PHOTOSYNTHESIS IN CELL-FREE SYSTEMS1 C. ALBERT KIND University of Connecticut, Storrs, Connecticut
.ONEof
the major problems of biology which has attracted the attention of investigators from many scientific disciplines is the unraveling of the complex process known as photosynthesis. I t is a problem which has been continually and vigorously attacked by chemists over a span of nearly two hundred years and, although their efforts have sometimes uncovered more problems than they have solved, the striking advances in the knowledge of the chemistry of photosynthesis which have been made in the past decade are a tribute t o their persistence and ingenuity. Not only has the chemist been plagued by various technical problems, but the very nature of the process, in particular its relation t o plant respiration, has introduced further difficulties. The latter point is illustrated by an equation which summarizes the processes of photosynthesis and respiration in the green plant.
the view that these materials are not primary products of photosynthesis but are produced from carbohydrate by secondary reactions. For example, it has been shown that albino seedlings, devoid of chlorophyll and therefore incapable of photosynthesis, may be grown to maturity in a medium containing carbohydrate as the sole carbon source. Additional evidence for the carbohydrate nature of the photosynthetic product has been gained by determinations of the relative volumes of carbon dioxide and oxygen exchanged during exposure of green plants to light. The ratio Ozproduced/ C02consumed, i. e., the photosynthetic quotient, is 1.0, determined over a wide range of plants and under a variety of conditions.? The corresponding values for proteinand fatwould be about 1.25and 1.4,respectively. The second, and more troublesome, aspect of the eqnation is indicated by the implication that photosynthesis is the "reverse" of respiration. The process respiration is not directly reversible in the sense that an increase in carbohydrate + 0% -‘ CO, + H,O photosynthesis the relative concentrations of carbon dioxide and water I n this equation respiration is pictured as a process in is alone sufficientto cause the synthesis of carbohydrate which carbohydrate is oxidized by molecular oxygen to in the absence of an external supply of energy. Howproduce carbon dioxide and water. Photosynthesis ever, it has generally been assumed that respiration prohas long been recognized as a process which produces ceeds unabated during photosynthesis and therefore carbohydrate (sugars, starch) from carbon dioxide the conventional methods of chemical analysis for and water, liberating molecular oxygen. The eqnation possible intermediates in the process do not permit emphasizes the fact that during photosynthesis carbon the clear distinction between such intermediates and dioxide is reduced, and that the over-all reaction appears those produced during the respiratory proress. Thus the fart that malic arid may be detected in extracts of to be a reversal of respiration. There are two aspects of this equation which require green leaves which have been undergoing active photofurther comment. The first is the use of the term synthesis does not permit the deduction that it is, of "carbohydrate" to describe the organic product of the necessity, an intermediate on the chemical pathway photosynthetic reaction. While it is true that photo- from carbon dioxide to carbohydrate. In point of fact synthetic organisms must also manufacture fat, pro- malic acid is known to be an intermediate in the retein, pigment, etc., there is ample evidence to support spiratory process of both plants and animals and is
.
'Based on a paper presented before a joint meeting of the Connecticut Valley Section of the American Chemical Society and the New England Association of Chemistry Teachers, St. Joseph College, Hartford, Connecticut, April 14, 1856.
This value demands that the product be a t the oxidation level of carbohydrate as represented in the equation: 6C02
+ 6HsO
-
+ 602
CsHlzOs
VOLUME 33, NO. 10, OCTOBER, 1956
probably derived therefore from the oxidative breakdown of carbohydrate. On the other hand, the sugar derivative fructose-6-phosphate has beendemonstrated t o he an intermediate not only in the respiratory process but also in the photosynthetic pathway. Although an understanding of the individual reactions leading to carbohydrate synthesis is, in theory, obscured by the possible connection with the reactions of carbohydrate breakdown, it is just this connection which has played a dominant role in the thinking of biochemists and, as will he seen, has been largely responsible for the rapid advances in understanding which have been made in recent years. Mention has been made of technical problems which have had to be overcome before definite progress toward an understanding of the detailed reactions involved in photosynthesis could be achieved. One of the most serious from the point of view of the biochemist has been the inability to duplicate the process in plant extracts or preparations free from intact living cells. Such a demonstration is necessary before the techniques of modern biochemistry can be fully exploited to separate and to study the individual reactions involved. About twenty years ago, however, the first promising lead in this direction vas reported by Hill (1) who was able to show that at least a part of the over-all reaction-namely, oxygen evolution, could be achieved by illuminating an aqueous suspension of chloroplasts.
531
characteristics of photosynthesis: dependence on light and oxygen production. Further it suggests that the over-all reaction may be experimentally divided into two processes, one of which is light-dependent. The light-dependent process produces molecular oxygen and, a t the same time, furnishes electrons which can he used for chemical reductions. The second process, now known not t o be directly dependent on light, consists of a series of reactions, catalyzed by specific enzymes, and responsible for the absorption and reduction of carbon dioxide. Much of the evidence for the nature of this process has been provided by the elegant experiments of Calvin and his collaborators at the University of Caliiornia (4, 6). THE FIXATION AND REDUCTION OF CARBON DIOXIDE
Calvin's experiments have dealt mostly with photosynthesis as carried out in unicellular algae. For present purposes they may be summarized as follows: Algal suspensions are illuminated in the presence of carbon dioxide containing radioactive carbon-14. After exposure for varying lengths of time the algae are quickly killed by immersion in hot alcohol and the alcohol extracts are analyzed qurtlitatively by the use of paper chromatography. Since all compounds which lie on the chemical pathway from carbon dioxide to carbohydrate will become labeled with isotopic carbon, the detection of such compounds may be carried out by placing a piece of X-ray film on the paper. When CHLOROPLASTS AND THE HILL REACTION the film is subsequently developed, the compounds The chloroplasts are clearly defined structures of which have derived their carbon from the carhon dioxide various sizes (diameter 3-10 microns) dispersed within may be identified by their positions on the radioautothe cytoplasm of plant cells. Some are ellipsoidal, gram. From such experiments a t least two important types some lens- or cup-shaped, and the number per cell varies from one to several hundred. The chloroplasts of information may be obtained. If the chemical in turn appear to be made up of a number of thin discs, pathway is represented by a scheme, where A, B, C, called grana, in which the chlorophyll is localized. A etc., represent intermediate products, suspension of chloroplasts may be prepared by grinding leaf material with sand, or homogenizing in a Waring blendor, in a sucrose solution, filtering out the cell then, not only will the chromatographic technique debris, and centrifuging down the chloroplast pellet. enable these intermediates to he identified but, as the When the pellet is resuspended in water and illuminated, time of exposure to the radioactive carbon dioxide is a weak evolution of oxygen gas may he detected. Hill progressively shortened, the number of intermediates and Scarisbrick (3)showed that the oxygen evolution which are labeled will be correspondingly lessened and could he greatly enhanced when an oxidant snch as the order in which they appear can be deduced. Ultiferric ion was also present in the medium. Under mately conditions can he established snch that the these conditions the reaction proceeds in accordance initial product of carbon dioxide fixation (Compound A) can be identified. These labeling experiments have with the following stoichiometry: demonstrated that many of the compounds which are 2H20 + 4Fet' 4FeC+ + 4H+ + 0, formed during photosynthesis lie also on the respiratory The fact that oxygen gas arises from the decomposition or oxidative pathway of carbohydrate metabolism. of water in snch systems, and not from a component of This pathway has been thoroughly studied in the past, the chloroplast, was shown by the H,018 experiments and the knowledge of the intermediate steps is perhaps of Holt and French (5). Although other oxidants the most extensive of all biochemical processes. Some (henzoquinone, Z,6-dichlorophenol indophenol) may of the key compounds in carbohydrate metabolism be used in the Hill reaction, carhon dioxide is not among which were detected on the radioautograms are given in them. The reaction does, however, show two of the the following abbreviated reaction sequence, arranged
-
JOURNAL OF CHEMICAL EDUCATION CEO
=
GH,,~~),
Starch
d o H
Glucose6-phosphate
CHIOPOJHl
Lo
s
Fruotose1,6-diphosphate
in the order in which they are known to occur during the oxidative breakdown of carbohydrate. Although the oxidative process goes beyond the phosphoglyceric acid stage-ultimately t o carbon dioxide and water-we need not consider the remxiniug compounds, since 3-phosphoglyceric acid turns out to be Compound A, the first detectable product of carbon dioxide fixation. As the equation indicates, these reactions are all known to be reversible, and so it becomes possible to account for the conversion of phosphoglyceric acid to starch. In order to carry out the conversion of phosphoglyceric acid t o starch by the mechanism postnlated, two important substances are required, in addition to the enzyme proteins. These are adenosine triphosphate (ATP) and reduced diphosphopyridine nucleotide (DPNH). The requirement for these additional factors has been established by reason of our understanding of the reactions of carbohydrate breakdown. The last reaction in the above scheme, in order to proceed as written, requires the related compounds adenosine diphosphate (ADP) and oxidized diphosphopyridine nucleotide (DPN+). The reaction may therefore be more clearly represented as follows:
CHO
-
COOH
=
HAOH
$coH
HAOPO~H~ H,AOPO~H, 6Phasph* glycerddehyde
3-Phosphoglyceric acid
the incorporation and reduction of carbon dioxide, and in light-dependent process producing electrons (reducing power) and molecular oxygen. Several important questions remain to be answered. There is, for example, the question of the mechanism by which the plant maintains adequate supplies of ATP and DPNH. Until quite recently the only knowu biochemical reactions which could lead t o the formation of these substances were those which involved the oxidation of tissue substances, carbohydrate, fat, protein, etc. These reactions do not furnish a realistic answer t o the problem since they inevitably result in a decrease in organic matter rather than an increase, and, in addition, they usually consume molecular oxygen, not liberate it. Attention has therefore been directed to the possibility that supplies of ATP and DPNH are maintained by the light-dependent process. THE PHOTOCHEMICAL PRODUCTION OF DPNH AND ATP
I n the earlier discussion of the Hill reaction it was pointed out that the reaction will proceed, with the liberation of molecular oxygen, only when an oxidant is present. If the chloroplast system could be shown to use DPN+ as an oxidant, then one of the requirements 3-phosphoglpeemldehyde + ADP + DPNt orthophosphate 3-phosphoglycerio acid + ATP + DI'NH + H + would be met. This problem was investigated by Vishniac and Ochoa (a), and in 1952 they demonThis is an oxidation-reduction reaction, the aldehyde strated that the systemdoes, in fact,rednce being oxidized to the acid by DPN. I t is clear ATP DPN+ in the light. ~h~ reaction may be formulated and DPNH are required to "drive" the reaction to the asfollows: left. If the hypothesis is correct, the provision of an chloroplasts adequate supply of these two factors to a cell-free 2H20 + 2DPNt , 2 D P N H + 2H+ + OI extract of plant material, which contains the requisite light enzymes, should he sufficient to demonstrate the prodnctiou of carbohydrate from carbon dioxide; pro- Although the mechanism of the reduction is not comviding the incorporation of COI into phosphoglyceric pletely understood, the evidence for the over-all reaction is ~onclusive.~ acid is also an enzymatic reaction. The formation of adenosine triphosphate by chloroAlthough Calvin's early experiments with intact cells had provided evidence that carbon dioxide could plast suspensions in a light-dependent process was he incorporated into phosphoglyceric acid by a "dark" discovered by Arnon and collaborators in 1954 (9). reaction, the first demonstration of this incorporation in Not only did the cell-free system form ATP from ADP cell-free extracts was reported by'Fager in 1952 (6). and orthophosphate, hut the process was shown to be I n 1955 Racker (7) achieved total fixation and reduc- anaerobic. I n fact the reaction proceeds more effition of carbon dioxide to carbohydrate in a cell-free ciently in the absence of oxygen than in its presence. enzyme system fortified with ATP and DPNH. Thus Nor is oxygen given off during ATP synthesis and, the postulated mechanism is supported by experimental 8 The ahilitv of numensiona of ta arournnlst,e ~ ~ DPNH ~ - ~ ~- "ernna" .~-. evidence. The over-all process of photosynthesis as well as the"r ~-d a t e d ' (triphasphopyridine ~ ~ ~ ~ nucleotide) has may be divided experimentally as well as conceptually recently been clearly shorn by A. SANPIETRO AND H. M. LANO into a series of enzymatic ("dark") reactions, involving (Science 124, 118 (1956)).
=
+
~~~
~~
~
~~
~~~
~
~
~
~
~
VOLUME 33, NO. 10, OCTOBER. 1956
therefore, the Hill reaction is not involved. Arnon has suggested the following reactions to describe this proress (10) : H1O ADP
--
chloroplasts light
2(H)
+ P,..,. + 2(H) + ( 0 )
+(0)
-
H20
+ ATP
gestion that the five-carbon sugar derivative, ribulose 1,5-diphosphate, is responsible for furnishing the a and P carbon atoms has been confirmed by the recent isolation and purification of the carboxylation enzyme (18, IS). The reaction as now understood is depicted by the following equation:
(The oxygen and hydrogen symbols in brackets represent as yet unidentified products of the photodecomposition of water, not molecular oxygen and hydrogen.) I According to this scheme the water molecule is deHCOH COOH composed by the light energy to give an oxidizing comI H*C0POIH* ponent (O), and a reducing component (H). The HAoH Ribulose-1,s. energy of the recombinationof these components to H2AoPoa9 diphosphate form water is linked to the synthesis of ATP. These recent experiments with the cell-free chlorplast systemc With somewhat longer exposure times (15 seconds) make it clear that two processes, essential for the reduction of carbon dioxide to carbohydrate, can occur the concentration of isotope in the a and p carbon atoms of phosphoglyceric acid rises to 50y0 which only a t the expense of light energy. Since the fixation of carbon dioxide into phospho- means that the ribulose 1,5-diphosphate is continually glyceric acid is a "dark" reaction and since, given ATP being regenerated during photosynthesis. The phosand DPNH, the acid may be reduced to carbohydrate phoglyceric acid formed by the carboxylation reaction by dark reactions, a somewhat clearer picture emerges. must therefore be utilized not only in the synthesis of The energy of the sunlight, in the presence of the the six-carbon units of starch, but also in the formation chloroplasts, "splits" the water into a reducing com- of the five-carbon acceptor molecule. The latter procponent (H) and an oxidizing component (0). The ess has been shown to involve the participation of reducing component may then undergo two types of four- and seven-carbon sugars, and reaction schemes reactions. Either it feeds into a system which ulti- leading t o the regeneration of the acceptor have been mately forms DPNH (Hill reaction), or into a system proposed by Benson (11) and Racker (7). in which it is reoxidized with the concomitant formation of ATP. The oxidizing component also has PHOTOSYNTHESIS IN CHLOROPtAST SYSTEMS alternate pathways. Either it finally appears as moleWe have seen that investigations using cell-free cular oxygen (Hill reaction) or reacts with the re- systems have permitted a rather extensive analysis of ducing component in ATP synthesis. During photo- many of the reactions which make up the photosynsynthesis both pathways are operative. thetic process. The chloroplast unit has been shown to he necessary for several of these. The final question
+
THE CARBON DIOXIDE ACCEPTOR SYSTEM
A great deal of information relative to the reactions involved in the formation of phosphoglyceric acid from carbon dioxide has been obtained from labeling experiments and has recently been discussed by Benson (11). For example, it has been possible to isolate quantities of phosphoglyceric acid large enough to permit the determination of the specific activities of the individual carbon atoms. Where the exposure to the radioactive carbon dioxide has been of relatively short duration (4 seconds), 90% of the isotope appears in the carboxyl carbon (5). The reaction is therefore a carboxylation, with the plant supplying an acceptor molecule to react with the GOz. The acceptor molecule thereby furnishes two of the three (or and P) carbon atoms of the acid as shown below. "Acceptor"
+ C*02
-
LIGHT
I
HeCBOPOIHn I
H ~ O H &*OOH
Evidence bearing on the nature of the acceptor molecule has been discussed by Benson (11) and the sug-
Lei?
The Hill reaction leading to the ~roductionof oxygen gaa and
DPNH. Riphe: The photolyais of water linked to the eynthesia of ATP. Cent": The reduction of carbon dioxide to carbohydrate. s procesa dependent on DPNH and ATP ( l o ) .
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JOURNAL OF CHEMICAL EDUCATION
to be considered here is whether the chloroplast can LITERATURE CITED carry out $he entire process. This question has been (1) HILL,R., Nature, 139, 881 (1937). answered in the affirmative by the recent work of Proc. R w 8% London, B, (2) HILL,R., AND R. SCARISBRICK, 129, 238 (1940). Arnon (10) who has demonstrated the conversion of (3) HOLT,A. S., AND C. S. FRENCH,Awh. Biochem., 19, 429 carbon dioxide to starch, accompanied by the evolution of stoichiometric amounts of oxygen, in a cell-free (4) ~ k(1948). v r N ; ~ J.. , CHEM.EDUC.,26, 639 (1949). chloroplast system. By the use of specific chemical (5) CALVIN,M., el al., Symposia Sac. Ezp. Bwl., 5 , 284 (1951). inhibitors the over-all process has been separated into (6) FAGER,E. W., Arch. Biochem., 41, 383 (1952). (7) RACKER, E., Nature, 175, 249 (1955). three main phases: the photolysis of water producing (8) V I ~ K N I A W., ~ , AND S. OCHOA,"Symposium on Phosphorus molecular oxygen (Hill reaction), photolysis producing Metabolism 11" (MCELROY and GLASS,editors), Johns ATP but not molecular oxygen, and carbon dioxide Hopkins Press, 1952, pp. 467-90. reduction. These are depicted in the accompanying (9) ARNON,D. I., F. R. WHATLEY, AND M. B. ALLEN, J. Am. figure. Althoueh it is still uerhaos uremature to Chem. Soc.. 76. 6324 (19541. , . accept this in k r o Dhotosvnthesis as iientical in all (10) ARNON,D. ~.;Sczknre,122,9 (1955). this major technical (11) BENSON,A. A., J. CHEM.E m . , 31.484 (1954). resp&s with the in;ivo A. L., B. L. HORECKER, AND J. HURWITZ, J. achievement has furnished the chemist with a repro- (12) WEISSBACH, Bwl. Chem., 218, 795 (1956). ducible system which could yield the final answer to (13) JAKOBY, W. B., D. O. BRUMMOND, AND S. OCHOA, J. B ~ O Z . one of nature's most fascinating secrets. C h . , 218, 811 (1956).
example: hexaquochromium I11 for the [Cr(H20),]+" ion. LEWIS POKRAS
P o r r r ~ c a ~INSTITUTE ~c OF BROOKLYN BROOKLYN, NEWYORK
To the Editor: The majority of the textbooks of high-school chemistry presently in use in Japan present Faraday's law in a two-law form. I wonder if that is the case in foreign countries? Provided that the definition of equivalent weight and that of quantity of electricity have been presented elsewhere, the two laws can well be summarized as one: the quantity of 96,500 coulombs is passed through an electrolyte, one gram-equivalent of substance is either deposited or dissolved at each
To the Editor: Would there be objections from Lewis Pokras and the reviewers of his paper, ''On the species present in aqueous solutions of 'salts' of polyvalent metals" (THIS JOURNAL, 33, 152 (1950)), t o using the term "cationic compound" or "ionic compound" to designate salt-like species such as [M (HzO)d]+" ? A N D RL. ~ JULIARD electrode,^, HOUDRY PROCESS CORPORATION This is a necessary and sufficient statement of what MARCUSHOOK,PENNSYLVANIA the laws involve. The main reason for the prevalence of two-law form may be mere convention. Yet this To the Editor: form obviously is confusing, not only to high-school The suggestion of Mr. Juliard that such species as students, but also to those in the general education [M(H20)d]+V be designated as "cationic compounds" course of the university. The presentation of any leaves me somewhat disturbed regarding the mislaw should be as concise as possible. conceptions to which the term may lead. I should I n connection with the above-mentioned fact, I very much regret the adoption of any nomenclature propose to call the law "Faraday's law of electrothat applied the term "compound" to a chemical chemical equivalence" rather than "Faraday's law It is my feeling entity bearing a net electrical charge. The former title sounds to me more of electrolysis." that the classical usage, which reserves the term "compound" t o electrically neutral species, and employs comprehensive than the latter, because the law deals the term "ion" (and its various derivatives) for charged only with the relation between the quantity of elec.a from tricity and that of each electrode product. It : species should be retained. this law that the concept of electrochemical equivalence To the extent that any generic name for such species is required, workers in the field appear to employ the originated. term "aquo-ions." This term has the advantage of being related t o the names of specific examples in the Stock and IUC schemes of nomenclature, as for
