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Photochemical reduction of oxygen in chloroplast preparations and in green plant cells. I. The study of oxygen exchanges in vitro and in vivo
Photochemical Reduction of Oxygen in Chloroplast Preparations and in Green Plant Cells. I. The Study of Oxygen Exchanges in vitro and in viva Allan H. Brown From
the Botany
Department,
and Norman
University
Good’
of Minnesota,
Minneapolis,
Minnesota
Received November 18, 1954
The ability of illuminated chloroplast preparations to form hydrogen peroxide with concomitant uptake of molecular oxygen was discovered by Mehler (1, 2). The stoichiometry observed was consistent with his hypothesis that molecular oxygen and hydrogen from the photolysis of water were the precursors of the hydrogen peroxide which he detected by the well-known trapping reaction with excess catalase and ethanol (3). That oxygen could be photochemically produced and consumed at the same time by chloroplast preparations was later confirmed by the use of tracer oxygen which permitted these simultaneous processes to be measured independently (4). These observations have been interpreted as proof that oxygen is functional as a “Hill oxidant”. The reactions postulated by Mehler were: 4HzO light 4(H) + 4(OH) (b) 4(OH) + 2HzO + 02 (c) 4(H) + 202 + 2H202 (cl) ~C~HE.OH + 2H202 catalase , BCH&HO (a)
Sum:
BC.ZH,OH
+ 02 -+ 2CHEHO
+ 4Hz0
+ 2H20
Reaction (a) is the photolysis of water usually postulated as the initial photochemical reaction of unspecified mechanism in which chlorophyll may participate. Reaction (b) summarizes the sequence of secondary processes leading to the production of molecular oxygen. Reaction 1 Present address: London, Ontario.
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(c) is the reduction of oxygen which, for the stoichiometry shown, proceeds at twice the rate of (b). The net effect is oxygen consumption. The over-all mechanism has come to be known as the “Mehler reaction.” The present paper reports further studies on the Mehler reaction. Additional evidence is provided confirming the mechanism postulated by the above reaction sequence. The stoichiometry is defined for the oxygen uptake and production under several relevant experimental conditions. A balanced photochemical oxygen exchange is demonstrated in the absence of oxidants other than molecular oxygen, and the mechanism of this exchange is elucidated. Data also are presented which have a bearing on the question of whether or not the Mehler reaction can occur in intact cells. METHODS
Chloroplast preparations were obtained from the leaves of: Spinacea olerucen (spinach), Beta vulgaris (chard), and Phytolacca decandra (pokeweed). Chloroplasts were isolated as described by Mehler (1). Several algal genera were used for experiments with whole cells. Chlorella pyrenoidosa (Emerson’s strain), Chlamydomonas moewusii (isolated by L. Provasoli), and Scenedesmus obliquua (Gaffron’s strain Da) were grown autotrophically at 28°C. in continuous white fluorescent light of 150 ft.-candles incident intensity. ,4 gas mixture consisting of 5% carbon dioxide in air was bubbled through the cultures. Anabaena sp. was grown without bubbling in a bicarbonate medium (5). Cells were harvested by centrifugation and resuspended, without washing, in phosphate or bicarbonatcx buffers. All experiments were performed at 20.9%. either in conventional Warburg manometer vessels or (in experiments involving tracer oxygen) in a rectangular Pyrex reaction vessel with a joint especially adapted for attachment to the mass spectrometer2 which was employed for continuously monitoring the gas phase within t,he reaction vessel. The vessel was attached to a manometer holder and shaken in a water bath in the same manner as were the ordinary Warburg vessels. The fashion in which the mass spectrometer was adapted and used in such experi ments has been described in detail (6-9). Light from either a bank of up to six 40-w. white fluorescent tubes or a pair of 500-w. tungsten filament reflector spot lamps was introduced into the bath from a side window and reflected onto the bottom of the vessel by an inclined mirror. Light intensity was varied by interposing calibrated screens between the window of the bath and the light source. At the beginning of an experiment the vessel containing buffer with appropriate addenda and the biological material was flushed with helium. Oxygen gas, highly enriched in the isotope with mass 34 (01601*), was then added to the desired concentration (usually from 0.5 to 3.0%). When appropriate, some carbon dioxide also was introduced as a gas or as bicarbonate buffer. During an experimental run * Consolidated
Engineering
Company.
Model
21-201.
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the vessel was closed (except for the very slow leak to the mass spectrometer). Relative measurements of the partial pressures of those mass components of the gas phase in the vessel which were of interest were recorded automatically. Absolute determinations of partial pressures of these gases were obtained by calibration with a known gas mixture of nearly the same composition as the experimental gas phase. Observed partial pressure changes may be converted to volumetric increments given the volume of the reaction vesssel, 28.8 ml. Computed volumes of gases produced or consumed were corrected for their respective solubilities in the liquid phase. Tracer oxygen was present in the gas phase of the reaction vessel in order to tag the oxygen being consumed. Oxygen produced photochemically from water would not contain significant amounts of tracer. By this device the processes of oxygen production and consumption could be observed separately but simultaneously. The actual production of oxygen, P, and the actual consumption, U, may be calculated from measurements of the pressure changes of tracer oxygen, Mad, and ordinary oxygen, M32, by the following formulas:
U= k[d&,dt(l+~)] P = k
dMsz/dt
-
dMsJdt
( 34 )I $
in which k is a calibration constant. Figures 1,3, and 4 present the isotope measurements directly while Figs. 2 and 5 show the calculated total oxygen exchange. The appropriate factor for conversion of the data (expressed in arbitrary units of relative partial pressure) into microliters (S.T.P.) of a particular gas is provided for each experiment. In order to achieve the purpose of these tracer experiments, it is essential that molecular oxygen should not exchange with the oxygen of water or of the biological material at a significant rate. This problem has been considered elsewhere (6) and there appears to be ample justification for the assumption that exchange reactions involving molecular oxygen do not occur to an appreciable extent under the conditions which are critical. Equally important for the present study is the matter of exchange reactions involving hydrogen peroxide. Dole et al. (10) and Cahill and Traube (11) were able to show that oxygen of hydrogen peroxide does not exchange with that of water. Therefore we avoid this possible complication in interpreting our results. The interpretation of our experiments also rests on the assumption that the uptake of oxygen, respiratory or otherwise, is partitioned between the oxygen isotopes of the gas phase in direct proportion to their partial pressures. This is supported by a variety of data (6, 9, 12). EXPERIMENTAL Exploring the implications of the Mehler raises a number of questions:
reaction
mechanism
proposed
by
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MINUTES FIG. 1. Equal production and consumption of oxygen by illuminated chloroplasts. The reaction vessel contained 5.0 ml. of a spinach chloroplast suspension (2.5 mg. chlorophyll) in phosphate buffer, pH 6.5. Light: fluorescent, about 150 ft. candles. Gas phase: helium, and oxygen enriched with mass 34. Temperature, 20.9”C. Ordinate X 0.64 = microliters of oxygen.
I. The concomitant production and consumption of oxygen was demonstrated by Mehler and Brown (4) with tracer oxygen only after the chloroplast preparation had photochemically reduced quinone. Is quinone reduction required to elicit the Mehler reaction and is the catalase-ethanol “trap” a necessary factor? Figure 1 shows an example of photoinduced production and consumption of oxygen by a chloroplast preparation to which no quinone had been added. The total oxygen pressure remained constant. Even though excess catalase and ethanol were not added, hydrogen peroxide apparently did not accumulate. Presumably it was dismuted by the endogenous catalase of t,he plastid preparation. Thus, if reaction (e) (e) 2H202 2 2HzO + 02 should be substituted for (d) in the mechanism delineated in the l&oduction, the over-all sequence of reactions would lead to no net change of oxygen, resulting instead in a one-for-one exchange of oxygen bet,ween
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gas phase and water of the reaction system. Experiments of which Fig. 1 is representative demonstrate the occurrence of a balanced oxygen exchange which would be undetectable without the use of isotopes. Such results indicate that the photochemical mechanism described by rea&ions (a), (b), and (c) may proceed in the absence of any oxidants other than oxygen itself, does not require stimulation by a preceding quinone reaction, and does not depend on the presence of ethanol or exogenous catalase. The role of endogenous catalase-reaction (e)-was tested by poisoning with cyanide. When 0.01 M KCN was added to a chloroplast reaction system, the uptake of tracer oxygen was enhanced. However, the increase was only tempoiary. After the light was turned off, dismutation of the accumulated peroxide presumably accounted for the observed after-production of oxygen. It was confirmed that 0.01 M KCN inhibited most but not quite all of the ability of chloroplasts to dismute hydrogen peroxide. II. If the catalaseethanol trap functions in the Mehler reaction in the manner proposed, its effect should be to prevent the return of oxygen to the gas phase-in effect, enhancing net oxygen consumption. Alternatively, the uptake observed manometrically could be the result of a reduction of the rate of oxygen evolution from the photolysis products of water. Which effect accounts for the preponderance of oxygen consumption over production when excess catalase and ethanol are added to the reaction system? Mehler had cogent reasons for rejecting the latter alternative, but, since the tracer method can distinguish enhanced consumption from inhibited production of oxygen, it is desirable to test directly which alternative prevails. Figure 2 illustrates the results of a representative experiment. Values for total oxygen change were obtained by adding the data for both isotopes. Production and consumption were calculated from the isotope data as described in the Methods section. The addition of catalase in this experiment caused a doubling of the oxygen consumption rate without changing the production rate. This is in accord with the mechanism postulated by Mehler. When ferric tartrate replaced ethanol and catalase in an experiment like the one featured in Fig. 2, the results were the same; i.e., oxygen consumption was doubled and oxygen production was unaltered. Ferric oxalate, ferric ammonium sulfate, and potassium tartrate, when used singly, were all without influence on the 1:l stoichiometry of the photoinduced oxygen exchange. The altered stoichiometry (stimulation of
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MINUTES FIG. 2. The effect of catalase and ethanol on the production and consumption of oxygen by illuminated chloroplasts. The reaction vessel contained 3.0 ml. of a PhytoZacca chloroplast preparation (0.7 mg. chlorophyll) in phosphate buffer, pR 6.5, with about 100 mg. ethanol. Side arm contained 1.5 mg. crystalline ratalasc in 1.0 ml. water. Light: incandescent tungsten with red filter (Corning ?Jo. 348), about 25 ft.-candles. Gas phase: helium, and oxygen enriched with mass 34. Temperature, 20.9”C. Ordinate X 39 = microliters of oxygen.
depended upon the simultaneous presence of tartrate and iron salts. The similarity of behavior between ethanol-catalase and ferric tartrate suggeststhat the latter may also function by “trapping” hydrogen peroxide, This was confirmed by showing that dilute solutions of hydrogen peroxide were decomposedby ferric tartrate without evolution of oxygen. III. Speculation concerning the mechanism of the Mehler reaction has been based entirely on the assumption that Hz02 is the product of oxygen reduction. Is Hz02 the sole product or is Hz0 produced under some conditions? It may be noted that Fig. 1 illustrates an exchange reaction the stoichiometry of which is compatible with the production of Hz0 rather than HgOz . However the efficacy of peroxide trapping devices and of cyanide in altering this stoichiometry argues strongly in favor of Hz02 . Nevertheless, model systems may be constructed which
uptake)
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I
The Efects of Cytochrome c or Catechol on the Consumption Production of Oxygen by Illuminated Chloroplasts
and
Reaction vessels contained 5.0 ml. of a suspension of spinach chloroplasts (2.5 mg. chlorophyll) in phosphate buffer, pH 6.5. Fluorescent light, about 150 ft.candles. Gas phase: helium, and oxygen enriched with mass 34 to permit determination of simultaneous production and consumption rates. Temperature 20.9”C. Oxygenexchange,rl./mia. Addenda
(final
concentration)
Cytochrome c (2.0 mg./ml.) Catechol (0.015 mg./ml.)
Chloroplasts alone Consumption Production
0.87 0.80
0.85 0.74
Chloroplasts Consumption
1.57 1.92
with
addendum Production
1.28 1.80
reduce oxygen through cytochrome or polyphenol oxidases, probably to water rather than to hydrogen peroxide. Table I shows representative experiments illustrating the stimulating effect of adding either catechol or cytochrome c to illuminated chloroplasts. Oxygen uptake and oxygen production, measured by the isotope method, were both increased by either of these addenda. Therefore a failure to observe net oxygen uptake in the presence of catalase and ethanol doesnot necessarily preclude oxygen reduction. IV. Can the Mehler reaction proceed in viva? The fact that cell-free preparations of chloroplasts reduced oxygen suggested that similar reactions might be important in the intact organism. The results of tracer experiments measuring oxygen consumption during photosynthesis (6) and comparisons of the efficiencies of photosynthesis and chloroplast reactions (13) indicate that ordinarily no significant photochemical oxygen uptake is superimposed on normal dark respiration. However, Mehler concluded that the reduction of oxygen by isolated chloroplasts became significant only when the supply of other oxidants was depleted. Experiments which are described in this paper were undertaken to determine whether this applied to intact cells. One approach to this objective was to deprive cells of carbon dioxide. Upon illumination no net gas exchange occurred, but when the tracer method was employed it was observed that an oxygen exchange proceeded approximately at the normal respiratory rate. Figure 3 showsan experiment with ChZoreZZa cells at a very low carbon dioxide tension in the presence of tracer oxygen. Gas-exchange rates were measured first in the dark. In the light photosynthesis quickly reduced the carbon dioxide tension to a very low constant value. (In another experiment alkali-soaked paper in the vessel side arm did not lower this level appreciably.) Oxygen uptake continued at the previous
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MINUTES FIG. 3. The effect of light on the uptake of oxygen by Chlorella cells at low carbon dioxide tension. Vessel contained 5.0 ml. Chlorella suspension in phosphate buffer (2.2% cells). Light: fluorescent, cu. 150 ft.-candles. Ordinates: mass 32 and mass 34 X 0.65 = microliters of oxygen; mass 44 X 0.62 = microliters of carbon dioxide.
respiratory rate. The light intensity employed corresponded to several times compensation. However, the rate of oxygen production declined until, just balancing the oxygen uptake, it was equivalent t,o the production rate of respiratory carbon dioxide. Apparently the latter portion of Fig. 3 is a demonstration of respiration and photosynthesis to compensation. There was no evidence of the extra oxygen exchange to be anticipated should an in vivo Mehler react#ion be superimposed on these processes. Net photosynthesis is completely suppressed by 0.01 M cyanide, and this offers the opportunity for another kind of approach to the problem of depleting natural oxidants derived from carbon dioxide. The gaseous metabolism of cyanide-poisoned suspensions of Chlorella and Chlamydomonas was studied by the tracer method. The concentration of cyanide used (0.01 M) has a negligible effect on the respiration of these species but effectively inhibits one or more of the dark reactions of photosynthesis. Figure 4 illustrates a typical experiment, with Chlorella. Upon
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4. The effect of light on the respiration of Chlorella in the presence of cyanide. The reaction vessel contained 6.0 ml. Chlorella suspension (1.7% cells) in phosphate buffer, pH 6.5, containing0.01 M potassium cyanide. Sidearm contained o-phenanthroline (0.3 mg.) in 0.5 ml. water. Ordinates: mass 32 and mass 34 X 0.62 = microliters of oxygen; mass 44 X 0.62 = microliters of carbon dioxide. FIG.
illumination, carbon dioxide production stopped completely. No carbon dioxide uptake occurred. Oxygen production commenced at a level roughly corresponding to compensation. Presumably a respiratory intermediate rather than free carbon dioxide served as the oxidant responsible for the compensating photosynthesis. Oxygen consumption increased only slightly. In the experiment illustrated, o-phenanthroline (which markedly inhibits the oxygen production of both chloroplast reactions and photosynthesis) was added before the second period of illumination. As expected, the presence of o-phenanthroline abolished most of the effect of illumination. ChEumydomonassuspensionsgave results qualitatively similar to those obtained with Chlorella. In other algal species,photosynthesis and respiration are both sensitive to 0.01 M cyanide. Examples are Scenedesmusand Anabaena. Adding cyanide to suspensionsof these organisms almost abolished the respiratory oxygen uptake. However, upon illumination of these poisoned suspensions, although no net increase or decrease of oxygen occurred, a balanced oxygen exchange was observed. The rate of oxygen consumption was then equal to or slightly in excessof the rate at which the cells
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90 MINUTES FIG. 5. The effect of light on the respiration of Anabaena in the presence of cyanide. The reaction vessel contained 5.0 ml. of Anabaena suspension (3.5yo by volume) in Warburg’s bicarbonate buffer No. 11. The vessel was opened at arrow to add 1.0 ml. of 0.01 M potassium cyanide and tracer oxygen. Light: incandescent tungsten. Unit of light intensity (indicated at top) about 130 ft.-candles. Left ordinate (before adding KCN) X 0.67 = microliters of oxygen. Right ordinate (after adding KCN) X 0.63 = microliters of oxygen. Temperature, 20.9%.
had respired in the absenceof cyanide. The behavior of Anabaena in such an experiment is illustrated by Fig. 5. Cells were suspendedin bicarbonate buffer and illuminated for a short time. Respiration was then followed in the dark. Addition of cyanide reduced respiration to 10% of the uninhibited rate. This slow net uptake of oxygen continued through intervals of illumination, but the actual oxygen consumption was raised by the light to about 140 % of the previous uninhibited respiratory level. The light-induced oxygen consumption was almost exactly balanced by a compensating production of oxygen. Except at low intensities these processeswere essentially independent of light intensity. Substitution of Scenedesmusfor Anabaena and of phosphate (pH 6.5) for bicarbonate buffer did not materially alter the results. DISCUSSION
Figure 6 presents an abbreviated schematic survey which encompasses the results described above and is a summary of current ideas regarding
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$ co,
$ (CH,O) + f H,O f Photosynthesis \
FIG.
6. Schematic review of the reactions of chloroplasts
discussed in this paper.
gross aspects of photosynthesis and chloroplast reactions. Most of the reactions designated by letters in this diagram are complex or multistep processes.Which of these reactions possesssteps in common is largely a matter for speculation. The central hydrogen atom in brackets represents the reduction product from the photolysis of water [reaction (a)], which leads to oxygen evolution (b) and, in photosynthesizing cells, to carbon dioxide reduction (g) ; but in cell-free chloroplast preparations, any of several reductions of suitable electron or hydrogen acceptors are observed (h, j). In the absence of competing oxidants, oxygen can react with this reduced photolysis product to form hydrogen peroxide (c) which can be reduced (d, e) or dismuted (f). Cytochrome c or catechol can also link the reduction product [H] to molecular oxygen (Ic), in which case water is formed rather than hydrogen peroxide. Indirect evidence (14-16) indicates that pyridine nucleotides apparently can function in like manner. If none of these reductions are possible in a particular experiment, then it has been suggested that the photolysis products may recombine (m) so that no gas uptake or production is observed. At present we are concerned with two questions: Is oxygen equivalent to other so called “Hill oxidants” in reactions of cell-free chloroplast preparations? And can the photochemical reduction of oxygen be a significant reaction in intact cells? In answer to the first question, available information, including data
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of this paper, indicate that the light-induced reduction of oxygen by chloroplasts under appropriate conditions is the over-all equivalent of similar reactions with other oxidants. No chloroplast preparations which were unable to reduce quinone or ferricyanide retained the ability to reduce oxygen. There was no oxygen uptake in the presence of inhibitors of conventional chloroplast reactions. o-Phenanthroline (5 X lo-+ M) depressedboth the rate of oxygen and of quinone reduction. However, there is reason to believe that the reduction of oxygen is not as direct as is the casewith an oxidant like quinone (vide paper No. II of this series). To the second question, a general answer cannot as yet be given. If during normal photosynthesis, photochemical oxygen reduction were superimposed upon regular respiratory and photosynthetic reactions, an enhancement of oxygen uptake should be detected by the tracer technique. Moreover, photochemical oxygen reduction, constituting a type of back reaction, might well diminish the rate of photosynthesis as compared to the rates of the reactions of isolated chloroplasts (which probably do not reduce oxygen in the presenceof other oxidants). In only one casehas the use of tracer oxygen demonstrated marked photostimulation of oxygen uptake under conditions of normal photosynthesis (S), and Ehrmantraut and Rabinowitch (13) have shown that in Chlorella the quantum yield of photosynthesis is essentially the sameas for the reduction of oxidants other than carbon dioxide. Furthermore, it now appears that light causes at best only small increases in the rate of oxygen consumption even in the absence of photosynthesis. It should be noted that the maximal velocity of oxygen uptake by illuminated living algae as described in this paper (Figs. 3, 4, and 5) does not much exceed the corresponding dark respiratory rate. However, the maximal rates of oxygen reduction by chloroplast preparations are comparable (per unit chlorophyll) to photosynthetic rates which are often twenty times more rapid than dark respiration. This quantitative comparison, showing that photoconsumption of oxygen can be more than an order of magnitude faster in vitro than in z)ivo, suggests that the rapid Mehler reaction in chloroplast preparations is an artifact and that the small, light-induced oxygen uptake by intact cells may not even proceed by the samemechanism. There remains the possibility that photochemical oxygen reduction during normal photosynthesis is not superimposed upon the respiratory metabolism but rather replaces it. This implies a relation between photosynthesis and respiration such as the existence of common reactions. For
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over a century cross links between respiration and photosynthesis have been a subject for speculation in terms of whether these processes have organic intermediates in common. Warburg’s observation that Chlorella photosynthesis is readily inhibited by cyanide only to the compensation point (17) can be interpreted by assuming that respiratory intermediates can be photochemically reduced rather than decarboxylated. Franck has recently cited observations of transient phenomena during photosynthetic inductions which support the assumption of such an interconnection (18). The discovery of rapid tagging of respiratory and glycolytic intermediates by brief exposures of photosynthesizing cells to tracer carbon dioxide has encouraged some investigators to expand these speculations even though their preliminary schemes (19) could not be maintained in all particulars in the light of our rapidly expanding knowledge of tracer carbon assimilation. Ochoa and co-workers have been particularly enthusiastic in their support of respiratory and photosynthetic cross linkage via Krebs cycle intermediates, and they have defended their view by experiments with isolated chloroplast preparations (14, 20) in which photolytic cleavage of water was coupled to reductive carboxylations by known enzyme systems in vitro. It now appears unlikely that photosynthesis achieves the back rotation of the Krebs cycle, and indeed indirect evidence suggests that during photosynthesis carbon metabolism via the Krebs cycle is suppressed (21). This should lead to a respiratory inhibition with regard to carbon dioxide production as claimed by Weigl et al. (22), but if the missing respiratory hydrogen is replaced by an approximately equivalent influx of photolytic “hydrogen,” no marked change in rate of oxygen uptake need be expected. The authors have not been able to formulate any hypothesis which could satisfactorily account for the apparent reversal by light of the cyanide inhibition of Anabaena and Scenedesmus respiration. There is evidence that the terminal oxidase responsible for Anabaena respiration is a cytochrome oxidase (5). As herein reported (Fig. 5), photostimulation of oxygen uptake occurs in this organism even when its normal respiration is severely inhibited by cyanide. Either the extra oxygen is consumed by a cyanide-resistant oxidase mechanism other than that responsible for the normal respiration, or cyanide directly or indirectly blocks respiration at some point other than the terminal oxidase. An alternative possibility, that light can reverse cyanide inhibition per se, is at present unsupported by any other evidence known to the authors.
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ACKNOWLEDGMENTS The script.
authors
are grateful
to Dr.
R.
Hill
for
his
helpful
criticism
of
the
manu-
The isotopically enriched oxygen was prepared by Dr. A. 0. Nier under a grant from the American Cancer Society through the Committee on Growth of the National Research Council. Financial assistance was obtained from the Graduate School of the University of Minnesota and the Rockefeller Foundation. These studies were aided by a contract between the Office of Naval Research, Department of t,he Navy, and the University of Minnesota (NR160-030). SUMMARY
1. Studies employing oxygen isotopes have shown that the stoichiometry of the consumption and production of oxygen by illuminated chloroplasts is that proposed by Mehler. Oxygen is reduced to hydrogen peroxide with a concurrent production of half as much oxygen originating from water. The reduction of oxygen in the Mehler reaction is thus analogous to t,he reduction of conventional chloroplast reagents such as quinone. 2. Spinach chloroplast preparations normally do not reduce oxygen direct,ly to s\-ater although polyphenol and cytochrome oxidases are present in the preparat,ions and catalyze an increased reduction of oxygen when either catechol or cytochrome c is added. 3. There is little photochemical reduction of oxygen superimposed on the respiratory oxygen uptake of living algae even when the cells are illuminated in the absenceof available carbon dioxide. 4. Light reversed the cyanide inhibition of oxygen uptake by Anabaena and Scenedesmuscell suspensions. 5. Evidence is presented which suggests that photochemically produced reductants may serve as hydrogen donors for the respiration of illuminated algae. Some possible interactions of photosynthesis and respiration are discussed. REFERENCES 1. MEHLER,
2. MEHLER, 3. KEILIN, 4. MEHLER,
A. A. D., A.
H., Arch. H., Arch.
Biochem. and Biophys. Biochem. and Biophys. AND HARTREE, E. F., Biochm. H., AND BROWN, A. H., Arch.
33, 65 (1951). 34, 339 (1951). J. 39, 293 (1945). Biochem. and Biophys.
(1952).
5. WEBSTER, 6. BROWN,
G. C., AND FRENKEL, A. W., Plant A. H., Am. J. Botany 40, 719 (1953).
Physiol.
28, 63
(1953).
38,
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7. BROWN, A. H., NIER, A. O., AND VAN NORMAN, R. W., Plant Physiol. 27, 320 (1952). 8. BROWN, A. H., AND WEBSTER, G., Am. J. Botany 40, 753 (1953). 9. JOHNSTON, J., AND BROWN, A. H., Plant Physiol. 29,177 (1954). 10. DOLE, M., RUDD, D. P., MUCHOW, G. R., AND COMTE, C., J. Chem. Phys.
20, 961 (1952). 11. CAHILL, A. E., AND TRAUBE, H. J., J. Am. Chem. Sot. 74, 2312 (1952). 12. DOLE, M., HAWKINS, R. C., AND BARKER, H. A., J. Am. Chem. Sot. 69, 226 (1947). 13. EHRMANTRAUT, H., AND RABINOWITCH, E., Arch. Biochem. and Biophys. 36, 67 (1952). 14. VISHNIAC, W., AND OCHOA, S., J. Biol. Clam. 196, 75 (1952). 15. VISHNIAC, W., AND OCHOA, S., J. Biol. Chem. 196, 501 (1952). 16. TOLMACH, L., Arch. Biochem. and Biophys. 33, 120 (1951). 17. WARBURG, O., Biochem. 2. 103, 188 (1920). 18. FRANCK, J., Arch. Biochem. and Biophys. 46, 190 (1953). 19. CALVIN, M., AND BENSON, A., Science 107, 467 (1948). 20. OCHOA, S., SALLES, J. B. V., AND ORTIZ, P. J., J. Biol. Chem. 167, 863 (1950). 21. BENSON, A., AND CALVIN, M., J. Exptl. Botany 1, 63 (1950). 22. WEIGL, J., WARRINGTON, P. M., AND CALVIN, M., J. Am. Chem. Sot. 73, 5058 (1951).
the Botany
Department,
and Norman
University
Good’
of Minnesota,
Minneapolis,
Minnesota
Received November 18, 1954
The ability of illuminated chloroplast preparations to form hydrogen peroxide with concomitant uptake of molecular oxygen was discovered by Mehler (1, 2). The stoichiometry observed was consistent with his hypothesis that molecular oxygen and hydrogen from the photolysis of water were the precursors of the hydrogen peroxide which he detected by the well-known trapping reaction with excess catalase and ethanol (3). That oxygen could be photochemically produced and consumed at the same time by chloroplast preparations was later confirmed by the use of tracer oxygen which permitted these simultaneous processes to be measured independently (4). These observations have been interpreted as proof that oxygen is functional as a “Hill oxidant”. The reactions postulated by Mehler were: 4HzO light 4(H) + 4(OH) (b) 4(OH) + 2HzO + 02 (c) 4(H) + 202 + 2H202 (cl) ~C~HE.OH + 2H202 catalase , BCH&HO (a)
Sum:
BC.ZH,OH
+ 02 -+ 2CHEHO
+ 4Hz0
+ 2H20
Reaction (a) is the photolysis of water usually postulated as the initial photochemical reaction of unspecified mechanism in which chlorophyll may participate. Reaction (b) summarizes the sequence of secondary processes leading to the production of molecular oxygen. Reaction 1 Present address: London, Ontario.
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(c) is the reduction of oxygen which, for the stoichiometry shown, proceeds at twice the rate of (b). The net effect is oxygen consumption. The over-all mechanism has come to be known as the “Mehler reaction.” The present paper reports further studies on the Mehler reaction. Additional evidence is provided confirming the mechanism postulated by the above reaction sequence. The stoichiometry is defined for the oxygen uptake and production under several relevant experimental conditions. A balanced photochemical oxygen exchange is demonstrated in the absence of oxidants other than molecular oxygen, and the mechanism of this exchange is elucidated. Data also are presented which have a bearing on the question of whether or not the Mehler reaction can occur in intact cells. METHODS
Chloroplast preparations were obtained from the leaves of: Spinacea olerucen (spinach), Beta vulgaris (chard), and Phytolacca decandra (pokeweed). Chloroplasts were isolated as described by Mehler (1). Several algal genera were used for experiments with whole cells. Chlorella pyrenoidosa (Emerson’s strain), Chlamydomonas moewusii (isolated by L. Provasoli), and Scenedesmus obliquua (Gaffron’s strain Da) were grown autotrophically at 28°C. in continuous white fluorescent light of 150 ft.-candles incident intensity. ,4 gas mixture consisting of 5% carbon dioxide in air was bubbled through the cultures. Anabaena sp. was grown without bubbling in a bicarbonate medium (5). Cells were harvested by centrifugation and resuspended, without washing, in phosphate or bicarbonatcx buffers. All experiments were performed at 20.9%. either in conventional Warburg manometer vessels or (in experiments involving tracer oxygen) in a rectangular Pyrex reaction vessel with a joint especially adapted for attachment to the mass spectrometer2 which was employed for continuously monitoring the gas phase within t,he reaction vessel. The vessel was attached to a manometer holder and shaken in a water bath in the same manner as were the ordinary Warburg vessels. The fashion in which the mass spectrometer was adapted and used in such experi ments has been described in detail (6-9). Light from either a bank of up to six 40-w. white fluorescent tubes or a pair of 500-w. tungsten filament reflector spot lamps was introduced into the bath from a side window and reflected onto the bottom of the vessel by an inclined mirror. Light intensity was varied by interposing calibrated screens between the window of the bath and the light source. At the beginning of an experiment the vessel containing buffer with appropriate addenda and the biological material was flushed with helium. Oxygen gas, highly enriched in the isotope with mass 34 (01601*), was then added to the desired concentration (usually from 0.5 to 3.0%). When appropriate, some carbon dioxide also was introduced as a gas or as bicarbonate buffer. During an experimental run * Consolidated
Engineering
Company.
Model
21-201.
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the vessel was closed (except for the very slow leak to the mass spectrometer). Relative measurements of the partial pressures of those mass components of the gas phase in the vessel which were of interest were recorded automatically. Absolute determinations of partial pressures of these gases were obtained by calibration with a known gas mixture of nearly the same composition as the experimental gas phase. Observed partial pressure changes may be converted to volumetric increments given the volume of the reaction vesssel, 28.8 ml. Computed volumes of gases produced or consumed were corrected for their respective solubilities in the liquid phase. Tracer oxygen was present in the gas phase of the reaction vessel in order to tag the oxygen being consumed. Oxygen produced photochemically from water would not contain significant amounts of tracer. By this device the processes of oxygen production and consumption could be observed separately but simultaneously. The actual production of oxygen, P, and the actual consumption, U, may be calculated from measurements of the pressure changes of tracer oxygen, Mad, and ordinary oxygen, M32, by the following formulas:
U= k[d&,dt(l+~)] P = k
dMsz/dt
-
dMsJdt
( 34 )I $
in which k is a calibration constant. Figures 1,3, and 4 present the isotope measurements directly while Figs. 2 and 5 show the calculated total oxygen exchange. The appropriate factor for conversion of the data (expressed in arbitrary units of relative partial pressure) into microliters (S.T.P.) of a particular gas is provided for each experiment. In order to achieve the purpose of these tracer experiments, it is essential that molecular oxygen should not exchange with the oxygen of water or of the biological material at a significant rate. This problem has been considered elsewhere (6) and there appears to be ample justification for the assumption that exchange reactions involving molecular oxygen do not occur to an appreciable extent under the conditions which are critical. Equally important for the present study is the matter of exchange reactions involving hydrogen peroxide. Dole et al. (10) and Cahill and Traube (11) were able to show that oxygen of hydrogen peroxide does not exchange with that of water. Therefore we avoid this possible complication in interpreting our results. The interpretation of our experiments also rests on the assumption that the uptake of oxygen, respiratory or otherwise, is partitioned between the oxygen isotopes of the gas phase in direct proportion to their partial pressures. This is supported by a variety of data (6, 9, 12). EXPERIMENTAL Exploring the implications of the Mehler raises a number of questions:
reaction
mechanism
proposed
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MINUTES FIG. 1. Equal production and consumption of oxygen by illuminated chloroplasts. The reaction vessel contained 5.0 ml. of a spinach chloroplast suspension (2.5 mg. chlorophyll) in phosphate buffer, pH 6.5. Light: fluorescent, about 150 ft. candles. Gas phase: helium, and oxygen enriched with mass 34. Temperature, 20.9”C. Ordinate X 0.64 = microliters of oxygen.
I. The concomitant production and consumption of oxygen was demonstrated by Mehler and Brown (4) with tracer oxygen only after the chloroplast preparation had photochemically reduced quinone. Is quinone reduction required to elicit the Mehler reaction and is the catalase-ethanol “trap” a necessary factor? Figure 1 shows an example of photoinduced production and consumption of oxygen by a chloroplast preparation to which no quinone had been added. The total oxygen pressure remained constant. Even though excess catalase and ethanol were not added, hydrogen peroxide apparently did not accumulate. Presumably it was dismuted by the endogenous catalase of t,he plastid preparation. Thus, if reaction (e) (e) 2H202 2 2HzO + 02 should be substituted for (d) in the mechanism delineated in the l&oduction, the over-all sequence of reactions would lead to no net change of oxygen, resulting instead in a one-for-one exchange of oxygen bet,ween
344
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gas phase and water of the reaction system. Experiments of which Fig. 1 is representative demonstrate the occurrence of a balanced oxygen exchange which would be undetectable without the use of isotopes. Such results indicate that the photochemical mechanism described by rea&ions (a), (b), and (c) may proceed in the absence of any oxidants other than oxygen itself, does not require stimulation by a preceding quinone reaction, and does not depend on the presence of ethanol or exogenous catalase. The role of endogenous catalase-reaction (e)-was tested by poisoning with cyanide. When 0.01 M KCN was added to a chloroplast reaction system, the uptake of tracer oxygen was enhanced. However, the increase was only tempoiary. After the light was turned off, dismutation of the accumulated peroxide presumably accounted for the observed after-production of oxygen. It was confirmed that 0.01 M KCN inhibited most but not quite all of the ability of chloroplasts to dismute hydrogen peroxide. II. If the catalaseethanol trap functions in the Mehler reaction in the manner proposed, its effect should be to prevent the return of oxygen to the gas phase-in effect, enhancing net oxygen consumption. Alternatively, the uptake observed manometrically could be the result of a reduction of the rate of oxygen evolution from the photolysis products of water. Which effect accounts for the preponderance of oxygen consumption over production when excess catalase and ethanol are added to the reaction system? Mehler had cogent reasons for rejecting the latter alternative, but, since the tracer method can distinguish enhanced consumption from inhibited production of oxygen, it is desirable to test directly which alternative prevails. Figure 2 illustrates the results of a representative experiment. Values for total oxygen change were obtained by adding the data for both isotopes. Production and consumption were calculated from the isotope data as described in the Methods section. The addition of catalase in this experiment caused a doubling of the oxygen consumption rate without changing the production rate. This is in accord with the mechanism postulated by Mehler. When ferric tartrate replaced ethanol and catalase in an experiment like the one featured in Fig. 2, the results were the same; i.e., oxygen consumption was doubled and oxygen production was unaltered. Ferric oxalate, ferric ammonium sulfate, and potassium tartrate, when used singly, were all without influence on the 1:l stoichiometry of the photoinduced oxygen exchange. The altered stoichiometry (stimulation of
PHOTOCHEMICAL
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MINUTES FIG. 2. The effect of catalase and ethanol on the production and consumption of oxygen by illuminated chloroplasts. The reaction vessel contained 3.0 ml. of a PhytoZacca chloroplast preparation (0.7 mg. chlorophyll) in phosphate buffer, pR 6.5, with about 100 mg. ethanol. Side arm contained 1.5 mg. crystalline ratalasc in 1.0 ml. water. Light: incandescent tungsten with red filter (Corning ?Jo. 348), about 25 ft.-candles. Gas phase: helium, and oxygen enriched with mass 34. Temperature, 20.9”C. Ordinate X 39 = microliters of oxygen.
depended upon the simultaneous presence of tartrate and iron salts. The similarity of behavior between ethanol-catalase and ferric tartrate suggeststhat the latter may also function by “trapping” hydrogen peroxide, This was confirmed by showing that dilute solutions of hydrogen peroxide were decomposedby ferric tartrate without evolution of oxygen. III. Speculation concerning the mechanism of the Mehler reaction has been based entirely on the assumption that Hz02 is the product of oxygen reduction. Is Hz02 the sole product or is Hz0 produced under some conditions? It may be noted that Fig. 1 illustrates an exchange reaction the stoichiometry of which is compatible with the production of Hz0 rather than HgOz . However the efficacy of peroxide trapping devices and of cyanide in altering this stoichiometry argues strongly in favor of Hz02 . Nevertheless, model systems may be constructed which
uptake)
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The Efects of Cytochrome c or Catechol on the Consumption Production of Oxygen by Illuminated Chloroplasts
and
Reaction vessels contained 5.0 ml. of a suspension of spinach chloroplasts (2.5 mg. chlorophyll) in phosphate buffer, pH 6.5. Fluorescent light, about 150 ft.candles. Gas phase: helium, and oxygen enriched with mass 34 to permit determination of simultaneous production and consumption rates. Temperature 20.9”C. Oxygenexchange,rl./mia. Addenda
(final
concentration)
Cytochrome c (2.0 mg./ml.) Catechol (0.015 mg./ml.)
Chloroplasts alone Consumption Production
0.87 0.80
0.85 0.74
Chloroplasts Consumption
1.57 1.92
with
addendum Production
1.28 1.80
reduce oxygen through cytochrome or polyphenol oxidases, probably to water rather than to hydrogen peroxide. Table I shows representative experiments illustrating the stimulating effect of adding either catechol or cytochrome c to illuminated chloroplasts. Oxygen uptake and oxygen production, measured by the isotope method, were both increased by either of these addenda. Therefore a failure to observe net oxygen uptake in the presence of catalase and ethanol doesnot necessarily preclude oxygen reduction. IV. Can the Mehler reaction proceed in viva? The fact that cell-free preparations of chloroplasts reduced oxygen suggested that similar reactions might be important in the intact organism. The results of tracer experiments measuring oxygen consumption during photosynthesis (6) and comparisons of the efficiencies of photosynthesis and chloroplast reactions (13) indicate that ordinarily no significant photochemical oxygen uptake is superimposed on normal dark respiration. However, Mehler concluded that the reduction of oxygen by isolated chloroplasts became significant only when the supply of other oxidants was depleted. Experiments which are described in this paper were undertaken to determine whether this applied to intact cells. One approach to this objective was to deprive cells of carbon dioxide. Upon illumination no net gas exchange occurred, but when the tracer method was employed it was observed that an oxygen exchange proceeded approximately at the normal respiratory rate. Figure 3 showsan experiment with ChZoreZZa cells at a very low carbon dioxide tension in the presence of tracer oxygen. Gas-exchange rates were measured first in the dark. In the light photosynthesis quickly reduced the carbon dioxide tension to a very low constant value. (In another experiment alkali-soaked paper in the vessel side arm did not lower this level appreciably.) Oxygen uptake continued at the previous
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MINUTES FIG. 3. The effect of light on the uptake of oxygen by Chlorella cells at low carbon dioxide tension. Vessel contained 5.0 ml. Chlorella suspension in phosphate buffer (2.2% cells). Light: fluorescent, cu. 150 ft.-candles. Ordinates: mass 32 and mass 34 X 0.65 = microliters of oxygen; mass 44 X 0.62 = microliters of carbon dioxide.
respiratory rate. The light intensity employed corresponded to several times compensation. However, the rate of oxygen production declined until, just balancing the oxygen uptake, it was equivalent t,o the production rate of respiratory carbon dioxide. Apparently the latter portion of Fig. 3 is a demonstration of respiration and photosynthesis to compensation. There was no evidence of the extra oxygen exchange to be anticipated should an in vivo Mehler react#ion be superimposed on these processes. Net photosynthesis is completely suppressed by 0.01 M cyanide, and this offers the opportunity for another kind of approach to the problem of depleting natural oxidants derived from carbon dioxide. The gaseous metabolism of cyanide-poisoned suspensions of Chlorella and Chlamydomonas was studied by the tracer method. The concentration of cyanide used (0.01 M) has a negligible effect on the respiration of these species but effectively inhibits one or more of the dark reactions of photosynthesis. Figure 4 illustrates a typical experiment, with Chlorella. Upon
348
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MINUTES
4. The effect of light on the respiration of Chlorella in the presence of cyanide. The reaction vessel contained 6.0 ml. Chlorella suspension (1.7% cells) in phosphate buffer, pH 6.5, containing0.01 M potassium cyanide. Sidearm contained o-phenanthroline (0.3 mg.) in 0.5 ml. water. Ordinates: mass 32 and mass 34 X 0.62 = microliters of oxygen; mass 44 X 0.62 = microliters of carbon dioxide. FIG.
illumination, carbon dioxide production stopped completely. No carbon dioxide uptake occurred. Oxygen production commenced at a level roughly corresponding to compensation. Presumably a respiratory intermediate rather than free carbon dioxide served as the oxidant responsible for the compensating photosynthesis. Oxygen consumption increased only slightly. In the experiment illustrated, o-phenanthroline (which markedly inhibits the oxygen production of both chloroplast reactions and photosynthesis) was added before the second period of illumination. As expected, the presence of o-phenanthroline abolished most of the effect of illumination. ChEumydomonassuspensionsgave results qualitatively similar to those obtained with Chlorella. In other algal species,photosynthesis and respiration are both sensitive to 0.01 M cyanide. Examples are Scenedesmusand Anabaena. Adding cyanide to suspensionsof these organisms almost abolished the respiratory oxygen uptake. However, upon illumination of these poisoned suspensions, although no net increase or decrease of oxygen occurred, a balanced oxygen exchange was observed. The rate of oxygen consumption was then equal to or slightly in excessof the rate at which the cells
PHOTOCHEMICAL
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90 MINUTES FIG. 5. The effect of light on the respiration of Anabaena in the presence of cyanide. The reaction vessel contained 5.0 ml. of Anabaena suspension (3.5yo by volume) in Warburg’s bicarbonate buffer No. 11. The vessel was opened at arrow to add 1.0 ml. of 0.01 M potassium cyanide and tracer oxygen. Light: incandescent tungsten. Unit of light intensity (indicated at top) about 130 ft.-candles. Left ordinate (before adding KCN) X 0.67 = microliters of oxygen. Right ordinate (after adding KCN) X 0.63 = microliters of oxygen. Temperature, 20.9%.
had respired in the absenceof cyanide. The behavior of Anabaena in such an experiment is illustrated by Fig. 5. Cells were suspendedin bicarbonate buffer and illuminated for a short time. Respiration was then followed in the dark. Addition of cyanide reduced respiration to 10% of the uninhibited rate. This slow net uptake of oxygen continued through intervals of illumination, but the actual oxygen consumption was raised by the light to about 140 % of the previous uninhibited respiratory level. The light-induced oxygen consumption was almost exactly balanced by a compensating production of oxygen. Except at low intensities these processeswere essentially independent of light intensity. Substitution of Scenedesmusfor Anabaena and of phosphate (pH 6.5) for bicarbonate buffer did not materially alter the results. DISCUSSION
Figure 6 presents an abbreviated schematic survey which encompasses the results described above and is a summary of current ideas regarding
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$ co,
$ (CH,O) + f H,O f Photosynthesis \
FIG.
6. Schematic review of the reactions of chloroplasts
discussed in this paper.
gross aspects of photosynthesis and chloroplast reactions. Most of the reactions designated by letters in this diagram are complex or multistep processes.Which of these reactions possesssteps in common is largely a matter for speculation. The central hydrogen atom in brackets represents the reduction product from the photolysis of water [reaction (a)], which leads to oxygen evolution (b) and, in photosynthesizing cells, to carbon dioxide reduction (g) ; but in cell-free chloroplast preparations, any of several reductions of suitable electron or hydrogen acceptors are observed (h, j). In the absence of competing oxidants, oxygen can react with this reduced photolysis product to form hydrogen peroxide (c) which can be reduced (d, e) or dismuted (f). Cytochrome c or catechol can also link the reduction product [H] to molecular oxygen (Ic), in which case water is formed rather than hydrogen peroxide. Indirect evidence (14-16) indicates that pyridine nucleotides apparently can function in like manner. If none of these reductions are possible in a particular experiment, then it has been suggested that the photolysis products may recombine (m) so that no gas uptake or production is observed. At present we are concerned with two questions: Is oxygen equivalent to other so called “Hill oxidants” in reactions of cell-free chloroplast preparations? And can the photochemical reduction of oxygen be a significant reaction in intact cells? In answer to the first question, available information, including data
PHOTOCHEMICAL
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of this paper, indicate that the light-induced reduction of oxygen by chloroplasts under appropriate conditions is the over-all equivalent of similar reactions with other oxidants. No chloroplast preparations which were unable to reduce quinone or ferricyanide retained the ability to reduce oxygen. There was no oxygen uptake in the presence of inhibitors of conventional chloroplast reactions. o-Phenanthroline (5 X lo-+ M) depressedboth the rate of oxygen and of quinone reduction. However, there is reason to believe that the reduction of oxygen is not as direct as is the casewith an oxidant like quinone (vide paper No. II of this series). To the second question, a general answer cannot as yet be given. If during normal photosynthesis, photochemical oxygen reduction were superimposed upon regular respiratory and photosynthetic reactions, an enhancement of oxygen uptake should be detected by the tracer technique. Moreover, photochemical oxygen reduction, constituting a type of back reaction, might well diminish the rate of photosynthesis as compared to the rates of the reactions of isolated chloroplasts (which probably do not reduce oxygen in the presenceof other oxidants). In only one casehas the use of tracer oxygen demonstrated marked photostimulation of oxygen uptake under conditions of normal photosynthesis (S), and Ehrmantraut and Rabinowitch (13) have shown that in Chlorella the quantum yield of photosynthesis is essentially the sameas for the reduction of oxidants other than carbon dioxide. Furthermore, it now appears that light causes at best only small increases in the rate of oxygen consumption even in the absence of photosynthesis. It should be noted that the maximal velocity of oxygen uptake by illuminated living algae as described in this paper (Figs. 3, 4, and 5) does not much exceed the corresponding dark respiratory rate. However, the maximal rates of oxygen reduction by chloroplast preparations are comparable (per unit chlorophyll) to photosynthetic rates which are often twenty times more rapid than dark respiration. This quantitative comparison, showing that photoconsumption of oxygen can be more than an order of magnitude faster in vitro than in z)ivo, suggests that the rapid Mehler reaction in chloroplast preparations is an artifact and that the small, light-induced oxygen uptake by intact cells may not even proceed by the samemechanism. There remains the possibility that photochemical oxygen reduction during normal photosynthesis is not superimposed upon the respiratory metabolism but rather replaces it. This implies a relation between photosynthesis and respiration such as the existence of common reactions. For
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over a century cross links between respiration and photosynthesis have been a subject for speculation in terms of whether these processes have organic intermediates in common. Warburg’s observation that Chlorella photosynthesis is readily inhibited by cyanide only to the compensation point (17) can be interpreted by assuming that respiratory intermediates can be photochemically reduced rather than decarboxylated. Franck has recently cited observations of transient phenomena during photosynthetic inductions which support the assumption of such an interconnection (18). The discovery of rapid tagging of respiratory and glycolytic intermediates by brief exposures of photosynthesizing cells to tracer carbon dioxide has encouraged some investigators to expand these speculations even though their preliminary schemes (19) could not be maintained in all particulars in the light of our rapidly expanding knowledge of tracer carbon assimilation. Ochoa and co-workers have been particularly enthusiastic in their support of respiratory and photosynthetic cross linkage via Krebs cycle intermediates, and they have defended their view by experiments with isolated chloroplast preparations (14, 20) in which photolytic cleavage of water was coupled to reductive carboxylations by known enzyme systems in vitro. It now appears unlikely that photosynthesis achieves the back rotation of the Krebs cycle, and indeed indirect evidence suggests that during photosynthesis carbon metabolism via the Krebs cycle is suppressed (21). This should lead to a respiratory inhibition with regard to carbon dioxide production as claimed by Weigl et al. (22), but if the missing respiratory hydrogen is replaced by an approximately equivalent influx of photolytic “hydrogen,” no marked change in rate of oxygen uptake need be expected. The authors have not been able to formulate any hypothesis which could satisfactorily account for the apparent reversal by light of the cyanide inhibition of Anabaena and Scenedesmus respiration. There is evidence that the terminal oxidase responsible for Anabaena respiration is a cytochrome oxidase (5). As herein reported (Fig. 5), photostimulation of oxygen uptake occurs in this organism even when its normal respiration is severely inhibited by cyanide. Either the extra oxygen is consumed by a cyanide-resistant oxidase mechanism other than that responsible for the normal respiration, or cyanide directly or indirectly blocks respiration at some point other than the terminal oxidase. An alternative possibility, that light can reverse cyanide inhibition per se, is at present unsupported by any other evidence known to the authors.
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ACKNOWLEDGMENTS The script.
authors
are grateful
to Dr.
R.
Hill
for
his
helpful
criticism
of
the
manu-
The isotopically enriched oxygen was prepared by Dr. A. 0. Nier under a grant from the American Cancer Society through the Committee on Growth of the National Research Council. Financial assistance was obtained from the Graduate School of the University of Minnesota and the Rockefeller Foundation. These studies were aided by a contract between the Office of Naval Research, Department of t,he Navy, and the University of Minnesota (NR160-030). SUMMARY
1. Studies employing oxygen isotopes have shown that the stoichiometry of the consumption and production of oxygen by illuminated chloroplasts is that proposed by Mehler. Oxygen is reduced to hydrogen peroxide with a concurrent production of half as much oxygen originating from water. The reduction of oxygen in the Mehler reaction is thus analogous to t,he reduction of conventional chloroplast reagents such as quinone. 2. Spinach chloroplast preparations normally do not reduce oxygen direct,ly to s\-ater although polyphenol and cytochrome oxidases are present in the preparat,ions and catalyze an increased reduction of oxygen when either catechol or cytochrome c is added. 3. There is little photochemical reduction of oxygen superimposed on the respiratory oxygen uptake of living algae even when the cells are illuminated in the absenceof available carbon dioxide. 4. Light reversed the cyanide inhibition of oxygen uptake by Anabaena and Scenedesmuscell suspensions. 5. Evidence is presented which suggests that photochemically produced reductants may serve as hydrogen donors for the respiration of illuminated algae. Some possible interactions of photosynthesis and respiration are discussed. REFERENCES 1. MEHLER,
2. MEHLER, 3. KEILIN, 4. MEHLER,
A. A. D., A.
H., Arch. H., Arch.
Biochem. and Biophys. Biochem. and Biophys. AND HARTREE, E. F., Biochm. H., AND BROWN, A. H., Arch.
33, 65 (1951). 34, 339 (1951). J. 39, 293 (1945). Biochem. and Biophys.
(1952).
5. WEBSTER, 6. BROWN,
G. C., AND FRENKEL, A. W., Plant A. H., Am. J. Botany 40, 719 (1953).
Physiol.
28, 63
(1953).
38,
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7. BROWN, A. H., NIER, A. O., AND VAN NORMAN, R. W., Plant Physiol. 27, 320 (1952). 8. BROWN, A. H., AND WEBSTER, G., Am. J. Botany 40, 753 (1953). 9. JOHNSTON, J., AND BROWN, A. H., Plant Physiol. 29,177 (1954). 10. DOLE, M., RUDD, D. P., MUCHOW, G. R., AND COMTE, C., J. Chem. Phys.
20, 961 (1952). 11. CAHILL, A. E., AND TRAUBE, H. J., J. Am. Chem. Sot. 74, 2312 (1952). 12. DOLE, M., HAWKINS, R. C., AND BARKER, H. A., J. Am. Chem. Sot. 69, 226 (1947). 13. EHRMANTRAUT, H., AND RABINOWITCH, E., Arch. Biochem. and Biophys. 36, 67 (1952). 14. VISHNIAC, W., AND OCHOA, S., J. Biol. Clam. 196, 75 (1952). 15. VISHNIAC, W., AND OCHOA, S., J. Biol. Chem. 196, 501 (1952). 16. TOLMACH, L., Arch. Biochem. and Biophys. 33, 120 (1951). 17. WARBURG, O., Biochem. 2. 103, 188 (1920). 18. FRANCK, J., Arch. Biochem. and Biophys. 46, 190 (1953). 19. CALVIN, M., AND BENSON, A., Science 107, 467 (1948). 20. OCHOA, S., SALLES, J. B. V., AND ORTIZ, P. J., J. Biol. Chem. 167, 863 (1950). 21. BENSON, A., AND CALVIN, M., J. Exptl. Botany 1, 63 (1950). 22. WEIGL, J., WARRINGTON, P. M., AND CALVIN, M., J. Am. Chem. Sot. 73, 5058 (1951).