FLASH SATURATION AND REACTION PERIODS I N PHOTOSYNTHESIS
F. F. RIEKE
AND
H. GAFFRON
Department of Chemistry, Fels Fund, University of Chicago, Chicago, Illinois Received December 4, 1948
Our present knowledge of the kinetics of the thermal, or dark, reactions in photosynthesis is in great part based on studies of intermittency effects. The type of experiment which has been easiest ,to interpret is that introduced by Emerson and Arnold, in which the illumination is by short intense flashes (1). We wished to extend these experimenh to another type of photosynthetic activity of green plants, namely, the photoreduction of carbon dioxide with molecular hydrogen (4). For this problem some features of Emerson and Arnold’s technique appeared to entail difficulties similar to those previously encountered in the study of cyanide-inhibited photosynthesis (9). By an alteration in the method, these difficulties can be avoided. The first section of this paper deals with the nature of the limitation referred to above and the modification in technique which we have introduced. The second section contains our results on cyanide-inhibited photosynthesis, and those on the photoreduction of carbon dioxide. PART I
In the original method, the rate of photosynthesis is observed as a function of the intensity and frequency of the flashes, which occur a t regular intervals. At low frequencies and sufficiently high intensities the rate has been found to be independent of intensity and proportional to the frequency; the constant of proportionality is the maximum yield per flash, or “flash saturation.” This part of the experiment offers no fundamental difficulties. The interpretation of the flash saturation which appears most acceptable is that given by Franck and Herzfeld (2). It is that the primary photoproduct is unstable and can contribute to photosynthesis only if immediately on formation it is stabilized by a reaction with a specific enzyme, referred to as “catalyst B.” The number of molecules of B supposed to be present is small-about 1/2000 of the number of chlorophyll molecules-and is measured by the absolute value of the flash saturation. The variation of the rate with the frequency of the flashes is of particular significance when the flashes are sufficiently intense to give flash saturation, so that the rate depends only on the frequency. Experiments have shown that, as the frequency is increased, the rate at first increases proportionally, then more slowly, and finally approaches a limit which is equal to the maximum rate in strong continuous light. The usual method of describing this phenomenon is to plot yield per flash against At, the time between flashes; the curve rises steeply at small values of At, and approaches a limit-the flash saturation-at 299
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F. F. RIEKE AND H. GAFFROS
large values of At. Under restricted conditions, which d l be discussed presently, the value r of At a t nhich the yield per flash is (1 - l / e ) t,imes its maximum value can be related to the velocity constant of the rate-limiting reaction. The restriction with Jyhich x e shall be concerned is connected with the fact that \\-het,herflashing or continuous light is used, the maximum rate of photosynthesis is the same, and that the rate of photosynthesis a t which the yield per flash declines strongly as the frequency of the flashes is increased, is substantially equal t o the rate in cont,iauous light a t nhich the efficiency (rate/intensity) decreases rapidly when the intensity is increased. There can thus be little doubt that the decrease in yield per flash v i t h increasing frequency and the decrease in efficiency with increasing intensity both arise from identical limitations of the photosynthetic mechanism. It is not obvious that this is the same factor as that Tyhich causes flash sat,uration, although it must be assumed that this i s so if the observed r is to be related to a velocity const'ant. The possibility that this assumption is not justified has been investigated by Franck and Herzfeld. They conclude that for photosynthesis under normal conditions (temperature about 20°C., no poison, etc.) it is generally justified, and that 1 / r is equal to the velocity constant of the rate-limiting reaction, which under these conditions is the decomposition of the complex B-substrate into B product. Holyever, for cyanide-inhibited photosynthesis the assumption is false; there an enzyme sensitive to cyanide and different from B (referred to as "Catalyst A") becomes the limiting factor, and the observed r has no direct relation to the velocity constants of either ,1or B. Thus an explanation is given for the observation that cyanide inhibition increases r and leaves the flash saturat,ion unaltered, contrary to t,he expectation t'hat it should act as a specific poison and decrease the number of active enzyme molecules but not influence greatly the velocity constant. This explanation is further substaiit'iated by Weller and Franck's observation that the dependence of yield per flash on AL is different in form for normal and for cyanide-inhibited photosynthesis (9). A situation somewhat similar to that just discussed may be anticipated for the photoreduction process u-ith hydrogen in certain algae. In this case the maximum rate is of the order of one-tenth that for photosynthesis and is a,ttained a t a rather low intensity; cont,inued irradiation vith intense light results in complete cessation of photoreduction and a return to normal photosynthesis. The change does not occur, hoiwver, if the intense illumination is continued for only a few seconds, Evidently the limitation on rate is here something quite different in nature from that which occurs in normal photosynthesis, although in many other respects the processes have striking similarities. We should expect that with flash illumination the rate of photoreduction would be subject t o the same limitation; that is, as the frequency of flashes is increased, the same maximum rate should be attained, and further increase in frequency should result in a return to oxygen production. Thus, Trhile the experiment might yield a value for T , this value could not be interpret'ed as having any simple relation to a velocity constant-for the reasons discussed above. The origin of the uncertainty in the interpretation of T and the measure
+
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adopted here to avoid it are illustrated in figure 1. These curves illustrate for some ideal cases the predictions based on the kinetics proposed by Franck and Herefeld. The concentration of the complex B-substrate is plotted as a function of time under the conditions of the flashing-light experiments. An exponential law of decay has been assumed in drawing these figures; the phenomenon would be qualitatively the same for other laws of decay. Likewise the argument is quite general, even though for concreteness it has been based on a specific kinetic scheme. It is assumed that in all cases the flashes are of saturating intensity. Since the decay is exponential, the yields per flash are proportional to the areas under the curves (shown shaded in a, b, and c). Conditions for the observation of flash saturation are represented by figure 1 (a). The case where the rate-limiting reaction is the decomposition of the complex and the interval At between flashes is equal to l / k , where k is the rate constant of the decomposition, is illustrated in figure l(b). The case where the rate is limited by some preceding reaction in the sequence, as supposed in the case of cyanide
C
FIG.1. Scheme explaining yield per flash as a function of the distribution of flashes.
inhibition, and At = l / k is illustrated in figure l(c). During the early part of the illumination the average rate (ordinate) falls (in c) to that of the limiting reaction and the yield per flash to that indicated by the shaded area, which is the valuc which mould be observed for an illumination period including thousands of flashes. For the yield per flash to be equal to (1 - l/e) X (maximum yield) as in case b, the interval between flashes would have to be much greater, and therrfore greater thanbl/k. In case c, k cannot be deduced from the observed 7 . The procedure followed here is demonstrated in figure l(d). The number of flashes per second is the same as in l(a); the interval between flashes is changed by spacing the flashes differently. Between the closely spaced pairs the rate-limiting reaction has time to recover and the observed reduction in yield per flash can be attributed entirely to the failure of B to recover completely in the time between the members of the pair, which is l / k for the drawing. The reduction in yield per flash from the maximum value is approximately one-half the area shaded in l(d). Instead of pairs, groups of several flashes may be formed to increase the sensi-
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tivity. If the groups are not too long, the ratio (yield per flash) X (maximum yield) for a group of n flashes with spacing 1/72 is sensibly equal to I - ( n - l)/ne. PART I1
A . Experimen,tal Flashing light n-as produced by cutting the light beam with a rotating disc having fifteen symmetrical openings. Any of these openings could be covered by black paper cemented to the disc. The disc rotated at a speed of eight revolutions per second. One open sector thus gave eight flashes per second, evenly spaced; three equally spaced sectors, twenty-four flashes per second, evenly spaced; two neighboring sectors, sixteen flashes unevenly spaced, etc. Since the duration of one flash was 0.004 see., the remaining dark times amounted respectively to 0.121, 0.037 sec., etc. With this one-sector disc the longest available dark period is of course 0.121 sec. Much longer dark intervals were produced by mounting on the same axis another sector with only one opening but rotating a t one-fourth or one-tenth the speed of the first. In this way any flash or combination of flashes produced by the first rotating disc illuminated the manometer vessels only every 1/4 or every 1.25 second. This dark interval is about a hundred times longer than the period used by Emerson and Arnold. The light source for intermittent illumination was a mercury arc (Type H 6 ) as used by Weller and Franck. After exclusion of the ultraviolet radiation by means of filters, the lamp gave an illumination of 160,000 lux, due mainly to the yellow and green mercury lines. For continuous illumination an incandescent lamp was used in order to prevent photooxidation, which can occur a t the ext,remelg high intensity provided by the mercury arc. The fact that the mercury arc itself radiates intermittent light a t twice the frequency of the alternating current n-as capitalized upon by using a synchronous motor to drive the rotating discs and synchronizing the light flashes with t.he maximum in t,he intensity curve of t,he mercury lamp. The strains of algae used were Chlorella pyrenoidosa (Strain Emerson) and Scanedesmus obliquus (Strain D3), grown a feiy days at 20°C. in mineral solution. A4ftera fractionated centrifugation, which yielded cells of uniform size and presumably of the same age, the cells were centrifuged doivn, Ivashed, and suspended either in carbonate buffer or in a dilute bicarbonate solution to be used in equilibrium with a gas phase containing 4 volume per cent carbon dioxide. Photosynthesis was measured by the gas exchange in a differential manometer ( 7 ) . The readings were made with a cathetometer. With 30 to 60 cmm. of cel!s in 15 cc. of suspension liquid, only 20 to 40 per cent of the incident radiation n-as absorbed. R. The action of cyanide on photosynthesis I n the following experiments me have changed dark interirals between flashes without altering the tot,al radiation per unit of time by gathering a number of evenly spaced flashes per second, say sixteen, in several groups, say four times
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four. The dark intervals within a group, then, are much smaller than those between the evenly spaced flashes, and the dark periods between the groups much longer. Biinging the flashes within a group closer together resulted in a diminished rate of photosynthesis, as shown in figure 2. The first flash of a group appearing after the long dark interval gives the normal high saturation yield. If a second follows it at an interval shorter than the saturation period, the corresponding yield is considerably smaller. Speaking in theoretical terms we may say that the photoproducts mill find only a certain fraction of the stabilizing catalyst molecules in a condition to react. More excess photoproduct is lost by back-reactions than with undisturbed flash saturation, and the total rate of photosynthesis drops in comparison n-ith that produced by the same number of flashes spaced farther apart. Figure 2 shows that each additional
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O F F L A S H E S IN G R O U P
FIG.2. Effect of adding flashes to a group upon the rate of photosynthesis. Centera of flashes 1/120 sec. apart. This is too short to obtain maximum yield per flash, which is found only with the first flash in each group. Groups 1/83 sec. apart.
flash in a group increases the rate of carbon dioxide reduction by about the same amount as the second flash. If cyanide should influence the course of the reaction which is responsible for the diminished yield in narrowly spaced groups of flashes, the yield of all flashes with the exception again of the very first one should drop still further. Table 1 presents the results of such an experiment. The cyanide dose was so high as to inhibit 80 per cent of photosynthesis a t saturation in continuous illumination. In flashing light with sixteen evenly spaced flashes per second, we still see an inhibiting effect of about 22 per cent. Evidently the dark intervals of 0.00 see. are too short to let the cyanide-inhibited reaction go to completion. Kext the sixteen flashes are placed in groups of four, with rather long dark times beheen the groups and much shorter intervals between the flashes of each group. The latter are made so short that the yield of the unpoisoned cella is somewhat diminished. On the other hand, the long dark periods between the groups are more than sufficient to allow the reaction which is doned down by cyanide to
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go to completion. Under these conditions the yield in the presence of cyanide remains exactly* the same as without cyanide. Xo influence of cyanide is observable. It has been pointed out in Part I that the enzyme responsible for the flash saturation has to recover its activity during the interval after each flash. The present experiments prove that this particular enzyme is not sensitire to cyanide. As to the nature of the cyanide-sensitive reaction, there remains a t present little doubt. The photochemical metabolism of purple bacteria, which do not produce oxygen, is sensitive to cyanide (3), as is also the photoreduction i n Scenedesinus (5). Ruben, Kamen, et al. (8) found that the reversible uptake of carbon dioxide by plants is inhibited by cyanide. We can safely assume, therefore, that it is this initial transformation of carbon dioxide into an assimilable form which is so sensitive to cyanide. Cyanide produces a scarcity of this bound carbon dioxide. The rate in the presence of cyanide is seen to be exactly the same in intense continuous light and with the sixteen flashes evenly spaced; TABLE 1 Influence of cyanide o n the “stabilization period” in Chlorella pyrenoidosa ( S t r a i n Emerson) Comparison of the rates in continuous and flashing illumination; thin suspension of algae in carbonate buffer (85 parts M/10 NaHCOs and 15 parts X/10 KsCOs); gas phase, a i r ; 20 per cent absorption of light of 0.578mp; intensity about twice t h a t needed forsatiiration; temperature, 20°C.
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this is eIidently the maximum rate allowed by the poisoned system. As soon a i the efficiency of the flashes is decreased below this value by spacing them in close groups, the diminished rate is no longer affected hy cyanide. These experiments allow us definitely to eliminate the earliest models of the photosynthetic unit as, for instance, presented by Gaffron and Kohl (6). As an explanation of the flash saturation, these authors assumed that each flash of saturating intensity completely exhausted the supply of assimilable carbon. Hence the effect of cyanide should “prolong” each and every dark period betneen flashes of saturation intensity, independent of any following dark interval. This, as \ve have seen, is not the case.
C. Flash saturatzon zn Scenedesmus under aerobac and anaerobic conditaons Like all plants hitherto investigated in flashing light, Scenedesmus shows a typical flash saturation curve (figure 3). Increasing the intensity of each flash hevond a certain limit does not increase the rate of carbon dioxide reduction, in
FLASH SATURATION A N D REACTION PERIODS I N PHOTOSYNTHESIS
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spite of interposed dark intervals of sufficient length to permit all dark reactions to run to completion. Repeating this experiment after adapting the algae to photoreduction with hydrogen, we encountered the difficulty that the algae upon prolonged illumination with flashing light began to turn back to normal photosynthesis. According to Gaffron (4), this turning back is due to the accumulation of intermediates which are not reduced in time by the hydrogentransporting system. The turn will occur whenever the average illumination, whether continuous or intermittent, is high enough to produce intermediates more rapidly than the hydrogenase system can take care of them. In other words, we observe a formal similarity to the case of the cyanide-poisoned algae. In photoreduction the reaction with hydrogen is the slowest in the chain; it is so slow that the maximum rate of carbon dioxide reduction compares better with the rate of respiration than with the maximum rate of photosynthesis (7). One must therefore reduce the total number of flashes per second till the effective intensity becomes smaller than the threshold value for the turning back. In
25
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INT E N S l l Y
FIG.3. Flash saturation of photosynthesis in the alga Scenedesmus. Eight flashes per second. 4 per cent carbon dioxide in air. the experiment shown in figure 4 the length of the interval between flashes was ten times that of the interval used in the aerobic experiments of figure 3. We have 0.8 instead of 8 flashes per second. In order to obtain the same scale, the rate of figure 4 is measured in millimeters of gas absorbed per 10 min. We see in both figures that flash saturation is reached a t the same intensity. Since two hydrogen molecules are equivalent to one molecule of oxygen, the hydrogen should be absorbed a t twice the rate of the oxygen evolution. Actually in this experiment the rate is only about 1.5 times faster. Yet we can state that the saturation value for the yield per flash is approximately the same as under aerobic conditions. This means that even on a purely empirical basis the experiments indicate a great similarity if not identity of the. photochemical processes. It becomes evident that the flash saturation has nothing to do with the process of oxygen evolution. If we call the dark reaction responsible for flash saturation the “stabilization period,” we can assume that reduced as well as oxidized intermediates are stabilized during its course. Apparently it makes
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F. F. RIEXE AND H. GAFFRnS
no difference for the course of the stabilizing reaction in which way the oxidized intermediates react further. The length of the stabilizing period was measured originally by Emerson and Arnold by bringing the single flashes closer and closer together till the yield per explained abol-e, this involves a considerable flash began to decrease.‘ i l ~ increase in total radiation per unit of time. For measuring the same reaction
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FIG.4. Flash saturation of photoreduction with hydrogen in the alga Sceaedesmzis. 0.8 flash per second. @OS
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FIG.5 , Measurement of the length of the “stabilizing period” in photoredurtion. Rate of photoreduction as a function of the time interval betxeen flashes making up n group of two.
period during photoreduction, any increase of integrated int,ensity had to be avoided. This vias done by using the same number of flashes per second but spacing them unevenly (see figure l(d)). For measuring the length of the “rtabilizing period” in photoreduction we actually need only a group of tlvo flashes. Depending on the dark interval between them, t’he yield of photosyntjhesis will increase from the value of one single flash (flashes coinciding) to doublc this value (flashes far apart). Figure 5 shows that, while a dark interval of l/200 1 The more elegant determination of the length of the period in comparing continuoiis and flash saturation cannot be used in the reduced state.
FLASH SATURATION AND REACTION PERIODS I N PHOTOSYNTHESIS
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sec. is too short for the maximum yield, 1/40 sec. is already long enough. The reaction time under anaerobic conditions is certainly not much different from that found for Scenedesmus under aerobic conditions. This proves that the reaction which makes photoreduction a much slower process than photosynthesis is not to be found among those determining the flash saturation, Le., the stabilization of photochemical products. This experiment presents, of course, further evidence for the conclusion drawn above that the type of the photochemical processes remain the same whether the oxidized intermediates yield oxygen or consume hydrogen. SUMMARY
1. Photosynthesis in flashing light was studied with the intention of settling the question of the action of cyanide on the length of the interposed dark period necessary to maintain flash saturation, and of measuring flash saturation and the corresponding reaction period during photoreduction with hydrogen in the alga Scenedesmus. 2. In all previous experiments with flashing light the spacing of the flashes determined the total energy of the irradiation per unit of time. The wider the intervals between flashes, the lower the average intensity. A method is described by which it is possible to change the intervals between flashes without varying the average intensity, simply by making the intervals of unequal length. 3. With this method it has been proved that the reaction responsible for flash saturation is not sensitive to cyanide. The apparent effect of cyanide upon photosynthesis in flashing light as commonly used is due to the fact that another reaction, not directly connected with the photochemical process as such, now becomes limiting. Probably this reaction concerns the initial fixation of carbon dioxide. 4. It is not possible to obtain saturation in continuous light with the alga Scenedesmus when the reduction of carbon dioxide proceeds with the absorption of hydrogen, because a t higher intensities photoreduction turns back to normal photosynthesis. With light flashes spaced far enough apart, no such reversion occurs. Under these conditions a flash saturation is observed which attains approximately the same value as the aerobic flash saturation. 5. In varying the interval between the flashes producing flash saturation in photoreduction it is found that the length of the dark interval between flashes necessary to maintain flash saturation is the same as that observed with normal photosynthesis by Emerson and Arnold. 6. The experiments described support the view that the truly photochemical processes remain unchanged whether carbon dioxide is reduced with the evolution of oxygen or with the absorption of hydrogen. REFERENCES (1) EMERSON, R., AND ARNOLD, W . : J. Gen. Physiol. 16,391 (1932); 16,191 (1932). (2) FRANCK, J., AND HERZFELD, K. F . : J. Phys. Chem. 46,978 (1941). (3) GAFFRON,H . : Biochem. Z. 260, 6 (1933); 279, 25 (1935).
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Y. M . ~ N I X G ,AND B. M. DUGGAR
GAFFRON, H . : Am. J. Botany87, 273 (1940). GAFFRON, H.: J. Gen. Physiol. 26, 195 (1942). GAFFRON, H., AND WOWL,K.: Naturwissenschaften 24, 87, 103 (1936). RIEKE, F. F.: Quantum efficiencies-to be published. RUBEN,S., KAMEN, M. D . , HASSID,W,Z , AXD D E ~ A U LD T ., C.: Science 90,570 (1939); J. Am. Chem. Soc. 62, 3447 (1940). (9) WELLER,S., AND FRANCK, J.: J. Phys. Chem. 46, 1359 (1941). (4) (5) (6) (7) (8)
CHLOROPHYLL FLUORESCENCE BND EXERGY TRANSFER IN T H E DIATOM NZTZSCHIA CLOXTERIUM' HERBERT J. DUTTOK,~WINSTOX hi. R.I.AXNING,~AND B. M. DUGGAR Departments of Botany and Limnology, University of Wisconsin, Madison, Wisconsin
Received December 4 , 1942
Recent experiments (4) indicate that carotenoid-sensitized photosynthesis occurs in the marine diatom Nikschia closterium. These experiments also showed that the same enzyme system is probably involved in the thermal reactions of photosynthesis, whether the effective light is absorbed by carotenoids or by chlorophyll. Emerson and Lewis (5, 6) have obtained evidence for partial carotenoid participation in photosynthesis in Chroococcus, a blue-green alga, and in Chlorella, a green alga. In Chroococcus, their evidence for phycocyaninsensitized photosynthesis was much more conclusive than the evidence for carotenoid-sensitized photosynthesis. Sone of these results showed whether energy absorbed by carotenoid molecules was transferred directly to subsequent reactions in the photos2.nthetic process or transferred to chlorophyll with subsequent reactions the same as though the energy had been originally absorbed by the chlorophyll. The fluorescence experiments described below were designed to give information regarding the method of utilization of energy absorbed by carotenoids. Earlier studies with the green alga Chlorella have shown that the quantum yield (and also the wave-length distribution) of fluorescence for light absorbed only by chlorophyll is nearly constant over a considerable range of wave lengths (10). For an organism rich in carotenoid pigments, e.g., Nitzschia, a constant quantum yield for chlorophyll fluorescence a t various wave lengths, despite wide variations in the proportion of exciting light absorbed by the various pigments, xould be good evidence for an efficient transfer of absorbed energy from other pigments to chlorophyll. If there Tvere no transfer of energy, the yield of chlorophyll fluorThis work vias supported by grants from the Wisconsin Alumni Research Foundation. Present address: Western Regional Research Laboratory, Bureau of Agricultural Chemistry and Engineering, C. S. Department of Agriculture, Albany, California. 3 Present address: Division of Plant Biology, Carnegie Institution of Washington, Stanford University, California. 1
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