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Evolution of photosynthesis
Comp. Biochem. Physiol., 1962, Vol. 4, pp. 205 to 216. Pergamon Press Ltd., London. Printed in Great Britain
EVOLUTION OF PHOTOSYNTHESIS HANS G A F F R O N Department of Biological Science (Fels Fund). Florida State University, Tallahassee, Florida, U.S.A. PRIMARY PHOTOCHEMISTRY ON EARTH FoR the purpose of this essay I shall assume that the Proceedings of the First International Symposium on the Origin of Life on the Earth and recent reviews relating to our topic (see References) are generally available and therefore may serve as sources for detailed references. Without further discussion of these points where minor disagreements still exist let us presuppose the following as given. We have had on earth a succession of conditions during which the atmosphere changed from a generally reducing to a strongly oxidizing one. This change of environment was brought about by a rapid loss of hydrogen and a gradual accumulation of oxygen until the present quasi steady state was reached. The main source of free energy on the surface of the earth has been and still is, of course, solar radiation. This was (and is) occasionally supplemented here and there by localized volcanic action, which produces heat and reactive chemicals. The change from a reducing to an oxidizing atmosphere was accompanied by an attenuation of the solar spectrum in the ultraviolet region, to the effect that whatever part photochemistry played in the successive creation and accumulation of organic substances and later of living organisms, had to rely in the course of time on the utilization of smaller and smaller light quanta, diminishing in energy from 9 to 1.3 eV/mole but absorbed in increasing amounts by living matter. This decrease in available energy per absorbed light quantum means that the direct photochemical dissociation of water molecules into radicals, as it happened in the beginning, gave way to a very complicated mechanism which in our time achieves the same result with a succession of quanta absorbed by the green pigment chlorophyll. Furthermore, the photodissociation of the constituents of the earliest atmosphere such as H~, H~O, NH3, CH4, H~S, HCN, (O, CO, COs) and the subsequent chance recombinations lead to the accumulation of large amounts of aliphatic acid (e.g. CHzCOOH), amino acids (e.g. CH~NHzCOOH) and of small amounts of oxygen and the oxides of carbon. By the simple means of thermal dehydration, condensation, dehydrogenation, decarboxylation and photochemical interconversion that were often catalysed by metallic or oxidic surfaces or metal ions in solution, the earth's surface became (perhaps only in selected spots) enriched with a host of substances which were very likely the precursors to the substances now existing as parts of living cells. During the last decade we have become convinced that dicarboxylic acids, urea, purines, carotenes, porphyrins, sterols and 205
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perhaps many more such "biochemicals" can arise in a sequence of spontaneous reactions. We are here mainly interested in the first appearance of colored organic substances which broadened the spectrum of possible photochemical activities. For example, we have learned that the biosynthesis of porphyrin out of acetic acid and glycin proceeds over intermediate stages that can also happen spontaneously. To a part this has been proven experimentally. The important, often-told and wellknown story needs no repetition. The appearance of colored, fluorescent substances like the porphyrins must have accelerated the interconversion of organic substances tremendously. We shall concentrate our attention on the porphyrins because these molecules are among the few that apparently have exerted their catalytic powers throughout the eons that it took life to develop. This does not mean that other substances absorbing visible light could not have played important roles. The photodissociation of acetone, CH2.CO.CH 3 at low temperatures by ultraviolet, for instance, leads to the formation of diacetyl, CH3.CO.CO.CH ~. The latter, a yellow substance, is activated in blue light and has been shown to sensitize further photochemical synthesis ; but, on the other hand, diacetyl decomposes quite easily. The porphyrin ring, once formed, has, however, enormous stability in comparison with other active pigments. This may be the reason why porphyrin metal complexes have become the most widely distributed biocatalysts on earth. With this background we can address ourselves to the question of the evolution of photosynthesis, defined as the photochemical assimilation and transformation of carbon dioxide carbon into carbohydrate and other constituents of living cells. The task is to find the path that leads smoothly from the simpler reactions which illuminated pure chlorophyll displays in the test tube to the achievement of the living cell. Fifty years of experimentation with model reactions have enabled us to describe with satisfying consistency how chlorophyll reacts in homogenous solution after it has absorbed a quantum of visible light. Many kinds of compounds of importance to the living cell have been so tested. A few such reactions may be mentioned. In presence of oxygen it sensitizes the photoxidation of amines, purines, tyrosin, sulfhydryl groups and ascorbic acid with great efficiency. In absence of oxygen it may catatyse the transfer of hydrogen from a hydrogen donor to an acceptor (from ascorbic acid to pyridine nucleotide). As the only solute in organic solvents it is usually bleached in the light. Such anaerobic reactions are often reversed in the dark. Without causing a permanent chemical change the excited chlorophyll may also induce the isomerization from a cis into a trans configuration of an unsaturated substance like carotene. As much as these reactions have been studied and satisfactorily explained, any one or any combination of these are insufficient to account for the role of chlorophyll in the plant. For one thing the reactions observed in vitro are not specific for chlorophyll a only but are shared by nearly all porphyrins and their derivatives. Yet we cannot ignore the fact that no matter what other substances may be found as constituents of the plastids, photosynthetic reactions occur only in presence of chlorophyll a, or the bacteriochlorophyll of the photosynthetic bacteria. And
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chlorophyll a is the same in the entire plant kingdom. This exclusiveness goes hand in hand with the prime achievement of the photosynthetic reaction--the release of molecular oxygen from water. It seems that during the transition from chemical evolution to the first organized cell this particular reaction, which requires the interlinked and interdependent co-operation of several light absorption acts, came into existence. So far no one has succeeded in imitating it in a model reaction, nor do we have a generally accepted theory. Under these circumstances the best approach to the problem is to start at the top with the fully developed mechanism of photosynthesis in green plants and to see whether, by analysing its parts, we find sufficient reasons to believe that there might be a correlation between partial functions and their appearance in the course of time. THE COMPLEXITY OF THE PHOTOSYNTHETIC MECHANISM Today, in 1961, we are certain that photosynthesis consists of a number of partially autonomous enzymatic reaction systems which cluster around a central pigment complex of high molecular weight having a definite and essential structure. Within this pigment complex happens the conversion of light energy into chemical energy. The result of the light reaction is a pair of reactive chemical species. The nature of these instable "primary products" is unknown and therefore the main subject of current speculation. The complete "primary process" consists of a sequence of two absorption acts which leads to the separation of H from OH in HOH. The achievement of this light reaction consists in the use of water as hydrogen donor for the production of a reducing agent capable of reducing triphosphopyridine nucleotide (TPN) very efficiently. The oxidized part of water, the equivalent to an hydroxyl-radical, is left as a waste product to be disposed of. The present proposals to explain the mechanism of the "photolysis of water" are not as different as the protagonists of various hypotheses try to make them appear. Suffice it to remark here that the pigmented mechanism which makes such an efficient use of absorbed visible light is in itself so complex that it must have had an evolution of its own. Unfortunately there are no clues as to how or when that happened, except that it probably was complete even before the first cell was formed. In contrast to our ignorance about the primary process we have a reasonably complete understanding of the various reactions following it. The parts of the photosynthetic mechanisms which can either be observed independently of any photochemistry or at least understood on the level of comparative biochemistry are: the fixation of carbon dioxide from the air, the reduction of the fixation product, the phosphorylation needed to make the first two steps possible, the elimination of the oxidized primary photoproducts either by spontaneous reduction with a rather wide number of hydrogen donors or by evolution of free oxygen. To these we can add a number of artificially induced reactions. The photochemical mechanism in algae and bacteria may, therefore, be compared to a motor for general purposes. The motor, the driving force, and the transmission belt remain the same, while different tools can be attached to perform
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different tasks as needed. We shall assume that the number of metabolic "attachments" has increased one at a time in the course of evolution; and it is uncertain whether we know them all. Let us now trace the evolution of photosynthesis backwards in an attempt to see each of the known partial reactions as a consequence of definite changes in environmental conditions. 1 would like to follow Pirie in keeping a clear separation between the second half of evolution--the Darwinian mechanism based on gene mutation in complete organisms--and the first half--the chemical evolution of organic molecules culminating in the inheritance of randomly acquired properties. Pirie showed for this purpose at the Moscow conference the nice diagram in the shape of an hourglass. The middle narrow region signifies the point in time where, at the end of the chemical evolution, the astonishing interdependence of all cellular reactions was finally accomplished. The first surprise on our trip down the evolutionary ladder is that during the entire epoch of the Darwinian evolution there was no essential change in the principles governing the photosynthetic mechanism. The most primitive organisms have it complete. This becomes much less surprising when we remember that photosynthesis with the evolution of oxygen had to be complete before Darwinian evolution could progress with the development of respiring multicellular organisms. No multicellular, differentiated organisms are known that are anaerobes. They either never existed or could not survive the turn to strongly aerobic conditions. However, our starting point is not at the very beginning of the Darwinian period because we are dealing with complete cells that are far from primitive eobionts (Pirie's word for pre-cellular self-reproducing entities). We should not forget (as Pringsheim has pointed out) that as far as fundamental life processes are concerned there are no primitive organisms left for us to study. But if life with oxygen is the last phase in evolution, the preceding one is life without oxygen. This trivial ascertainment permits us to conclude that the mechanism for the release of oxygen is the most recent acquisition of the photosynthetic apparatus. Can we make this plausible ? And what is left when we take it away ? The evolution of oxygen from water requires at least three steps. During the study of herbicidal chemicals on the metabolism of algae, in particular Scenedesmus obliquus, substances have been found that stop plant growth by preventing the release of oxygen in the light. Among these inhibitors some work so specifically that no other metabolic process of importance is touched. Not only do the poisoned algae continue to respire or ferment as usual, but also their photochemical mechanism remains perfectly normal. The test consists in letting them use hydrogen in conjunction with the assimilation of carbon dioxide. The same effect, a specific inhibition of oxygen release only, can be achieved in certain algae by growing them in absence of the customary manganous ions. Both these observations make it extremely plausible that whatever else had to change to make the release of free oxygen possible--an indispensable part of this last evolutionary step must have been the addition of certain catalysts to an already existing complete
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mechanism for the fixation of carbon dioxide, for photophosphorylation, and for photoreduction with hydrogen. In place of the reaction leading to molecular oxygen there has been originally a coupling to hydrogenases or dehydrogenases. With the elimination of oxygen evolution we have arrived on the level of the anaerobic purple and green bacteria. The idea to put them in this place on the phylogenetic scale has been accepted ever since van Niel first proposed it. Let me remark that as long as an organism which contains only chlorophyll a or bacteriochlorophyll (and a minimum number of other pigments) can achieve complete photosynthesis with the evolution of oxygen or the oxidation of various hydrogen donors respectively, it is unnecessary for our purposes to ponder why related plants may have developed additional light-absorbing pigments or specificities for other hydrogen donors. Our attitude would be different if together with other pigments we would have found an altogether new type of photosynthesis. This is not the case. We have a certain freedom to interpret comparable data obtained from different forms of photosynthetic micro-organisms in favor of any evolutionary theory because it is not to be expected that the algae and bacteria we study today have remained unchanged, just as they were at the start of the aerobic epoch. After all, a billion years must have left a mark even upon organisms which did not evolve much further. We should not see in the present-day purple bacteria, for instance, the direct ancestors of our present-day green algae. To understand better the way photosynthesis evolved we should give more weight to the traits which these organisms have in common than to deviations which can be understood as due to later developments. It is more important that some green and blue-green algae can still perform partial reactions typical for the purple bacteria than that these reactions cannot any more be found in the majority of higher plants. The following are some known examples of common metabolic processes: absorption or evolution of hydrogen gas during the anaerobic photometabolism via an oxygen-sensitive hydrogenase; fixation of molecular nitrogen; photochemical utilization of acetate and, finally, assimilation of carbon dioxide. The reduction of carbon dioxide is an effect twice removed from the primary process. First, a carboxylic acid has to be photochemically reduced to an aldehyde which can be transformed into various carbohydrates. Second, at least one among these sugars has to be photochemically phosphorylated to function as an acceptor for carbon dioxide in a spontaneous, exergonic carboxylation in order to re-form the same carboxylic acid that started the cycle. This means from an evolutionary viewpoint that carbon dioxide reduction could have been perfected only after the ways for storing light energy had first been properly developed. It is of interest in this respect that the purple bacteria, as well as some algae, are not well adapted to the production or utilization of carbohydrates, but prefer to orient their metabolism around aliphatic acids, preferably acetic, one of the primary compounds in chemical evolution. Nevertheless, the purple bacteria fix free carbon dioxide by a mechanism very similar, if not identical, to that of the green algae. Hence we assume that an exergonic carboxylation was added I4
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to an already existing simpler photochemical process by developments within an organism antedating both the present day algae and present day purple bacteria. The newest support for such a view is the existence of some rare green algae that refuse to touch anything else but acetate and behave like obligate photoorgano-troph bacteria. This brings us to the next step down the evolutionary scale, to organisms that neither evolve oxygen nor assimilate carbon dioxide but use light for the assimilation of simple organic molecules into suitable cell material. There seem to be no organisms left that manage to live purely on this reaction and nothing else. The algae just mentioned, while having remained primitive with respect to the de novo synthesis of organic compounds, have undergone two mutations, leading to the formation of green chlorophyll and to the respiratory use of oxygen (evolved by other plants). The next move downward in complexity is quite considerable, but it is well supported by experiment. If we eliminate from the photochemical system all synthetic reactions involving carbon compounds, which experimentally can be done quite easily, we remove the reason for the existence of an organized cell. We are left with the naked chlorophyll complex. Stripped of the cell and of all enzymes necessary for the synthesis of organic cell constituents, it has yet not lost the power to capture light energy for a number of chemical uses. There is no reason why we should not believe that such a complex evolved ahead of the organized cells and existed for a while independently by itself. CELL-FREE PHOTOSYNTHETIC PARTICLES An intact chloroplast has many millions of chlorophyll molecules which cover the lamellae shown in microphotographs. But even small fractions of chloroplasts comprising no more than a few hundred chlorophyll molecules are still able to release oxygen from water when given the opportunity to reduce numerous physiological and unphysiological compounds in the light (Hill reaction). These reactions often go faster when coupled to a typical phosphorylating system that saves part of the light energy in simple polyphosphates or in the form of the best known among the ubiquitous biological phosphate compounds, adenosine triphosphate (ATP}. This kind of storing light energy proceeds most efficiently when the substance to be reduced in the Hill reaction is an electron transport catalyst which can be reoxidized on the spot by a coupling with intermediates on the path of photosynthetic oxygen. This is Arnon's cyclic photophosphorylation. The ability to evolve oxygen and that to fix carbon dioxide can both be easily abolished without damage to the photo-oxido-reduction system. Thus it is understandable that the conversion of light energy into ATP could be shown to be independent of the other two reactions. The question whether photoactive (or photosynthetic, if you wish) pigment complexes of the kind extracted from algae or purple bacteria ever existed for themselves in the pre-cellular stage of evolution we can answer with assurance only after they have been artificially compounded in the laboratory. But if they existed
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it is easy to see that they fulfilled very useful tasks in an organic soup containing most of the building blocks for a future cell. What about simplifying the system further ? The phosphorylation mechanism can be uncoupled, or the enzyme destroyed, this does not touch the original oxido-reduction mechanism--it makes it only a little more difficult to demonstrate that it is functioning and to find out what the light is doing. We shift, therefore, to physical methods which permit us to observe the primary process directly--the events in the pigment complex following the absorption of light quanta. The automatic recordings of spectral changes are accumulating fast in many laboratories. In such preparations stripped free to the bone of the metabolic reactions which are characteristic for a living cell, we see interesting oxido-reductions going on, for instance, between chlorophyll a.and cytochromes. Such light-driven reactions will proceed even at low temperatures where ordinary biochemical reactions stop. Because the reacting pigments are known, the spectral changes look familiar--they are similar to what has been seen in test tube models. But we should not forget that the pigment complex still contains macromolecules--proteins and phosphatides. The observed reactions depend on substances which are very advanced on the scale of the purely chemical evolution; and more than that, the structure of this association determines the nature and efficiency of the primary photochemistry. Attempts to remove or inactivate the remnants of proteins and enzymes of the original photosynthetic mechanism while retaining some specific photochemistry have so far been without success. Our methods are still too crude. When we try to simplify the photochemical redox system still further at least three basic characteristics disappear together. First: the specificity of chlorophyll a for what it does in the natural complex. It now reacts, when illuminated, exactly like other related or even unrelated dyestuffs. Second: the secret of a multi-quantum mechanism geared to work as a unit. All remaining photoreactions are of the known one quantum type. Third: the possibility of using water as a hydrogen donor. Some may argue that this was lost at an earlier stage of our analysis. I shall deal with this argument on another occasion. Be it as it may, too many things have changed at once, and instead of continuing our descent step by step into the past of the living photochemistry while ta,king note of an understandable evolutionary sequence, we have suddenly fallen through a dark hole, as it were, and landed on familiar grounds, namely the photochemistry of assorted pigments mentioned in the beginning. This obscure passage from oxido-reductions in natural pigment complexes to related reactions in artificially constructed systems is now the front line of research in photosynthesis. It seems that nowhere else in the field of biology are the chances as good as here to discover and understand the step by step transition from an entirely chemical (and artificial) reaction mechanism to one that has first regulated and later determined the evolution of living things. Some progress has also been made here. We know already that the chlorophyll pigment complex is surprisingly durable in the dried state, and that not all coloured components are equally indispensable as is chlorophyll a. /~-Carotene is not needed for photosynthesis
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except in the presence of oxygen. Under aerobic conditions it protects the cells against photo-oxidation. And yet carotenes are already present in those anaerobic organisms, the purple bacteria, which we like to regard as the nearest relatives of those anaerobic organisms that lived in the time before there was any oxygen. In retracing the developmental steps of the photochemical mechanism in living organisms we lost the path in that region of the time scale where also the traces of other biological reaction mechanisms (if they can be followed so far back) disappear w a t the time of the eobionts, when the macromolecules became the means to remove certain basic reagents from the homogenous solution. The two-dimensional arrangement on proteins or interfaces between hydrophylic and hydrophobic compounds produced effects that in many cases were decidedly more than just an acceleration of reactions known to proceed in the same way, though more slowly, in solution. This seems particularly true in the case of photosynthesis, with its amazing overall efficiency. QUANTUM NUMBERS AND EFFICIENCY Some ways to trap electrons or protons set in motion by the absorbed light energy will be more efficient than others. Photosynthesis does not depend on random collisions in solutions where it is next to impossible to separate the primary products so as to prevent a recombination. In the living cell a few selected chemicals permanently surround the light absorbing pigment. The chlorophyll is embedded in a structure composed of protein, phospholipids, carotene, quinone, cytochrome and a varying number of other pigments. Whenever this arrangement is disturbed or broken down to the point where less than 200 chlorophyll molecules remain together nothing is left but exergonic one quantum processes. No one has seen the accumulation of reactive intermediates belonging to the photochemical phase whenever inhibitors block the formation of stable, already recognized products at either end of the photochemical system. In other words, the energy-rich primary intermediates disappear in msec by back or side reactions at the least disturbance of the energy (or electron-proton) transfer system. It came as a surprise and has remained a puzzle that quantum number measurements on the various forms of photosynthetic reactions either in green plants, the normal or hydrogen-adapted variety, or in bacteria utilizing hydrogen donors of vastly differing free energy content, or in chloroplast preparations, always gave the same answer. Regardless of the overall free energy changes indicated by the chemistry of the process, two quanta are uniformly needed for the final transfer of one hydrogen or of its equivalent. This means that the stoichiometry of the process has remained unchanged as far back in time as we have been able to follow it. On the other hand, we can make a case for an evolution of photochemical efficiency in terms of light energy stored as chemical energy. The anaerobic bacteria achieve barely half a per cent efficiency for the reduction of carbon dioxide with free hydrogen and little more when using organic compounds as hydrogen donors. In the green plants the same reduction now coupled to the evolution of oxygen amounts to an efficiency of 35 per cent. One-third of the
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absorbed light energy is stored as carbohydrate. This increase in efficiency was gained by an extension of the enzymatic system coupled to the original pigmented apparatus. It is certainly true that complete photosynthesis in the green plants is more complex than that in the bacteria, and that, again, fewer steps and catalysts are needed if these micro-organisms dispense also with the reactions leading to the reduction of carbon dioxide. BACK-REACTIONS, PHOSPHORYLATION AND RESPIRATION A light absorption act increases the free energy of a molecule by the amount of energy contained in the absorbed light quantum. In order to obtain chemical work from such an increase in free energy (the excited state) it is necessary to prevent the rapid and complete dissipation of the potential electronic energy into thermal vibrations while the excited molecule reverts to its ground state. This immediate heat loss can be postponed by coupling the return to the ground state with a mechanism suitable to convert the short-lived free electronic energy of the excited state into potential chemical energy, in other words coupling it with the synthesis of one or more chemicals which retain a good part of the energy originally delivered by the absorbed light quantum. The efficiency with which the energy captured in the first pair of oxidizedreduced photoproducts can be utilized in cellular metabolism depends on the number and nature of further intermediate steps before the original energy level of the entire system has again been reached. The evolution of photosynthesis (the later part which we begin to understand) is therefore the story of ever more prolonged and subdivided oxido-reductions and back reactions. Figure 1 is a scheme illustrating the hypothesis that the various types of photosynthetic reactions now known came about by an increase of the number of coupled oxido-reductions following the primary process. The argument makes use of the known differences in the pattern of light-induced phosphorylation in bacteria and algae. Since three intermediate steps are the minimum to produce free oxygen the scheme is extended in three stages, numbered 1, 2, 3. Simply for reasons of symmetry, we allot also three steps, a, b, c to the formation of reduced pyridine nucleotide. The increasing complexity of a cyclic phosphorylation is indicated by three levels II, III, IV. In reality any intermediate on the right may be able to connect with any one on the left according to affinities and potentials of the factors available. The interpretation of the scheme goes as follows: Step I, the primary process, leads in 10-7 sec or less to the photoproducts •H-Chl. and .Cyt..(OH). with a much longer lifetime. How many unsolved problems are to be considered here we shall not discuss. A back reaction inside the pigment complex means loss of the light energy as heat. Step II provides the first opportunity to avoid the loss of energy by extending the oxidation or reduction to outside molecules (photoreduction) or by offering a faster back reaction, coupled to phosphorylation. This is meant to correspond to the "shorter" phosphorylation cycle in bacteria, adapted algae and bacterial
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chromatophores which is not inhibited by D C M U oxygen releasing step.
I
II
III
Primary Process
and similar poisons of the
h v + C h l ; C y t . - ~ H C h l ( = a ) ; C y t . O H ( = 1)-+ Chl;Cyt.(heat HOH H O H loss)
Short cyclic ~-~ P not inhibited by DCMU second step Cyclic , ~ P inhibited by DCMU
<--a
,
~
1 - ~
Factor <--~ 4~
~.~p +-- b + - a
inhibit. 1 !--~ 2 --~
;
• ~ Factors < I ~,,
~.~p IV
Higher efficiency ~-~ P via Pyridinenucleotide
+- c <- b <-- a (Pyr. Nuc.) T
;
1 -+ 2 -+ 3 -+ [02]
> Factors <-,¢
~P ¥
Oxidative Photophosphorylation
+- c <---b -<- a (Pyr. Nuc.) t
;
1 -+ 2 -~- 3 -.'- O2 Photosynthetic
~ Factors ~
O2 (air)
3,1
~P VI
Respiration ~
p/o
-- 3
H2 D o n o r -+ Pyr. Nuc. -+ Factors +~
Oz (air) ~
FIG. 1. Partial evolution of respiration from back reactions in a photochemical system reacting with water. Step I I I is different from I I because the phosphorylation is now sensitive to D C M U , as found in green plants and chloroplasts. Step I V has been added because we know that T P N need not be a part of the phosphorylation cycle. W h e n not engaged in storing energy-rich phosphate the reactions I I - I I I promote the various forms of photochemical reduction described in the beginning of this article. U p to here photosynthesis is an anaerobic process, because, as has been known since 1953, oxygen is not necessary for any of the
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normal photosynthetic reactions also in green plants. Recently Krogman, Vennesland et al. discovered that there are phosphorylations in chloroplast preparations that require not only light' but free molecular oxygen. What happens is an exchange of oxygen. The oxygen of photosynthesis escapes while the oxygen of the air is taken up. The advantage of our scheme is that in it oxidative photophosphorylation finds quite naturally a place as the next evolutionary advance. Step V is most conveniently explained as a break in the original cycle at points 2 or 3 so that photosynthetic oxygen is evolved, while oxygen of the air becomes the ultimate electron acceptor in the phosphorylation mechanism. This rather odd state of affairs may have once upon a time been of the greatest importance, namely as the transition phase to the appearance of respiration in the living world. Step VI is a non-photochemical oxidation system. It comes about when a second break on the reducing side in the mechanism of step V separates the entire phosphorylation chain from the photochemical mechanism. For this to happen all that is needed is a shift of specificities or an exchange of pyridine-nucleotides. This leads to the substitution of independent organic hydrogen donors for those made photochemically. The result is the oxidation of organic compounds by oxygen coupled to the storage of phosphate bond energy, or respiration. Considering that a complete respiratory mechanism could come into being only after photosynthesis had reached its latest development, andthat photophosphorylation is very likely mediated by a chain of catalysts very similar to the oxygencytochrome-flavin-pyridine-nucleotide part of many respiratory mechanisms this hypothetical extension of the evolution of photosynthesis seems worth a discussion. TRANSITION TO AEROBIC CONDITIONS The transition from anaerobic to aerobic conditions did not conspicuously alter the set of chemicals promoting metabolic reactions. The survival and adaptation to the presence of oxygen required first of all new means to render autoxidations harmless by removing the highly reactive hydrogen peroxide. Some of the iron porphyrins thus became peroxidases and catalases. An important aspect of this is that the very reaction, which was the cause for the accumulation of more and more oxygen, was the one most directly threatened by it. Fluorescent porphyrin derivatives sensitize photoxidation of organic compounds with excellent efficiency at very low oxygen concentrations and iron porphyrins lower the activation energy barrier against spontaneous oxidations by molecular oxygen. In order to keep functioning, the photochemical part in particular had to be protected against oxygen. Perhaps there have been numerous photosynthetic mechanisms up to this point--and only the chlorophyll a mechanism we know survived and flourished actively. The one present in the green and red bacteria survived but could continue its activity only under anaerobic conditions. We have good evidence that the photosynthetic organisms which did survive the shift to oxygen in the atmosphere have apparently been those that happened to contain carotenes in their chromophores; I say happened since the beautiful experiments
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of Cohen-Bazire, Stanier and Doudoroff and further new evidence have shown that these carotenes have no ascertained primary function in the photosynthetic mechanism. But in their absence the illuminatedchlorophyllcomplexis extremely sensitive to molecularoxygenand rapidly destroyedby photooxidation. There seem to bc no organisms around that arc photosyntheticyet able to grow anaerobically in the dark by means of fermentation. The green plants arc unable to grow in the light in the absence of oxygen--that is respiration--even if they may continue to reduce carbon dioxide quite efficiently. Onlya few purple bacteria arc sitting on the fence--they grow in the light without oxygen--or with it in the dark like the majorityof unpigmcntcdbacteria. The microbes known as strict autotrophs--the photolitho and chcmolithotrophs--werc once considered the most primitive organisms yet they have to perform the most difficulttask of all. So difficultis it indeed to convert carbon dioxide (as the sole carbon source) in contact only with the elements and their inorganic compounds into living matter and to start life in this special way that Ha]dane and Oparin eliminatedthe problem by convincingus that it never existed. But why then do we have a few (very few) such complete chcmoautotrophs ? The answer is that these must bc later evolutionarysidelines that came up where conditions wcrc favorable and that they had no chance for a quick further evolution after one line of complete autotrophs--thc photo-lithotrophicgreen plants-took over the earth. Our present information on living processes lets us believe that any kind of energy source available on this planet has earlier or later been tentatively put to some use for the differentiationand growth of living things. REFERENCES I.U.B. Symposium I, Moscow 1957. Pergamon Press,
The Origin of Life on the Earth. London, 1959. The Evolution of Life. Vol. 1 of Evolution after Darwin. University of Chicago Press, 1960. Photosynthesis and Chemosynthesis. Vol. 1B, Plant Physiology (edited by F. C. STF.WARD). Academic Press, New York, London, 1960. Problems of Photosynthesis. Reports of Second All Union Conference, U.S.S.R. Academy of Sciences, Moscow, 1959. Comparative Biochemistry of Photoreactive Systems. Symp. Comparative Biology. Academic Press, New York, London, 1960. The Photochemical Apparatus. Brookhaven Symposium in Biology I I, Associate Universities Inc. Office Tech. Service, Commercial Dept. Washington, 1959. Rhythmic and Synthetic Processes in Growth. 15th Symposium Society for Study of Development and Growth. Princeton University Press, 1957.
EVOLUTION OF PHOTOSYNTHESIS HANS G A F F R O N Department of Biological Science (Fels Fund). Florida State University, Tallahassee, Florida, U.S.A. PRIMARY PHOTOCHEMISTRY ON EARTH FoR the purpose of this essay I shall assume that the Proceedings of the First International Symposium on the Origin of Life on the Earth and recent reviews relating to our topic (see References) are generally available and therefore may serve as sources for detailed references. Without further discussion of these points where minor disagreements still exist let us presuppose the following as given. We have had on earth a succession of conditions during which the atmosphere changed from a generally reducing to a strongly oxidizing one. This change of environment was brought about by a rapid loss of hydrogen and a gradual accumulation of oxygen until the present quasi steady state was reached. The main source of free energy on the surface of the earth has been and still is, of course, solar radiation. This was (and is) occasionally supplemented here and there by localized volcanic action, which produces heat and reactive chemicals. The change from a reducing to an oxidizing atmosphere was accompanied by an attenuation of the solar spectrum in the ultraviolet region, to the effect that whatever part photochemistry played in the successive creation and accumulation of organic substances and later of living organisms, had to rely in the course of time on the utilization of smaller and smaller light quanta, diminishing in energy from 9 to 1.3 eV/mole but absorbed in increasing amounts by living matter. This decrease in available energy per absorbed light quantum means that the direct photochemical dissociation of water molecules into radicals, as it happened in the beginning, gave way to a very complicated mechanism which in our time achieves the same result with a succession of quanta absorbed by the green pigment chlorophyll. Furthermore, the photodissociation of the constituents of the earliest atmosphere such as H~, H~O, NH3, CH4, H~S, HCN, (O, CO, COs) and the subsequent chance recombinations lead to the accumulation of large amounts of aliphatic acid (e.g. CHzCOOH), amino acids (e.g. CH~NHzCOOH) and of small amounts of oxygen and the oxides of carbon. By the simple means of thermal dehydration, condensation, dehydrogenation, decarboxylation and photochemical interconversion that were often catalysed by metallic or oxidic surfaces or metal ions in solution, the earth's surface became (perhaps only in selected spots) enriched with a host of substances which were very likely the precursors to the substances now existing as parts of living cells. During the last decade we have become convinced that dicarboxylic acids, urea, purines, carotenes, porphyrins, sterols and 205
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perhaps many more such "biochemicals" can arise in a sequence of spontaneous reactions. We are here mainly interested in the first appearance of colored organic substances which broadened the spectrum of possible photochemical activities. For example, we have learned that the biosynthesis of porphyrin out of acetic acid and glycin proceeds over intermediate stages that can also happen spontaneously. To a part this has been proven experimentally. The important, often-told and wellknown story needs no repetition. The appearance of colored, fluorescent substances like the porphyrins must have accelerated the interconversion of organic substances tremendously. We shall concentrate our attention on the porphyrins because these molecules are among the few that apparently have exerted their catalytic powers throughout the eons that it took life to develop. This does not mean that other substances absorbing visible light could not have played important roles. The photodissociation of acetone, CH2.CO.CH 3 at low temperatures by ultraviolet, for instance, leads to the formation of diacetyl, CH3.CO.CO.CH ~. The latter, a yellow substance, is activated in blue light and has been shown to sensitize further photochemical synthesis ; but, on the other hand, diacetyl decomposes quite easily. The porphyrin ring, once formed, has, however, enormous stability in comparison with other active pigments. This may be the reason why porphyrin metal complexes have become the most widely distributed biocatalysts on earth. With this background we can address ourselves to the question of the evolution of photosynthesis, defined as the photochemical assimilation and transformation of carbon dioxide carbon into carbohydrate and other constituents of living cells. The task is to find the path that leads smoothly from the simpler reactions which illuminated pure chlorophyll displays in the test tube to the achievement of the living cell. Fifty years of experimentation with model reactions have enabled us to describe with satisfying consistency how chlorophyll reacts in homogenous solution after it has absorbed a quantum of visible light. Many kinds of compounds of importance to the living cell have been so tested. A few such reactions may be mentioned. In presence of oxygen it sensitizes the photoxidation of amines, purines, tyrosin, sulfhydryl groups and ascorbic acid with great efficiency. In absence of oxygen it may catatyse the transfer of hydrogen from a hydrogen donor to an acceptor (from ascorbic acid to pyridine nucleotide). As the only solute in organic solvents it is usually bleached in the light. Such anaerobic reactions are often reversed in the dark. Without causing a permanent chemical change the excited chlorophyll may also induce the isomerization from a cis into a trans configuration of an unsaturated substance like carotene. As much as these reactions have been studied and satisfactorily explained, any one or any combination of these are insufficient to account for the role of chlorophyll in the plant. For one thing the reactions observed in vitro are not specific for chlorophyll a only but are shared by nearly all porphyrins and their derivatives. Yet we cannot ignore the fact that no matter what other substances may be found as constituents of the plastids, photosynthetic reactions occur only in presence of chlorophyll a, or the bacteriochlorophyll of the photosynthetic bacteria. And
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chlorophyll a is the same in the entire plant kingdom. This exclusiveness goes hand in hand with the prime achievement of the photosynthetic reaction--the release of molecular oxygen from water. It seems that during the transition from chemical evolution to the first organized cell this particular reaction, which requires the interlinked and interdependent co-operation of several light absorption acts, came into existence. So far no one has succeeded in imitating it in a model reaction, nor do we have a generally accepted theory. Under these circumstances the best approach to the problem is to start at the top with the fully developed mechanism of photosynthesis in green plants and to see whether, by analysing its parts, we find sufficient reasons to believe that there might be a correlation between partial functions and their appearance in the course of time. THE COMPLEXITY OF THE PHOTOSYNTHETIC MECHANISM Today, in 1961, we are certain that photosynthesis consists of a number of partially autonomous enzymatic reaction systems which cluster around a central pigment complex of high molecular weight having a definite and essential structure. Within this pigment complex happens the conversion of light energy into chemical energy. The result of the light reaction is a pair of reactive chemical species. The nature of these instable "primary products" is unknown and therefore the main subject of current speculation. The complete "primary process" consists of a sequence of two absorption acts which leads to the separation of H from OH in HOH. The achievement of this light reaction consists in the use of water as hydrogen donor for the production of a reducing agent capable of reducing triphosphopyridine nucleotide (TPN) very efficiently. The oxidized part of water, the equivalent to an hydroxyl-radical, is left as a waste product to be disposed of. The present proposals to explain the mechanism of the "photolysis of water" are not as different as the protagonists of various hypotheses try to make them appear. Suffice it to remark here that the pigmented mechanism which makes such an efficient use of absorbed visible light is in itself so complex that it must have had an evolution of its own. Unfortunately there are no clues as to how or when that happened, except that it probably was complete even before the first cell was formed. In contrast to our ignorance about the primary process we have a reasonably complete understanding of the various reactions following it. The parts of the photosynthetic mechanisms which can either be observed independently of any photochemistry or at least understood on the level of comparative biochemistry are: the fixation of carbon dioxide from the air, the reduction of the fixation product, the phosphorylation needed to make the first two steps possible, the elimination of the oxidized primary photoproducts either by spontaneous reduction with a rather wide number of hydrogen donors or by evolution of free oxygen. To these we can add a number of artificially induced reactions. The photochemical mechanism in algae and bacteria may, therefore, be compared to a motor for general purposes. The motor, the driving force, and the transmission belt remain the same, while different tools can be attached to perform
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different tasks as needed. We shall assume that the number of metabolic "attachments" has increased one at a time in the course of evolution; and it is uncertain whether we know them all. Let us now trace the evolution of photosynthesis backwards in an attempt to see each of the known partial reactions as a consequence of definite changes in environmental conditions. 1 would like to follow Pirie in keeping a clear separation between the second half of evolution--the Darwinian mechanism based on gene mutation in complete organisms--and the first half--the chemical evolution of organic molecules culminating in the inheritance of randomly acquired properties. Pirie showed for this purpose at the Moscow conference the nice diagram in the shape of an hourglass. The middle narrow region signifies the point in time where, at the end of the chemical evolution, the astonishing interdependence of all cellular reactions was finally accomplished. The first surprise on our trip down the evolutionary ladder is that during the entire epoch of the Darwinian evolution there was no essential change in the principles governing the photosynthetic mechanism. The most primitive organisms have it complete. This becomes much less surprising when we remember that photosynthesis with the evolution of oxygen had to be complete before Darwinian evolution could progress with the development of respiring multicellular organisms. No multicellular, differentiated organisms are known that are anaerobes. They either never existed or could not survive the turn to strongly aerobic conditions. However, our starting point is not at the very beginning of the Darwinian period because we are dealing with complete cells that are far from primitive eobionts (Pirie's word for pre-cellular self-reproducing entities). We should not forget (as Pringsheim has pointed out) that as far as fundamental life processes are concerned there are no primitive organisms left for us to study. But if life with oxygen is the last phase in evolution, the preceding one is life without oxygen. This trivial ascertainment permits us to conclude that the mechanism for the release of oxygen is the most recent acquisition of the photosynthetic apparatus. Can we make this plausible ? And what is left when we take it away ? The evolution of oxygen from water requires at least three steps. During the study of herbicidal chemicals on the metabolism of algae, in particular Scenedesmus obliquus, substances have been found that stop plant growth by preventing the release of oxygen in the light. Among these inhibitors some work so specifically that no other metabolic process of importance is touched. Not only do the poisoned algae continue to respire or ferment as usual, but also their photochemical mechanism remains perfectly normal. The test consists in letting them use hydrogen in conjunction with the assimilation of carbon dioxide. The same effect, a specific inhibition of oxygen release only, can be achieved in certain algae by growing them in absence of the customary manganous ions. Both these observations make it extremely plausible that whatever else had to change to make the release of free oxygen possible--an indispensable part of this last evolutionary step must have been the addition of certain catalysts to an already existing complete
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mechanism for the fixation of carbon dioxide, for photophosphorylation, and for photoreduction with hydrogen. In place of the reaction leading to molecular oxygen there has been originally a coupling to hydrogenases or dehydrogenases. With the elimination of oxygen evolution we have arrived on the level of the anaerobic purple and green bacteria. The idea to put them in this place on the phylogenetic scale has been accepted ever since van Niel first proposed it. Let me remark that as long as an organism which contains only chlorophyll a or bacteriochlorophyll (and a minimum number of other pigments) can achieve complete photosynthesis with the evolution of oxygen or the oxidation of various hydrogen donors respectively, it is unnecessary for our purposes to ponder why related plants may have developed additional light-absorbing pigments or specificities for other hydrogen donors. Our attitude would be different if together with other pigments we would have found an altogether new type of photosynthesis. This is not the case. We have a certain freedom to interpret comparable data obtained from different forms of photosynthetic micro-organisms in favor of any evolutionary theory because it is not to be expected that the algae and bacteria we study today have remained unchanged, just as they were at the start of the aerobic epoch. After all, a billion years must have left a mark even upon organisms which did not evolve much further. We should not see in the present-day purple bacteria, for instance, the direct ancestors of our present-day green algae. To understand better the way photosynthesis evolved we should give more weight to the traits which these organisms have in common than to deviations which can be understood as due to later developments. It is more important that some green and blue-green algae can still perform partial reactions typical for the purple bacteria than that these reactions cannot any more be found in the majority of higher plants. The following are some known examples of common metabolic processes: absorption or evolution of hydrogen gas during the anaerobic photometabolism via an oxygen-sensitive hydrogenase; fixation of molecular nitrogen; photochemical utilization of acetate and, finally, assimilation of carbon dioxide. The reduction of carbon dioxide is an effect twice removed from the primary process. First, a carboxylic acid has to be photochemically reduced to an aldehyde which can be transformed into various carbohydrates. Second, at least one among these sugars has to be photochemically phosphorylated to function as an acceptor for carbon dioxide in a spontaneous, exergonic carboxylation in order to re-form the same carboxylic acid that started the cycle. This means from an evolutionary viewpoint that carbon dioxide reduction could have been perfected only after the ways for storing light energy had first been properly developed. It is of interest in this respect that the purple bacteria, as well as some algae, are not well adapted to the production or utilization of carbohydrates, but prefer to orient their metabolism around aliphatic acids, preferably acetic, one of the primary compounds in chemical evolution. Nevertheless, the purple bacteria fix free carbon dioxide by a mechanism very similar, if not identical, to that of the green algae. Hence we assume that an exergonic carboxylation was added I4
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to an already existing simpler photochemical process by developments within an organism antedating both the present day algae and present day purple bacteria. The newest support for such a view is the existence of some rare green algae that refuse to touch anything else but acetate and behave like obligate photoorgano-troph bacteria. This brings us to the next step down the evolutionary scale, to organisms that neither evolve oxygen nor assimilate carbon dioxide but use light for the assimilation of simple organic molecules into suitable cell material. There seem to be no organisms left that manage to live purely on this reaction and nothing else. The algae just mentioned, while having remained primitive with respect to the de novo synthesis of organic compounds, have undergone two mutations, leading to the formation of green chlorophyll and to the respiratory use of oxygen (evolved by other plants). The next move downward in complexity is quite considerable, but it is well supported by experiment. If we eliminate from the photochemical system all synthetic reactions involving carbon compounds, which experimentally can be done quite easily, we remove the reason for the existence of an organized cell. We are left with the naked chlorophyll complex. Stripped of the cell and of all enzymes necessary for the synthesis of organic cell constituents, it has yet not lost the power to capture light energy for a number of chemical uses. There is no reason why we should not believe that such a complex evolved ahead of the organized cells and existed for a while independently by itself. CELL-FREE PHOTOSYNTHETIC PARTICLES An intact chloroplast has many millions of chlorophyll molecules which cover the lamellae shown in microphotographs. But even small fractions of chloroplasts comprising no more than a few hundred chlorophyll molecules are still able to release oxygen from water when given the opportunity to reduce numerous physiological and unphysiological compounds in the light (Hill reaction). These reactions often go faster when coupled to a typical phosphorylating system that saves part of the light energy in simple polyphosphates or in the form of the best known among the ubiquitous biological phosphate compounds, adenosine triphosphate (ATP}. This kind of storing light energy proceeds most efficiently when the substance to be reduced in the Hill reaction is an electron transport catalyst which can be reoxidized on the spot by a coupling with intermediates on the path of photosynthetic oxygen. This is Arnon's cyclic photophosphorylation. The ability to evolve oxygen and that to fix carbon dioxide can both be easily abolished without damage to the photo-oxido-reduction system. Thus it is understandable that the conversion of light energy into ATP could be shown to be independent of the other two reactions. The question whether photoactive (or photosynthetic, if you wish) pigment complexes of the kind extracted from algae or purple bacteria ever existed for themselves in the pre-cellular stage of evolution we can answer with assurance only after they have been artificially compounded in the laboratory. But if they existed
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it is easy to see that they fulfilled very useful tasks in an organic soup containing most of the building blocks for a future cell. What about simplifying the system further ? The phosphorylation mechanism can be uncoupled, or the enzyme destroyed, this does not touch the original oxido-reduction mechanism--it makes it only a little more difficult to demonstrate that it is functioning and to find out what the light is doing. We shift, therefore, to physical methods which permit us to observe the primary process directly--the events in the pigment complex following the absorption of light quanta. The automatic recordings of spectral changes are accumulating fast in many laboratories. In such preparations stripped free to the bone of the metabolic reactions which are characteristic for a living cell, we see interesting oxido-reductions going on, for instance, between chlorophyll a.and cytochromes. Such light-driven reactions will proceed even at low temperatures where ordinary biochemical reactions stop. Because the reacting pigments are known, the spectral changes look familiar--they are similar to what has been seen in test tube models. But we should not forget that the pigment complex still contains macromolecules--proteins and phosphatides. The observed reactions depend on substances which are very advanced on the scale of the purely chemical evolution; and more than that, the structure of this association determines the nature and efficiency of the primary photochemistry. Attempts to remove or inactivate the remnants of proteins and enzymes of the original photosynthetic mechanism while retaining some specific photochemistry have so far been without success. Our methods are still too crude. When we try to simplify the photochemical redox system still further at least three basic characteristics disappear together. First: the specificity of chlorophyll a for what it does in the natural complex. It now reacts, when illuminated, exactly like other related or even unrelated dyestuffs. Second: the secret of a multi-quantum mechanism geared to work as a unit. All remaining photoreactions are of the known one quantum type. Third: the possibility of using water as a hydrogen donor. Some may argue that this was lost at an earlier stage of our analysis. I shall deal with this argument on another occasion. Be it as it may, too many things have changed at once, and instead of continuing our descent step by step into the past of the living photochemistry while ta,king note of an understandable evolutionary sequence, we have suddenly fallen through a dark hole, as it were, and landed on familiar grounds, namely the photochemistry of assorted pigments mentioned in the beginning. This obscure passage from oxido-reductions in natural pigment complexes to related reactions in artificially constructed systems is now the front line of research in photosynthesis. It seems that nowhere else in the field of biology are the chances as good as here to discover and understand the step by step transition from an entirely chemical (and artificial) reaction mechanism to one that has first regulated and later determined the evolution of living things. Some progress has also been made here. We know already that the chlorophyll pigment complex is surprisingly durable in the dried state, and that not all coloured components are equally indispensable as is chlorophyll a. /~-Carotene is not needed for photosynthesis
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except in the presence of oxygen. Under aerobic conditions it protects the cells against photo-oxidation. And yet carotenes are already present in those anaerobic organisms, the purple bacteria, which we like to regard as the nearest relatives of those anaerobic organisms that lived in the time before there was any oxygen. In retracing the developmental steps of the photochemical mechanism in living organisms we lost the path in that region of the time scale where also the traces of other biological reaction mechanisms (if they can be followed so far back) disappear w a t the time of the eobionts, when the macromolecules became the means to remove certain basic reagents from the homogenous solution. The two-dimensional arrangement on proteins or interfaces between hydrophylic and hydrophobic compounds produced effects that in many cases were decidedly more than just an acceleration of reactions known to proceed in the same way, though more slowly, in solution. This seems particularly true in the case of photosynthesis, with its amazing overall efficiency. QUANTUM NUMBERS AND EFFICIENCY Some ways to trap electrons or protons set in motion by the absorbed light energy will be more efficient than others. Photosynthesis does not depend on random collisions in solutions where it is next to impossible to separate the primary products so as to prevent a recombination. In the living cell a few selected chemicals permanently surround the light absorbing pigment. The chlorophyll is embedded in a structure composed of protein, phospholipids, carotene, quinone, cytochrome and a varying number of other pigments. Whenever this arrangement is disturbed or broken down to the point where less than 200 chlorophyll molecules remain together nothing is left but exergonic one quantum processes. No one has seen the accumulation of reactive intermediates belonging to the photochemical phase whenever inhibitors block the formation of stable, already recognized products at either end of the photochemical system. In other words, the energy-rich primary intermediates disappear in msec by back or side reactions at the least disturbance of the energy (or electron-proton) transfer system. It came as a surprise and has remained a puzzle that quantum number measurements on the various forms of photosynthetic reactions either in green plants, the normal or hydrogen-adapted variety, or in bacteria utilizing hydrogen donors of vastly differing free energy content, or in chloroplast preparations, always gave the same answer. Regardless of the overall free energy changes indicated by the chemistry of the process, two quanta are uniformly needed for the final transfer of one hydrogen or of its equivalent. This means that the stoichiometry of the process has remained unchanged as far back in time as we have been able to follow it. On the other hand, we can make a case for an evolution of photochemical efficiency in terms of light energy stored as chemical energy. The anaerobic bacteria achieve barely half a per cent efficiency for the reduction of carbon dioxide with free hydrogen and little more when using organic compounds as hydrogen donors. In the green plants the same reduction now coupled to the evolution of oxygen amounts to an efficiency of 35 per cent. One-third of the
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absorbed light energy is stored as carbohydrate. This increase in efficiency was gained by an extension of the enzymatic system coupled to the original pigmented apparatus. It is certainly true that complete photosynthesis in the green plants is more complex than that in the bacteria, and that, again, fewer steps and catalysts are needed if these micro-organisms dispense also with the reactions leading to the reduction of carbon dioxide. BACK-REACTIONS, PHOSPHORYLATION AND RESPIRATION A light absorption act increases the free energy of a molecule by the amount of energy contained in the absorbed light quantum. In order to obtain chemical work from such an increase in free energy (the excited state) it is necessary to prevent the rapid and complete dissipation of the potential electronic energy into thermal vibrations while the excited molecule reverts to its ground state. This immediate heat loss can be postponed by coupling the return to the ground state with a mechanism suitable to convert the short-lived free electronic energy of the excited state into potential chemical energy, in other words coupling it with the synthesis of one or more chemicals which retain a good part of the energy originally delivered by the absorbed light quantum. The efficiency with which the energy captured in the first pair of oxidizedreduced photoproducts can be utilized in cellular metabolism depends on the number and nature of further intermediate steps before the original energy level of the entire system has again been reached. The evolution of photosynthesis (the later part which we begin to understand) is therefore the story of ever more prolonged and subdivided oxido-reductions and back reactions. Figure 1 is a scheme illustrating the hypothesis that the various types of photosynthetic reactions now known came about by an increase of the number of coupled oxido-reductions following the primary process. The argument makes use of the known differences in the pattern of light-induced phosphorylation in bacteria and algae. Since three intermediate steps are the minimum to produce free oxygen the scheme is extended in three stages, numbered 1, 2, 3. Simply for reasons of symmetry, we allot also three steps, a, b, c to the formation of reduced pyridine nucleotide. The increasing complexity of a cyclic phosphorylation is indicated by three levels II, III, IV. In reality any intermediate on the right may be able to connect with any one on the left according to affinities and potentials of the factors available. The interpretation of the scheme goes as follows: Step I, the primary process, leads in 10-7 sec or less to the photoproducts •H-Chl. and .Cyt..(OH). with a much longer lifetime. How many unsolved problems are to be considered here we shall not discuss. A back reaction inside the pigment complex means loss of the light energy as heat. Step II provides the first opportunity to avoid the loss of energy by extending the oxidation or reduction to outside molecules (photoreduction) or by offering a faster back reaction, coupled to phosphorylation. This is meant to correspond to the "shorter" phosphorylation cycle in bacteria, adapted algae and bacterial
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chromatophores which is not inhibited by D C M U oxygen releasing step.
I
II
III
Primary Process
and similar poisons of the
h v + C h l ; C y t . - ~ H C h l ( = a ) ; C y t . O H ( = 1)-+ Chl;Cyt.(heat HOH H O H loss)
Short cyclic ~-~ P not inhibited by DCMU second step Cyclic , ~ P inhibited by DCMU
<--a
,
~
1 - ~
Factor <--~ 4~
~.~p +-- b + - a
inhibit. 1 !--~ 2 --~
;
• ~ Factors < I ~,,
~.~p IV
Higher efficiency ~-~ P via Pyridinenucleotide
+- c <- b <-- a (Pyr. Nuc.) T
;
1 -+ 2 -+ 3 -+ [02]
> Factors <-,¢
~P ¥
Oxidative Photophosphorylation
+- c <---b -<- a (Pyr. Nuc.) t
;
1 -+ 2 -~- 3 -.'- O2 Photosynthetic
~ Factors ~
O2 (air)
3,1
~P VI
Respiration ~
p/o
-- 3
H2 D o n o r -+ Pyr. Nuc. -+ Factors +~
Oz (air) ~
FIG. 1. Partial evolution of respiration from back reactions in a photochemical system reacting with water. Step I I I is different from I I because the phosphorylation is now sensitive to D C M U , as found in green plants and chloroplasts. Step I V has been added because we know that T P N need not be a part of the phosphorylation cycle. W h e n not engaged in storing energy-rich phosphate the reactions I I - I I I promote the various forms of photochemical reduction described in the beginning of this article. U p to here photosynthesis is an anaerobic process, because, as has been known since 1953, oxygen is not necessary for any of the
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normal photosynthetic reactions also in green plants. Recently Krogman, Vennesland et al. discovered that there are phosphorylations in chloroplast preparations that require not only light' but free molecular oxygen. What happens is an exchange of oxygen. The oxygen of photosynthesis escapes while the oxygen of the air is taken up. The advantage of our scheme is that in it oxidative photophosphorylation finds quite naturally a place as the next evolutionary advance. Step V is most conveniently explained as a break in the original cycle at points 2 or 3 so that photosynthetic oxygen is evolved, while oxygen of the air becomes the ultimate electron acceptor in the phosphorylation mechanism. This rather odd state of affairs may have once upon a time been of the greatest importance, namely as the transition phase to the appearance of respiration in the living world. Step VI is a non-photochemical oxidation system. It comes about when a second break on the reducing side in the mechanism of step V separates the entire phosphorylation chain from the photochemical mechanism. For this to happen all that is needed is a shift of specificities or an exchange of pyridine-nucleotides. This leads to the substitution of independent organic hydrogen donors for those made photochemically. The result is the oxidation of organic compounds by oxygen coupled to the storage of phosphate bond energy, or respiration. Considering that a complete respiratory mechanism could come into being only after photosynthesis had reached its latest development, andthat photophosphorylation is very likely mediated by a chain of catalysts very similar to the oxygencytochrome-flavin-pyridine-nucleotide part of many respiratory mechanisms this hypothetical extension of the evolution of photosynthesis seems worth a discussion. TRANSITION TO AEROBIC CONDITIONS The transition from anaerobic to aerobic conditions did not conspicuously alter the set of chemicals promoting metabolic reactions. The survival and adaptation to the presence of oxygen required first of all new means to render autoxidations harmless by removing the highly reactive hydrogen peroxide. Some of the iron porphyrins thus became peroxidases and catalases. An important aspect of this is that the very reaction, which was the cause for the accumulation of more and more oxygen, was the one most directly threatened by it. Fluorescent porphyrin derivatives sensitize photoxidation of organic compounds with excellent efficiency at very low oxygen concentrations and iron porphyrins lower the activation energy barrier against spontaneous oxidations by molecular oxygen. In order to keep functioning, the photochemical part in particular had to be protected against oxygen. Perhaps there have been numerous photosynthetic mechanisms up to this point--and only the chlorophyll a mechanism we know survived and flourished actively. The one present in the green and red bacteria survived but could continue its activity only under anaerobic conditions. We have good evidence that the photosynthetic organisms which did survive the shift to oxygen in the atmosphere have apparently been those that happened to contain carotenes in their chromophores; I say happened since the beautiful experiments
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of Cohen-Bazire, Stanier and Doudoroff and further new evidence have shown that these carotenes have no ascertained primary function in the photosynthetic mechanism. But in their absence the illuminatedchlorophyllcomplexis extremely sensitive to molecularoxygenand rapidly destroyedby photooxidation. There seem to bc no organisms around that arc photosyntheticyet able to grow anaerobically in the dark by means of fermentation. The green plants arc unable to grow in the light in the absence of oxygen--that is respiration--even if they may continue to reduce carbon dioxide quite efficiently. Onlya few purple bacteria arc sitting on the fence--they grow in the light without oxygen--or with it in the dark like the majorityof unpigmcntcdbacteria. The microbes known as strict autotrophs--the photolitho and chcmolithotrophs--werc once considered the most primitive organisms yet they have to perform the most difficulttask of all. So difficultis it indeed to convert carbon dioxide (as the sole carbon source) in contact only with the elements and their inorganic compounds into living matter and to start life in this special way that Ha]dane and Oparin eliminatedthe problem by convincingus that it never existed. But why then do we have a few (very few) such complete chcmoautotrophs ? The answer is that these must bc later evolutionarysidelines that came up where conditions wcrc favorable and that they had no chance for a quick further evolution after one line of complete autotrophs--thc photo-lithotrophicgreen plants-took over the earth. Our present information on living processes lets us believe that any kind of energy source available on this planet has earlier or later been tentatively put to some use for the differentiationand growth of living things. REFERENCES I.U.B. Symposium I, Moscow 1957. Pergamon Press,
The Origin of Life on the Earth. London, 1959. The Evolution of Life. Vol. 1 of Evolution after Darwin. University of Chicago Press, 1960. Photosynthesis and Chemosynthesis. Vol. 1B, Plant Physiology (edited by F. C. STF.WARD). Academic Press, New York, London, 1960. Problems of Photosynthesis. Reports of Second All Union Conference, U.S.S.R. Academy of Sciences, Moscow, 1959. Comparative Biochemistry of Photoreactive Systems. Symp. Comparative Biology. Academic Press, New York, London, 1960. The Photochemical Apparatus. Brookhaven Symposium in Biology I I, Associate Universities Inc. Office Tech. Service, Commercial Dept. Washington, 1959. Rhythmic and Synthetic Processes in Growth. 15th Symposium Society for Study of Development and Growth. Princeton University Press, 1957.