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Carbon dioxide metabolism in hydrogen-adapted Scenedesmus
ARCHIVES
OF
Carbon
BIOCHEMISTRY
Dioxide
GABRIEL
AND
178184
100,
Metabolism
GINGRAS,
From the Department
BIOPHYSICS
in Hydrogen-Adapted
RICHARD
of Chemistry
(1963)
A. GOLDSBY
and Lawrence Radiation Berkeley, California3 Received
October
ASD
Laboratory,
Scenedesmus’ MELVIN University
CALVIN of California,
8, 1962
1. Photoreduction of Cl402 by adapted Scenedesmus gives the same carbon fixation pattern as photosynthesis. 2. Photoreduction is susceptible to progressive inhibition by increasing oxygen tension. The inhibition is parallel. Hydrogenase activity is inhibited in a parallel manner by oxygen tension as measured by tritium exchange. 3. Photoreduction is relatively insensitive to CMU inhibition. Photoreduction at low light intensities is much faster than photosynthesis at the same light intensities. These resu1t.s are consistent with the ideas that hydrogen contributes electrons directly to CO2 photoreduction and possibly ATP as well. INTRODUCTION
A few organisms dispersed among three phyla have the ability to use molecular hydrogen for their metabolism. This is a very strange faculty, for in nature hydrogen gas is certainly uncommon. Even more surprising, perhaps, is the fact that green algae such as Scenedesmus or Chlamydomonas have hydrogenase as a constitutive enzyme. Scenedesmus, for example, shows hydrogenase activity (as measured by the isotopeexchange reaction) after a few minutes of incubaGon under hydrogen (1). The enzyme, moreover, is inhibited by small concentrations of oxygen (2). The obvious question, then, is what is the role of hydrogenase in an aerobic organism such as Scenedesmus? This work is a contribution to our understanding of two aspects of this problem: the path of carbon, and the sources of energy for carbon fixation under hydrogen. The carbon reduction cycle is now known to be so common among autotrophs as to 1 Abstracted from thesis submitted by R. A. Goldsby, June 1961, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of California, Berkeley, Calif. 2 Present address: Research Department, E. I. duPont de Nemours and Co., Wilmington, Del. 3 The work described in this paper was sponsored by the U. S. Atomic Energy Commission. 178
appear to be the rule for these organisms. It would therefore be very surprising if, as the available evidence suggests (3), CO2 entered mainly through malic acid in hydrogen-adapted Scenedesmus. This would, moreover, be in contrast to what has been found to occur in hydrogen-adapted bacteria (4, 5). Since the evidence for the malic acid route is not unequivocal, we decided to reinvestigate the problem. METHODS Scenedesmus obliquus grown under conditions of constant temperature, pH, illumination, etc. (6J was used throughout the work. After harvest, the cells were centrifuged and resuspended in growth medium (7), diluted fivefold. The suspension contained 2.5 ml. of packed cells (centrifugation for 15 min. at 600 X g) per 100 ml. The experiments were carried out in special air-tight vessels whose ground-joint tops were fitted with gas inlet and outlet closed by stopcocks, and another inlet closed by a rubber policeman through which liquids or gases could be injected. Each vessel contained 4.0 ml. (i.e., 0.1 ml. of packed cells) of algal suspension at pH 7.0. The cells were hydrogen-adapted in complete darkness for 7 hr. with constant shaking under a flow mixture of 99% HZ and 1% CO*. Before reaching the algae, this gas mixture was passed through three CrClz (oxsorbent) traps followed by two water traps, to remove the traces of 02 it contained. At the completion of the adaptation
CARBON
DIOXIDE TABLE
Total
fixation
G&S Total fixation Mono-P sugars Di-P sugars PGA PEPA Citric acid Malic acid Aspartic acid Glutamic acid Serine Glycine Alanine Unaccounted for
179
METABOLISM I
CO2 FIXATION PATTERN IN THE LIGHT (1250 mx) 30 MIN. given in counts/min. The rest of the data are in percentage of the soluble
H? 21.6 X IO6 15.6 0.8 32.9 2.6 0.4 6.3 7.3 1.2 4.3 0.5 2G.2 1.7
Hz 0 1 % 02 23.2 X 10” 20.2 1.1 32.0 1.9 0.6 3.3 12.2 2.3 4.3 0.5 18.8 2.5
Hz 0.5 7002 21.7 x 106 16.7 0.7 32.5 1.8 0.4 4.1 14.4 3.2 4.3 0.4 19.3 3.5
period, the vessels were swept with either 99% air and 1% COZ or 99% Hz and 170 Con. Depending on the experiment, the cells were then subjected to various treatments (preillumination, preincubation with inhibitors, etc.) prior to the injection of 200 yc. (7.8 rmoles) of NaHC1403. Incubation in the presence of NaHC1403 was conducted at 21-23°C. with constant shaking. The reaction was stopped by removing the top of the vessel and rapidly adding 16 ml. methanol. This operation can easily be carried out within 5 sec. An aliquot of the alcoholic suspension was counted, and the rest of the cells were extracted twice in 80% methanol at 60°C. and twice in 20y0 methanol at room temperature. Aliquots of the combined extracts were chromatographed on paper in two dimensions (phenolwater and butanol-propionic acid-water) ; the resulting spots were mapped by radioautography identified by with and cochromatography authentic samples. The radioactivities were determined by direct counting on the paper; the figures are reported as per cent of the soluble radioactivity and were calculated by taking the ratio of the radioactivity in one compound to the total radioactivit,y found on the chromatogram and multiplying by 100. RESULTS
1. C1402 FIXATIOK
PATTERN THE LIGHT
IN
The algae were adapted, then preilluminated for 5 min. prior to the addition of T\‘aHC1403. Toward the end of the preillumination period, various volumes of air were injected into the vessels so as to give
Hz 1.5% 02 24.G X 10” 17.8 0.5 27.8 1 G 0.7 4.0 12.5 3.0 4.2 0.8 17.0 7.8
H3
5 70 02 14.8 x 106 23.6 0.3 30.6 3.6 1.7 1.G 13.0 6.2 3.6 0.8 9.3 5.4
activity. Air
8.8 X lo6 29.2 3.0 20.3 3.5 1.0 2.5 14.2 4.5 2.2 0.3 11.8 7.2
the O2 concentrations shown in Table I. Incubation period in the presence of NaHC1403: 30 min.; light intensity at the surface of the vessels: 12<50lux. Table I shows that the total carbon fixation under hydrogen is about 2.5 times that under air. In contrast to the dark fixation (see Table II), the light fixation under hydrogen is unaffected by oxygen except at the 5% level, where there is inhibition. This inhibition can best be explained by an oxygen inactivation of the hydrogenase (2). Table I also shows that under air the percentage of CL4in the mono- and diphosphosugars is higher than under hydrogen. The reverse is true for PGA4 and alanine, which contain a greater share of the Cl4 under hydrogen. Only alanine, malic, aspartic, and glutamic acids are affected by increasing the oxygen concentration under hydrogen. This can be explained by a decrease in the ratio of reduced coenzyme to oxidized coenzyme by respiration and inhibition of hydrogenase, which slows down reductive carboxylation leading to malic acid. A similar argument may be used to explain the stimulating effect of oxygen on the labeling of glutamic acid. If we assume that the rate-limiting step in the over-all synthesis of glutamate is the formation of oc-keto4 Abbreviations used: ATP, adenosine triphosphate; DNP, dinitrophenol; CMU, 3-&chlorophenyl).1-dimethylurea; DCMU, 3-(2,4-dichlorophenyl)-1-l-dimethylurea; PGA, phosphoglyceric acid; PEPA, phosphoenolpyruvic acid.
180
GINGRAS,
GOLDSBY
AND CALVIN
TABLE II COZ FIXATION PATTERN IN THE DARK (30 MIN.) Total fixation given in counts/min. The rest of the data are given in percentage of the soluble activity.
Total fixation Mono-P sugars Di-P sugars PGA PEPA Citric acid Malic acid Aspartic acid Glutamic acid Serine Glycine Alanine Unaccounted for
1.1 x 106 16.3 7.1 6.6 1.0 1.9 11.2 1.3 0 0.5 0.6 50.3 0
Hz 0.1 % 02
Hz 0.5 % 02
Ht 1.5 % 02
1.5 x 106 16.1 9.7 7.4 0.3 0.3 10.3 0.6 0 0.2 0.2 54.5 0
2.9 x 106 13.6 6.2 18.5 1.8 0.1 8.2 1.6 0 1.5 0.4 47.0 0.9
6.6 x 106 18.6 2.6 23.3 2.7 0.8 8.0 3.1 0.9 2.6 0.9 28.2 6.6
glutarate by the Krebs cycle, decrease in the ratio of reduced to oxidized coenzyme will favor glutamate formation. We have observed relatively large amounts of CXketoglutaric acid under similar conditions when an oxidant such as methylene blue is added to the cells in the dark; ar-ketoglutaric acid is usually not seen under our normal experimental conditions. The effect of added oxygen on the labeling of alanine is rather striking, but it is even more so in the dark fixation pattern and will be discussed in the next section. 2. U402 FIXATION PATTERN IN THE DARK
This experiment was carried out at the same time and with the same population of cells as the light CO2 fixation experiment. Conditions were the same in both cases, except that here the vessels were wrapped with masking tape so as to keep their contents in complete darkness. Table II shows that under hydrogen, C402 fixation is again about three times as high as under air. When oxygen is added to the hydrogen, a remarkable phenomenon occurs: The CY402 fixation increases with oxygen concentration until, at 5% 02, it reaches a level comparable to that obtained by photoreduction (Table I). This is the oxyhydrogen reaction studied by Gaffron (8). Not only are photoreduction and oxyhydrogen reactions similar in the rates, but
HZ 5 %O% 16. X lo6 19.5 1.8 25.7 1.6 0.8 5.0 8.7 2.2 4.7 0.9 25.8 3.2
Air
0.38 x 106 3.5 0 17.4 1.0 9.2 9.5 37.9 20.5 0 0 0.8 0
also in the patterns of CO2 fixation. Compare, for example, the 5 % 02 dark fixation pattern with the 0.5% 02 light fixation pattern. So far as carbon dioxide is concerned, we have achieved photosynthesis in the dark. We think these results clearly indicate that molecular hydrogen is utilized for the reduction of a coenzyme which can either directly reduce carbon compounds or reduce oxygen via an oxidative electron-transport chain with the production of high-energy phosphates. Table II shows that as the concentration of oxygen is raised, the proportion of Cl4 in the sugar diphosphates and in alanine decreases and, instead, goes up in PGA and in aspartate. The high sensitivity of alanine to oxygen is especially interesting. It indicates that this amino acid is formed mostly by a reductive process. This process could be a direct reductive amination of pyruvate or a reductive amination of ar-ketoglutarate or of oxalacetate followed by transamination with pyruvate. The first reaction has been found to occur in bacteria (lo), but has not been reported in plants. The present evidence does not permit a choice between these mechanisms. 3. KINETIC
EXPERIMENTS
In order to learn what is the route of carbon fixation in Scenedesmus during photoreduction or the oxyhydrogen reaction,
CARBON
DIOXIDE
we applied kinetic methods which were so successful in the elucidation of the path of carbon in photosynthesis. The experimental procedure was the same as described before. The sampling technique was different, however. After the NaHC1403 had been introduced in a tuberculin syringe through the policeman, the cells were rapidly shaken by hand, and a 1.0.ml. sample was taken in the syringe and rapidly injected in methanol. The first two points were l.O-ml. samples; the following were 0.5.ml. samples. The cells were either preilluminated or preincubated with 1.5 % O2 for 5 min. before addition of NaHCYO 3. (a) Light CO, Fixation Figure 1 shows that under air at 1250 lux, COZ enters mainly through PGA and is reduced via the carbon reduction cycle (9). At the same light intensity, but under hydrogen (Fig. 2), most of the COZ is fixed in PGA, but an appreciable fraction is fixed in malic or oxalacetic acids. Observe (Fig. 2) the rapid rise in the activity of malic and aspartic acids, and compare with fixation rates in these compounds under air. It is of interest also that the decrease in the per cent of Cl4 in PGA is more rapid under hydrogen than under air and is accompanied by a faster increase in alanine and the sugar monophosphates. This we take to agree with the conclusion reached in the previous experiment that hydrogen is used in the
181
METABOLISM
reduction of PGA and in the transamination or the reductive aminat,ion of pyruvate. (b) Dark CO2 Fixation In the dark under hydrogen (Fig. 3), COZ seems to enter mainly through aspartic acid and malic acid, although a small percentage probably enters through PGA. An unidentified spot was observed below aspartic acid on our radioautograms, but showed no activity in the liquid scintillation counter. We think the compound probably decomposed and volatilized in the period between chromatography and counting. Such behavior would be consistent with oxalacetic acid. Figure 4 shows that the oxyhydrogendriven CO2 fixation occurs mainly via PGA at least in I.:, R oxygen, the concentration
FIG. 2. Kinetics of CO2 fixation in photoreduction at low light intensity. Gas phase: hydrogen. Light intensity: 1250 lux.
100 F
FIG. 1. Kinetics of CO* fixation in photosynthesis at low light intensity. Gas phase: air. Light intensity: 1250 lux.
TIME Ired
FIG. 3. Kinetics under hydrogen.
of COI fixation
in the dark
GINGRAS,
182
GOLDSBY
AND CALVIN
Tz in the gas phase with Hz0 in the medium: Hz0
l, TIME bed
FIG. 4. Kinetics of CO2 fixation in the oxyhydrogen reaction. Gas phase: hydrogen plus 1.5y0 oxygen. Dark.
we used in this experiment. Badin (3) reported evidence suggestive of a predominance of malic acid over PGA as an entry of COz in hydrogen-adapted Xcenedesmus at very low (490 lux) light intensity or in the oxyhydrogen reaction (0.5 % 02). In those experiments, however, the algae were not killed anaerobically but, were exposed to air for 3-4 min. before killing. For these first, minutes, critical ones, exposure to air could cause a large difference. 4. CO2 FIXATION
AND HYDROGENASE ACTIVITY
The rate of CO2 fixation in the oxyhydrogen reaction depends on two opposite factors: (a) the energy-yielding reaction and (b) the inactivation of hydrogenase by oxygen. Increasing the oxygen tension will accelerate the energy-yielding reaction up to a certain limit; on the other hand, it will also inhibit the hydrogenase and this will tend to decrease the oxyhydrogen reaction. In order to measure hydrogenase activity under different oxygen tensions, we had recourse to the isotope-exchange reactions discovered by Farkas et al. (12) and used by Hoberman and Rittenberg (13) as an assay method for hydrogenase : DzO
+
HI.
hydrogenase
> HD
+
DOH
Instead of following the rate of exchange of D,O with Hz, we followed the exchange of
+
Tz
hydrogenase
) HT
+
HOT
The algae are simply incubated in the dark in the presence of a tracer amount of Tz in the hydrogen atmosphere. After a certain period of time, the reaction is stopped by methanol. and the suspension is swept with Hz to remove dissolved tritium. An aliquot sample of the total mixture is counted directly in the scintillation counter (1). A double-labeling experiment was carried out as follows: The algae were hydrogen adapted for 7 hr. as usual. Each vessel contained 80 ~1. of packed cells in a volume of 3.0 ml. of growth medium diluted 1:5. The atmosphere of the vessels was then replaced with a mixture of Hz and TP, the algae left in contact with this mixture for 35 min., then 30 PC. (25 pmoles) of NaHC1403 injected, followed by various volumes of air, and a 1 .O-ml. sample was immediately taken. Ten minutes later the rest of the algae were killed and sampled again. The samples were counted in a liquid scintillation counter where, because of the low Cl4 fixation, it was possible to discriminate between activity due to Cl4 and activity due to tritium. Figure 5 shows the fixation during the 10 min. following injection of air. This
experiment
relationship hydrogenase
shows
between activity.
%o;,
the
expected
CO, fixation and There must be an
MU-22812
FIG. 5. The effect of oxygen on the rate of COP fixation and on hydrogenase activity. Ten-minute fixation time and exposure time in the presence of COP.
CARBON
DIOXIDE
optimal point where the enzyme is active enough and the oxygen pressure high enough to permit the oxyhydrogen reaction to proceed at maximal rate. This point appears to be between 0 and 0.5% O2 under the conditions of this experiment. Carbon dioxide fixation is shown to attain a maximum between 0 and 0.5% 02 and to decline as oxygen tension is increased. This is in contrast to the experiment reported in Table II where CO2 fixation increased almost linearly with O2 tension, up to 5 % 02. We think this apparent discrepancy is due to the smaller volume of the suspension and to the faster rate of shaking in the present experiment, as well as its shorter duration (10 min. vs. 30 min.). In t,he first experiment the rate of diffusion of and permitted the oxygen was limiting hydrogenase to go on working, even at 5 % 02, as witnessed by the almost linear relationship between CO2 fixation and oxygen concentration. In the present experiment, the rate of diffusion of 02 is much faster so that the enzyme is inactivated (Fig. 5) completely at 5 % OZ. The fact that 0%diffusion rate was limiting in the first experiment does not invalidate the conclusions reached; it means that the curves shown in Fig. 2 are artificially shifted to the right; their shape would not be changed by a higher rate of diffusion of oxygen. 5. INHIBITOR
STUDIES
In the hope of learning more about the mechanism of the photoreduction and the oxyhydrogen reactions, we studied next the effect of various inhibitors on the CO2 fixation. The usual experimental procedure was followed with the exception that the cells were preincubated with the inhibitors for a period of 60 min. before addition of KaHC?“03. Table III shows the effect of high concentrations of menadione (vitamin IQ, 2,4-dinitrophenol, and CMU [3-(4chlorophenyl)-1 , 1-dimethylurea] on photosynthesis and on photoreduction. Photosynthesis appears to be the more sensitive to all three inhibitors. The effect of CMU, a Hill reaction inhibitor, is particularly striking: It inhibits photosynthesis twice as much as it inhibits photoreduction.
METABOLISM
183 TABLE
EFFECT
OF INHIBITORS
PHOTOSYNTHESIS
III ON
AND
Cl402
FIXATION
IN
PHOTOREDUCTION
Light intensity: 1250 lux; final concentration of inhibitors indicated in parentheses; preincubation with the inhibitors: 60 min.; incubation time with NaHC140d: 30 min. Gas phase
-_
Per cent inhibition
Inhibitor
counts/ min.x lo-”
Hydrogen
None Menadione (saturated) CMU (saturated) 2,4-DNP(lOF M) ,
--Air
None Menadione (saturated) CMU (saturated) 2,4-DNP(lO+ M)
TABLE EFFECT
OF INHIBITORS
18.1 5.9 9.7 7.2 3.8 0.08 0.3 0.05
65 45 GO 98 92 99
1
IV ON THE
DARK
C1402
FIXATION
Preincubation with inhibitors: 60 min., incubation with NaHCY403: 30 min.; final concentration of inhibitor in parentheses. Gas phase
Inhibitor ited
Hydrogen Menadione (saturated) Hydrogen CMU (saturated) Hydrogen 2,4-DNP (1OF K) Air Menadione (saturated) Air CMU (saturated) Air 2,4-DNP (lo+ M) Hydrogen + 1.5% Menadione (saturated) 03 Hydrogen + 1.50j0 CMU (saturated) 02 Hydrogen + 1.5y0 2,4-DNP (IO+ M) 02
0 0 90 97 0 70 80 0 98
Table IV shows the effect of the three inhibitors on CY402 dark fixation under hydrogen, air, and under a mixture of hydrogen and oxygen. Menadione inhibits the fixation under air and under hydrogen plus oxygen; this is consistent with an inhibition of respiration as reported elsewhere
184
GINGRAS,
GOLDSBY
(14-17). Both the air and the oxyhydrogen fixations are more sensitive to menadione than photoreduction. Likewise, the hydrogen and the oxyhydrogen fixations are more sensitive to dinitrophenol than photoreduction. CMU does not appreciably affect any of the dark fixations. Since these observations have been made and this paper written, a report by Gaffron has appeared (18) describing a mutant of Scenedesmus which can grow heterotrophitally or by photoreduction without evolution of oxygen. This is insensitive to DCMU as well. The conclusions reached by Gaffron are very similar to those described here. DISCUSSION
The data are consistent with the proposal that there is a reduction of a coenzyme by molecular hydrogen. When oxygen is added in the dark, some of the hydrogen is combined with oxygen to yield water and highenergy phosphates; the reduced coenzyme and the high-energy phosphate can be used to reduce CO2 via the carbon reduction cycle. This oxyhydrogen reaction exhibits respiration-like sensitivity to menadione and to 2,4-dinitrophenol. Carbon dioxide fixation under hydrogen at the light intensity used here is from two to four times faster than under air. It is clear from this that molecular hydrogen contributes electrons and probably ATP as well for the reduction of carbon compounds. At the concentrations used, the three inhibitors block photosynthetic fixation almost completely, but photoreduction even in the presence of these inhibitors
AND
CALVIN
still fixes more CY402 than does photosynthesis at these low light intensities. REFERENCES 1. GOLDSBY, R. A., Thesis, Univ. of California, 1961. UCRL 9806. 2. FISHER, H. F., KRASNA, A. E., AND RITTENBERG, D., J. Biol. Chem. 209, 569 (1954). 3. BADIN, E., AND CALVIN, M., J. grn. Chem. Sot. 72, 5266 (1950). 4. STOPPANI, A. 0. M., FULLER, R. C., AND CALVIN, M., J. Bacte?ioZ. 69, 491 (1955). 5. MILHAUD, G., AUBERT, J. P., AND MILLET, J., Corn@. rend. 243, 102 (1956). 6. BASSHAM, J. A., AND CALVIN, M., “The Path of Carbon in Photosynthesis,” pp. 2833. Prentice Hall, Englewood Cliffs, N. J., 1957. 7. MYERS, J., Plant Physiol. 22, 590 (1947). 8. GAFFRON, H. A., J. Gen. Physiol. 26,241(1942). 9. BASSHAM, J. A., BENSON, A. A., KAY, L. D., HARRIS, A. Z., WILSON, A. T., AND CALVIN, M., J. Am. Chem. Sot. 76, 1760 (1954). 10. WIAME, J. M., AND PIERARD, A., h’ature 176, 1073 (1955). 11. BISHOP, N. I., Biochim. et Biophys. Acta 27, 205 (1958) . 12. FARKAS, A., FARKAS, L., AND YUDKIN, Proc. Roy. Sot. (London) B155, 372 (1934). 13. HOBERMAN, H. D., AND RITTENBERG, D., J. Biol. Chem. 147, 211 (1943). 14. GAFFRON, H. A., J. Gen. Physiol. 28, 259 (1944). 15. ARNON, D. I., Nature 184, 10 (1959). 66, 16. HORWITZ, L., Arch. Biochem. Biophys. 23 (1957). 17. HORWITZ, L., AND ALLEN, F. L., Arch. Biothem. Biophys. 66, 45 (1957). in Biochemistry” 18. GAFFRON, H., in “Horizons (B. Pullman and M. Kasha, eds.), p. 78. Academic Press, New York, 1962.
OF
Carbon
BIOCHEMISTRY
Dioxide
GABRIEL
AND
178184
100,
Metabolism
GINGRAS,
From the Department
BIOPHYSICS
in Hydrogen-Adapted
RICHARD
of Chemistry
(1963)
A. GOLDSBY
and Lawrence Radiation Berkeley, California3 Received
October
ASD
Laboratory,
Scenedesmus’ MELVIN University
CALVIN of California,
8, 1962
1. Photoreduction of Cl402 by adapted Scenedesmus gives the same carbon fixation pattern as photosynthesis. 2. Photoreduction is susceptible to progressive inhibition by increasing oxygen tension. The inhibition is parallel. Hydrogenase activity is inhibited in a parallel manner by oxygen tension as measured by tritium exchange. 3. Photoreduction is relatively insensitive to CMU inhibition. Photoreduction at low light intensities is much faster than photosynthesis at the same light intensities. These resu1t.s are consistent with the ideas that hydrogen contributes electrons directly to CO2 photoreduction and possibly ATP as well. INTRODUCTION
A few organisms dispersed among three phyla have the ability to use molecular hydrogen for their metabolism. This is a very strange faculty, for in nature hydrogen gas is certainly uncommon. Even more surprising, perhaps, is the fact that green algae such as Scenedesmus or Chlamydomonas have hydrogenase as a constitutive enzyme. Scenedesmus, for example, shows hydrogenase activity (as measured by the isotopeexchange reaction) after a few minutes of incubaGon under hydrogen (1). The enzyme, moreover, is inhibited by small concentrations of oxygen (2). The obvious question, then, is what is the role of hydrogenase in an aerobic organism such as Scenedesmus? This work is a contribution to our understanding of two aspects of this problem: the path of carbon, and the sources of energy for carbon fixation under hydrogen. The carbon reduction cycle is now known to be so common among autotrophs as to 1 Abstracted from thesis submitted by R. A. Goldsby, June 1961, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of California, Berkeley, Calif. 2 Present address: Research Department, E. I. duPont de Nemours and Co., Wilmington, Del. 3 The work described in this paper was sponsored by the U. S. Atomic Energy Commission. 178
appear to be the rule for these organisms. It would therefore be very surprising if, as the available evidence suggests (3), CO2 entered mainly through malic acid in hydrogen-adapted Scenedesmus. This would, moreover, be in contrast to what has been found to occur in hydrogen-adapted bacteria (4, 5). Since the evidence for the malic acid route is not unequivocal, we decided to reinvestigate the problem. METHODS Scenedesmus obliquus grown under conditions of constant temperature, pH, illumination, etc. (6J was used throughout the work. After harvest, the cells were centrifuged and resuspended in growth medium (7), diluted fivefold. The suspension contained 2.5 ml. of packed cells (centrifugation for 15 min. at 600 X g) per 100 ml. The experiments were carried out in special air-tight vessels whose ground-joint tops were fitted with gas inlet and outlet closed by stopcocks, and another inlet closed by a rubber policeman through which liquids or gases could be injected. Each vessel contained 4.0 ml. (i.e., 0.1 ml. of packed cells) of algal suspension at pH 7.0. The cells were hydrogen-adapted in complete darkness for 7 hr. with constant shaking under a flow mixture of 99% HZ and 1% CO*. Before reaching the algae, this gas mixture was passed through three CrClz (oxsorbent) traps followed by two water traps, to remove the traces of 02 it contained. At the completion of the adaptation
CARBON
DIOXIDE TABLE
Total
fixation
G&S Total fixation Mono-P sugars Di-P sugars PGA PEPA Citric acid Malic acid Aspartic acid Glutamic acid Serine Glycine Alanine Unaccounted for
179
METABOLISM I
CO2 FIXATION PATTERN IN THE LIGHT (1250 mx) 30 MIN. given in counts/min. The rest of the data are in percentage of the soluble
H? 21.6 X IO6 15.6 0.8 32.9 2.6 0.4 6.3 7.3 1.2 4.3 0.5 2G.2 1.7
Hz 0 1 % 02 23.2 X 10” 20.2 1.1 32.0 1.9 0.6 3.3 12.2 2.3 4.3 0.5 18.8 2.5
Hz 0.5 7002 21.7 x 106 16.7 0.7 32.5 1.8 0.4 4.1 14.4 3.2 4.3 0.4 19.3 3.5
period, the vessels were swept with either 99% air and 1% COZ or 99% Hz and 170 Con. Depending on the experiment, the cells were then subjected to various treatments (preillumination, preincubation with inhibitors, etc.) prior to the injection of 200 yc. (7.8 rmoles) of NaHC1403. Incubation in the presence of NaHC1403 was conducted at 21-23°C. with constant shaking. The reaction was stopped by removing the top of the vessel and rapidly adding 16 ml. methanol. This operation can easily be carried out within 5 sec. An aliquot of the alcoholic suspension was counted, and the rest of the cells were extracted twice in 80% methanol at 60°C. and twice in 20y0 methanol at room temperature. Aliquots of the combined extracts were chromatographed on paper in two dimensions (phenolwater and butanol-propionic acid-water) ; the resulting spots were mapped by radioautography identified by with and cochromatography authentic samples. The radioactivities were determined by direct counting on the paper; the figures are reported as per cent of the soluble radioactivity and were calculated by taking the ratio of the radioactivity in one compound to the total radioactivit,y found on the chromatogram and multiplying by 100. RESULTS
1. C1402 FIXATIOK
PATTERN THE LIGHT
IN
The algae were adapted, then preilluminated for 5 min. prior to the addition of T\‘aHC1403. Toward the end of the preillumination period, various volumes of air were injected into the vessels so as to give
Hz 1.5% 02 24.G X 10” 17.8 0.5 27.8 1 G 0.7 4.0 12.5 3.0 4.2 0.8 17.0 7.8
H3
5 70 02 14.8 x 106 23.6 0.3 30.6 3.6 1.7 1.G 13.0 6.2 3.6 0.8 9.3 5.4
activity. Air
8.8 X lo6 29.2 3.0 20.3 3.5 1.0 2.5 14.2 4.5 2.2 0.3 11.8 7.2
the O2 concentrations shown in Table I. Incubation period in the presence of NaHC1403: 30 min.; light intensity at the surface of the vessels: 12<50lux. Table I shows that the total carbon fixation under hydrogen is about 2.5 times that under air. In contrast to the dark fixation (see Table II), the light fixation under hydrogen is unaffected by oxygen except at the 5% level, where there is inhibition. This inhibition can best be explained by an oxygen inactivation of the hydrogenase (2). Table I also shows that under air the percentage of CL4in the mono- and diphosphosugars is higher than under hydrogen. The reverse is true for PGA4 and alanine, which contain a greater share of the Cl4 under hydrogen. Only alanine, malic, aspartic, and glutamic acids are affected by increasing the oxygen concentration under hydrogen. This can be explained by a decrease in the ratio of reduced coenzyme to oxidized coenzyme by respiration and inhibition of hydrogenase, which slows down reductive carboxylation leading to malic acid. A similar argument may be used to explain the stimulating effect of oxygen on the labeling of glutamic acid. If we assume that the rate-limiting step in the over-all synthesis of glutamate is the formation of oc-keto4 Abbreviations used: ATP, adenosine triphosphate; DNP, dinitrophenol; CMU, 3-&chlorophenyl).1-dimethylurea; DCMU, 3-(2,4-dichlorophenyl)-1-l-dimethylurea; PGA, phosphoglyceric acid; PEPA, phosphoenolpyruvic acid.
180
GINGRAS,
GOLDSBY
AND CALVIN
TABLE II COZ FIXATION PATTERN IN THE DARK (30 MIN.) Total fixation given in counts/min. The rest of the data are given in percentage of the soluble activity.
Total fixation Mono-P sugars Di-P sugars PGA PEPA Citric acid Malic acid Aspartic acid Glutamic acid Serine Glycine Alanine Unaccounted for
1.1 x 106 16.3 7.1 6.6 1.0 1.9 11.2 1.3 0 0.5 0.6 50.3 0
Hz 0.1 % 02
Hz 0.5 % 02
Ht 1.5 % 02
1.5 x 106 16.1 9.7 7.4 0.3 0.3 10.3 0.6 0 0.2 0.2 54.5 0
2.9 x 106 13.6 6.2 18.5 1.8 0.1 8.2 1.6 0 1.5 0.4 47.0 0.9
6.6 x 106 18.6 2.6 23.3 2.7 0.8 8.0 3.1 0.9 2.6 0.9 28.2 6.6
glutarate by the Krebs cycle, decrease in the ratio of reduced to oxidized coenzyme will favor glutamate formation. We have observed relatively large amounts of CXketoglutaric acid under similar conditions when an oxidant such as methylene blue is added to the cells in the dark; ar-ketoglutaric acid is usually not seen under our normal experimental conditions. The effect of added oxygen on the labeling of alanine is rather striking, but it is even more so in the dark fixation pattern and will be discussed in the next section. 2. U402 FIXATION PATTERN IN THE DARK
This experiment was carried out at the same time and with the same population of cells as the light CO2 fixation experiment. Conditions were the same in both cases, except that here the vessels were wrapped with masking tape so as to keep their contents in complete darkness. Table II shows that under hydrogen, C402 fixation is again about three times as high as under air. When oxygen is added to the hydrogen, a remarkable phenomenon occurs: The CY402 fixation increases with oxygen concentration until, at 5% 02, it reaches a level comparable to that obtained by photoreduction (Table I). This is the oxyhydrogen reaction studied by Gaffron (8). Not only are photoreduction and oxyhydrogen reactions similar in the rates, but
HZ 5 %O% 16. X lo6 19.5 1.8 25.7 1.6 0.8 5.0 8.7 2.2 4.7 0.9 25.8 3.2
Air
0.38 x 106 3.5 0 17.4 1.0 9.2 9.5 37.9 20.5 0 0 0.8 0
also in the patterns of CO2 fixation. Compare, for example, the 5 % 02 dark fixation pattern with the 0.5% 02 light fixation pattern. So far as carbon dioxide is concerned, we have achieved photosynthesis in the dark. We think these results clearly indicate that molecular hydrogen is utilized for the reduction of a coenzyme which can either directly reduce carbon compounds or reduce oxygen via an oxidative electron-transport chain with the production of high-energy phosphates. Table II shows that as the concentration of oxygen is raised, the proportion of Cl4 in the sugar diphosphates and in alanine decreases and, instead, goes up in PGA and in aspartate. The high sensitivity of alanine to oxygen is especially interesting. It indicates that this amino acid is formed mostly by a reductive process. This process could be a direct reductive amination of pyruvate or a reductive amination of ar-ketoglutarate or of oxalacetate followed by transamination with pyruvate. The first reaction has been found to occur in bacteria (lo), but has not been reported in plants. The present evidence does not permit a choice between these mechanisms. 3. KINETIC
EXPERIMENTS
In order to learn what is the route of carbon fixation in Scenedesmus during photoreduction or the oxyhydrogen reaction,
CARBON
DIOXIDE
we applied kinetic methods which were so successful in the elucidation of the path of carbon in photosynthesis. The experimental procedure was the same as described before. The sampling technique was different, however. After the NaHC1403 had been introduced in a tuberculin syringe through the policeman, the cells were rapidly shaken by hand, and a 1.0.ml. sample was taken in the syringe and rapidly injected in methanol. The first two points were l.O-ml. samples; the following were 0.5.ml. samples. The cells were either preilluminated or preincubated with 1.5 % O2 for 5 min. before addition of NaHCYO 3. (a) Light CO, Fixation Figure 1 shows that under air at 1250 lux, COZ enters mainly through PGA and is reduced via the carbon reduction cycle (9). At the same light intensity, but under hydrogen (Fig. 2), most of the COZ is fixed in PGA, but an appreciable fraction is fixed in malic or oxalacetic acids. Observe (Fig. 2) the rapid rise in the activity of malic and aspartic acids, and compare with fixation rates in these compounds under air. It is of interest also that the decrease in the per cent of Cl4 in PGA is more rapid under hydrogen than under air and is accompanied by a faster increase in alanine and the sugar monophosphates. This we take to agree with the conclusion reached in the previous experiment that hydrogen is used in the
181
METABOLISM
reduction of PGA and in the transamination or the reductive aminat,ion of pyruvate. (b) Dark CO2 Fixation In the dark under hydrogen (Fig. 3), COZ seems to enter mainly through aspartic acid and malic acid, although a small percentage probably enters through PGA. An unidentified spot was observed below aspartic acid on our radioautograms, but showed no activity in the liquid scintillation counter. We think the compound probably decomposed and volatilized in the period between chromatography and counting. Such behavior would be consistent with oxalacetic acid. Figure 4 shows that the oxyhydrogendriven CO2 fixation occurs mainly via PGA at least in I.:, R oxygen, the concentration
FIG. 2. Kinetics of CO2 fixation in photoreduction at low light intensity. Gas phase: hydrogen. Light intensity: 1250 lux.
100 F
FIG. 1. Kinetics of CO* fixation in photosynthesis at low light intensity. Gas phase: air. Light intensity: 1250 lux.
TIME Ired
FIG. 3. Kinetics under hydrogen.
of COI fixation
in the dark
GINGRAS,
182
GOLDSBY
AND CALVIN
Tz in the gas phase with Hz0 in the medium: Hz0
l, TIME bed
FIG. 4. Kinetics of CO2 fixation in the oxyhydrogen reaction. Gas phase: hydrogen plus 1.5y0 oxygen. Dark.
we used in this experiment. Badin (3) reported evidence suggestive of a predominance of malic acid over PGA as an entry of COz in hydrogen-adapted Xcenedesmus at very low (490 lux) light intensity or in the oxyhydrogen reaction (0.5 % 02). In those experiments, however, the algae were not killed anaerobically but, were exposed to air for 3-4 min. before killing. For these first, minutes, critical ones, exposure to air could cause a large difference. 4. CO2 FIXATION
AND HYDROGENASE ACTIVITY
The rate of CO2 fixation in the oxyhydrogen reaction depends on two opposite factors: (a) the energy-yielding reaction and (b) the inactivation of hydrogenase by oxygen. Increasing the oxygen tension will accelerate the energy-yielding reaction up to a certain limit; on the other hand, it will also inhibit the hydrogenase and this will tend to decrease the oxyhydrogen reaction. In order to measure hydrogenase activity under different oxygen tensions, we had recourse to the isotope-exchange reactions discovered by Farkas et al. (12) and used by Hoberman and Rittenberg (13) as an assay method for hydrogenase : DzO
+
HI.
hydrogenase
> HD
+
DOH
Instead of following the rate of exchange of D,O with Hz, we followed the exchange of
+
Tz
hydrogenase
) HT
+
HOT
The algae are simply incubated in the dark in the presence of a tracer amount of Tz in the hydrogen atmosphere. After a certain period of time, the reaction is stopped by methanol. and the suspension is swept with Hz to remove dissolved tritium. An aliquot sample of the total mixture is counted directly in the scintillation counter (1). A double-labeling experiment was carried out as follows: The algae were hydrogen adapted for 7 hr. as usual. Each vessel contained 80 ~1. of packed cells in a volume of 3.0 ml. of growth medium diluted 1:5. The atmosphere of the vessels was then replaced with a mixture of Hz and TP, the algae left in contact with this mixture for 35 min., then 30 PC. (25 pmoles) of NaHC1403 injected, followed by various volumes of air, and a 1 .O-ml. sample was immediately taken. Ten minutes later the rest of the algae were killed and sampled again. The samples were counted in a liquid scintillation counter where, because of the low Cl4 fixation, it was possible to discriminate between activity due to Cl4 and activity due to tritium. Figure 5 shows the fixation during the 10 min. following injection of air. This
experiment
relationship hydrogenase
shows
between activity.
%o;,
the
expected
CO, fixation and There must be an
MU-22812
FIG. 5. The effect of oxygen on the rate of COP fixation and on hydrogenase activity. Ten-minute fixation time and exposure time in the presence of COP.
CARBON
DIOXIDE
optimal point where the enzyme is active enough and the oxygen pressure high enough to permit the oxyhydrogen reaction to proceed at maximal rate. This point appears to be between 0 and 0.5% O2 under the conditions of this experiment. Carbon dioxide fixation is shown to attain a maximum between 0 and 0.5% 02 and to decline as oxygen tension is increased. This is in contrast to the experiment reported in Table II where CO2 fixation increased almost linearly with O2 tension, up to 5 % 02. We think this apparent discrepancy is due to the smaller volume of the suspension and to the faster rate of shaking in the present experiment, as well as its shorter duration (10 min. vs. 30 min.). In t,he first experiment the rate of diffusion of and permitted the oxygen was limiting hydrogenase to go on working, even at 5 % 02, as witnessed by the almost linear relationship between CO2 fixation and oxygen concentration. In the present experiment, the rate of diffusion of 02 is much faster so that the enzyme is inactivated (Fig. 5) completely at 5 % OZ. The fact that 0%diffusion rate was limiting in the first experiment does not invalidate the conclusions reached; it means that the curves shown in Fig. 2 are artificially shifted to the right; their shape would not be changed by a higher rate of diffusion of oxygen. 5. INHIBITOR
STUDIES
In the hope of learning more about the mechanism of the photoreduction and the oxyhydrogen reactions, we studied next the effect of various inhibitors on the CO2 fixation. The usual experimental procedure was followed with the exception that the cells were preincubated with the inhibitors for a period of 60 min. before addition of KaHC?“03. Table III shows the effect of high concentrations of menadione (vitamin IQ, 2,4-dinitrophenol, and CMU [3-(4chlorophenyl)-1 , 1-dimethylurea] on photosynthesis and on photoreduction. Photosynthesis appears to be the more sensitive to all three inhibitors. The effect of CMU, a Hill reaction inhibitor, is particularly striking: It inhibits photosynthesis twice as much as it inhibits photoreduction.
METABOLISM
183 TABLE
EFFECT
OF INHIBITORS
PHOTOSYNTHESIS
III ON
AND
Cl402
FIXATION
IN
PHOTOREDUCTION
Light intensity: 1250 lux; final concentration of inhibitors indicated in parentheses; preincubation with the inhibitors: 60 min.; incubation time with NaHC140d: 30 min. Gas phase
-_
Per cent inhibition
Inhibitor
counts/ min.x lo-”
Hydrogen
None Menadione (saturated) CMU (saturated) 2,4-DNP(lOF M) ,
--Air
None Menadione (saturated) CMU (saturated) 2,4-DNP(lO+ M)
TABLE EFFECT
OF INHIBITORS
18.1 5.9 9.7 7.2 3.8 0.08 0.3 0.05
65 45 GO 98 92 99
1
IV ON THE
DARK
C1402
FIXATION
Preincubation with inhibitors: 60 min., incubation with NaHCY403: 30 min.; final concentration of inhibitor in parentheses. Gas phase
Inhibitor ited
Hydrogen Menadione (saturated) Hydrogen CMU (saturated) Hydrogen 2,4-DNP (1OF K) Air Menadione (saturated) Air CMU (saturated) Air 2,4-DNP (lo+ M) Hydrogen + 1.5% Menadione (saturated) 03 Hydrogen + 1.50j0 CMU (saturated) 02 Hydrogen + 1.5y0 2,4-DNP (IO+ M) 02
0 0 90 97 0 70 80 0 98
Table IV shows the effect of the three inhibitors on CY402 dark fixation under hydrogen, air, and under a mixture of hydrogen and oxygen. Menadione inhibits the fixation under air and under hydrogen plus oxygen; this is consistent with an inhibition of respiration as reported elsewhere
184
GINGRAS,
GOLDSBY
(14-17). Both the air and the oxyhydrogen fixations are more sensitive to menadione than photoreduction. Likewise, the hydrogen and the oxyhydrogen fixations are more sensitive to dinitrophenol than photoreduction. CMU does not appreciably affect any of the dark fixations. Since these observations have been made and this paper written, a report by Gaffron has appeared (18) describing a mutant of Scenedesmus which can grow heterotrophitally or by photoreduction without evolution of oxygen. This is insensitive to DCMU as well. The conclusions reached by Gaffron are very similar to those described here. DISCUSSION
The data are consistent with the proposal that there is a reduction of a coenzyme by molecular hydrogen. When oxygen is added in the dark, some of the hydrogen is combined with oxygen to yield water and highenergy phosphates; the reduced coenzyme and the high-energy phosphate can be used to reduce CO2 via the carbon reduction cycle. This oxyhydrogen reaction exhibits respiration-like sensitivity to menadione and to 2,4-dinitrophenol. Carbon dioxide fixation under hydrogen at the light intensity used here is from two to four times faster than under air. It is clear from this that molecular hydrogen contributes electrons and probably ATP as well for the reduction of carbon compounds. At the concentrations used, the three inhibitors block photosynthetic fixation almost completely, but photoreduction even in the presence of these inhibitors
AND
CALVIN
still fixes more CY402 than does photosynthesis at these low light intensities. REFERENCES 1. GOLDSBY, R. A., Thesis, Univ. of California, 1961. UCRL 9806. 2. FISHER, H. F., KRASNA, A. E., AND RITTENBERG, D., J. Biol. Chem. 209, 569 (1954). 3. BADIN, E., AND CALVIN, M., J. grn. Chem. Sot. 72, 5266 (1950). 4. STOPPANI, A. 0. M., FULLER, R. C., AND CALVIN, M., J. Bacte?ioZ. 69, 491 (1955). 5. MILHAUD, G., AUBERT, J. P., AND MILLET, J., Corn@. rend. 243, 102 (1956). 6. BASSHAM, J. A., AND CALVIN, M., “The Path of Carbon in Photosynthesis,” pp. 2833. Prentice Hall, Englewood Cliffs, N. J., 1957. 7. MYERS, J., Plant Physiol. 22, 590 (1947). 8. GAFFRON, H. A., J. Gen. Physiol. 26,241(1942). 9. BASSHAM, J. A., BENSON, A. A., KAY, L. D., HARRIS, A. Z., WILSON, A. T., AND CALVIN, M., J. Am. Chem. Sot. 76, 1760 (1954). 10. WIAME, J. M., AND PIERARD, A., h’ature 176, 1073 (1955). 11. BISHOP, N. I., Biochim. et Biophys. Acta 27, 205 (1958) . 12. FARKAS, A., FARKAS, L., AND YUDKIN, Proc. Roy. Sot. (London) B155, 372 (1934). 13. HOBERMAN, H. D., AND RITTENBERG, D., J. Biol. Chem. 147, 211 (1943). 14. GAFFRON, H. A., J. Gen. Physiol. 28, 259 (1944). 15. ARNON, D. I., Nature 184, 10 (1959). 66, 16. HORWITZ, L., Arch. Biochem. Biophys. 23 (1957). 17. HORWITZ, L., AND ALLEN, F. L., Arch. Biothem. Biophys. 66, 45 (1957). in Biochemistry” 18. GAFFRON, H., in “Horizons (B. Pullman and M. Kasha, eds.), p. 78. Academic Press, New York, 1962.