume of O evolved and CO 22used up is almost unity. 1862-64 Julius Sachs (from Breslau) investigated the synthesis of starch under the influence of light and in relation to chlorophyll. He worked out the overall equation of photosynthesis: 6 CO + 6 HO + solar energy ; CHO + O. 22612621869-71 K.A. Timirjazev (from St. Petersburg) developed a method to investigate the spectrum of light and against the background of the law of energy conservation he stressed the pivotal role of green plants for the transformation of light energy into chemical energy. 1872 W. Pflüger defined respiration as a process located on the cellular level. 1883 The replication of chloroplasts was observed by F. Schmitz and A.F.W. Schimper. 1883 An elegant experiment was first performed by the German botanist Thomas Engelmann. He illuminated a filamentous alga with light that had been passed through a prism, thus exposing different segments of the alga to different wavelengths of light. Engelmann used aerobic bacteria, wich seek oxygen, to determine which segments of the plant were releasing the most O. 2Bacteria congregated in greatest density around the parts of the alga illuminated with red and blue light. He also demonstrated the correspondence between the action spectrum of photosynthesis and the absorption spectrum of chlorophyll. 1887 Sachs reviewed the current knowledge and concluded: the most definite proof that ... the chlorophyll body [chloroplast] itself is the organ which decomposes carbon dioxide and consequently assimilates this organic substance, is afforded by the fact ... that the first recocnizable products of assimilation [starch] appear not in any haphazard place in the green cell, but in the chlorophyll body itself. Until the findings of Hill and van Niel scientists now believed that chloroplasts are the site of

complete photosynthesis because it was still thought that oxygen evolution and CO assimilation were inseparable events. 21888 Pfeffer: Handbuch zum Stoffwechsel und Kraftwechsel in der Pflanze 1900 R.Höber establishes the measurement of pH-values. 1901 K.A. Timirjazev studied drought tolerance and was the first to reveal the antagonism between transpiration and assimilation. 1902 Max Rubner was able to prove that th first law of thermodynamics (energy conservation) is also applicable to organisms (Gesetze des Energieverbrauchs im Organismus). 1905 The British plant physiologist Blackman interpreted the shape of the light-saturation curves by suggesting that photosynthesis is a two-step mechanism involving a photochemical or light-dependent reaction and a non-photo-chemical or light-independent reaction. th1895-06 Until the end of the 19 century scientists still assumed that the synthesis and degradation of intracellular substances would require the living protoplasm of intact cells. However, this changed with the discovery of isolated biological enzymes which were still active (Bertrand, 1895 and Buchner, 1897). Thus, the enzyme theory of metabolism was established and the field of Plant Biochemistry developed rapidly. Among the founding fathers were F. Czapek (Biochemie der Pflanzen, 1905) and F.F. Blackman (1906) who wrote about the function of the protoplasm: [It is] ... a complicated congeries of catalytic agents, adapted to the metabolic work that the cell has to do. 1905 F.F. Blackman and G.L.C. Matthaei investigated the influence of the factors light and temperature on the assimilation of CO 2and were able to distinguish between a temperature-dependent but light-independent and a light-dependent but temperature-independent reaction. They suggested a two-step mechanism involving a photochemical or light reaction and a non-photochemical with a high temperature coefficient (indicative of an enzymatic reaction). 1905 K.S. Merezkovskij first claimed an endosymbiotic origin of chloroplasts. 1905-36 Between 1905 and 1916 Albert Einstein published his theory of relativity while the uncertainty principle was formulated in 1927 by the German Werner Heisenberg, and consequently the mathematical formulation of quantum theory was developed in the preceding years which also enabled physicists to calculate and predict the emission of energy from the sun. Accordingly the sun is considered to be a giant thermonuclear reactor. The energy it emits comes from fusion reactions much like those that occur in a hydrogen bomb. Four hydrogen atoms fuse to form one helium, which has a mass less than the total mass of the hydrogen atoms. The lost mass is converted to energy, and by means of Albert Einstein?s famous formula (E=mc?) it was calculated that each minute about 120 million tons of solar matter are converted to a colossal amount of energy that radiates out into space. A small fraction of that energy reaches Earth in the form of electromagnetic energy (waves/particles), taking only a few minutes to make the trip (the speed of light is about 300000 km/sec). 1906 M.S. Cvet (or Tswett from Italy) introduced chromatography by separating the pigments of leaves. 1909 Michaelis first invented electrophoresis. 1909 C. Correns identified inheritable factors in plastids. 1913 R. Willstätter determined the overall structure of chlorophyll. 1915 Dixon established his hypothesis on transpiration and the ascent of sap in plants (Kohäsonstheorie). 1923 Hevesy was the first to use to apply the tracer technique to plant physiology as he followed the passage of radioactive isotopes through plants. 1926 E.Münch studied source-sink-relation and proposed his hypothesis (Druckstromtheorie). 1926 Warburg postulated that CO molecules remain adsorbed on the chloroplast surface until succesive step-wise reduction by 2light-activated chlorophyll converts them to glucose and liberates oxygen. 1927 Osterhout is the first to measure a membrane potential. 1927 Warburg and Negelein developed spectrophotometry. 1929 ATP described by K. Lohmann. 1930 Prior to about 1930, many investigators in the field believed that the primary reaction in photosynthesis was splitting of CO by 2light to carbon and O, while the carbon would subsequently be reduced to carbohydrates by water in a different set of 2reactions. 1930-41 It was found that some bacteria can assimilate CO and synthesize carbohydrates without the use of light energy. Subsequently, 2the Dutch microbiologist van Niel who worked mostly in California showed that some bacteria can assimilate CO in light without 2evolving O. After manifold experiments with these sulphur bacteria C.B. van Niel concluded that the basic principle of 2photosynthesis was a light-driven exchange of hydrogen from a donator (HA) which was to be oxidised to CO (acceptor) 22which would consequently be reduced. On the basis of this he postulated that in plants the hydrogen had to be derived from the splitting of water. Formerly, many investigators believed that the primary reaction in photosynthesis was splitting of CO by light 2to Carbon and O. 21932 Emerson and Arnold measure the length of the light-independent reaction with light flashes lasting less than a millisecond. Flash saturation occurred in normal cells when one molecule of O evolved from 2500 chlorophyll molecules. Emerson and 2Arnold concluded that the maximum yield of photosynthesis was not determined by the number of chlorophyll molecules capturing the light but by the number of enzyme molecules that carry out the light-independent reactions. 1935 Danielli and Davson?s first model of a membrane containing proteins. 1937 R. Hill successfully isolated chloroplasts and separated them from the respiratory particles (the first mitochondria were purified in 1951 by A. Millerd) by differential centrifugation. By using the affinity of haemoglobin to oxygen he was able to measure the release of oxygen from isolated chloroplasts by the use of spectroscopy in the presence of suitable electron acceptors (oxidants) which became reduced. This light-driven transfer of electrons from water to non-physiological oxidants is now known as the Hill reaction. He proved that chloroplasts were able to produce oxygen (split water) in the absence of CO. However, the 2O-evolving chloroplasts prepared by Hill failed to photoreduce CO, the natural electron acceptor of photosynthesis. It was 22then thought that CO assimilation is impossible in a cell-free system. 21937 H. Krebs: The citric acid cycle. 1938 R. Hill observed the splitting of water with isolated chloroplasts. 1939 H. Fischer and W. Wenderoth determined the chemical structure of the chlorophylls and their conjugated double bonds. 181939 Kamen and S. Ruben and co-workers established the decomposition of HO and the resulting liberation of O by using O. 22214Furthermore, they were the first to use the tracer technology for work with CO. 21925-53 A number of other enabling techniques for research in plant physiology like climatized growth chambers, ultracentrifugation, electron microscopy, x-ray-diffraction, TL- and GL-chromatography, fluorescence spectrophotometry or the use of aphids to study the phloem became available. 1941 Fritz Lippmann and Hermann Kalckar defined the general metabolic function of ATP. 1943 The first comprehensive hypothesis for a role of ATP in photosynthesis was formulated by Ruben in 1943 who postulated that photosynthetic CO assimilation is a dark process, dependent on photochemically generated ATP and reduced pyridine 2nucleotides. However, at the time there was no experimental evidence for this and Ruben?s hypothesis did not fare well in the succeeding years. It was deemed incorrect on both theoretical and experimental grounds. 1944-45 R.L. Emmerson also postulated a key role for ATP during photosynthesis based on experiments with Chlorella: The sole function of light energy in photosynthesis is the formation of energy-rich phosphate bonds. This proposal was strongly opposed by Rabinowitch (1945) who found the supporting evidence inadequate and considered the idea theoretically unsound: What good can be served by converting light quanta (even those of red light, which amount to about 43 kcal per Einstein) into phosphate quanta of only 10 kcal per mol ? This appears to start in the wron direction - toward dissipation rather

than toward accumulation of energy. 1944 R.Consden and co-workers adapted paper chromatography to the analysis of amino acids and it is now also becoming useful for other compounds. 1945-57 In the year 1945 Melvin Calvin, Benson and colleagues had started a series of investigations that resulted in the elucidation of the light-independent reactions of photosynthesis. They used the unicellular green alga Chlorella in their work because it was easy to let these organisms grow consistently. Their findings later proved to be pertinent to a wide variety of organisms ranging from photosynthetic bacteria to higher plants.The aim of their work was to determine the pathway by which CO becomes fixed 21414into carbohydrate. The experimental strategy was to use radioactive carbon (C) as a tracer. CO was injected into a 2suspension of algae that had been carrying out photosynthesis with normal CO. The algae were killed after a certain time (5-60 2seconds) by dropping the suspension into alcohol. The cells were homogenized and the radioactive compounds in the algae were separated by paper chromatography. The paper chromatogram was then pressed against photographic film, which became black where the paper contained a radioactive spot. In his Nobel Lecture (1957), Calvin noted that their primary data resided in the number, position, and intensity - that is, radioactivity - of the blackened areas [on paper chromatograms]. The paper ordinarily does not print out the names of these compounds, unfortunately, and our principal chore for the succeeding ten years was to properly label those blacked areas on the film. The radiochromatogram of the algal suspension after sixty seconds of illumination was so complex that it was not feasible to detect the earliest intermediate in the fixation of CO. However, the pattern after only five seconds of illumination was 2much simpler. In fact, there was just one prominent radioactive spot, which proved to be 3-phosphoglycerate. The formation of 3-phosphoglycerate as the first detectable radioactive intermediate suggested that a two-carbon compound is the acceptor for the CO. This proved not to be so. The actual reaction sequence is more complex. The CO molecule condenses with 22ribulose 1,5-bisphosphate to form a transient six-carbon compound, which is rapidly hydrolysed to two molecules of 3-phosphoglycerate. This reaction is driven by a protein called ribulose 1,5-bisphosphate carboxylase (commonly called Rubisco). Rubisco is located on the stromal surface of the thylakoid membrane and it is very abundant in chloroplasts, comprising more than 16% of their total protein. In fact, Rubisco is probably the most abundant protein in the biosphere, and it is one of only two proteins in the world which are able to bind CO and thus fix it into organic moleculesThe first detectable 2. product of this reaction is 3-phosphoglycerate. 1948 Current knowledge of the occurrence of quinones in the photosynthetic apparatus began with the demonstration that the vitamin K of leaves is localized in chloroplasts by Dam. 1321948-52 The first experiments of Aronoff and Calvin (1948) with the sensitive P technique used to test the ability of isolated chloroplasts to form ATP (on illumination) gave negative results and seemed to contradict the hypotheses of Rubens and Emmerson (s. above). The most plausible model for ATP formation in photosynthesis became one that envisaged a +collaboration between chloroplast and mitochondria. Chloroplasts would, in that scheme, reduce NAD photochemically and mitochondria would reoxidize it with oxygen and form ATP via oxidative phosphorylation according to the model of Vishniac and Ochoa (1952). But this model also posed a serious physiological problem as photosynthesis in saturating light can proceed at a rate almost 30 times greater than the rate of respiration. It was difficult to see, therefore, how the respiratory mechanisms of mitochondria could cope with the ATP requirement in photosynthesis. 14 1948-56 Brown and Frank (1948) thought they had proved by experiments with CO that isolated chloroplasts were not able to fix 2CO and together with the findings of van Niel and Hill that the photoproduction of oxygen by chloroplasts was basically 2independent of CO assimilation this led scientists ti conclude that the chloroplast was an: incomplete machine ... [which] can 2evolve O from HO in light provided an external substance B is present ... [and that] this requirement for B can be attributed 22to a deficiency in the enzymatic mechanism operative in various steps by which CO is converted into the normal assimilation 2+product. (van Niel, 1956). A few years earlier, when the oxygen evolution by chloroplasts was linked (via NADP) to CO 2assimilation (s.a.) by a malic enzyme of cytoplasmic origin in 1951 Arnon similarly concluded that isolated chloroplasts lack either either the appropriate hydrogen carriers or enzymes, or both, required for the reduction of carbon dioxide by the hydrogen derived from the photolysis of water, and that in the intact cell these factors are found chiefly or wholly outside the chloroplasts. In sum, it contrast to the prediction of Sachs in 1887 (s.a.) it now seemed well-established that the chloroplast was indeed a system much simpler than that required for photosynthesis and was the site of only the light absorbing and water splitting reactions of the overall photosynthetic process as Lumry put it in 1954. Thus at midcentury, the concept of CO assimilation by chloroplasts was abandoned (for a few months/years). 21949 Katz was the first proponent of the idea that photon energy is used in photosynthesis to transfer electrons rather than cumbersome atoms. 1950 The Society for Experimental Biology held Symposium on Carbon Dioxide Fixation and Photosynthesis at Sheffield, England, and the Proceedings of that Symposium (published in 1951) provide a reliable record of some of the main experimental and conceptual realities that characterized photosynthetic research up to the midtwentieth century. 1951 Calvin finally proved that phosphoglycerate is the first product of carbon dioxide assimilation (see above). 1951 The photosynthetic reduction of NADP to NADPH by isolated chloroplasts with simultaneous evolution of O was reported in 221951 by three different laboratories, including Arnon?s. Thus the first link between oxygen evolution by chloroplasts (via +NADP) to CO assimilation had been found, although the significance of this was not clear to the majority of researchers. 21951 Hill confirmed that the photoproduction of oxygen by chloroplasts was basically independent of CO assimilation. In 1951 Hill 2wrote: If we break the green cell, it is possible to separate the fluid containing the chloroplast and chloroplast fragments from the tissue. This green juice can no longer assimilate carbon dioxide, but in the case of many plants the insoluble material, for a time at least, is still capable of giving oxygen in light ... . This evolution of oxygen takes place in the presence of soluble substances contained in the plant juice or better by the addition of reagents which can act as hydrogen acceptors... . The cell preparations then though not showing photosynthesis, can still convert light into a form of chemical energy ... . We may very well call this the chloroplast reaction. This chloroplast reaction became to be known as Hill Reaction. 1951 Burk and Warburg divided the photosynthetic energy conversion process into two parts: (1) a one-quantum light reaction which liberates oxygen and converts a bound species of CO into carbohydrate; and (2) a dark oxidative reaction which 2provides the rest of the total energy needed for CO assimilation. However, subsequent work with isolated chloroplasts soon 2yielded evidence that one-quantum light reactions in photosynthesis are concerned not with CO assimilation and terminal 2events in oxygen evolution but with intermediate light-induced electron transfer steps. 1952-73 Cytochromes were described and characterised as electron carriers by a number of research groups (among them: Hill, Lundegard, Arnon, Anderson and Bendall). 1953 In the early 1950s, when it appeared that isolated chloroplasts did not assimilate CO, photosynthesis came to be regarded, like 2fermentation in the days of Pasteur, as a process that could not be separated from the structural and functional complexity of whole cells. Thus, Rabinowitch (1953) concluded that the task of separating it [photosynthesis] from other life processes in the cell and analysing it into its essential chemical reactions has proved to be more difficult than was anticipated. The photosynthetic process like certain other groups of reactions in living cells seems to be bound to the structure of the cell. It cannot be repeated outside that structure. 1954 Frenkel observed the formation of ATP from ADP and Pi on illumination of membrane preparations. 1954 The principle of photophosphorylation (i.e. light-induced synthesis of ATP from ADP and Pi) was proposed and demonstrated by Arnon. Isolated chloroplasts were thus able to convert light energy into chemical energy and trap it in the pyrophosphate bonds of ATP. Several uinque features distinguished this photosynthetic phosphorylation (photophosphorylation) from

substrate-level phosphorylation in fermentation and oxidative phosphorylation in respiration: (1) ATP formation occured in the chlorophyll-containing lamellae and was independent of other enzyme systems or organelles; (2) no energy-rich substrate, other than absorbed photons, served as a source of energy; (3) no oxygen was produced or consumed; (4) ATP formation was not accompanied by a measurable electron transport involving any external electron donor or acceptor. The next objective in this avenue of research was to explain the mechanism of photophosphorylation as it seemed unlikely that light was directly involved in the formation of ATP itself because ATP formation was already known to be a reaction which universally occured in all cells independently of photosynthesis. Light ernergy, therefore, had to be used in photophosphorylation before ATP synthesis and in a manner unrelated to CO assimilation or oxygen evolution and the most 2probable mechanism for such a role seemed to be a light-induced electron flow (Levitt) which was known to be involved in the ATP formation in nonphotosynthetic cells. 1954 D.I. Arnon and co-workers provided direct evidence for the long standing hypothesis that CO assimilation takes place in 2chloroplasts. 1954-56 Arnon, Trebst, Losada and Tsujimoto succeeded in distinguishing light-dependent from light-independent reactions by separating the chloroplasts into a granal and stromal fraction and demonstrating ATP and NADPH formation in the grana in 2light, and the light-independent CO reduction by enzymes of the stroma. 2Later the group discovered cyclic photophosphorylation, which produced only ATP, and proposed its role for providing additional ATP for CO fixation in chloroplasts. For this reason, prior to the discovery of noncyclic photophosphorylation 2(1957-61), cyclic photophosphorylation was regarded as the only source of ATP for CO assimilation. 2141955 Arnon: The ability of whole isolated chloroplasts to carry out the synthesis of starch from CO and water without the aid of 2external enzyme systems and organic substrates provided incontrovertible evidence for concluding that chloroplasts are indeed, as was asserted but not documented in earlier periods the sites of both O evolution and CO assimilation. 221956 The sraw-coloured chloroplast extract (stroma) obtained after osmotic shock treatment of chloroplasts was found to contain all the necessary components for CO fixation (Whatley). 21956 The key role of pyridine nucleotides was confirmed by A. San Pietro and H.M. Lang 1956 The existence of photochemical reaction centres was proved for bacteria and higher plants (L.N.M. Duysen and B. Kok) 1957 R. Emerson confirmed the existence of two photosystems in the thylakoid membrane which was later verified by Döring et al. in 1967. 1957-61 James and Das (1957) and Lundegard (1961) confirmed that chloroplasts cannot respire and lack the terminal enzyme of respiration (cytochrome oxidase). This feature ensured that which was generated had to be produced photochemically and not formed by respiration. 1957-61 Arnon and co-workers (1957) isolated a soluble protein, first named the TNP-reducing factor, which when added to low ++concentrations of broken chloroplasts (grana) catalysed the photoreduction of substrate amounts of NADP and not NAD. This facto later become recognized as a synonym for ferredoxin, an electron carrier protein that interacts with the enzyme ferredoxin-NADP reductase. However this reduction was first thought to be wholly unrelated or even antagonistic to photophosphorylation. It was therefore a surprise when the group of Arnon provided direct experimental evidence for a +coupling between photoreduction of NADP and the synthesis of ATP in 1958. ATP formation was stoichiometrically coupled +with a light-driven transfer of electrons from water to NADP. It was envisaged that a chlorophyll molecule excited by a captured +photon transfers an electron to NADP. It was postulated that electrons thus removed from chlorophyll are replaced by -+electrons from water with a resultant evolution of oxygen. In this manner, light would induce an electron flow from OH to NADP and a coupled phosphorylation. Because of the unidirectional or noncyclic nature of this electron flow, this process was named noncyclic photophosphorylation (Arnon, 1960 and 1961). 1958 The experiment of Trebst, Tsujimoto and Arnon (1958, Nature 182) directly substantiated the validity of the concept proposing a light-dependent and a light-independent phase of photosynthesis through a physical separation of the two phases after fractionating isolated chloroplasts. First, they were able to show that chloroplasts produced oxygen, ATP and ATP in a light-dependent manner in the absence of CO. Then, after the lamellar (membranous) portion had been discarded and the 2remaining chlorophyll-free stroma was provided with CO, ATP and NADPH the fixation of CO proceeded in a 22light-independent manner. 1959 Robertson?s concept of a unit membrane. 1959-61 A hypothesis was put forward by Arnon that a chlorophyll molecule, on absorbing a quantum of light, becomes excited and promotes an electron to an outer orbital with a higher enrgy level. This high-energy electron would then be transferred to an adjacent electron acceptor molecule, a catalyst (A) with a strongly electronegative oxidation-reduction potential. Therfore, the transfer of an electron from excited chlorophyll to this first acceptor would be the proper energy conversion step and thus terminate the photochemical phase of the process. By transforming a flow of photons into a flow of electrons it would constitute a mechanism for generating a strongly electronegative reductant at the expense of the excitation energy of chlorophyll. Several of the exergonic electron transfer steps, particularly those involving cytochromes, were thought to be coupled with phosphorylation. At the end of one cycle, an electron would be returned to the electron-deficient chlorophyll molecule and the quantum absorption process could be repeated. 1960 The groups of Losada and Trebst identified and characterized the CO-fixing enzymes. 21960 Emerson and co-workers in Illinois measured the quantum yields of photosynthesis in algae and observed that the average quantum yield obtained by using two superimposed light beams of different wavelengths was higher than the average quantum yield obtained by using the two beams separately. To explain this enhancement of quantum yield, Emerson and Rabinowitch in 1960 postulated the existence of two light reactions in photosynthesis. 1960 R. Hill and F. Bendall reviewed the photochemical and thermodynamic studies of the preceding years and put forward the Z-Scheme of electron transport showing the operation of two photosystems, in a series, in photosynthetic electron transport and phosphorylation. These led to the development of the idea of two photosystems operating in a series; PS II in which O 2evolution occurs, and PS I in which ferredoxin is photoreduced and NADPH is formed. 21960-61 Plastocyanin, a copper-containing protein, was discovered by Katoh in Chlorella and later in leaf tissue and chloroplasts. 1961-62 Losada and co-workers experimentally separated noncyclic noncyclic photophosphorylation by isolated chloroplasts into two light reactions: one light reaction photooxidised water, yielded oxygen, and reduced dichlorophenol indophenol (DCIP). A +second light reaction gave a photoreduction of NADP by reduced DCIP and a coupled photophosphorylation. Duysens and Amesz proposed that photosynthesis in higher plants consisted of two light reactions operating in series: a long-wavelength, +photosystem I, which oxidizes cytochrome f and photoreduces NADP, and a short-wavelength, photosystem II, which reduces cytochrome f and produces oxygen by dehydrogenation of water. 1961-66 P. Mitchell (Nature 191, p.144-148) published his work on Coupling of phosphorylation to electron transfer by a chemiosmotic type of mechanism. +Even after the mechanisms of carbon fixation via ribulose 1,5-bisphosphate was known the fate of protons (H) released after the splitting of water and the exact mechanism for the synthesis of ATP via the usage of high energy electrons from the electron transport chains remained an enigma for a long time. For some time scientists assumed that excited electrons might be trapped by an activated protein which could then use their power to drive the synthesis of ATP. Investigators in many laboratories tried for several decades to isolate these putative energy-rich proteins, but none were found. A radically different mechanism, the chemiosmotic hypothesis, was postulated by Peter Mitchell in 1961. He proposed that electron transport and ATP synthesis are coupled by a proton gradient across the membrane rather than mediated by an activated protein. According to this model the

+splitting of water and the electron transport chains induce an increase of the H-concentration in the thylakoid space. As a consequence a proton gradient across the thylakoid membrane is generated and finally the movement of protons along this gradient (back to the stroma) can be used to synthesize ATP. Thus, Peter Mitchell speculated that a so called proton-motive force drives the activity of the ATP synthase. A revealing experiment which was performed by André Jagendorf in the year 1966 provided strong support for Mitchell?s hypothesis that ATP synthesis is driven by a proton-motive force. Jagendorf was able to create an artificial pH gradient across thylakoid membranes of isolated chloroplasts. In order to achieve this he first soaked isolated chloroplasts in a pH 4 buffer for several hours. These chloroplasts were then rapidly mixed with a pH 8 buffer containing ADP and P. As a result of this the pH of the stroma suddenly increased to 8, whereas the pH of the thylakoid space remained at 4. A burst of ATP synthesis then accompanied the disappearance of the pH gradient across the thylakoid membrane. Thus, the thylakoid membrane makes ATP as hydrogen protons diffuse from the thylakoid compartment back to the stroma through the ATP synthase complex, whose catalytic heads use this proton-motive force to synthesize ATP on the stroma side of the membrane, where it is used to help drive the sugar synthesis during the Calvin cycle (light independent reactions). 1961 DNA found in plastids (Ris and Plaut) 1962-65 Mortenson described a nonheme-iron-containing protein they isolated from Clostridium pasteurianum and other bacteria which was later found to function as an electron carrier and named it ferredoxin. A connetion between ferredoxin and +photosynthesis was established when Tagawa and Arnon crystallized it and found it to mediate the photoreduction of NADP by spinach chloroplasts. Nonetheless, in 1963 Shin and co-workers demonstrated that ferredoxin could not react directly with +NADP, and Shin and Arnon confirmed in 1965 that a photochemically reduced ferredoxin transferred electrons to a +flavoprotein enzyme called ferredoxin-NADP reductase. Thus, the first possible sequence of events leading from photoxidised chlorophyll via electron carriers to NADPH had been unravelled. 1965/66 Kortschak et al. and Hatch & Slack described a very efficient CO binding mechanism which is used in some tropical grasses 2before the calvin cycle (C4-plants). 1965 Pressmann identified ionophores in membranes. 1965 The ultrastructure of chloroplasts was revealed by A. Frey-Wyssling and K. Mühlethaler and their structure was found to agree well with physiological data and hypotheses. 1965 Arnon and Crane identified seven benzoquinones as normal constituents of chloroplasts (plastoquinones A, B, C and D and α-, β-, and γ-tocopherolquinone. 1970 David Walker was able to demonstrate in isolated `intact? chloroplasts the obligatory coupling of photosynthetic CO uptake 2and O evolution and later he also confirmed the required movement of sugars (and phosphate) in and out of the chloroplast for 2CO fixation to occur. 21970 The theory of endosymbiosis was first formulated by Lynn Margulis 1971 Vernon summarized the current concept of the pigment assemblies in thylakoids including accessory pigments and reaction center chlorophyll molecules. 1972 The Fluid-Mosaic-Model by Singer and Nicholson. 1988 The most notable achievement in the photosynthesis field in recent years was the crystallization of the reaction centre from a purple photosynthetic bacterium and the determination of its structure by X-ray crystallographic analysis by Deisenhofer, Huber and Michel in Germany for which they were awarded the Nobel Prize in 1988. Crystallization of the light-harvesting complex and photosynthetic reaction centres from selected plants and cyanobacteria have been achieved. High-resolution X-ray analysis of these crystals in the dark and light phases, which is in progress, would help to unravel the structure-function relationships og the reaction centre components and to understand the molecular processes associated with O evolution. 2

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