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Photosynthetic evolution of oxygen by flashes of light
Photosynthetic Evolution of Oxygen by Flashes of Light’ F. L. Allen and J. Franck From
the Research
Institutes,
University
of Chicago,
(Fels
Fund),
Chicago,
Illinois
Received March 21, 1955
I. LITERATURE
AND INTERPRETATION
The efliciency of utilization of light energy by the photosynthetic machinery depends on whether this energy arrives in a continuous stream or in short pulses separated by relatively long dark intervals. As is the case with other problems in the field of photosynthesis, the reason for this is not only improperly understood, but apparent contradictions even exist between sets of experimental data from different laboratories. Neither can any of these observations be rejected nor has a satisfactory unifying scheme been developed which will obviate all the difficulties. It might be of interest, therefore, to inquire whether perhaps a way exists in which some of the discrepancies can be avoided along lines which will either confirm, reject, or preferably clarify the present status. Warburg (1) was the first2 to expose algae to short, equal periods of light and dark. He observed at high intensities a net increase in oxygen yield over that which would obtain due to illuminating the algae half the total time with the same but continuous intensity. However, it was in 1932 that Emerson and Arnold (3, 4) performed what is today the classical experiment in flash photosynthesis. Using very short flashes (cu. lO+ sec.) and variable dark intervals between the flashes, they were able to show that if this dark interval was made about 0.01 sec., all the photochemical products created during the brief light flash could be 1 This work was made possible through financial assistance from the Fels Fund (University of Chicago); the Office of Naval Research, Department of the Navy (Contract No. 119-272) ; and the National Science Foundation. * Prior to Warburg (I), Brown and Escombe (2) used the method of rotating sectors to provide flashing light for photosynthesis, being unaware of intermittency effects. 124
PHOTOSYNTHETIC
EVOLUTION
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OXYGEN
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handled during the subsequent dark period. A shorter dark interval reduced the yield per flash, a longer one did not contribute anything more. Further, the maximum yield per flash, at their highest light intensity, was independent of the temperature, although at low temperatures the dark interval required to attain this maximum was greater. Their yield per flash showed a surprising constancy relative to the number of chlorophyll molecules present; only about one oxygen molecule was liberated per 2000 chlorophyll molecules. Now, entirely in agreement with Emerson and Arnold, Tamiya and Chiba (5) confirmed the period of 0.01 sec. and the maximum yield of about l/2000 independent of temperature. However, when Tamiya increased his light intensity above a certain limit, the whole character of his curves became different. Not only did the dark period become much longer (up to 0.1 sec.), but the yield per flash rose and became strongly temperature dependent. Tamiya, therefore, concluded that Emerson and Arnold had used flashes of low total intensity, and thus had not achieved real flash saturation. In order to attain his presumed flash saturation, Tamiya used flashes which were very long compared with those of Emerson and Arnold-between 0.6 X UPa and 8 X 10-3 sec. at halfwidth compared with about 1P sec. of the latter authors. We will return to this point later. Faced with this discrepancy, Clendenning and Ehrmantraut (6), as well as Ehrmantraut and Rabinowitch (7), repeated the work of Emerson and Arnold, again using short flashes of about UP sec., and extended it to the case of the Hill reaction with quinone. Their combined results indicated that the period of 0.01 sec. and a yield of about l/2000 occur in the Hill reaction as well as in photosynthesis. The yield of l/2000 remained constant for photosynthesis even if the flash intensity was raised stepwise to a factor of four times the intensity previously used by Emerson and Arnold. In studies of the kinetics of photosynthesis, Gilmour, Lumry, Spikes, and Eyring (8) measured the reduction rates of ferricyanide ions by sugar-beet chloroplasts, both in continuous and flashing light. These authors used flash times even longer than Tamiya’s, in the range of 2.8-16.7 X lo-3 sec. With the shorter flashes, the curves they obtained indicate a period of about 0.01 sec., both at low (31,000 lux) and high (250,000 lux) light intensities, but in the latter case the curves resemble Tamiya’s. In fact, their thorough kinetic analysis of the data revealed
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AND
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FRANCK
the existence of five different reaction periods,3 depending on the external conditions. Gilmour et al. suggest that it is the difference in flash duration and not flash intensity which must be responsible for the discrepancy between the results of Tamiya and those of Emerson and Arnold. They therefore assume that a reservoir of photosynthetic products must be filled to obtain the results of Tamiya, and that a time of W6 sec., as used by Emerson and Arnold, is too short to fill it, irrespective of flash intensity. To explain the results of Emerson and Arnold, Franck and Herzfeld (9) suggested that the early photochemical products created during the flash were unstable and disappeared by back reactions unless stabilized by an enzyme. The dark period of about 0.01 sec. (required to obtain the maximum yield) was thus its working period. The limited number of these enzyme molecules (called at that time catalyst B) was responsible for flash saturation. This was the over-all picture as it existed several years ago. The present status of flash photosynthesis, including the observations of Tamiya and of Gilmour et al., can be reconciled with the original explanation of Franck and Herzfeld by making a minor modification. We need only assume that the working period of the stabilizing enzyme is in the order of 1W4 sec., in place of about 0.01 sec. This means that the enzyme can work several times during a flash lasting, say 1W3 sec., but only once during a flash lasting lO+ sec. The period of about 0.01 sec., for instance in the case of Emerson and Arnold, would then indicate that the total amount of material made by the flash can be worked up within this time by all the enzymes involved. $n the controversy on quantum yields, we agree with those who come to the conclusion that two quanta are necessary to effect the transfer of one hydrogen in normal photosynthesis. This, as well as other considerations, has led one of us (J. F.) to propose the following picture (lo), which we discuss here briefly: The first absorption act produces a metastable state of the chlorophyll. This metastable state has a biradical character-its absorption spectrum has been found and studied by Livingston et al. (11). Since this metastable state is relatively longlived, sufficient time exists to permit, by virtue of its biradical character, a complexing with the photosynthetic oxidant and the enzyme which plays the role of OH acceptor (possibly cytochrome F?). Neither the 3 Weller and Franck the existence of longer sec. duration.
(23) as well as Rieke Blackman (enzymatic)
and Gaffron periods
(24) had previously shown than the one of about 0.01
PHOTOSYNTHETIC
EVOLUTION
OF OXYGEN
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energy of the first excited state nor the energy of the lower-lying metastable state is sufhcient to effect the transfer of a hydrogen to the photosynthetic oxidant with the concomitant dissociation of water. However, as a result of the shift in the absorption spectrum of the metastable state of chlorophyll toward longer wavelengths,4 this chlorophyll in the metastable state can collect quanta by sensitized fluorescence from other excited chlorophyll molecules. An excited triplet state (11) can therefore be quickly formed, which relative to the singlet ground state now possessessufficient energy to transfer (a) an H from water to the photosynthetic oxidant and (b) an OH to an enzyme. It is to this enzyme that we can ascribe a working period of 1W4 sec. Of course, the energy transfer from an excited chlorophyll molecule to a chlorophyll molecule in its metastable state must be quite efficient-as we know sensitized fluorescence is, provided the overlapping of the corresponding absorption and emission spectra is favorable-since flash saturation already occurs when approximately l/100 of the chlorophyll molecules perform an initial absorption act. This modified picture, then, seemsto remove the discrepanciesapparently existing between different observations on flash photosynthesis. II. EXPERIMENTAL All of the contributions on flash photosynthesis discussedin Sec. I have been made with repetitive flashes, and the yield per flash has been deduced from an integrated effect. It would be of considerable interest to study the yields from single isolated flashes of light. No technique of sufficient sensitivity has been developed which will allow this under aerobic conditions. However, Franck, Pringsheim, and Lad (12) have shown that the method of phosphorescence quenching permits, under anaerobic conditions, the measurement of the evolution of oxygen from a single flash. Their experiments were, however, very preliminary. As a source of light they used photographic flashbulbs, the half-width being about 20 X 1W3 sec. Using this brief intense flash, they were able to demonstrate the evolution of oxygen from algae previously in total 4 The absorption spectra of chlorophyll in the triplet state have not as yet been measured in the red region of the spectrum. However, the strong similarity between the absorption spectrum of the chlorophyll phase test intermediate throughout the visible, as measured by Weller (22) r and the absorption spectrum of chlorophyll in the triplet state as measured by Livingston et al. (11) makes such a prediction reasonable.
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darkness and under an anaerobic state of about lW-” mm. Hg. Their yield of oxygen molecules per chlorophyll molecule for a single flash was approximately l/10,000 in contrast to Emerson and Arnold’s l/2000. This discrepancy was assumed not to be significant and easily explained by the inhibition which they observed under the conditions of their experiments. A repetition and extension of these experiments has been carried out and, particularly, a comparison between cells capable of performing photosynthesis and cells in the presence of the Hill reagent quinone. The oxygen production was determined using the method of Pollack, Pringsheim, and Terwoord (13). In order to have oxygen evolutions per flash large enough to be measured with comparative ease, the sample holder described previously (14) was replaced by a larger one (Fig. 1) having a volume of 20 cc. Agitation of the sample was provided by the gas flow of nitrogen (plus 2% carbon dioxide) at the rate of 10 cc./min. rising through the liquid after being broken up into fine
FIG. 1. The sample-holder (filled with ink for the photograph) position inside the flash coil. The short flash, therefore, illuminates from all sides.
is shown in the sample
PHOTOSYNTHETIC
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FIN. 2. Shape and duration of the two types of light flashes used in the experiments described. A. Single short flash. Time scale: 0.15 milliseconds per division, B. Single long flash. Time scale: 10.0 milliseconds per division. C. Short and long flash superimposed, showing manner in which timing was determined for the twoflash technique. The peak of the short flash is considerably off scale. Time scale: 10.0 milliseconds per division. bubbles by the fritted glass bottom. Needless to say, we were working under strict anaerobiosis-the pressure of oxygen in the carrier gas with the sample in total darkness was at most We mm. Hg. The present experiments were all performed with the algae, Scenedesmus obliquus strain Da . Procedures for growing the algae and their handling were identical with those described previously (14). The concentrations used in these experiments were, however, much higher-in the neighborhood of 0.40/o. Therefore, the algae were never centrifuged and seldom diluted, but transferred directly from the culture tube8 to the sample holder and anaerobiosis established. The suspending solution was always the culture medium at pH 8.2 except in the cases where quinone was used as a Hill oxidant. The algae were in that case suspended either in culture medium or in distilled water. Quinone solutions were made up just before use from freshly sublimed reagent. Owing to the large volume of the present samples, about 2 hr. was required to establish an anaerobic state of IO+ mm. Hg after placing the sample in the holder and beginning flushing with the carrier gas. The experiments were thus performed after approximately 3 hr. of anaerobiosis, obviously not all this time at the final pressure of less than 10-B mm. Hg of oxygen. Two types of flashes were ueed, short flashes of extremely high intensity and long flashes of a lower intensity, which will subsequently be referred to merely as short flashes and long flashes. The short flashes were provided by an Amglo 1TZ discharge lamp,6 shown in Fig. 1, together with a 1OOmicrofarad (pf.) condenser and a variable voltage power supply. The shape and duration of such a flash is shown in Fig. 2, the half-width being about half a millisecond. The hold-off voltage for this lamp was over 4OCil v., 80 that the discharge required initiation by an ionizing spark from a Tesla coil activated by a small condenser. The suspension holder fitted inside the flash lamp coil and, therefore, was surrounded by the 6 Amglo Corporation,
4234 Lincoln
Avenue, Chicago 18, Illinois.
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F. L. ALLEN
AND J. FRANCK
flash. For this reason, and others as well, it is difficult to ascertain the exact intensity. The manufacturer rates the lamp at approximately 35 lumens/watt-second. At 2000 v. and 100 pf. this would mean7O@l lumen-sec. From theshape of the light intensity vs. time curve, one can estimate the peak value under these conditions to be about 25 million lumens. For higher values of the voltage the total intensity would be higher, roughly proportional to the square of the voltage. Spectral analysis of the actual flash indicated a strong concentration of the emission toward the blue end of the visible spectrum. The long flashes were provided by a camera shutter in combination with a 1000-w. tungsten filament lamp, heat absorber, collimating lenses, etc. The halfwidth of the flash was about 25 X 10es sec., i.e., some 50 times longer than the short flashes, and the peak intensity some 250,000 lux. This was varied by changing the voltage on the lamp, and no attempt was made to measure the intensity with any accuracy. The superimposition of the two flashes, when desired, was accomplished by triggering the shutter with a solenoid and activating the Tesla coil for the discharge tube both with thyratrons. The timing was provided by the old but dependable method of shorting contacts with a freely falling plumb. An oscilloscope sweep was tripped at the beginning of the sequence and a photocell registered on it the phasing of the two flashes, an example of which is shown in Fig. 3. In order to eliminate uncertainties, the events as registered on the oscilloscope were recorded permanently by photographing the trace. Although the burst of oxygen evolved from a short flash may itself last a very short time, the large volume of the liquid sample prevents the instantaneous flushing out by the carrier gas. Therefore, on the Brown recorder one observes a burst having a half-width of about 1 min., as in Fig. 4. This burst is transformed to rate of oxygen evolution by means of the calibration of the instrument, and graphical integration under the resulting curve yields the total amount of oxygen evolved by the flash.
III.
THE
HILL
REACTION
WITH QUINONE
As is well known, the Hill reaction is simpler than photosynthesis inasmuch as the complicated carbon dioxide reduction cycle is replaced by the more straightforward reduction of a substitute oxidant. This simplification is made particularly evident by the reported (6) absence of induction periods in the Hill reaction. We therefore discuss first our observations on the Hill reaction with quinone. With a suspension of Scenedesmusin the presence of approximately 5 x 1O-4M quinone, it is very easy to observe and measurethe evolution of oxygen from a single short flash. Such a burst is shown in Fig. 4 and corresponds to about one oxygen molecule per 30,000 chlorophyll molecules. This value is low compared to the yield of l/2000 obtained by Ehrmantraut and Rabinowitch for the case of repetitive flashes. Our flash intensity could be varied over wide limits without changing the flash
PHOTOSYNTHETIC
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FIG. 3. Top: Inhibition of low background rate of photosynthesis by a very strong, short light flash. The small spike just before the oxygen burst is an electrical disturbance caused by the flash. Middle left: The extremely small yield observed as a result of a short flash if the algae are in total darkness previous to the flash. Actually, the effect observed in this case was caused by the lack of complete shielding of the sample from the room lights at the time the flash was applied. Middle right : Enrichment of the carrier gas with a small amount of oxygen prior to passing through the sample does not cause a short flash to evolve oxygen from the algae. Bottom left: Large burst evolved by the 25.millisecond flash from algae previously in total darkness. Bottom right: The sample has been illuminated with a weak background light and is photosynthesizing at a very low rate. The short flash is now able to evolve oxygen. Time scale: Each accented division on the abscissa is 1 min. Flashes came under the number that indicates the voltage.
132
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AND
J.
FRANCK
I
6
FIG. 4. Oxygen evolution by short flashes from cells suspended in 5 X lo-’ M quinone. Large burst is the evolution from two short flashes spaced about 1 sec. apart. Small burst is the result of a single short flash. The total amount of oxygen represented by the large burst is 30% greater than twice that represented by the small burst. Time scale: Each accented division on the abscissa is 1 min.
length. This was done by interposing suitable absorbers between the sample holder and the coil of the discharge lamp, and by changing the discharge voltage. It was thus possible to show that this yield of about l/30,000 was not low as a result of failure to reach flash saturation. Likewise, since it was possible to increase the flash energy by a factor of, roughly, ten without decreasingthe yield (once flash saturation had been reached), it could not be the result of having gone excessively beyond flash saturation. We take this value, therefore, as the order of magnitude of oxygen evolution to be expected from a single, short, isolated flash of saturating intensity under the conditions of our experiments. Considerable care had to be exercised with the use of quinone in these
PHOTOSYNTHETIC
EVOLUTION
TABLE Interval between the two flashes SCG. 1
250
OF
OXYGEN
133
I Combined oxygen ield from the two flas ii es cc.
5.5 4.2
x 10-7 X 10-7
flash experiments, since it was found that, especially with strong flashes, the algae were fairly rapidly poisoned, perhaps as a result of photodecomposition products of quinone. This is not a new observation; quinone inhibitions of one sort or another have been reported before (6). When this was the case, the yield of l/30,000 was no longer attainable. It was, therefore, necessary to perform each experiment quickly and with a minimum number of short flashes. Also, the cells remained in the presence of quinone for about 2 hr. before measurements could be made. This may have been detrimental and might be responsible for the low yield observed with single flashes. Of greater interest is the observation that the combined yield obtained from two short saturating flashes increases as the interval of time between the two flashes is reduced. Table I illustrates this effect. Thus, there is a net gain of some 30 9%as a result of applying two short flashes a second rather than a few minutes apart. This result, in which apparently something from the first flash carries over to the subsequent one, is further exemplified by the use of a teehnique of superimposing two flashes, one short flash and one long flash, and observing their combined yield as a function of their phasing, i.e., as a function of the position of the short flash relative to the long one. The general characteristics of the two flashes were described earlier. As stated before, the short flash was saturating. The long flash was not saturating, its yield depending on the voltage on the 1000-w. lamp, which was, however, kept constant during any given series of measurements. The result of such an experiment is shown in Fig. 5. The yield is plotted on the ordinate as cubic centimeters of oxygen per flash, meaning the combined yield of the short and long flash (their yield cannot be resolved separately, of course). The abscissa indicates the phasing of the short flash relative to the long one (the approximate shape and duration of the long flash is represented by the shaded trapezoid). The experimental points give the yield, at the time relative to the long flash, indicated by their position with reference to the trapezoid. For example, for the results of the particular experiment illustrated in Fig. 5, if the short
134
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FRANCK
I % ; 0” ::
-----&--------------~
,
----------------~ I
-50
-40
-30
-20
-10
0
+I0
+20
+30
+40
+50
MILLISECONDS
FIG. 5. Results of the two-flash technique with cells in the presence of 5 X 10-4 M quinone. The points at the extreme right are the values for which the horizontal lines were drawn. For explanation see text.
flash arrived 30 X KF3 sec.before the middle of the long flash, the oxygen yield observed for both together was about 2.8 X lO--’ cc. Separately, each flash was capable of producing oxygen also, and these yields are indicated by the horizontal lines located at the proper position on the ordinate: the lower dotted line indicates the yield per short flash alone; the upper dashed line indicates the yield per long flash alone. Both flashes combined, irrespective of their phasing, produce more oxygen than either alone. The more striking feature, however, is the higher yield obtained if the short flash is early. The data presented in Table I indicate that either the first light flash produces something which requires a second light flash to evolve oxygen, or that a substance produced by the first flash and which normally leads to oxygen evolution doesso at such a slow rate that it cannot be detected by our present method-the second flash merely hastening this evolution. In either case,the second light flash leads to additional oxygen evolution which is observed. We are inclined to think that there is some connection between the formation of this long-living substance and the photochemical reaction whose occurrence is suggested by the quenching of chlorophyll fluorescence in the presence of quinone (15, 16). The quinone concentration in our experiments is not known exactly because some of the quinone is
PHOTOSYNTHETIC
EVOLUTION
OF
135
OXYGEN
flushed away by the carrier gas during the hours preceding the experiments. We estimate a concentration of 1F4 M, which would give a quenching of about 10-30 % (15). While the bulk of the quinone reduction may proceed with the utilization of two quanta per H atom transferred, in the manner indicated in Sec. I, the percentage of quinone reduction associated with fluorescence quenching might, in principle, be achieved in a one-quantum process. We indicate a chlorophyll with its ring V in a hydrated state, viz., C ;: H-ClOe.--.-
OH
I/
I COOCHS
“i OH
OH /
by the symbol
H-Cph
. An impact
\
between
quinone
and
‘OH OH /
excited by light absorption
H-Cph* \
to the first excited state
OH t OH /
and indicated
by H-Cph*
permits energetically \
the reaction:
OH
OH
OH
/ quinone
/
+ H-Cph*
+ \
semiquinone
+
-Cph \
‘OH
‘OH
I’his process might be identical with the one observed by Linschitz and Rennert (17), i.e., a photochemical reversible bleaching of chlorophyll in the presence of quinone in a rigid glass. One of the hydroxyls of this OH / radical could be removed by an enzyme and utilized for -CPh. \
OH
136
F. L.
O2 evolution,
ALLEN
AND
J. FRANCK
because the reaction OH
-Cph
/ \
where Cph-OH
+ enzyme
4
Cph-OH
+ enzyme-OH
OH
represents
the enol form of chlorophyll
C=C-OH
i should be slightly exothermic. However, the reaction would require a considerable heat of activation for the opening of a C-OH bond and the closing of the double bond. Thus it would proceed very slowly in the dark. OH / On account of the radical character of -Cph the enzyme might \ ’ OH be adsorbed on it; and, if now a light quantum is absorbed, it will provide ample energy for the heat of activation. We may think of this overall process as an “abnormal” mechanism. At high light intensity by far the major portion of the quinone reaction might again proceed as the two-quanta processdiscussedin Sec. I, while at very low light intensities the heat of activation might be provided by thermal fluctuations before the arrival of the next light quantum. The results obtained with the short and long flashes superimposedon each other can be explained along the same lines. There is competition OH / between the process creating the longer-lived species -Cph ‘\ OH formed by an impact between quinone and a chlorophyll molecule in its first excited state (the “abnormal” mechanism), and the two-quanta process via the excited metastable state of chlorophyll (the “normal” mechanism) as discussedin Sec. I. If the short flasharrives early, the long flash finds a relatively high concentration of the longer-lived radical to “work” on, and the oxygen evolved is therefore larger. If the short 5ash arrives late, most of the oxygen evolution is the result of the twoquanta process during the long flash, and the oxygen evolution as the
PHOTOSYNTHETIC
EVOLUTION
OF OXYGEN
137
result of the “abnormal” mechanism from the short flash is not observed, at least to any significant extent. IV. PHOTOSYNTHESIS WITH 2% CARBON DIOXIDE In sharp contrast to the observations discussed in the preceding section, namely, the ability of a single short flash to evolve oxygen cells suspended in 5 X 10-* M quinone, cells in the presence of 2% carbon dioxide and, therefore, capable of performing normal photosynthesis, do not evolve oxygen as the result of a single short flash. The upper limit of the yield under these conditions can be estimated to be about one oxygen molecule per lo6 or lo6 chlorophyll molecules. In spite of surrounding the flash coil with aluminum foil to increase the intensity and at the highest flash energy (5000 v.) we could provide, no oxygen evolution was observed. Further evidence that this was not the result of insufficient flash intensity is given by the following observation. When the sample was irradiated with a very low but continuous light and then the flash applied, a significant burst of oxygen was evolved, as shown in Fig. 3, corresponding to a yield of about l/50,000. In this particular case, the weak continuous illumination was sufficient to produce from the sample a steady rate of only lO-* vol. oxygen/hr./vol. algae. However, it must be borne in mind that this figure is deceptive because, with suspensions in the order of 0.4% and the low background light intensities used, not all the algae are illuminated equally or at the same time. In any case, this gives an indication of the low level of background light required. The pressure of oxygen in the carrier gas after passing through the sample and picking up the oxygen was, for the particular experiment illustrated in Fig. 3, 2.2 X KP mm. Hg; and the concentration of oxygen dissolved in the algal suspension (expressed in terms of the pressure of oxygen in an atmosphere above the sample which would be in equilibrium with this concentration) was shown by the method discussed previously (14) to be in the order of 1w3 mm. Hg. When the intensity of the low background light is increased, the yield per flash is increased also, as shown in Fig. 7. This is to be expected, if for no other reason than that in view of the rather dense algal suspensions used, an increase in the background light results in more algae receiving light and, therefore, being able to evolve oxygen when the short flash is applied. It is extremely improbable that one can ascribe these observations to the presence of the very low concentration of oxygen produced by the
138
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FFtANCK
weak background illumination, as can be seen from some experiments in which oxygen was mixed into the nitrogen carrier gas before it passed through the sample. The short flash was then applied, the sample being previously in total darkness. Figure 3 illustrates such an experiment in which the carrier gas was enriched with 1W4 mm. Hg of oxygen besides the usual 2 % carbon dioxide. Not the slightest trace of oxygen evolution can be observed as the result of the short flashes. Evidently, the low background illumination is able to provide some sort of “priming,” and the question immediately arises: After this low background light is removed, how long does this “priming” survive? In other words, how long does this ability of short flashes to evolve oxygen in the presence of a low continuous illumination persist after this illumination is turned off? The answer is given in Fig. 6. At zero time the continuous light was removed, and at various times after this zero time the short flash was applied and its yield measured. The half-life obtained is in the neighborhood of 20 sec. Figure 7 shows that the yield per flash in photosynthesis is strongly dependent on the flash energy and the background illumination. This is especially true if the flash energy greatly exceeds that required for obtaining maximum evolution per flash at a given fixed value of the background illumination. It must be remembered that the values plotted in I
I IO
FIQ. 6. The persistence the low background light
I
I
1 I 20 30 SECONDS of the ability of the short is turned off.
I
I 40 flash
to evolve
oxygen
after
PHOTOSYNTHETIC
EVOLUTION
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139
FIG. 7. Effect on the yield per short flash (when superimposed on a low back ground light) as a function of the energy of the flash, at two different values of the intensity of the background illumination. The oxygen pressures by the curves indicate the rate of photosynthesis and, therefore, the intensity of the background light. The energy units are arbitrary.
Fig. 7 are obtained by superimposing the short flash on a low background of light, i.e., on a low rate of photosynthesis. At low flash energies no effect is observed on this background rate as the result of the short flash. However, if the intensity of the flash is made very great, the background rate of photosynthesis is partially inhibited for a matter of minutes immediately following the flash. It slowly regains its original value as shown in Fig. 3. In contrast to this, for the case of the Hill reaction with quinone, no particular influence of either the short flash on the steady evolution, or of the steady evolution of oxygen on the yield per short flash, was observed. A burst of oxygen was merely superimposed on the steady background rate. While it was observed that a short flash alone was unable to result in the evolution of oxygen, the burst of oxygen due to a long flash alone was quite large. One such burst is shown in Fig. 3. The long flash, some25 X UP3 sec. at half-width, was not saturating because of the rather dense suspensionsused: 0.345%. However, the total amount of light falling on the sample was estimated to be about the samein both the short and the long flashes. This was probably true within an order of magnitude,
140
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FRANCK
MILUSECONDS
8. Results of the two-flash technique with cells in the presence of 2% carbon dioxide and, therefore, capable of performing photosynthesis. Two sets of data are plotted. The points on the extreme right are the values for which the horizontal line is drawn. For explanation see text. FIG.
the short flash possibly being the stronger. Yet the 25 X KV sec. flash was able to evolve oxygen while the 0.5 X 1P sec.was not. This observation confirms the result of Franck et al. (12) that the ignition of a photographic flashbulb resulted in a burst of oxygen from algae previously in the dark. We conclude, therefore, that as far as a single flash is concerned, it is not merely a question of the number of quanta involved, but also the space of time over which they arrive. The technique of superimposing two flashes, a long and a short one, described previously for the Hill reaction, was used also in the case of Scenedesmuswith 2% carbon dioxide. The results are shown in Fig. 8. The graphing is the sameas described previously with the exception that a horizontal line has not been drawn for the oxygen due to a short flash alone becausenone is evolved. Again, if the short flash arrives early, the yield is greater than that from the long flash alone; likewise if it arrives late. But when they occur together the yield is decreased.This was always the casewhen the two flashes were superimposedand is not the same as the behavior observed with cellsin the presenceof quinone. This decrease in yield is reminiscent of the inhibition of the low background rate by a short flash, discussedabove.
PHOTOSYNTHETIC
EVOLUTION
OF OXYGEN
141
These results have to be considered in the light of various inhibitions present. Reasonshave been presented before (12,14,18) for believing that under prolonged anaerobic conditions, the enzyme or enzymes concerned with the liberation of oxygen become inhibited. If, therefore, one short flash does not yield any observable oxygen, it only means that sufficient removal of the inhibition of this enzyme has not occurred. That a single short flash has a preparatory action is evident from the results with pairs of flashes. In a similar manner, a low background illumination activates the system sufficiently so that the effect of a single short flash becomes observable as a burst of oxygen. But this is true only if the short flash is not too intense. At the highest intensities6 we see in Fig. 3 that the oxygen evolution produced by the short flash is followed by a period in which the rate of photosynthesis from the continuous irradiation is temporarily depressedfor some 10 or 15 min. The net influence of the superimposition of the short flash is a reduction of the oxygen evolution. This is another example of the type which led Franck (19) to the assumption of a so-called “narcotic”; the photooxidation of sugars results in the production of substanceswhich “blanket” the chlorophyll and lower the photosynthetic activity. This same type of phenomenon can account for the observations made with the short and the long flashessuperimposed.When the short flash arrives before the long one, it serves to remove some of the inhibition of the oxygen-liberating enzyme and therefore prepares the photosynthetic machinery so that it can make better use of the long flash. The same applies if the short flash arrives late, except that in this casethe long flash now serves to remove some of the inhibition of the oxygen-liberating enzyme. If, however, the short flash arrives directly superimposedon the long one, it serves only to inhibit in the manner suggested previously. As a result, the portion of the long flash (subsequent to the arrival of the short one) is not effectively utilized. Conditions are different when quinone is present, for there is no metabolism. The cells are intact only in a morphological sense, and we assume that any inhibitory products are prevented from forming, as well as being removed. Noack et al. (20) observed that algae previously inhibited by long anaerobiosis were able to evolve oxygen copiously upon the addition of a small amount of quinone. Warburg (21) subsequently showed that quinone itself was being reduced, but, nevertheless, 6 Rough estimates indicate that at the highest flash intensities, were available to excite every chlorophyll molecule.
sufficient quanta
142
F. L. ALLEN
AND J. FRANCK
the rapid evolution of oxygen indicates the removal of the inhibitions. Shiau and Franck (15) have shown that the addition of quinone to anaerobically inhibited algae reduces the fluorescene and removes the induction anomalies connected with it. Thus, ample evidence leads one to expect that a single short flash alone will result in the evolution of oxygen from cells in the presence of quinone but not from cells in the presence of carbon dioxide. SUMMARY
An attempt is made to reconcile the apparently contradictory observations in flash photosynthesis using short flashes (cu. lO+ sec.) and long flashes (cu. W3 sec.). It is shown that the introduction of a new period of ca. W4 sec. not only obviates these previous difficulties, but also is not in contradiction with any existing observations. In addition, a mechanism for the primary photochemical process, suggested by one of us (J. F.) and discussedin detail elsewhere, is presented. Using reversible phosphorescencequenching as a means of measuring very small concentrations of oxygen, we have also studied the evolution of oxygen under anaerobic conditions by single flashes of light. The biological material was the algae XcenedesmusobZ~@~sin the presence of either carbon dioxide or the Hill reagent quinone. It has been observed that while a single, intense, half-millisecond flash evolves oxygen in the Hill reaction, it does not evolve oxygen in photosynthesis. A flash 50 times as long and of much lower intensity results in the evolution of oxygen in both systems. Further, in the Hill reaction, the yield from an intense half-millisecond flash is increased by a previous similar flash preceding it by one full second. This observation suggests that certain photoproducts are capable of surviving longer than the generally assumed 1W2 sec. Various other observations on the effect of overlapping short and long flashes, and some plausible explanations, are described. REFERENCES 1. WARBURG,O.,B~~&~~.Z. 100,230 (1919). 2. BROWN, H. T., AND ESCOMBE, F.,Proc. Roy.Soc. (London) B76, 29 (1905). 3. EMERSON, R., AND ARNOLD, W.,J.Gen. Physiol. 16,391 (1932). 4. EMERSON, R., AND ARNOLD, W., J. Gen. Physiol. 16, 191 (1932). 5. TAMIYA, H., AND CHIBA, Y., Studies Tokugawa Znstitue 6, parts I and II (1949). 6. CLENDENNING, K.A., ANDEHRMANTRAUT, H. C.,Arch.Biochem.29,387 (1950). 7. EHRMANTRAUT, H., AND RABINOWITCH, E., Arch. Biochem. and Biophys. 36, 67 (1952).
PHOTOSYNTHETIC
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OXYGEN
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8. GILMOUR, H. S. A., LUMRY, R., SPIKES, J., AND EYRING, H., “Studies of Photosynthetic Processes, Technical Report XI.” University of Utah, 1953. See also Nature 1’73, 31 (1954). 9. FRANCK, J., AND HERZFELD, K. F., J. Phys. Chem. 46,978 (1941). 10. FRANCK, J., Daedalus 1 (1955). 11. LIVINGSTON, R., PORTER, G., AND WINDSOR, M., Nature 1’73, 485 (1954). 12. FRANCK, J., PRINGSHEIM, P., AND LAD, D., Arch. Biochem. 7,103 (1945). 13. POLLACK, M., PRINGSHEIM, P., AND TERWOORD, D., J. Chem. Phys. 12, 295 (1944). 14. ALLEN, F. L., Arch. Biochem. and Biophys. 66, 38 (1956). 15. SHIAU, Y., AND FRANCK, J., Arch. Biochem. 14,253 (1947). 16. BRUGGER, J., Thesis, Univ. of Chicago, 1954. 17. LINSCHITZ, H., AND RENNERT, J., Nature 169, 193 (1952). 18. GAFFRON, H., Biochem. 2. 280, 337 (1935). 19. FRANCK, J., Chap. 16. “PHOTOSYNTHESIS IN PLANTS,” The Iowa State College Press, Ames, Iowa, 1949. 20. NOACK, K., PIRSON, A., AND MICIIAELS, H., Naturwissenschuften 27,645 (1939). 21. WARBURG, O., “Heavy Metal Prosthetic Groups and Enzyme Action” (Trans. by Alexander Lawson), Chap. 20. Clarendon Press, Oxford, 1949. 22. WELLER, A., J. Am. Chem. Sot. 70, 5819 (1954). 23. WELLER, J., AND FRANCK, J., J. Phys. Chem. 46, 1359 (1941). 24. RIEKE, F. F., AND GAFFRON, H., J. Phys. Chem. 47,299 (1943).
the Research
Institutes,
University
of Chicago,
(Fels
Fund),
Chicago,
Illinois
Received March 21, 1955
I. LITERATURE
AND INTERPRETATION
The efliciency of utilization of light energy by the photosynthetic machinery depends on whether this energy arrives in a continuous stream or in short pulses separated by relatively long dark intervals. As is the case with other problems in the field of photosynthesis, the reason for this is not only improperly understood, but apparent contradictions even exist between sets of experimental data from different laboratories. Neither can any of these observations be rejected nor has a satisfactory unifying scheme been developed which will obviate all the difficulties. It might be of interest, therefore, to inquire whether perhaps a way exists in which some of the discrepancies can be avoided along lines which will either confirm, reject, or preferably clarify the present status. Warburg (1) was the first2 to expose algae to short, equal periods of light and dark. He observed at high intensities a net increase in oxygen yield over that which would obtain due to illuminating the algae half the total time with the same but continuous intensity. However, it was in 1932 that Emerson and Arnold (3, 4) performed what is today the classical experiment in flash photosynthesis. Using very short flashes (cu. lO+ sec.) and variable dark intervals between the flashes, they were able to show that if this dark interval was made about 0.01 sec., all the photochemical products created during the brief light flash could be 1 This work was made possible through financial assistance from the Fels Fund (University of Chicago); the Office of Naval Research, Department of the Navy (Contract No. 119-272) ; and the National Science Foundation. * Prior to Warburg (I), Brown and Escombe (2) used the method of rotating sectors to provide flashing light for photosynthesis, being unaware of intermittency effects. 124
PHOTOSYNTHETIC
EVOLUTION
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OXYGEN
125
handled during the subsequent dark period. A shorter dark interval reduced the yield per flash, a longer one did not contribute anything more. Further, the maximum yield per flash, at their highest light intensity, was independent of the temperature, although at low temperatures the dark interval required to attain this maximum was greater. Their yield per flash showed a surprising constancy relative to the number of chlorophyll molecules present; only about one oxygen molecule was liberated per 2000 chlorophyll molecules. Now, entirely in agreement with Emerson and Arnold, Tamiya and Chiba (5) confirmed the period of 0.01 sec. and the maximum yield of about l/2000 independent of temperature. However, when Tamiya increased his light intensity above a certain limit, the whole character of his curves became different. Not only did the dark period become much longer (up to 0.1 sec.), but the yield per flash rose and became strongly temperature dependent. Tamiya, therefore, concluded that Emerson and Arnold had used flashes of low total intensity, and thus had not achieved real flash saturation. In order to attain his presumed flash saturation, Tamiya used flashes which were very long compared with those of Emerson and Arnold-between 0.6 X UPa and 8 X 10-3 sec. at halfwidth compared with about 1P sec. of the latter authors. We will return to this point later. Faced with this discrepancy, Clendenning and Ehrmantraut (6), as well as Ehrmantraut and Rabinowitch (7), repeated the work of Emerson and Arnold, again using short flashes of about UP sec., and extended it to the case of the Hill reaction with quinone. Their combined results indicated that the period of 0.01 sec. and a yield of about l/2000 occur in the Hill reaction as well as in photosynthesis. The yield of l/2000 remained constant for photosynthesis even if the flash intensity was raised stepwise to a factor of four times the intensity previously used by Emerson and Arnold. In studies of the kinetics of photosynthesis, Gilmour, Lumry, Spikes, and Eyring (8) measured the reduction rates of ferricyanide ions by sugar-beet chloroplasts, both in continuous and flashing light. These authors used flash times even longer than Tamiya’s, in the range of 2.8-16.7 X lo-3 sec. With the shorter flashes, the curves they obtained indicate a period of about 0.01 sec., both at low (31,000 lux) and high (250,000 lux) light intensities, but in the latter case the curves resemble Tamiya’s. In fact, their thorough kinetic analysis of the data revealed
126
F.
L.
ALLEN
AND
J.
FRANCK
the existence of five different reaction periods,3 depending on the external conditions. Gilmour et al. suggest that it is the difference in flash duration and not flash intensity which must be responsible for the discrepancy between the results of Tamiya and those of Emerson and Arnold. They therefore assume that a reservoir of photosynthetic products must be filled to obtain the results of Tamiya, and that a time of W6 sec., as used by Emerson and Arnold, is too short to fill it, irrespective of flash intensity. To explain the results of Emerson and Arnold, Franck and Herzfeld (9) suggested that the early photochemical products created during the flash were unstable and disappeared by back reactions unless stabilized by an enzyme. The dark period of about 0.01 sec. (required to obtain the maximum yield) was thus its working period. The limited number of these enzyme molecules (called at that time catalyst B) was responsible for flash saturation. This was the over-all picture as it existed several years ago. The present status of flash photosynthesis, including the observations of Tamiya and of Gilmour et al., can be reconciled with the original explanation of Franck and Herzfeld by making a minor modification. We need only assume that the working period of the stabilizing enzyme is in the order of 1W4 sec., in place of about 0.01 sec. This means that the enzyme can work several times during a flash lasting, say 1W3 sec., but only once during a flash lasting lO+ sec. The period of about 0.01 sec., for instance in the case of Emerson and Arnold, would then indicate that the total amount of material made by the flash can be worked up within this time by all the enzymes involved. $n the controversy on quantum yields, we agree with those who come to the conclusion that two quanta are necessary to effect the transfer of one hydrogen in normal photosynthesis. This, as well as other considerations, has led one of us (J. F.) to propose the following picture (lo), which we discuss here briefly: The first absorption act produces a metastable state of the chlorophyll. This metastable state has a biradical character-its absorption spectrum has been found and studied by Livingston et al. (11). Since this metastable state is relatively longlived, sufficient time exists to permit, by virtue of its biradical character, a complexing with the photosynthetic oxidant and the enzyme which plays the role of OH acceptor (possibly cytochrome F?). Neither the 3 Weller and Franck the existence of longer sec. duration.
(23) as well as Rieke Blackman (enzymatic)
and Gaffron periods
(24) had previously shown than the one of about 0.01
PHOTOSYNTHETIC
EVOLUTION
OF OXYGEN
127
energy of the first excited state nor the energy of the lower-lying metastable state is sufhcient to effect the transfer of a hydrogen to the photosynthetic oxidant with the concomitant dissociation of water. However, as a result of the shift in the absorption spectrum of the metastable state of chlorophyll toward longer wavelengths,4 this chlorophyll in the metastable state can collect quanta by sensitized fluorescence from other excited chlorophyll molecules. An excited triplet state (11) can therefore be quickly formed, which relative to the singlet ground state now possessessufficient energy to transfer (a) an H from water to the photosynthetic oxidant and (b) an OH to an enzyme. It is to this enzyme that we can ascribe a working period of 1W4 sec. Of course, the energy transfer from an excited chlorophyll molecule to a chlorophyll molecule in its metastable state must be quite efficient-as we know sensitized fluorescence is, provided the overlapping of the corresponding absorption and emission spectra is favorable-since flash saturation already occurs when approximately l/100 of the chlorophyll molecules perform an initial absorption act. This modified picture, then, seemsto remove the discrepanciesapparently existing between different observations on flash photosynthesis. II. EXPERIMENTAL All of the contributions on flash photosynthesis discussedin Sec. I have been made with repetitive flashes, and the yield per flash has been deduced from an integrated effect. It would be of considerable interest to study the yields from single isolated flashes of light. No technique of sufficient sensitivity has been developed which will allow this under aerobic conditions. However, Franck, Pringsheim, and Lad (12) have shown that the method of phosphorescence quenching permits, under anaerobic conditions, the measurement of the evolution of oxygen from a single flash. Their experiments were, however, very preliminary. As a source of light they used photographic flashbulbs, the half-width being about 20 X 1W3 sec. Using this brief intense flash, they were able to demonstrate the evolution of oxygen from algae previously in total 4 The absorption spectra of chlorophyll in the triplet state have not as yet been measured in the red region of the spectrum. However, the strong similarity between the absorption spectrum of the chlorophyll phase test intermediate throughout the visible, as measured by Weller (22) r and the absorption spectrum of chlorophyll in the triplet state as measured by Livingston et al. (11) makes such a prediction reasonable.
128
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AND
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FRANCK
darkness and under an anaerobic state of about lW-” mm. Hg. Their yield of oxygen molecules per chlorophyll molecule for a single flash was approximately l/10,000 in contrast to Emerson and Arnold’s l/2000. This discrepancy was assumed not to be significant and easily explained by the inhibition which they observed under the conditions of their experiments. A repetition and extension of these experiments has been carried out and, particularly, a comparison between cells capable of performing photosynthesis and cells in the presence of the Hill reagent quinone. The oxygen production was determined using the method of Pollack, Pringsheim, and Terwoord (13). In order to have oxygen evolutions per flash large enough to be measured with comparative ease, the sample holder described previously (14) was replaced by a larger one (Fig. 1) having a volume of 20 cc. Agitation of the sample was provided by the gas flow of nitrogen (plus 2% carbon dioxide) at the rate of 10 cc./min. rising through the liquid after being broken up into fine
FIG. 1. The sample-holder (filled with ink for the photograph) position inside the flash coil. The short flash, therefore, illuminates from all sides.
is shown in the sample
PHOTOSYNTHETIC
EVOLUTION
OF
OXYGEN
129
FIN. 2. Shape and duration of the two types of light flashes used in the experiments described. A. Single short flash. Time scale: 0.15 milliseconds per division, B. Single long flash. Time scale: 10.0 milliseconds per division. C. Short and long flash superimposed, showing manner in which timing was determined for the twoflash technique. The peak of the short flash is considerably off scale. Time scale: 10.0 milliseconds per division. bubbles by the fritted glass bottom. Needless to say, we were working under strict anaerobiosis-the pressure of oxygen in the carrier gas with the sample in total darkness was at most We mm. Hg. The present experiments were all performed with the algae, Scenedesmus obliquus strain Da . Procedures for growing the algae and their handling were identical with those described previously (14). The concentrations used in these experiments were, however, much higher-in the neighborhood of 0.40/o. Therefore, the algae were never centrifuged and seldom diluted, but transferred directly from the culture tube8 to the sample holder and anaerobiosis established. The suspending solution was always the culture medium at pH 8.2 except in the cases where quinone was used as a Hill oxidant. The algae were in that case suspended either in culture medium or in distilled water. Quinone solutions were made up just before use from freshly sublimed reagent. Owing to the large volume of the present samples, about 2 hr. was required to establish an anaerobic state of IO+ mm. Hg after placing the sample in the holder and beginning flushing with the carrier gas. The experiments were thus performed after approximately 3 hr. of anaerobiosis, obviously not all this time at the final pressure of less than 10-B mm. Hg of oxygen. Two types of flashes were ueed, short flashes of extremely high intensity and long flashes of a lower intensity, which will subsequently be referred to merely as short flashes and long flashes. The short flashes were provided by an Amglo 1TZ discharge lamp,6 shown in Fig. 1, together with a 1OOmicrofarad (pf.) condenser and a variable voltage power supply. The shape and duration of such a flash is shown in Fig. 2, the half-width being about half a millisecond. The hold-off voltage for this lamp was over 4OCil v., 80 that the discharge required initiation by an ionizing spark from a Tesla coil activated by a small condenser. The suspension holder fitted inside the flash lamp coil and, therefore, was surrounded by the 6 Amglo Corporation,
4234 Lincoln
Avenue, Chicago 18, Illinois.
130
F. L. ALLEN
AND J. FRANCK
flash. For this reason, and others as well, it is difficult to ascertain the exact intensity. The manufacturer rates the lamp at approximately 35 lumens/watt-second. At 2000 v. and 100 pf. this would mean7O@l lumen-sec. From theshape of the light intensity vs. time curve, one can estimate the peak value under these conditions to be about 25 million lumens. For higher values of the voltage the total intensity would be higher, roughly proportional to the square of the voltage. Spectral analysis of the actual flash indicated a strong concentration of the emission toward the blue end of the visible spectrum. The long flashes were provided by a camera shutter in combination with a 1000-w. tungsten filament lamp, heat absorber, collimating lenses, etc. The halfwidth of the flash was about 25 X 10es sec., i.e., some 50 times longer than the short flashes, and the peak intensity some 250,000 lux. This was varied by changing the voltage on the lamp, and no attempt was made to measure the intensity with any accuracy. The superimposition of the two flashes, when desired, was accomplished by triggering the shutter with a solenoid and activating the Tesla coil for the discharge tube both with thyratrons. The timing was provided by the old but dependable method of shorting contacts with a freely falling plumb. An oscilloscope sweep was tripped at the beginning of the sequence and a photocell registered on it the phasing of the two flashes, an example of which is shown in Fig. 3. In order to eliminate uncertainties, the events as registered on the oscilloscope were recorded permanently by photographing the trace. Although the burst of oxygen evolved from a short flash may itself last a very short time, the large volume of the liquid sample prevents the instantaneous flushing out by the carrier gas. Therefore, on the Brown recorder one observes a burst having a half-width of about 1 min., as in Fig. 4. This burst is transformed to rate of oxygen evolution by means of the calibration of the instrument, and graphical integration under the resulting curve yields the total amount of oxygen evolved by the flash.
III.
THE
HILL
REACTION
WITH QUINONE
As is well known, the Hill reaction is simpler than photosynthesis inasmuch as the complicated carbon dioxide reduction cycle is replaced by the more straightforward reduction of a substitute oxidant. This simplification is made particularly evident by the reported (6) absence of induction periods in the Hill reaction. We therefore discuss first our observations on the Hill reaction with quinone. With a suspension of Scenedesmusin the presence of approximately 5 x 1O-4M quinone, it is very easy to observe and measurethe evolution of oxygen from a single short flash. Such a burst is shown in Fig. 4 and corresponds to about one oxygen molecule per 30,000 chlorophyll molecules. This value is low compared to the yield of l/2000 obtained by Ehrmantraut and Rabinowitch for the case of repetitive flashes. Our flash intensity could be varied over wide limits without changing the flash
PHOTOSYNTHETIC
EVOLUTION
OF
OXYGEN
131
FIG. 3. Top: Inhibition of low background rate of photosynthesis by a very strong, short light flash. The small spike just before the oxygen burst is an electrical disturbance caused by the flash. Middle left: The extremely small yield observed as a result of a short flash if the algae are in total darkness previous to the flash. Actually, the effect observed in this case was caused by the lack of complete shielding of the sample from the room lights at the time the flash was applied. Middle right : Enrichment of the carrier gas with a small amount of oxygen prior to passing through the sample does not cause a short flash to evolve oxygen from the algae. Bottom left: Large burst evolved by the 25.millisecond flash from algae previously in total darkness. Bottom right: The sample has been illuminated with a weak background light and is photosynthesizing at a very low rate. The short flash is now able to evolve oxygen. Time scale: Each accented division on the abscissa is 1 min. Flashes came under the number that indicates the voltage.
132
F.
8
L.
ALLEN
AND
J.
FRANCK
I
6
FIG. 4. Oxygen evolution by short flashes from cells suspended in 5 X lo-’ M quinone. Large burst is the evolution from two short flashes spaced about 1 sec. apart. Small burst is the result of a single short flash. The total amount of oxygen represented by the large burst is 30% greater than twice that represented by the small burst. Time scale: Each accented division on the abscissa is 1 min.
length. This was done by interposing suitable absorbers between the sample holder and the coil of the discharge lamp, and by changing the discharge voltage. It was thus possible to show that this yield of about l/30,000 was not low as a result of failure to reach flash saturation. Likewise, since it was possible to increase the flash energy by a factor of, roughly, ten without decreasingthe yield (once flash saturation had been reached), it could not be the result of having gone excessively beyond flash saturation. We take this value, therefore, as the order of magnitude of oxygen evolution to be expected from a single, short, isolated flash of saturating intensity under the conditions of our experiments. Considerable care had to be exercised with the use of quinone in these
PHOTOSYNTHETIC
EVOLUTION
TABLE Interval between the two flashes SCG. 1
250
OF
OXYGEN
133
I Combined oxygen ield from the two flas ii es cc.
5.5 4.2
x 10-7 X 10-7
flash experiments, since it was found that, especially with strong flashes, the algae were fairly rapidly poisoned, perhaps as a result of photodecomposition products of quinone. This is not a new observation; quinone inhibitions of one sort or another have been reported before (6). When this was the case, the yield of l/30,000 was no longer attainable. It was, therefore, necessary to perform each experiment quickly and with a minimum number of short flashes. Also, the cells remained in the presence of quinone for about 2 hr. before measurements could be made. This may have been detrimental and might be responsible for the low yield observed with single flashes. Of greater interest is the observation that the combined yield obtained from two short saturating flashes increases as the interval of time between the two flashes is reduced. Table I illustrates this effect. Thus, there is a net gain of some 30 9%as a result of applying two short flashes a second rather than a few minutes apart. This result, in which apparently something from the first flash carries over to the subsequent one, is further exemplified by the use of a teehnique of superimposing two flashes, one short flash and one long flash, and observing their combined yield as a function of their phasing, i.e., as a function of the position of the short flash relative to the long one. The general characteristics of the two flashes were described earlier. As stated before, the short flash was saturating. The long flash was not saturating, its yield depending on the voltage on the 1000-w. lamp, which was, however, kept constant during any given series of measurements. The result of such an experiment is shown in Fig. 5. The yield is plotted on the ordinate as cubic centimeters of oxygen per flash, meaning the combined yield of the short and long flash (their yield cannot be resolved separately, of course). The abscissa indicates the phasing of the short flash relative to the long one (the approximate shape and duration of the long flash is represented by the shaded trapezoid). The experimental points give the yield, at the time relative to the long flash, indicated by their position with reference to the trapezoid. For example, for the results of the particular experiment illustrated in Fig. 5, if the short
134
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L.
ALLEN
AND
J.
FRANCK
I % ; 0” ::
-----&--------------~
,
----------------~ I
-50
-40
-30
-20
-10
0
+I0
+20
+30
+40
+50
MILLISECONDS
FIG. 5. Results of the two-flash technique with cells in the presence of 5 X 10-4 M quinone. The points at the extreme right are the values for which the horizontal lines were drawn. For explanation see text.
flash arrived 30 X KF3 sec.before the middle of the long flash, the oxygen yield observed for both together was about 2.8 X lO--’ cc. Separately, each flash was capable of producing oxygen also, and these yields are indicated by the horizontal lines located at the proper position on the ordinate: the lower dotted line indicates the yield per short flash alone; the upper dashed line indicates the yield per long flash alone. Both flashes combined, irrespective of their phasing, produce more oxygen than either alone. The more striking feature, however, is the higher yield obtained if the short flash is early. The data presented in Table I indicate that either the first light flash produces something which requires a second light flash to evolve oxygen, or that a substance produced by the first flash and which normally leads to oxygen evolution doesso at such a slow rate that it cannot be detected by our present method-the second flash merely hastening this evolution. In either case,the second light flash leads to additional oxygen evolution which is observed. We are inclined to think that there is some connection between the formation of this long-living substance and the photochemical reaction whose occurrence is suggested by the quenching of chlorophyll fluorescence in the presence of quinone (15, 16). The quinone concentration in our experiments is not known exactly because some of the quinone is
PHOTOSYNTHETIC
EVOLUTION
OF
135
OXYGEN
flushed away by the carrier gas during the hours preceding the experiments. We estimate a concentration of 1F4 M, which would give a quenching of about 10-30 % (15). While the bulk of the quinone reduction may proceed with the utilization of two quanta per H atom transferred, in the manner indicated in Sec. I, the percentage of quinone reduction associated with fluorescence quenching might, in principle, be achieved in a one-quantum process. We indicate a chlorophyll with its ring V in a hydrated state, viz., C ;: H-ClOe.--.-
OH
I/
I COOCHS
“i OH
OH /
by the symbol
H-Cph
. An impact
\
between
quinone
and
‘OH OH /
excited by light absorption
H-Cph* \
to the first excited state
OH t OH /
and indicated
by H-Cph*
permits energetically \
the reaction:
OH
OH
OH
/ quinone
/
+ H-Cph*
+ \
semiquinone
+
-Cph \
‘OH
‘OH
I’his process might be identical with the one observed by Linschitz and Rennert (17), i.e., a photochemical reversible bleaching of chlorophyll in the presence of quinone in a rigid glass. One of the hydroxyls of this OH / radical could be removed by an enzyme and utilized for -CPh. \
OH
136
F. L.
O2 evolution,
ALLEN
AND
J. FRANCK
because the reaction OH
-Cph
/ \
where Cph-OH
+ enzyme
4
Cph-OH
+ enzyme-OH
OH
represents
the enol form of chlorophyll
C=C-OH
i should be slightly exothermic. However, the reaction would require a considerable heat of activation for the opening of a C-OH bond and the closing of the double bond. Thus it would proceed very slowly in the dark. OH / On account of the radical character of -Cph the enzyme might \ ’ OH be adsorbed on it; and, if now a light quantum is absorbed, it will provide ample energy for the heat of activation. We may think of this overall process as an “abnormal” mechanism. At high light intensity by far the major portion of the quinone reaction might again proceed as the two-quanta processdiscussedin Sec. I, while at very low light intensities the heat of activation might be provided by thermal fluctuations before the arrival of the next light quantum. The results obtained with the short and long flashes superimposedon each other can be explained along the same lines. There is competition OH / between the process creating the longer-lived species -Cph ‘\ OH formed by an impact between quinone and a chlorophyll molecule in its first excited state (the “abnormal” mechanism), and the two-quanta process via the excited metastable state of chlorophyll (the “normal” mechanism) as discussedin Sec. I. If the short flasharrives early, the long flash finds a relatively high concentration of the longer-lived radical to “work” on, and the oxygen evolved is therefore larger. If the short 5ash arrives late, most of the oxygen evolution is the result of the twoquanta process during the long flash, and the oxygen evolution as the
PHOTOSYNTHETIC
EVOLUTION
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result of the “abnormal” mechanism from the short flash is not observed, at least to any significant extent. IV. PHOTOSYNTHESIS WITH 2% CARBON DIOXIDE In sharp contrast to the observations discussed in the preceding section, namely, the ability of a single short flash to evolve oxygen cells suspended in 5 X 10-* M quinone, cells in the presence of 2% carbon dioxide and, therefore, capable of performing normal photosynthesis, do not evolve oxygen as the result of a single short flash. The upper limit of the yield under these conditions can be estimated to be about one oxygen molecule per lo6 or lo6 chlorophyll molecules. In spite of surrounding the flash coil with aluminum foil to increase the intensity and at the highest flash energy (5000 v.) we could provide, no oxygen evolution was observed. Further evidence that this was not the result of insufficient flash intensity is given by the following observation. When the sample was irradiated with a very low but continuous light and then the flash applied, a significant burst of oxygen was evolved, as shown in Fig. 3, corresponding to a yield of about l/50,000. In this particular case, the weak continuous illumination was sufficient to produce from the sample a steady rate of only lO-* vol. oxygen/hr./vol. algae. However, it must be borne in mind that this figure is deceptive because, with suspensions in the order of 0.4% and the low background light intensities used, not all the algae are illuminated equally or at the same time. In any case, this gives an indication of the low level of background light required. The pressure of oxygen in the carrier gas after passing through the sample and picking up the oxygen was, for the particular experiment illustrated in Fig. 3, 2.2 X KP mm. Hg; and the concentration of oxygen dissolved in the algal suspension (expressed in terms of the pressure of oxygen in an atmosphere above the sample which would be in equilibrium with this concentration) was shown by the method discussed previously (14) to be in the order of 1w3 mm. Hg. When the intensity of the low background light is increased, the yield per flash is increased also, as shown in Fig. 7. This is to be expected, if for no other reason than that in view of the rather dense algal suspensions used, an increase in the background light results in more algae receiving light and, therefore, being able to evolve oxygen when the short flash is applied. It is extremely improbable that one can ascribe these observations to the presence of the very low concentration of oxygen produced by the
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weak background illumination, as can be seen from some experiments in which oxygen was mixed into the nitrogen carrier gas before it passed through the sample. The short flash was then applied, the sample being previously in total darkness. Figure 3 illustrates such an experiment in which the carrier gas was enriched with 1W4 mm. Hg of oxygen besides the usual 2 % carbon dioxide. Not the slightest trace of oxygen evolution can be observed as the result of the short flashes. Evidently, the low background illumination is able to provide some sort of “priming,” and the question immediately arises: After this low background light is removed, how long does this “priming” survive? In other words, how long does this ability of short flashes to evolve oxygen in the presence of a low continuous illumination persist after this illumination is turned off? The answer is given in Fig. 6. At zero time the continuous light was removed, and at various times after this zero time the short flash was applied and its yield measured. The half-life obtained is in the neighborhood of 20 sec. Figure 7 shows that the yield per flash in photosynthesis is strongly dependent on the flash energy and the background illumination. This is especially true if the flash energy greatly exceeds that required for obtaining maximum evolution per flash at a given fixed value of the background illumination. It must be remembered that the values plotted in I
I IO
FIQ. 6. The persistence the low background light
I
I
1 I 20 30 SECONDS of the ability of the short is turned off.
I
I 40 flash
to evolve
oxygen
after
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FIG. 7. Effect on the yield per short flash (when superimposed on a low back ground light) as a function of the energy of the flash, at two different values of the intensity of the background illumination. The oxygen pressures by the curves indicate the rate of photosynthesis and, therefore, the intensity of the background light. The energy units are arbitrary.
Fig. 7 are obtained by superimposing the short flash on a low background of light, i.e., on a low rate of photosynthesis. At low flash energies no effect is observed on this background rate as the result of the short flash. However, if the intensity of the flash is made very great, the background rate of photosynthesis is partially inhibited for a matter of minutes immediately following the flash. It slowly regains its original value as shown in Fig. 3. In contrast to this, for the case of the Hill reaction with quinone, no particular influence of either the short flash on the steady evolution, or of the steady evolution of oxygen on the yield per short flash, was observed. A burst of oxygen was merely superimposed on the steady background rate. While it was observed that a short flash alone was unable to result in the evolution of oxygen, the burst of oxygen due to a long flash alone was quite large. One such burst is shown in Fig. 3. The long flash, some25 X UP3 sec. at half-width, was not saturating because of the rather dense suspensionsused: 0.345%. However, the total amount of light falling on the sample was estimated to be about the samein both the short and the long flashes. This was probably true within an order of magnitude,
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8. Results of the two-flash technique with cells in the presence of 2% carbon dioxide and, therefore, capable of performing photosynthesis. Two sets of data are plotted. The points on the extreme right are the values for which the horizontal line is drawn. For explanation see text. FIG.
the short flash possibly being the stronger. Yet the 25 X KV sec. flash was able to evolve oxygen while the 0.5 X 1P sec.was not. This observation confirms the result of Franck et al. (12) that the ignition of a photographic flashbulb resulted in a burst of oxygen from algae previously in the dark. We conclude, therefore, that as far as a single flash is concerned, it is not merely a question of the number of quanta involved, but also the space of time over which they arrive. The technique of superimposing two flashes, a long and a short one, described previously for the Hill reaction, was used also in the case of Scenedesmuswith 2% carbon dioxide. The results are shown in Fig. 8. The graphing is the sameas described previously with the exception that a horizontal line has not been drawn for the oxygen due to a short flash alone becausenone is evolved. Again, if the short flash arrives early, the yield is greater than that from the long flash alone; likewise if it arrives late. But when they occur together the yield is decreased.This was always the casewhen the two flashes were superimposedand is not the same as the behavior observed with cellsin the presenceof quinone. This decrease in yield is reminiscent of the inhibition of the low background rate by a short flash, discussedabove.
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These results have to be considered in the light of various inhibitions present. Reasonshave been presented before (12,14,18) for believing that under prolonged anaerobic conditions, the enzyme or enzymes concerned with the liberation of oxygen become inhibited. If, therefore, one short flash does not yield any observable oxygen, it only means that sufficient removal of the inhibition of this enzyme has not occurred. That a single short flash has a preparatory action is evident from the results with pairs of flashes. In a similar manner, a low background illumination activates the system sufficiently so that the effect of a single short flash becomes observable as a burst of oxygen. But this is true only if the short flash is not too intense. At the highest intensities6 we see in Fig. 3 that the oxygen evolution produced by the short flash is followed by a period in which the rate of photosynthesis from the continuous irradiation is temporarily depressedfor some 10 or 15 min. The net influence of the superimposition of the short flash is a reduction of the oxygen evolution. This is another example of the type which led Franck (19) to the assumption of a so-called “narcotic”; the photooxidation of sugars results in the production of substanceswhich “blanket” the chlorophyll and lower the photosynthetic activity. This same type of phenomenon can account for the observations made with the short and the long flashessuperimposed.When the short flash arrives before the long one, it serves to remove some of the inhibition of the oxygen-liberating enzyme and therefore prepares the photosynthetic machinery so that it can make better use of the long flash. The same applies if the short flash arrives late, except that in this casethe long flash now serves to remove some of the inhibition of the oxygen-liberating enzyme. If, however, the short flash arrives directly superimposedon the long one, it serves only to inhibit in the manner suggested previously. As a result, the portion of the long flash (subsequent to the arrival of the short one) is not effectively utilized. Conditions are different when quinone is present, for there is no metabolism. The cells are intact only in a morphological sense, and we assume that any inhibitory products are prevented from forming, as well as being removed. Noack et al. (20) observed that algae previously inhibited by long anaerobiosis were able to evolve oxygen copiously upon the addition of a small amount of quinone. Warburg (21) subsequently showed that quinone itself was being reduced, but, nevertheless, 6 Rough estimates indicate that at the highest flash intensities, were available to excite every chlorophyll molecule.
sufficient quanta
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the rapid evolution of oxygen indicates the removal of the inhibitions. Shiau and Franck (15) have shown that the addition of quinone to anaerobically inhibited algae reduces the fluorescene and removes the induction anomalies connected with it. Thus, ample evidence leads one to expect that a single short flash alone will result in the evolution of oxygen from cells in the presence of quinone but not from cells in the presence of carbon dioxide. SUMMARY
An attempt is made to reconcile the apparently contradictory observations in flash photosynthesis using short flashes (cu. lO+ sec.) and long flashes (cu. W3 sec.). It is shown that the introduction of a new period of ca. W4 sec. not only obviates these previous difficulties, but also is not in contradiction with any existing observations. In addition, a mechanism for the primary photochemical process, suggested by one of us (J. F.) and discussedin detail elsewhere, is presented. Using reversible phosphorescencequenching as a means of measuring very small concentrations of oxygen, we have also studied the evolution of oxygen under anaerobic conditions by single flashes of light. The biological material was the algae XcenedesmusobZ~@~sin the presence of either carbon dioxide or the Hill reagent quinone. It has been observed that while a single, intense, half-millisecond flash evolves oxygen in the Hill reaction, it does not evolve oxygen in photosynthesis. A flash 50 times as long and of much lower intensity results in the evolution of oxygen in both systems. Further, in the Hill reaction, the yield from an intense half-millisecond flash is increased by a previous similar flash preceding it by one full second. This observation suggests that certain photoproducts are capable of surviving longer than the generally assumed 1W2 sec. Various other observations on the effect of overlapping short and long flashes, and some plausible explanations, are described. REFERENCES 1. WARBURG,O.,B~~&~~.Z. 100,230 (1919). 2. BROWN, H. T., AND ESCOMBE, F.,Proc. Roy.Soc. (London) B76, 29 (1905). 3. EMERSON, R., AND ARNOLD, W.,J.Gen. Physiol. 16,391 (1932). 4. EMERSON, R., AND ARNOLD, W., J. Gen. Physiol. 16, 191 (1932). 5. TAMIYA, H., AND CHIBA, Y., Studies Tokugawa Znstitue 6, parts I and II (1949). 6. CLENDENNING, K.A., ANDEHRMANTRAUT, H. C.,Arch.Biochem.29,387 (1950). 7. EHRMANTRAUT, H., AND RABINOWITCH, E., Arch. Biochem. and Biophys. 36, 67 (1952).
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8. GILMOUR, H. S. A., LUMRY, R., SPIKES, J., AND EYRING, H., “Studies of Photosynthetic Processes, Technical Report XI.” University of Utah, 1953. See also Nature 1’73, 31 (1954). 9. FRANCK, J., AND HERZFELD, K. F., J. Phys. Chem. 46,978 (1941). 10. FRANCK, J., Daedalus 1 (1955). 11. LIVINGSTON, R., PORTER, G., AND WINDSOR, M., Nature 1’73, 485 (1954). 12. FRANCK, J., PRINGSHEIM, P., AND LAD, D., Arch. Biochem. 7,103 (1945). 13. POLLACK, M., PRINGSHEIM, P., AND TERWOORD, D., J. Chem. Phys. 12, 295 (1944). 14. ALLEN, F. L., Arch. Biochem. and Biophys. 66, 38 (1956). 15. SHIAU, Y., AND FRANCK, J., Arch. Biochem. 14,253 (1947). 16. BRUGGER, J., Thesis, Univ. of Chicago, 1954. 17. LINSCHITZ, H., AND RENNERT, J., Nature 169, 193 (1952). 18. GAFFRON, H., Biochem. 2. 280, 337 (1935). 19. FRANCK, J., Chap. 16. “PHOTOSYNTHESIS IN PLANTS,” The Iowa State College Press, Ames, Iowa, 1949. 20. NOACK, K., PIRSON, A., AND MICIIAELS, H., Naturwissenschuften 27,645 (1939). 21. WARBURG, O., “Heavy Metal Prosthetic Groups and Enzyme Action” (Trans. by Alexander Lawson), Chap. 20. Clarendon Press, Oxford, 1949. 22. WELLER, A., J. Am. Chem. Sot. 70, 5819 (1954). 23. WELLER, J., AND FRANCK, J., J. Phys. Chem. 46, 1359 (1941). 24. RIEKE, F. F., AND GAFFRON, H., J. Phys. Chem. 47,299 (1943).