4.6 Cursory Elements of Photobiology
4.6.1 Photosynthesis
As it is well known, the history of the chemistry occurring in chlorophyll photo- synthesis [166–168] is intimately bound to that of chemistry itself, in particular to the discovery by Priestly, Scheele, Lavoisier, and others that air contained a gas, oxygen, that was necessary for life of animals and burning of a candle, and was produced by green plants. The photoinduced activity of leaves was studied in detail by Senebier in the eighteenth century [169–171]. He measured the gas development when green plants were illuminated, depending on the “quality” of air used (in modern terms, oxygen—dephlogisticated air—was evolved and CO2—fixed air—was absorbed), and ascertained that this phenomenon was due to light, not to heat. He further attempted to determine which range of visible light was more effective by coloring the water bath with dyes that filtered a part of the spectrum, but had not sufficient pieces of evidence for drawing conclusions (Fig.4.12).
Fig. 4.12 The experiments by Senebier [169], who measured the quantity and quality (allowing or no respiration) of air liberated around a leaf illuminated by light
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In the following decades, the absorption of carbon dioxide was demonstrated by several scientists and was generally referred to as “carbon assimilation.” In 1862, Sachs [170,171] observed the formation of starch in the granules of chlorophylls.
However, in 1893 Barnes pointed out that in animal physiology the term “assim- ilation” was largely used with reference to the transformation of food into tissues.
To eliminate any confusion, he proposed the use of new terms for designing the function of green plants and proposed photosyntax or photosynthesis. Although he preferred the first, the latter one came into general use [172,173].
In an advanced work, Timiriazeff in 1877 exposed five reversed test tubes to light dispersed by a prism, respectively, to the furthest part of the red, to red in correspondence to the chlorophyll absorbance maximum, to orange, yellow, and green [174]. The green organs to be examined were placed in the tube, along with a measured volume of air added with 5 % CO2. After exposure to dispersed solar light, the amount of CO2 consumed was determined and shown to increase in passing from green to yellow, orange, and the red light corresponding to the chlorophyll maximum, whereas extreme red (that was not absorbed) showed no consumption, but rather some formation of CO2, due to respiration. Thus, provided that it was absorbed, light energy was converted into chemical energy via reduction of carbon anhydride.
As for the active pigment, extraction with alcohols of dried leaves gave a dark green, resinous material. The properties of this material (soluble in alcohol, ether, alkalis, cold sulfuric acid, reacting with chlorine, nitric acid) suggested that this had little relation with vegetal resins and rather resembled vegetal dyes. In view of “its properties and the role that it plays in plants economy” such a compound deserved
“to be considered as an immediate principle of plants” and the name “chlorophyll”
was proposed in 1818 by Pelletier and Caventou [175]. That different dyes were present in leaves was later recognized, but separation was no easy task. An in-depth study by Willsta¨tter based on preferential distribution of the colored compounds present in organic solvents vs. acids of various concentration led to the isolation of chlorophyll a and b and to a reasonable attribution of the chemical structure [176]. The significance of chlorophyll photosynthesis in global energy exchange was clearly evidenced by Mayer in his formulation of the first principle of thermo- dynamics [177]. Nature, he thought, has given itself the mission to capture solar light and to transform this, which is the most rapidly moving force in a solid form.
With this target, it has populated the Earth surface of organisms able to transform this force into a chemical difference. These areplants. “The plants word builds a reservoir, in which the solar rays are fixed and are transformed into forms suitable for their use. An economical preoccupation, to which the physical existence of manhood is necessarily linked, and that causes an instinctive good feeling when anyone looks to a rich vegetation,” testifies how deeply mankind recognizes the importance of chlorophyll photosynthesis. The question has to be confronted, whether plants during their life transformed chemicals or were able to create them must be answered, and it was actually clear that the whole of the matter plants accumulated arose from the environment. Every gram of a tree weighing several tons has been taken from there. Matter could not be generated, but only
108 4 Some Paradigmatic Topics
transformed. More precisely, carbon dioxide was reduced. Plants were able to exert their chemical function only under the influence of light, and no such result could be obtained by heating. Light, he thought, seemed to be the only physical force (perhaps along with electricity) able to be transformed into a difference in chemical work.
By using the experimental setup shown in Fig.4.13, it was found that the amount of carbon dioxide “assimilated” at equal light intensity falling on the same surface was the same for leaves of different plants [178,179], but this value increased with temperature, more or less rapidly for different leaves. This fact was attributed to the intervening of some thermal activated process. In every case, an optimal value of assimilation was attained and was not overcome by a further increase either in light intensity or in CO2concentration.
The way in which carbon anhydride from the atmosphere was absorbed and chemically transformed was the subject of many studies. The reactions involved could be rationalized as redox processes, as was demonstrated by Wurmser [180], but the chemical reactions actually involved required much work for the
Fig. 4.13 The energy exchange was assessed by Blackman and Matthaei in an thermal bath [178, 179]. This was exposed to solar light on the roof of the institute. The temperature was controlled by running water (entering through C and outflowing from D) and measured by thermometer G. Carbon dioxide from a generator and air were introduced from A, which led to a thrice bent tube ensuring that it equilibrated the temperature with the bath. The gas leaving the chamber through B was dried through a tube of calcium chloride and the CO2present was determined by passing through a baryta tube
4.6 Cursory Elements of Photobiology 109
identification. A rationalization was presented by Willsta¨tter and was based on the reaction of a complex that carbonate anion formed with chlorophyll. Irradiation was proposed to cause a rearrangement to a peroxidic structure that then decayed spontaneously with production of oxygen and formaldehyde that then polymerized to sugars (Scheme4.22) [181].
In Willsta¨tter’s mechanism, this would have required that chlorophyll was constantly associated with carbon anhydride; otherwise the short lifetime of exited state would have caused a large loss. Traces of formaldehyde were detected from illuminated leaves, but a relation with oxygen developed was not demonstrated, and at any rate the process occurring in chloroplast was not duplicated in solution, suggesting a role of something more complex than a single molecule of chlorophyll in chloroplasts function.
The way in which carbon anhydride from the atmosphere was absorbed and chemically transformed was the still quite far from rationalization. The reactions involved could be rationalized as redox processes, as was demonstrated by Wurmser [180], but the chemical reactions actually involved required much work for the identification. Scientists had to confront the problem of how one of the most stable molecules, carbon dioxide, came to be reduced and from where the required hydrogen came. Discarded, after 1850, the role of “vital force,” various hypotheses were put forward. Liebig [182] proposed that various organic acids, such as oxalic, malic, tartaric and citric acids, were the intermediates in the stepwise reduction of CO2 to carbohydrates: “If one considers that unripe fruit, for example, grapes, cannot be enjoyed due to their high acid content; that in sunlight these fruits behave in the same way as leaves, namely, that they are capable of absorbing carbonic acid and releasing oxygen (de Saussure); that at the same time as the acids decrease, the sugars increase: in view of these points, one cannot reject the idea that the carbon of the organic acids in unripe fruit becomes part of the sugars in ripe fruit; that, therefore, the acid is transformed into sugar, effected by the release of oxygen and the components absorption of water” [183]. Another great chemist, Baeyer, opposed this view and suggested that rather “when sunlight strikes chlorophyll, which is surrounded by CO2, the carbon dioxide appears to undergo the same Scheme 4.22 Proposed
mechanism for
photosynthesis involving bonding of carbon dioxide and rearrangement to a peroxidic species
110 4 Some Paradigmatic Topics
dissociation as at higher temperatures: oxygen escapes and carbon monoxide remains bound to the chlorophyll (or rather forms a chlorophyll–CO2complex).
The simplest reduction of carbon monoxide is to the aldehyde of formic acid—it only needs to take up hydrogen, CO + H2¼COH2.
Under the influence of the contents of the cells, as well as through the alkalines, this aldehyde is then converted into sugar. [. . .] Glycerol could, in addition, be formed by the condensation of three molecules and the subsequent reduction of the thus formed glyceric aldehyde” [184]. As Baeyer observed, direct conversion of CO2to sugars was to be preferred to the other hypothesis, because actually organic acids were not accumulated in plants, or at least their content varied among different species and type of cells. The direct conversion of carbon dioxide into formaldehyde seemed to find support with the development of the chemistry of sugars by Emil Fisher and others at the end of the century, with the demonstration that homologation by reaction with formaldehyde was indeed possible for forming the most abundant sugars, hexoses [185]. However, repeated attempts definitely demonstrated that enhancing the formaldehyde content in the plant environment did not cause any increase in the rate of photosynthesis. Furthermore, formaldehyde was strongly toxic, and, at least in retrospect, it is difficult to understand why this mechanism, clearly too simplified, was universally accepted. As for the oxygen atom detached at the beginning of the process, this may lead to a peroxide:
unfortunately, again a toxic compound.
After the war, important advancements took place. In particular Otto Warburg introduced new experimental techniques [186]. The use of a sensitive manometer allowed us to measure microliters rather than milliliters of gas and thus to examine much smaller samples, over which it was easier to control the spatial and temporary uniformity of irradiation. The manometers were mounted on a thermostat, so that the vessels could be illuminated with light bulbs from below and were oscillated by an electric motor. Rather than leaves or whole plants, he used the easily grown unicellular green algae Chlorella. This allowed a much better control of experi- mental conditions, the use of small samples (the proportion of photosynthetic material was higher in this alga), and dispensed with competing paths, such as reflection and filtering of light by other tissues generally occurring with leaves.
Under such controlled conditions, suspensions of cells were illuminated, the light intensity measured by a sensitive vacuum bolometer (developed by the father of Otto, Emil Warburg [187,188]), and the development of oxygen was measured.
The actual photochemical aspects were explored in a new perspective, in particular the use of rotating sectors allowed us to study reactions where light intensity was the rate-limiting step, the action of inhibitors was recognized, as well as the dependence on carbon dioxide concentration and lamp intensity and wavelength.
On the basis of such experiments, Warburg proposed that the primary photochem- ical process involved the pigments of the cell and produced a strongly reducing agent. Other thermal reactions occurred at the surface of cells and caused the formation of transients complexes with CO2 and liberation of oxygen [186]
(Fig.4.14).
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Consideration of the energy involved in the process and of the energy of the quantum absorbed by chlorophyll showed that interaction with at least three excited molecules was required for each molecule of CO2formed [186]. The investigations by Emil Warburg, fostered also by the formulation of the Einstein equivalence law (1905 in the first formulation; see Chap.2), and by his son Otto gave a quantitative framework to photosynthesis substituting well-defined chemical measurements to qualitative observations [188].
The path toward the full rationalization was still long, however, key problems being whether chlorophyll not only absorbed light but participated into the reaction (e.g., by forming initially a hydrogenated, or dehydrogenated, form, both hypoth- eses were considered) that complexed carbon dioxide, the role of water as hydrogen source, the mechanism of oxygen liberation (generally assumed to result from enzymatic splitting of hydrogen peroxide), the number of quanta required for the liberation of one molecule of oxygen, and how the energy could be stored. The measurement of quantum yield of photosynthesis became one of the most investi- gated quantities, and the object of a long controversy between Warburg (who found 4–5 quanta to be required for the reduction of one molecule of CO2, and Emerson, who found 8–12) [189,190], a controversy that was resolved only when Emerson showed that, contrary to naı¨ve expectation, the proportion of CO2and O2formed was not 1 and not constant during photosynthesis, so that the amount of gas evolved could not be directly used for measuring of quantum yield. Actually, their propor- tion varied according to the time of irradiation and intensity of the light beam, and Fig. 4.14 Apparatus used by O. Warburg for measuring oxygen evolution during photosynthesis
112 4 Some Paradigmatic Topics
futher flashing experiments proved that a high number of chlorophyll molecules (2400) were involved in such a reaction [189,190] and the participation of a second quantum of light. This extraordinary work that required the combined effort of biologists, physiologists, chemists, and physics and was concluded in mid-twentieth century is well known and cannot be summarized here, but detailed accounts are available.
Studies on organisms different from green plants showed that assimilation of carbon promoted by light could proceed through different reactions (in Fig.4.15a
“light cabinet” is shown where illumination is ensured by four incandescent lamps, which made easier to carry out experiments under uniform conditions).
Thus, in purple and green sulfur bacteria the photosynthetic reaction occurred according to the general equation 4.3 (A, sulfur or a sulfur compound)
CO2þ2 H2A!CH2OþH2Oþ2 A ð4:3Þ Green bacteria dehydrogenated hydrogen sulfide to sulfur, while under favorable conditions purple bacteria dehydrogenated hydrogen sulfide, sulfur, sulfite, and thiosulfate all the way to sulfate. This suggested that, if photosynthesis conformed to a single scheme, in green plants oxygen was liberated from water, not from CO2. (see eq. 4.4)
CO2þ H2O!CH2OþO2 ð4:4Þ Fig. 4.15 Light cabinet
used for the artificial illumination of plants
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The all-important issue of the timescale involved was confronted by various scientists by using intermittent irradiation. By using a neon arc pulsed by rectified current at 50 Hz, Emerson and Arnold showed that “photosynthesis involved a light reaction not affected by temperature, and capable of proceeding at great speed (in less than 105s), and a dark reaction dependent on temperature,” which required less than 0.04 s for completion at 25C and about 0.4 s at 1.1C. The light reaction was shown to depend on carbon dioxide concentration and to be inhibited by narcotics, neither of which characteristics were observed with the dark reaction, which was rather inhibited by cyanide. In subsequent experiments using cells varying in chlorophyll content, it was found that “the amount of chlorophyll present per molecule of carbon dioxide reduced per single flash was about 2480 molecules”
and “the time required for one unit in the photosynthetic mechanism to complete the cycle of photochemical” and thermal reaction was about 0.02 s at 25C. These findings were the basis from which the “trick” nature uses for capturing short-lived excited states via a multiple molecules antenna was discovered [189,190] and over the years developed into the system based on two photosynthetic units as is presently envisaged [191,192].