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Light and Life-Photosynthesis CORNELIS B . VAN NIEL Professor o f M i c r o b i o l o g y , Stanford University, Stanford, Calif. T h e m a t e r i a l s o f Tvhich l i v i n g o r g a n i s m s a r e c o m p o s e d , th.e s o - c a O e d " o r g a n i c s u b s t a n c e s " , b e l o n g to t h e u n s t a b l e f o r m s o f m a t t e r , a n d a r e , t h e r e f o r e , i n a c o n s t a n t flux of t r a n s f o r m a t i o n . Im order t h a t so precarious a condition m a y persist, t h e living organism h a s t o b e r e g u l a r l y p r o v i d e d witfca a f r e s h s u p p l y o f u n s t a b l e m a t e r i a l s
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M AN'S senses have made h i m familiar with the material aspects of t h e world he lives in, and his mind has continually striven to grasp and interpret t h e interrelations between these attributes of oixr environment. Among the fundamental achievements in this category, designated a s "natural science", must be rated the concept, that matter consists of an aggregate of elementary particles which occur i n various combinations. In this manner a great diversity of substances arise which differ in part b y their degree o f stability. There is also an undeniable tendency for the less stable combinations t o become transformed into more stable ones. Laving organisms must have a regular and a fresh supply of unstable materials. T h e best known example of this necessity is the food requirement of animals ; we know that they sooner or later perish without it. I t is also common experience that such food consists o f the substance of other animals and of plants. Searching for the ultimate source of foodstuffs one is invariably led t o t h e plants; they furnish the organic matter on which the plant-eating animals feed, and these, in turn, serve as nourishment for other m e m bers of the animal kingdom. And the plants themselves? B e i n g largely composed of unstable, organic substances, they, too, should have access to a constant supply of unstable aggregates of matter if they are t o fulfill their function as primary food source. Several observable and reproducible facts have, since van Helmont's discovery more than 300 years ago that the main food for plants does not come from the soil, a n d Stephen Hales' hunch a century later that s o m e of i t might come from the air, perV O L U M E
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mitted increasingly specific interpretations of the manner in which the supply i s brought about. I t was Ingen Housz' experiments (1779) on the effect of light which furnished the basis for the current concept. Investigations on the effect o f light o n matter have led to the belief that a substance, upon absorption of light, may thereby become converted into a much more unstable state. The* imaginative studies of Kngelmaun emphasized the effective absorption of light by the plant pigments, especially leaf-green or chlorophyll, and this can now be paraphrased b y saying that in such a way is brought about the supply of plants with highly unstable forms of matter. In the plant these unstable entities exert* a secondary effect upon normally stable substances whereby is achieved the formation of those materials which serve a s food for animals. Consequently, from a material viewpoint, the living world as we know i t depends ultimately on light which thus appears a s the prime mover of life. The process b y which the elaboration of organic matter occurs i n plants under the influence of light is referred to as photosynthesis. A clearer understanding of the composition and properties of matter, due to the efforts of the chemists, has gradually made it possible to describe the changes involved in more precise terms. Early i n the 19th century it was deduced, from quantitative measurements, that photosynthesis consists of a conversion by green plants, under the influence of light, of carbon dioxide and water to a corresponding quantity of sugar and oxygen. About 100 years later, Blackman demonstrated that this transformation should b e looked upon as composed of at least t w o different stages, one of which is
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photochemical, the other not. With the elucidation, by Willstatter and StolL, of the chemical nature of chlorophyll, the pigment common t o all green plants, the chief components of the photosynthetic process seemed to be known, and the first classical attempt was made t o formulate a hypothesis concerning the manner in which they interact t o accomplish the final result. Based upon a tremendous body of experimental evidence, in which they verified the quantitative relationship between carbon dioxide used and oxygen produced, and convincingly ascertained that chlorophyll, t h o u g h an obvious reaction partner, i s not permanently changed during the process, Willstatiter and Stoll, in 1918, postulated the following sequence of events. First, carbon dioxide combines with chlorophyll ajid water. In a photochemical reaction light then effects a rearrangement of the complex to chlorophyll-formaldehyde peroxide. In a subsequent nonphotochemical or dark reaction the new complex is broken up into chlorophyll, formaldehyde, and oxygen. This frees the chlorophyll for again combining with carbon dioxide; the formaldehyde is meanwhile converted into sugar. So well did this hypothesis account for the observed facts that for more than a decade it governed the thoughts of students of photosynthesis and their experimental approach toward t h e problems. Warburg and Negelein's studies, presumably demonstrating that four light quanta absorbed b y chlorophyll can cause the conversion into sugar of one carbon dioxide molecule, were not at variance with it, and resulted in a number of more or less successful attempts to define the changes in the chlorophyll-carbon dioxide complex caused b y each consecutive quantum. 1363
Nevertheless, the concept has l>een abandoned. One reason was that experi mental evidence for the existence o f a chlorophyll—carbon dioxide complex could never be produced. More decisive, how ever, was the result of studies by Emerson and Arnold which showed that the requi site number of quanta absorbed by any four of some 2,500 chlorophyll molecules could achieve the formation of sugar from one molecule of carbon dioxide. This most effectively ended all attempts at "explain ing" photosynthesis as the gradual trans formation of a chlorophyll-carbon dioxide complex b y successive quantum iiits; Kohn pointed out that a t low light intensi ties it might require years before the same chlorophyll complex could absorb 4 suc cessive quanta, while observably photo synthesis can start off within a fraction of a second under such conditions. An additional reason for reconsidering the mechanism of photos3mthesis was furnished by the study of photosyntlietic bacteria. This revealed the existence of photosynthetic processes which a r e in some respects fully comparable to that of green plants, while in others they exhibit characteristic differences. With the aid of a chlorophyllous pigment these bacteria, when illuminated, convert carbon dioxide into organic matter, as do the green plants. Uniike the latter, however, they d o not produce oxygen, and their photosynthetic activity is rigorously dependent u p o n the presence of reducing substances among which hydrogen sulfide and hydrogen gas are particularly instructive. From quanti tative determinations it appeared t h a t one specific case may be represented b y the chemical equation: C 0 2 + 2H 2 S —• (CH 2 0) + H 2 0 + 2 S
(1 )
Comparison with the reaction equation for green plant photosynthesis C 0 2 + 2 H 2 0 — (CH 2 0) + H 2 0 -f O a
(2)
shows a striking resemblance; and i t was this similarity which gave rise to t h e hy pothesis that photosynthesis should b e considered as a partly photochemical proc ess in which carbon dioxide is reduced t o organic matter with hydrogen derived from specific hydrogen donors, rather t h a n as a photochemical rearrangement of a puta tive chlorophyll-carbon dioxide complex. The most general formulation of this con cept can be expressed in the equation:
(CH 2 0) + H 2 0 -f 2 Α
(3)
H 2 A here stands for the specific hydro gen donors which different photosynthetic organisms require or can utilize to accomp lish the reduction of carbon dioxide to or ganic matter, the latter being summarily denoted by the symbol (CH 2 0). From this formulation it follows that the oxygen evolution in green plant photo synthesis would be the logical consequence
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o f the participation of H 2 0 as hydrogen donor for the carbon dioxide reduction; whenever some other substance* fulfills this role, its dehydrogenation yields cor respondingly different dehydrogenation products. The formation of sulfur from sulfide, of selenium from selenide, of tetrathionate from thiosulfate, or o f sulfate from sulfite is a typical example. Because some bacterial photosynthèses proceed in the presence of simple organic substances as the only reductants, it was e^en considered possible that such compounds, instead of serving the organisms as sa carbon source for the elaboration of tlieir cell constituents, might function a s special hydrogen donors for a photochemical carbon dioxide reduction. Incontrovertible evidence for this viewpoint was supplied when Foster demonstrated that the carbon skeleton of secondary alcohols is not modified by photosynthesizing bacteria, and that these alcohols are quantitatively dehydrogenated to the corresponding ketones, while carbon dioxide is the only carbonaceous building stone for the synthesis of the organic cell materiaUs: C0 2 -f 2
1N
>CHOH-*
τα/ (CH 2 0) + H 2 0 + 2
W* \ c
« 0
(4)
Occasional attempts have been made to ascribe the lack of oxygen production in bacterial photosynthèses t o the fact that the process occurs in the presence of reducing substances. Any oxygen evolution could thus be jbscured b y its secondary utilization for their oxidation. But this interpretation implies that oxygen production should then become observable as soon as the supply of reductants has been exhausted—which is not the case. Also, whatever doubts may have existed as to the fundamental sirmilarity of bacterial and green plant photosynthèses have been dissipated, largely avs a result of Gaffron's demonstration that by special treatment green plants can be s o modified that their photosynthetic activity becomes dependent upon an external sufpply of reducing substances, and now proceeds without the evolution of oxygeaa. This i s equivalent to saying that their metabolic activities have been experimentally converted into those characteristic of the photosynthetic bacteria. At present there are, conseqgruently, n o serious arguments against tfcae concept that photosynthesis represents an oxidation-reduction process, induced by light, and proceeding with the participation o f special pigments, in which caroon dioxide is the ultimate hydrogen acceptor, while a variety of substances can serve as hydrogen donors. Photosynthesis of green plants, characterized by the liberation of oxygen, results from the exclusive use of H 2 0 in this capacity. As an afterthought one may consider
the studies on the bacterial photosynthèses with hydrogen sulfide as a first "tracer" experiment from which the origin of oxygen could be deduced. That it is derived entirely from water and not par*ially from carbon dioxide—as the earlier WillstatterStoll hypothesis required—has later been clinched by the beautiful experiments of Ruben and co-workers, and by Winogradov and Teis, in which oxygen isotopes were used. If photosynthesizing plants are provided with water containing heavy oxygen and with carbon dioxide containing the light isotope, the oxygen evolved consists of heavy oxygen; conversely, if carbon dioxide with the heavy oxygen isotope is used together with water containing the light-weight variety, the gas produced is composed of the latter isotope: CCV e +• 2 H 2 0 1 8 — > (CH 2 O ie ) + H 2 O i e + CV8 18 C0 2 +- 2 H 2 0 1 6 — > (CH 2 0 1 8 ) + H 2 0 1 8 + 0 2 "
(5a) (5b)
I t may appear trivial to debate whether photosynthesis is t o be more adequately considered as a rearrangement of a carbon dioxide—chlorophyll complex or as a special oxidation-reduction process. However, the latter interpretation implies consequences which can be of considerable aid in guiding future efforts to comprehend more fully this aspect of the material basis of life. A glimpse of some of these may, therefore, serve to obviate the criticism of futility. In spite of many extensive and intensive investigations on photosynthesis the information thus far secured is entirely insufficient to develop an idea of the mechanism which is more than a general outline or a vague speculation. B y comparison the current interpretations of other biochemical processes, such as oxidative and fermentative decompositions, stand out as impressively detailed and well-founded concepts. Two reasons in particular can be adduced to account for this marked difference. First, it has been possible to study reactions of the latter type as more or less elaborate chains composed of relatively simple steps. In the early stages this was achieved by the use of poisons, b y the substitution of postulated intermediate products for the substrate undergoing decomposition, or b y the addition of substances which could be expected to interact in a ; predictable manner with special groups of intermediate products whereby these could be "trapped" in sufficient quantities to make them accessible to exact chemical analysis. Later, this approach was considerably advanced by the use of extracts from cells with which certain types of decompositions could still b e accomplished. Its ultimate development has resulted in the isolation, in some* cases in a chemically pure form, of highly, active biological catalysts or enzymes, each one capable of performing a specific link in the chain. And the utter simplicity
CH E M I C A L A N D
ENGINEERING
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of the chemical nature of such steps has brought our comprehension of the completed chain practically on the same level with that of more ordinary chemical reactions. As applied to photosynthesis this method of approach is still in its infancy. T o be sure, it is known that a number of substances can act as poisons for the process; however, an evaluation of this information in terms of specific inhibitions is as yet difficult. Furthermore, a few trapping experiments have been conducted in the hope of sidetracking important intermediate products; but the results are inconclusive at best. And the attempts a t reproducing the photosynthetic process or parts of it with mashed cells or extracts have been so disappointing as to rather discourage further experimentation in this direction. The only promising lead has been the demonstration that those structures of green plant cells in which the green pigments are concentrated, the socalled chloroplasts, may, under special conditions still liberate oxygen upon illumination. Nevertheless, we are as y e t far from the realization of a reproduction of photosynthesis with systems that would permit a ready analysis of component reaction steps. Comparative
Biochemistry
The second approach which has been extraordinarily fruitful in t h e interpretation of the mechanism of biochemical reactions is the consistent application of the principles of "comparative biochemistry". Gradually arrived a t through the pioneer investigations of Neuberg, Warburg, Wieland, Myerhof, and others, on the mechanism· of biological decompositions by means of the above-mentioned methods, these principles have been most succinctly enunciated by Kluyver in the form that all biochemical reactions can be resolved into series of steps, each one representing a simple case of an enzymatic hydrogen transfer. Hereby was revealed a fundamental unity in the chemical behavior of living organisms so great that it is no longer mere speculation to postulate that the formation of a certain product, by no matter what organism, involves very similar if not identical step reactions. Although the present is not an apt occasion to elaborate the various reasons for accepting this conclusion, i t is most appropriate t o follow its implications for the study of photosynthesis. Until rather recently the comparative biochemical reasoning was not applied to this process which, because of its striking differences from all other biochemical reactions, must have seemed t o represent a quite different and completely isolated phenomenon. As will be apparent, however, the formulation of photosynthesis as a partly photochemical transfer of hydrogen from some "donor" to carbon dioxide consti-
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tutes a direct application of the comcept that in essence all biochemical reactions conform to this pattern. An immeKiiate and most important consequence is that thus photosynthesis is no longer a process unique in all its aspects, but incorporates features encountered in other biochesnical reactions. By trying to understand more clearly what these common aspects are it is reasonable to expect important contributions to our comprehension of photosynthesis from studies with nonphotosynthetic organisms. T h e process is thereby removed from the position of strict isolation which it has so long occupied. Concomitantly, this provides opportunities for a fertile interchange of ideas and experimental methods. Photosynthesis
a CO2
Reduction
Especially in this connection the modified formulation offers hope for adva-nces. I»io longer considered as a photocheanical rearrangement of a carbon dioxide-cbalorophyll complex but instead as a carbo>n dioxide reduction, the photosynthetic process thus becomes linked with a numtaer of other biochemical reactions which aire instances of carbon dioxide reduction- At present several cases are known in which, a conversion of carbon dioxide into organic substances is accomplished b y different organisms without the interference of light. Already a number of simila-rities in such reactions have become apparent, and i t does not seem too great a flight of the imagination to expect that a further study of those phenomena will elucidiate a. common denominator which also per-tains to photosynthesis. At first sight it m a y appear startli Jig to divorce the carbon dioxide redutctioni proper from the photochemical reaction. in photosynthesis. Nevertheless, there are sound reasons for emphasizing this as t h e most probable situation. «Chief among them is the fact that photosynthetic bacteria, as well a s Gaffron's modified algae, can manufacture their or-ganie constituents entirely from carbon dLoxide in complete darkness. Since this convincingly shows that the organisms possess the enzyme systems for a nonphotocîiemical carbon dioxide reduction, it becomes superfluous, hence scientifically unjustified, to postulate t h e occurrence of additional enzymes by which this conversion would be achieved only i n light. Present knowledge of the "dark" carbon dioxide reductions makes i t likely that some of the steps involved can be steadied with isolated enzyme systems. In consideration of what such s ladies haves contributed to the understanding of otheJ biochemical processes the great significance of this possibility will be clear. One of the exciting discoveries o
