Twenty years ago it was not possible to discuss satisfactorily the “comparative” biochemistry of photosynthesis. The reason is a simple one: there was only one kind of photosynthesis known. This is the basic food-forming process carried out by green plants when they are properly illuminated. The main features of photosynthesis had been discovered in the course of the nineteenth century, and these can briefly be summarized as follows.
In 1804 de Saussure established, on the basis of quantitative chem- ical measurements, that photosynthesis by green plants can be repre- sented by the equation:
CO2 + H2O —Light (CH2O) + O2
The symbol (CH20) in this equation represents a reduction product of carbon dioxide, in general, “organic matter.
” It is used here in this wide sense for the sake of simplicity, and not in an attempt to designate any one particular type of organic molecule, least of all formaldehyde.
For the rest, the equation shows that from one molecule each of carbon dioxide and water one equivalent of this “organic matter” is produced together with one molecule of oxygen.
The next important contribution to an understanding of photosynthesis was made by Engelmann. In the years around 1880 he was able to demonstrate, by a most ingenious methodology, that light absorbed specifically by chlorophyll, the green coloring matter of plants, can be used effectively for photosynthesis. Thus it was made clear that photosynthesis can be accomplished with chlorophyll as the only light-absorbing agent.
Nevertheless, photosynthesis is a process that involves more than a light-catalyzed reaction between chlorophyll, carbon dioxide, and water molecules. The classical experiments of Blackman and Matthaei, published in the early years of this century, demonstrated that photosynthesis is the net result of a combination of photochemical (“light”) and non-photochemical (“dark”) reactions. The observed effects of carbon dioxide concentration, of temperature, and of light intensity on the rate of oxygen production by green plants could best be interpreted by postulating that the velocity of the photosynthetic process may be governed cither by the rate of penetration of carbon dioxide into the cells, by the rate at which the temperature-dependent dark reactions proceed, or by the rate of a photochemical reaction.
This concept of “limiting factors” has greatly aided in the analysis of photosynthesis. Although F.ngelmann’s contributions had emphasized the important function of chlorophyll, even before 1880, it was not until some thirty years later that the chemistry of this pigment was clearly understood. As a result of the careful, painstaking, and imaginative work of Willstatter and Stoll, chlorophyll was then obtained as a chemically pure compound which, upon analysis and by a series of specific degradation procedures, was found to represent a substance with a tetra-pyrrolc nucleus, containing a number of characteristic and specific sidc-chains, and binding a magnesium atom between the nitrogen atoms of the pyrrole groups.
With the newly gained insight into the structure of chlorophyll, amplified by an impressive array of experiments on the photosynthetic process in various plants under controlled conditions, these two scien- tists later developed an integrated concept of its mechanism. It was suggested that carbonic acid is bound by the magnesium atom of the chlorophyll; that under the influence of light the atoms of this complex
are reshuffled to form a new compound, chlorophyll-formaldehyde- peroxide; and that, subsequently, the latter is broken up by a dark reaction into its component units, chlorophyll, formaldehyde, and oxygen. The chlorophyll thus liberated is then ready to bind another carbonic acid molecule, the formaldehyde is condensed to sugar, and the oxygen makes its appearance as the conspicuous gaseous product of pliotosyn- thctic activity.
Finally, about twenty-five years ago, Warburg and Ncgelein deter- mined that the absorption of a minimum of four light quanta by- chlorophyll sufficed for the production of one molecule of oxygen by photosynthesis. This, in turn, made it possible to postulate more or less specific reactions that would be induced by each separate quantum hit. These developments had led to a general acceptance of the Willstatter and Stoll concept of the photosynthetic mechanism at the end of the twenties.
At that time, results of a study of the physiology of certain colored bacteria led to the conclusion that these organisms display a photosynthetic activity which is different from that of green plants. Hereby’ was opened the possibility of investigating photosynthesis by the “comparative biochemical” approach that had been developed by Kluyver and Donkcr. As a consequence a rather different view of the mechanism of this process emerged which will be traced in the following pages.
Concept of Bacterial Photosynthesis
It was observed that various types of bacteria, green, brown, red, or purple in color, can grow in strictly mineral media, in the absence of air, but only when the cultures are illuminated. Carbon dioxide being the only carbon compound present in the environment, growth of the bacteria implies that organic matter is produced from that substance. And, because this carbon dioxide assimilation depends upon proper illumination, it is evident that the process possesses the characteristics of carbon dioxide assimilation by green plants; in other words, represents a photosynthetic activity.
Supporting this conclusion is the fact that such bacteria contain a pigment system in which a green component, now known to be a magnesium porphyrin chemically very similar to chlorophyll, pre- dominates. Furthermore, light that is most strongly absorbed by thisSupporting this conclusion is the fact that such bacteria contain a pigment system in which a green component, now known to be a magnesium porphyrin chemically very similar to chlorophyll, pre- dominates. Furthermore, light that is most strongly absorbed by thisgreen pigment is the most effective in permitting growth, just as in the case of green plants.
But it was equally evident that bacterial photosynthesis differs in two major respects from green plant photosynthesis. In the first place, the conversion of carbon dioxide into organic matter under the influence of light appeared to be strictly dependent upon the presence of sulfidein the medium. Secondly, the most sensitive and specific test that is known for demonstrating the presence of oxygen—the “luminous bac- teria method” developed by Bcycrinck, which permits the detection of this element in quantities of the order of magnitude of a millionth ofa microgram—fails completely to reveal the liberation of this gas in illuminated cultures of photosynthesizing bacteria.
By means of quantitative analyses it was then shown that the process performed by illuminated cultures of green and purple bacteria can be expressed, as a close approximation, by simple equations:
CO2 + 2H2S —Light (CH2O) + H2O + 2S, or
2C02 + H2S + 2H2O—Light 2(CH2O) + H2SO4;
the symbol (CH20) again standing for “organic matter” in general.
Now let us start by comparing the equation of de Saussurc, very slightly modified, with the first of the equations for bacterial photo- synthesis:
C)a 4- 2H2O —->Light (CH2O) + HwO + O2;
CO2 4- 2H2S —-»Light (CH2O) + H2O + 2S.
The similarity is striking, and the comparison suggests that the place of HoO in green plant photosynthesis might, in the case of the bacterial activity, be taken over by H2S. Since the latter compound is oxidized, or dehydrogenated, to sulfur, this suggested that the photosyntheticbacteria carry out a reduction of carbon dioxide, under the influence of light, by means of a specific reducing agent, H2S. In that event pho- tosynthesis can be regarded as a process in which carbon dioxide is photochemically reduced to organic matter with the simultaneous oxida-tion of a reducing agent. In its most, general formulation photosynthesis may then be represented by the equation:
CO2 + 2″H2A”—-> Light CH2O) + H2O + 2″A”.
“H2A” is here used to denote any one of a number of reducing agents, or “hydrogen donors,” and “A” the oxidation, or dehydrogenation product.
Applied to green plant photosynthesis, the above interpretation sug- gests that the specific reducing agent for the photochemical carbon dioxide reduction is H2O. Such photosynthesis would thus consist in an oxidation of HoO with concomitant carbon dioxide reduction. The oxygen evolved would, therefore, represent the dchydrogcnation product of H20. If this were so, the oxygen should be derived exclusively from the water molecules, not entirely or in part from carbon dioxide, as required by the Willstatter-Stoll theory.
This consequence was tested experimentally in 1941 by Ruben and collaborators. They determined the isotopic composition of oxygen evolved by green plants in systems composed of:
(a) H2180 + C16O2, and
(*) H216O + C18O2.
The results showed, indeed, that the oxygen comes exclusively from H2O, and not in whole or in part from C02.
Additional support for this view has been furnished by experiments on oxygen production by chloroplast suspensions. In 1937 R. Hill had shown that isolated chloroplasts, incapable of carrying out a normal photosynthesis, can yet be made to evolve oxygen upon illumination.
This is possible provided that the medium in which the chloroplasts are suspended contains a reducible compound, namely, ferric oxalate. French and collaborators, in subsequent studies, found that various dyes may be substituted for the ferric oxalate. Finally, the experiments of Warburg and Liittgens have provided a beautifully simple and convincing demonstration of the photochemical activity of chloroplast suspensions by illuminating them in a solution of quinone. Under these conditions the result can be expressed by the equation
2C0H4O2 + 2HsO—Light 2C6H4(OH)2 + O2.
This equation shows the reducing action of water, under the influence of illuminated chloroplasts, whereby the quinone is converted into hydroquinone with the simultaneous liberation of oxygen, obviously the dchydrogenation product of H20.
These photochemical reactions of chloroplasts may all be considered as specific examples of a general type, represented by the equation.
By analogy one might suppose that in these eases the specific photochemical reaction would consist in a photodecomposition, not of H20, but rather of “H2A”. Nevertheless, from the point of view of comparative biochemistry it is more reasonable to expect that the specific photochemical reaction in all photosynthèses is the same. And there arc sound arguments that can be advanced to strengthen this view.
It is true that H20 does not occur among the reaction partners of the equations for bacterial photosyntheses. But it should be remembered that these equations merely represent the over-all changes that can be observed as a result of photosynthetic activity. And because H2O appears as one of the reaction products, derived from the reduc- tion of carbon dioxide, it would be difficult to detect its participation as a reactant. Hcncc it is entirely possible that a more adequate expression of the events would be contained in the equation.
Problem of the Hydrogen Acceptor
An excellent starting point for comprehending this situation is provided by the above-noted equivalence of oxygen and light in the metabolic activities of phoiosynthctic bacteria. What does this equivalence mean? Obviously that during illumination the organisms produce something that can function in like manner as oxygen. Yet the substance cannot be oxygen as such; the negative results of tests for this element with luminous bacteria rule this out.
Of course, it need not be oxygen. It could be any substance that can serve as hydrogen acceptor so as to permit the dehydrogenation of the oxidizable substrate through the proper, non-photochemical enzyme systems. Some thirteen years ago Roelofscn showed that the oxidation-reduction potential of a suspension of photosynthetic bacteria undergoes a sudden change upon illumination in a direction that indicates the formation of an oxidizing substance. This is exactly what appears to be needed.
Therefore I have recently attempted to obtain more direct evidence for the production of a hydrogen acceptor in a photochemical reaction that does not involve carbon dioxide or “H2A”. Cultures of photosynthetic bacteria were illuminated in the absence of oxygen, carbon dioxide, and external hydrogen donors. It could be expected that the addition of an appropriate EUA-compound at the moment the light is turned of! would result in the oxidation of a certain amount of HoA, proportional to the amount of hydrogen acceptor formed.
The results of these experiments have so far been entirely negative. This docs not necessarily invalidate the above deductions; it could simply mean that the oxidizing substance does not accumulate in detectable amounts. If it were produced in minute quantity, the methods employed would not be sensitive enough to demonstrate its presence. The very fact that there is so little present at any one time is rather suggestive of an enzyme system. By considering this suggestion in combination with the experiments on chloroplast suspensions, a consistent interpretation can now be developed.
Wc have seen that the oxygen production by such suspensions depends strictly on the addition of an adequate hydrogen acceptor (ferric oxalate, redox dyes, quinone). Why does it stop in the absence of these substances? The most reasonable answer is that the formation of oxygen is coupled with the simultaneous production of a reduced substance, probably a reduced enzyme. If this hydrogenated enzyme cannot relieve itself of the accumulated hydrogen atoms by transferring them to another acceptor, present in excess, it cannot itself operate as acceptor in the photochemical reaction. Then, the latter is blocked.
Is it not logical to draw the parallel, and conclude that for the bac- terial photosynthèses an external hydrogen donor is required for the continuous reduction of another enzyme system which becomes oxidized in the photochemical reaction: This enzyme in its oxidized form would then represent the oxidizing substance, or hydrogen acceptor, produced during illumination, yet never present in more than minute amounts. To be sure, such substances should also arise in green plant photosynthesis. But in this case oxygen is evolved. And thus would be accomplished a reformation of the enzyme in its reduced state, by some sort of self-regeneration. If, for some reason, this self-regeneration cannot occur, the continued participation of the enzyme system in the photochemical reaction to form the oxidation product must needs cease, and photosynthesis no longer is possible. In that case the presence of external reducing agents becomes compulsory. Can wc account for the funda- mental difference between green plant and bacterial photosyntheses on this basis? I believe so.
The foregoing arguments have forced us to conclude that in all pho- tosynthèses the primary photochemical reaction is concerned with a transformation of HoO, under the influence of pigments and enzymes, into a reducing and an oxidizing compound. Now this sort of decom- position requires energy. It is supplied in the form of light quanta absorbed by the pigment system. The chlorophyll of green plants is phorosynthctically functional at wave lengths up to 680 m/i, where the energy per mole-quantum amounts to about 42,000 calorics.
With this amount of energy the photochemical decomposition of H20, resulting in the formation of a reducing system that can reduce carbon dioxide, and of an oxidizing system that can liberate molecular oxygen, both in subsequent dark reactions, is thermodynamically just possible. But the green pigment of the photosynthetic bacteria effectively absorbs radiant energy for photosynthesis at considerably longer wave lengths, up to about 950 ni/*. Here, however, the energy per mole-quantum is only about 30,000 calories—insufficient to yield the same decomposition products. Since a reducing system capable of carbon dioxide reduction must be formed, the implication would seem to be that the other product, the “oxidized compound,” is a different one: a more stable substance, no longer capable of self-rcgcneration by transformations that lead to oxygen production.
If the enzyme systems postulated to operate in the photochemical reaction be designated as E’ and E” to indicate those that give rise to the reducing and oxidizing systems, respectively, it is possible to indicate the various types of reactions in the photosynthetic mechanism.
This sketchy but integrated concept can also be used as a basis for some speculations on the physiological evolution of photosynthesis. At the time when it first became possible to think about this process along comparative biochemical lines, it was generally held that the first living organisms appearing on the earth must have found them- selves in a strictly inorganic environment. This entailed the consequence that the organisms should have been cither chcmo- or photosynthetic. For lack of pertinent argument it was, however, impossible to decide
between these alternatives.
Within the past decade, however, it has been recognized that such a concept implies a serious difficulty. It is especially the advance in our knowledge concerning the mechanism of biological syntheses that has emphasized this. More and more clearly it has been demonstrated that these syntheses, like the degradation of substrates by oxidation and fermentation, must be considered as enzyme-controlled scries of consecutive step reactions; and the more limited the variety of substrate molecules in the environment in which growth takes place, the larger must be the number of biosynthetic reactions, hence also of enzyme systems in the organisms. The chemo- and photo-autotrophs, performing all their biosyntheses ultimately from carbon dioxide and other minerals, must therefore be equipped with a greater variety of synthetic mechanisms than, for example, organisms that can grow only when supplied with diverse vitamins and amino acids.
Now, it is difficult indeed to conceive of the genesis of the former type of cells from nothing blit inorganic matter. A much more rational concept concerning the origin of life has been developed by Haldane, Oparin, and Horowitz. Their basic assumption is that, long before living creatures appeared on the earth, organic matter was present in relatively large quantity, and comprising an enormous variety of molecular types. These could have originated as the result of purely photochemical and non-photochemical reactions, first yielding such substances as carbon monoxide, hydrocarbons, and unsaturated compounds with the potentiality of undergoing further substitutions and condensations, and ultimately giving rise to molecules of increasing complexity. An important aspect of this concept is the fact that the stability of these organic substances would be very different in a lifeless environment from what it is known to be when all sorts of organisms arc around, carrying out processes by which the organic molecules are destroyed. This difference in stability is amply attested to by the success of the modern canning industry!
By postulating, then, that before the appearance of life the earth represented a “hot dilute soup,” as Haldane expresses it, it is possible to imagine interactions of complex organic compounds in such a manner that new complexes originate which would possess the property of selectively absorbing from the environment molecules like those they are composed of, thus increasing the mass, and splitting into new units. Because this kind of “living system” operates without profound chemical transformations of the chemicals provided by the environment, biosynthetic processes would be limited to a minimum. By simple changes, equivalent to single-gene mutations, enzyme systems could now be initiated capable of carrying out the last steps in a biosynthetic chain. In an important contribution Horowitz has pointed out that such “mutations” have eminent survival value and may lead, stepwise, to the evolution of an entire scries of biosynthetic reactions.
In this manner one can rationally conceive of a physiological evolu- tion of living organisms characterized by an ever increasing potentiality for biosynthesis. Ultimately, this would thus result in the appear- ance of organisms capable of synthesizing all their cellular constituents from carbon dioxide as the carbon source, i.e. chemosynthetic organisms. The type of physiological evolution involved represents an increasing tendency towards independence from organic molecules supplied by the environment; and with the cheino-autotrophs the extreme limit has been reached.
It must, however, be realized that the necessary transformations of carbon dioxide arc, in fact, reductions and that consequently the organisms, though independent of organic substances, still require a supply of reducing compounds. Herein the appearance of a photosynthetic mechanism produces an important change. By means of an enzyme- controlled photochemical process the photosynthesizing cell can use H20 as a reducing substance.
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