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Photochemical reduction of oxygen in chloroplast preparations. II. Mechanisms of the reaction with oxygen
Photochemical Reduction of Oxygen in Chloroplast Preparations. II. Mechanisms of the Reaction with Oxygen Norman From the Biochemistry
Good’ and Robert Hill Cambridge University,
Depaf-tment, Received
November
Cambridge, England
18, 1954
INTRODUCTION Mehler’s investigation (l-3) showed that oxygen could play a dual role in chloroplast reactions, serving simultaneously as hydrogen acceptor in one part of the system and as ultimate product in another part of the system. The experiments described in the earlier paper in this series (4) confirmed Mehler’s observations. Oxygen was reduced to hydrogen peroxide by illuminated chloroplasts with a concurrent production of half as much oxygen, presumably from water: Hz0
-
2H
2H + 02 Over-all
Hz0
+
302
+
+O,
Hz02 -+
H202
Thus the net result of the reduction of one mole of oxygen is the uptake of half a mole of oxygen, while the result of the reduction of an equivalent amount of another hydrogen acceptor is the production of half a mole of oxygen. For this reason, given the same photolytic activity, the production of oxygen with other oxidants should be equal to the consumption of oxygen when oxygen acts as the oxidant. However, the data presented by Mehler indicated that oxygen reduction was often much slower than the reduction of other chloroplast reagents such as p-benzoquinone or ferricyanide. Moreover, it was shown in the previous communication that the direct reduction of oxygen by chloroplasts does not ordinarily occur to a significant extent in viva. It therefore seemedreasonable to supposethat the reaction with oxygen might be indirect, inL Present address: London, Ontario.
Science
Service
Laboratory,
355
University
Sub.
Post
Office,
356
NORMAN
COOD AND ROBERT HILL
volving intermediate hydrogen transport systems not necessary for the reduction of other chloroplast reagents and not available to the chloroplast in situ. The present paper provides additional reasons for accepting this interpretation of oxygen reduction. It has now been demonstrated that many chloroplast preparations reduce oxygen very slowly if at all. However, soluble, heat-stable factors from the leaves of some plants and a variety of known substances stimulate the light-dependent oxygen uptake. Most, if not all, of the catalysts fall into one of the two classes: those constituting autoxidizable oxidation-reduction systems and those reacting in the preparations to produce such systems. Since it is likely that these substances owe their catalytic activity to the fact that they are reduced in the chloroplast reaction and reoxidized by molecular oxygen, it is here suggested that the reduction of oxygen may be used to measure the effectiveness with which the catalysts function as chloroplast reagents. MATERIALS
AND METHODS
Chloroplast preparations were obtained from the leaves of Spinacea oleracea (spinach), Beta vulgaris (chard), Stellaria media (chickweed), Pisum sativum (pea), and Chenopodium bonus-henricus (Good King Henry). Leaves from these plants were ground in a chilled mortar with buffer containing from 6 to 8 g. glucose or in a Waring blendor with buffer and ice. The homogenates were filtered through glass wool or cheesecloth, and the chloroplasts were separated by centrifugation either at 2300 X g or at 20,000 X g. In the former case the chloroplasts were largely intact, bgt, in the latter case the material used contained many fragmented plastids and other particulate matter. Phosphate, tris(hydroxymethyl)aminomethanemaleic, tris-chloride, and tris-acetate buffers were used as suspending media at pH’s bstween 6.5 and 7.4. A11 these buffers contained 0.05 M potassium chloride. Oxygen reduction was measured as oxygen uptake in Warburg manometers. The hydrogen peroxide formed in the reaction was “trapped” by using 2% ethanol and excess catalase. The catalase was prepared from horse liver by the method of Keilin and Hartree (5) and was used at the rate of 0.025 mg. hematin per vessel. The reductions of other chloroplast reagents were measured as oxygen production. A standard constant-temperature bath was lined with mirrors, a cooling coil was added, and light was supplied from above by three 40-w. fluorescent lamps and four 60-w. incandescent tungsten lamps. Reaction temperatures were between 16 and 21°C. Each vessel had alkali in its center well, and the gas phase consisted of air. RESULTS
1. The Rate of Reduction of Oxygen Compared to the Rate of Reduction of Quinone or Fe&cyanide (Table I)
Chloroplast preparations differed in their ability to reduce conventional chloroplast reagents such as quinone and ferricyanide. However,
TABLE The Rate
of Oxygen
Reduction
I
Compared with and Fe&cyanide
the Rote
of Reduction
of Q&none
Oxygen reduction was measured as oxygen uptake in the presence of catalase and ethanol. Fcrricyanide and quinone reduction were measured as oxygen production. The chloroplast suspensions contained from 0.1 to 0.3 mg. chlorophyll per manometer vessel. The suspension media consisted of various buffers from nH 6.5 to 7.4. Temperatures were from 16 to 21°C.
E3iF. 1.
Chard
2.
Chard
3.
Chard
4.
Spinach
5.
Spinach
Plant
Intact
I .2
1.8
-
Intact
1.2
1.8
-
Fragmented Fragmcnted Intact
1.8
1.8
1.4
1.6
-
1.4
2.8
-
1.8
2.2
-
0.1 1.6
-. -
1.8 4.0
Mortar Mortar Mortar Mortar
Fragmented Intact Leaf Homogenate Intact Intact Intact Intact
1.2 0 1 0.0 0.0
-
3.6 2.4 1.2 2.8
Mortar
Intact
0.0
-
1.2
Mortar
Intact
0.0
-
2.4
Mortar Waring blendor Mortar
Intact Intact
0.0 0.1
-
1.8 1.2
0.1
-
3.0
Mortar
Fragmented Intact
0.0
-
2.4
Mortar Mortar
Intact Intact
0.0 0.1
2.2 5.0
2.6
of leaves
6b. Spinach 7. Chickweed 8a. Chard
Mortar Mortar
8b. 9. 10. 11. 12. 13. 14. 15.
Chard Chickweed Pea Chenopodium bonus-hen&u Chenopodium bonus-hen&x Chenopodium bonus-henricu Pea Pea
16a. Chenopodium bonus-henricu 16b. Chenopodium bonus-henricu 17. Chard (young) 18. Chard (young)
-
1
irinding
Waring blendor Waring blendor Waring blendor Waring blendor Waring blendor Waring blendor Mortar
6s. Spinach
E
Description Chloroplasts
Intact
357
of
F deduction cIf Oxygen
kduction I Quinone
Reduction of Ferricyanide with ferric o&date)
P 1. Oa/min.
1. 02/&n.
Ll. Ol/rnb.
1 .o
2.4
-
358
NORMAN
GOOD
AND
ROBERT
HILL
they varied a great deal more in their ability to reduce oxygen. Almost half of the preparations did not reduce oxygen to an extent which could be detected manometrically. The rate of oxygen reduction depended, primarily, on the species of the plant and on the physiological condition of the leaves from which the chloroplast had been obtained. Most preparations from spinach and chard were particularly active in this respect, although leaves from very young chard plants yielded preparations unable to reduce oxygen. The reactivity toward oxygen also depended to a smaller extent on the method of preparation of the chloroplast suspensions. The results of a number of experiments are collected in Table I. The rate of oxygen reduction never exceeded the rate of reduction of ferricyanide or quinone, but these activities were not otherwise correlated. 2. The Catalysis of Oxygen Reduction from the Leaves of Chenopodium
by Soluble, Heat-Stable Factors bonus-henricus (Fig. 1)
A thick homogenate was made by grinding leaves of Chenopodium in a minimum of phosphate buffer. It was filtered through glass wool and centrifuged for 10 min. at 2000 X g. The chloroplasts were resuspended in the same buffer, and the supernatant was centrifuged again for 30 min. at 20,000 X g. The second supernatant was a clear yellow solution almost free of chlorophyll. Part of this solution was heated to 100°C. for about 1 min. The suspended chloroplasts (0.28 mg. chlorophyll per vessel) were illuminated in the presence of catalase and alcohol, with or without the addition of the leaf extract, or with potassium ferricyanide. Oxygen was reduced slowly in the absence of leaf extract but rapidly in its presence. The catalytic factors from the leaves were not destroyed when the extract was heated. It is noteworthy, however, that the oxygen consumption only attained maximum velocity after some minutes of illumination. bonus-henricus
3. The Stimulation of Oxygen Reduction by Treatment Preparations with Quinone (Figs. 2-4)
of Chloroplasf
Mehler showed that spinach chloroplasts reduced oxygen more rapidly after they had reduced quinone (2). Our preparations from spinach and Chenopodium bonus-henricus did not show this effect (Fig. 3, vessels 1 and 2). However, the quinone stimulation was very marked with chard preparations (Fig. 2, vessels 1 and 2). Moreover, a chlorophyll-free portion of the quinone-treated chard suspensions (obtained by centrifu-
PHOTOCHEMICAL
OXYGEN
10
0
REDUCTION.
359
II
20 MINUTES
FIG. 1. Chenopodium
hen&us and
The effect of leaf extracts on the reduction of oxygen by chloroplasts of bonus-henricus. Each vessel contained chloroplasts from C. bonus(0.28 mg. chlorophyll) suspendedinphosphatebuffer, pH 7.2, withcatalase
ethanol.
Vessel
1 contained
in
addition
13.0
mg.
potassium
ferricganide;
vessel 2, no addenda; vessel 3, 1.5 ml. leaf extract; and vessel 4, 1.5 ml. boiled leaf extract. Total volume of liquid in each vessel, 3.0 ml. Temperature, 20°C. gation of the reaction mixture from vessel 1, Fig. 2) was effective in stimulating the reduction of oxygen by chloroplasts from Chenopodium bonus-henricus (Fig. 3, vessel 4). Since it was also possible to show that, systems catalyzing the reduction of oxygen by ascorbic acid are sometimes formed when quinone-treated chloroplasts are illuminated (Fig. 4), it seemslikely that autoxidizable substances capable of oxidizing either the chloroplast system or ascorbic acid are photochemically produced
from quinone derivatives and substancespresent in chard leaves.
4. The Stimulation of Oxygen Reduction by Nanganous Ions (Figs. 2 and 3) Mehler also reported that manganous ions accelerated the reduction of oxygen by illuminated chloroplasts. This observation has been confirmed and extended to preparations from chard and Chenopodium
360
NORMAN
GOOD
AND
IO
ROBERT
20
HILL
30
MINUTES
FIG. 2. The effect of quinone and manganous chloride on the reduction of oxygen by illuminated chard chloroplasts. Each vessel contained chloroplasts from young chard plants suspended in tris(hydroxymethyl)aminomethane-maleic buffer, pH 6.7, with catalase and ethanol. Vessel 1 contained in addition 0.5 mg. quinone; vessel 2, no addenda; and vessel 3, manganous chloride (6.0 X lo-4 M). Total volume of liquid in each vessel, 3.0 ml. Temperature, 20°C.
bonus-hen&us (Fig. 2, vessel 3; and Fig. 3, vessel 3). The increased oxygen uptake must have been due to hydrogen peroxide formation since it did not occur when the peroxide-trapping catalase and ethanol were omitted (Fig. 3, vessel 5). The manganous-ion effect has not been further investigated. 5. The Stimulation of Oxygen Reduction by Chloroplast ReagentsWhose ReducedForms are A&oxidizable (Table II) A large number of substances readily undergo reversible reduction when treated with appropriate reagents. The reduced forms of many of these react spontaneously with molecular oxygen to give hydrogen peroxide and the reoxidized substances. Such autoxidizable materials should catalyze the reduction of oxygen by chloroplasts if they function as chloroplast reagents, and if they fail to act catalytically it is probable
PHOTOCHEMICAL
I
1 IO
OXYGEN
I 20 MINUTES
REDUCTION.
361
II
1 30
I 40
FIG. 3. The effect of quinone and manganous chloride on the reduction of oxygen by chloroplasts of Chenopodium bonus-henricus. Each vessel contained chloroplasts of C. bonus-henricus suspended in tris-maleic buffer, pH 7.2. All the vessels except vessel 5 contained catalase and ethanol. Vessel 1 contained in addition 0.5 mg. quinone; vessel 2, no addenda; vessel 3, manganous chloride (6.0 X lo-* M); vessel 4, 1.0 ml. of a chlorophyll-free portion of the reaction mixture from vessel 1, Fig. 2; and vessel 5, manganous chloride (6.0 X IO-’ M) but no catalase and ethanol.
that they are not reduced by the chloroplasts. Thus Horowitz (6), using the isotope method described in the previous communication (4), found that benzylviologen caused a fivefold increase in t’he rate of reduction of oxygen by spinach chloroplasts. The effect of various other additions have been investigated, and the results of the survey are presented in Table II. Many substances stimulated oxygen uptake, some at very low concentrations. With most chloroplasts, flavines saturated the syst’em (caused oxygen to be reduced at the same rate as quinone or ferricyanide) when about 1O-6 M, but, surprisingly, the chloroplast,s of Chenopodium bonus-henricus scarcely responded to riboflavine or its derivatives. Ant,hocyanins, janus green, and litmus were all effective catalysts. Methylviologen and benzylviologen were less act,ive and did not saturate the system at any concentration. Methylene blue, pyocyanin, and antho xanthines were inactive. The pyridine nucleotides (DPK and TPN) are not themselves autoxidizable nor did they stimulate the reduction of oxygen by the chloroplast preparations. However, DPK was st,ill inactive
362
NORMAN
GOOD
AND
ROBERT
HILL
MINUTES
FIG.
4. The production of a catalyst of ascorbic acid oxidation by the illumination of quinone-treated chard chloroplasts. The vessel contained chard chloroplasts (0.2 mg. chlorophyll) suspended in phosphate buffer, pH 6.5. Quinone (0.5 mg.) and then ascorbic acid (3.5 mg.) were added in the dark. Illumination by fluorescent lights, about 1.50 ft.-candles. Temperature, 20.9”C.
in the presence of a system which mediates its oxidation oxygen (diaphorase and methylene blue).
by molecular
DISCUSSION It has been found that the reduction of molecular oxygen by chloroplast preparations is not simply dependent upon the activity of the preparations as measured by oxygen output with other hydrogen acceptors. Photochemically active preparations with no detectable ability to reduce oxygen have been obtained. When certain additions were made to these preparations, it was found that the rate of oxygen reduction became equal to the rate of reduction of conventional chloroplast reagents. In some cases it was demonstrated that the substance responsible for the increased capacity to reduce oxygen was itself reduced by illuminated chloroplasts and reoxidized in air. It seems probable, therefore, that most of the reduction of oxygen by chloroplasts is indirect in the sense that intermediate hydrogen transport systems are required, systems which are not required for the reduction of chloroplast reagents such as quinone or ferricyanide. Many plant products constitute appropriate autoxidizable oxidation-reduction systems, and it has been shown that some of these can couple the chloroplast reaction with oxygen re-
PHOTOCHEMICAL
The Egects of Various
OXYGEN
REDUCTIOK.
II
TABLE II Substances on the Rate of Reduction by Illuminated Chloroplasts
of
Oxygen
=
Active
catalysts
of oxygen
reduction
Ribollavine Riboflavine-5-phosphate @MN) Triacetylriboflavine-5-phosphate Flavine-adenine-dinucleotide @‘AD)
Anthocyaninsa (delphinidin) (cyanin) Janus greena Litmus Manganous ion p-Benzoquinoneh only)
Inefficient oxygen
catalysts reduction
of
Inactive
Benzyl viologen Methyl viologen
Anthoxanthines (quercetin) (apigenin) Thionine, hiethylene
blue
Diphosphopyridine-nucleotide (DPN) DPN + diaphorase -+ methylene blue Triphosphopyridine-nucleotide (TPN) Pyocyanin p-Benzoquinone Hydroquinone Ferric ion Cupric ion Flavines (with C. bonus-henricus only)
(with chard
I L
a Unstable; catalytic substance not definitely known. * Associated with the photoproduction of oxidation catalysts?
duction. However, the endogenous systems mediating the reaction of chloroplasts with molecular oxygen have not been identified. The lag in oxygen uptake which is often encountered at the beginning of a period of illumination (see most of the figures) suggests that some of these “endogenous” intermediates may be produced by photochemical reactions. Apparently an enhanced photoproduction of oxidation catalysts occurs when quinone-treated chard chloroplasts are illuminated. Although much of the ability of chloroplasts to reduce oxygen is assoiiated with the presence of soluble autoxidizable substances, some preparations continue to reduce oxygen slowly even after repeated washings. Moreover, treatments which have not yet been related to t,he formation of autoxidizable hydrogen transport systems, for instance the addition of manganous ions, stimulate the reduction of oxygen. It is
364
NORMAN
GOOD
AND
ROBERT
HILL
conceivable, therefore, that certain conditions may bring about the “exposure” of reactive sites not ordinarily available to oxygen. Since it is difficult to distinguish between true catalysis and such noncatalytic activations, the possibility that oxygen may occassionally react directly with the photolysis reduction product [H] cannot be excluded. However, there is no unequivocal evidence that oxygen is thus directly reduced photochemically in living plants (4). Therefore we might suppose that the frequently observed inactivity of the isolated chloroplasts toward molecular oxygen represents the state of the photochemical system as it exists in the cell. But if during the process of isolation the chloroplasts lost a component which normally prevents the “shortcircuiting” of the reducing system by moIecular oxygen, the resulting preparation might reduce oxygen directly. Analogous reductions of oxygen by light-activated pigment systems are well known, and the discovery of such reactions in chloroplasts could make a considerable contribution to our knowledge of the photochemistry of the chloroplast reaction. The reduction of autoxidizable chloroplast reagents may be conveniently followed by utilizing Mehler’s method of measuring oxygen reduction. The great disadvantage of studying chloroplast reactions by this indirect method is the element of ambiguity introduced by the fact that the proposed mechanisms may sometimes by questioned; it is well to recognize that some addenda, for instance quinone and manganous ions, stimulate oxygen reduction in an unknown manner. However, conclusions derived from observations of oxygen reduction have been confirmed in several instances by experiments in which the reduction of the supposed chloroplast reagent was measured in the absence of oxygen. Under sufficiently anaerobic conditions, benzylviologen and FAD were reduced, the latter almost completely, while pyocyanin was not reduced at all. Details of these experiments will be published in another paper. Under the same conditions methylene blue, which does not enhance the Mehler reaction, was also slowly but completely reduced. This apparent discrepancy with regard to methylene blue perhaps reflects the great difference in the sensitivities of the optical and manometric techniques employed, since both the reduction and the reoxidation of the dye were slow. Also, feeble anaerobic reductions are relatively difficult to interpret because they may be indirect. The failure of DPN to stimulate a Mehler reaction, even in the presence of a specific oxidase, may mean that this substance was also slowly (and perhaps indirectly) reduced. Thus
PHOTOCHEMICAL
OXYGEN
REDUCTION.
II
365
the participation of the pyridine nucleotides in chloroplast reactions, reported by Vishniac and Ochoa and by others (7, 8) is probably less immediate than the participation of the flavine nucleotides. Since the flavines are very active in mediating oxygen reduction and are rapidly reduced in VCKUOby illuminated chloroplasts, they certainly may be added to the list of biologically important substances which have been found to react with the isolated photochemical system of photosynthetic organisms. However, the authors do not wish to imply that the observed reductions of the flavine nucleotides in vitro constitute evidence that similar reactions in viva play a part in the photosynthetic process. The fact that potentially active chloroplast preparations from Chenopodium bonus-henricus apparently do not react with either class of coenzyme suggests that the known coenzymes may not be directly concerned in normal chloroplast reductions. ACKNOWLEDGMENTS The authors are grateful to Dr. Allan Brown for his helpful criticism of this paper. The riboflavine derivatives used in these studies were kindly supplied by the late Dr. S. M. H. Christie. This research was supported by the Agricultural Research Council of Great Brit,ain.
SUMMARY
1. The reduction of oxygen by illuminated chloroplast preparations represents a reaction which corresponds t’o t,he reduction of other oxidants such as quinone but is probably dependent on additional hydrogen transport systems. Chloroplast preparations which reduce oxygen very slowly, if at all, have been obtained from the leaves of all five of t,he plant speciestested. 2. Many substances whose reduced forms are reoxidized by molecular oxygen stimulate the reduction of oxygen by chloroplasts. Some of t,hese substancesare normal components of leaves. Unidentified, soluble, heatstable factors from leaves of Chenopodiumbonus-henricuscatalyze oxygen reduction. 3. Usually the reduction of oxygen associated with endogenous oxidation catalysts only begins after several minutes of illumination. Moreover, the illumination of quinone-treated chard chloroplasts causesthe formation of substances capable of catalyzing the oxidation of either ascorbic acid or the chloroplast system by molecular oxygen. It is therefore suggested that much of the ability of chloroplasts to reduce oxygen depends on the photoproduction of autoxidizable hydrogen acceptors.
366
NORMAN
GOOD AND ROBERT
HILL
4. If the reduction of oxygen measures the reduction of autoxidizable chloroplast reagents, it may be concluded that the flavines are rapidly reduced by most chloroplasts. Reduction of diphosphopyridine nucleotide (DPN) could not be detected by this method although diaphorase and methylene blue were added in order to catalyze the reaction with molecular oxygen. REFERENCES 1. MEHLER, A. H., Arch. Biochem. and Biophys. 33, 65 (1951). 2. MEHLER, A. H., Arch. Biochem. and Biophys. 34, 339 (1951). 3. MEHLER, A. H., AND BROWN, A. H., Arch. Biochem. and Biophys. 38,365 (1952). 4. BROWN, A. H., AND GOOD, N., Arch. Biochem. and Biophys. 67,340 (1955). 5. KEILIN, D., AND HARTREE, E. F., Biochem. J. 39, 148 (1945). 6. HOROWITZ, L., Dissertation, University of Minnesota, 1952. 7. VISHNIAC, W., AND OCHOA, S., J. Biol. Chem. 196, 75 (1952). 8. ARNON, D. I., Nature 187, 1008 (1951).
Good’ and Robert Hill Cambridge University,
Depaf-tment, Received
November
Cambridge, England
18, 1954
INTRODUCTION Mehler’s investigation (l-3) showed that oxygen could play a dual role in chloroplast reactions, serving simultaneously as hydrogen acceptor in one part of the system and as ultimate product in another part of the system. The experiments described in the earlier paper in this series (4) confirmed Mehler’s observations. Oxygen was reduced to hydrogen peroxide by illuminated chloroplasts with a concurrent production of half as much oxygen, presumably from water: Hz0
-
2H
2H + 02 Over-all
Hz0
+
302
+
+O,
Hz02 -+
H202
Thus the net result of the reduction of one mole of oxygen is the uptake of half a mole of oxygen, while the result of the reduction of an equivalent amount of another hydrogen acceptor is the production of half a mole of oxygen. For this reason, given the same photolytic activity, the production of oxygen with other oxidants should be equal to the consumption of oxygen when oxygen acts as the oxidant. However, the data presented by Mehler indicated that oxygen reduction was often much slower than the reduction of other chloroplast reagents such as p-benzoquinone or ferricyanide. Moreover, it was shown in the previous communication that the direct reduction of oxygen by chloroplasts does not ordinarily occur to a significant extent in viva. It therefore seemedreasonable to supposethat the reaction with oxygen might be indirect, inL Present address: London, Ontario.
Science
Service
Laboratory,
355
University
Sub.
Post
Office,
356
NORMAN
COOD AND ROBERT HILL
volving intermediate hydrogen transport systems not necessary for the reduction of other chloroplast reagents and not available to the chloroplast in situ. The present paper provides additional reasons for accepting this interpretation of oxygen reduction. It has now been demonstrated that many chloroplast preparations reduce oxygen very slowly if at all. However, soluble, heat-stable factors from the leaves of some plants and a variety of known substances stimulate the light-dependent oxygen uptake. Most, if not all, of the catalysts fall into one of the two classes: those constituting autoxidizable oxidation-reduction systems and those reacting in the preparations to produce such systems. Since it is likely that these substances owe their catalytic activity to the fact that they are reduced in the chloroplast reaction and reoxidized by molecular oxygen, it is here suggested that the reduction of oxygen may be used to measure the effectiveness with which the catalysts function as chloroplast reagents. MATERIALS
AND METHODS
Chloroplast preparations were obtained from the leaves of Spinacea oleracea (spinach), Beta vulgaris (chard), Stellaria media (chickweed), Pisum sativum (pea), and Chenopodium bonus-henricus (Good King Henry). Leaves from these plants were ground in a chilled mortar with buffer containing from 6 to 8 g. glucose or in a Waring blendor with buffer and ice. The homogenates were filtered through glass wool or cheesecloth, and the chloroplasts were separated by centrifugation either at 2300 X g or at 20,000 X g. In the former case the chloroplasts were largely intact, bgt, in the latter case the material used contained many fragmented plastids and other particulate matter. Phosphate, tris(hydroxymethyl)aminomethanemaleic, tris-chloride, and tris-acetate buffers were used as suspending media at pH’s bstween 6.5 and 7.4. A11 these buffers contained 0.05 M potassium chloride. Oxygen reduction was measured as oxygen uptake in Warburg manometers. The hydrogen peroxide formed in the reaction was “trapped” by using 2% ethanol and excess catalase. The catalase was prepared from horse liver by the method of Keilin and Hartree (5) and was used at the rate of 0.025 mg. hematin per vessel. The reductions of other chloroplast reagents were measured as oxygen production. A standard constant-temperature bath was lined with mirrors, a cooling coil was added, and light was supplied from above by three 40-w. fluorescent lamps and four 60-w. incandescent tungsten lamps. Reaction temperatures were between 16 and 21°C. Each vessel had alkali in its center well, and the gas phase consisted of air. RESULTS
1. The Rate of Reduction of Oxygen Compared to the Rate of Reduction of Quinone or Fe&cyanide (Table I)
Chloroplast preparations differed in their ability to reduce conventional chloroplast reagents such as quinone and ferricyanide. However,
TABLE The Rate
of Oxygen
Reduction
I
Compared with and Fe&cyanide
the Rote
of Reduction
of Q&none
Oxygen reduction was measured as oxygen uptake in the presence of catalase and ethanol. Fcrricyanide and quinone reduction were measured as oxygen production. The chloroplast suspensions contained from 0.1 to 0.3 mg. chlorophyll per manometer vessel. The suspension media consisted of various buffers from nH 6.5 to 7.4. Temperatures were from 16 to 21°C.
E3iF. 1.
Chard
2.
Chard
3.
Chard
4.
Spinach
5.
Spinach
Plant
Intact
I .2
1.8
-
Intact
1.2
1.8
-
Fragmented Fragmcnted Intact
1.8
1.8
1.4
1.6
-
1.4
2.8
-
1.8
2.2
-
0.1 1.6
-. -
1.8 4.0
Mortar Mortar Mortar Mortar
Fragmented Intact Leaf Homogenate Intact Intact Intact Intact
1.2 0 1 0.0 0.0
-
3.6 2.4 1.2 2.8
Mortar
Intact
0.0
-
1.2
Mortar
Intact
0.0
-
2.4
Mortar Waring blendor Mortar
Intact Intact
0.0 0.1
-
1.8 1.2
0.1
-
3.0
Mortar
Fragmented Intact
0.0
-
2.4
Mortar Mortar
Intact Intact
0.0 0.1
2.2 5.0
2.6
of leaves
6b. Spinach 7. Chickweed 8a. Chard
Mortar Mortar
8b. 9. 10. 11. 12. 13. 14. 15.
Chard Chickweed Pea Chenopodium bonus-hen&u Chenopodium bonus-hen&x Chenopodium bonus-henricu Pea Pea
16a. Chenopodium bonus-henricu 16b. Chenopodium bonus-henricu 17. Chard (young) 18. Chard (young)
-
1
irinding
Waring blendor Waring blendor Waring blendor Waring blendor Waring blendor Waring blendor Mortar
6s. Spinach
E
Description Chloroplasts
Intact
357
of
F deduction cIf Oxygen
kduction I Quinone
Reduction of Ferricyanide with ferric o&date)
P 1. Oa/min.
1. 02/&n.
Ll. Ol/rnb.
1 .o
2.4
-
358
NORMAN
GOOD
AND
ROBERT
HILL
they varied a great deal more in their ability to reduce oxygen. Almost half of the preparations did not reduce oxygen to an extent which could be detected manometrically. The rate of oxygen reduction depended, primarily, on the species of the plant and on the physiological condition of the leaves from which the chloroplast had been obtained. Most preparations from spinach and chard were particularly active in this respect, although leaves from very young chard plants yielded preparations unable to reduce oxygen. The reactivity toward oxygen also depended to a smaller extent on the method of preparation of the chloroplast suspensions. The results of a number of experiments are collected in Table I. The rate of oxygen reduction never exceeded the rate of reduction of ferricyanide or quinone, but these activities were not otherwise correlated. 2. The Catalysis of Oxygen Reduction from the Leaves of Chenopodium
by Soluble, Heat-Stable Factors bonus-henricus (Fig. 1)
A thick homogenate was made by grinding leaves of Chenopodium in a minimum of phosphate buffer. It was filtered through glass wool and centrifuged for 10 min. at 2000 X g. The chloroplasts were resuspended in the same buffer, and the supernatant was centrifuged again for 30 min. at 20,000 X g. The second supernatant was a clear yellow solution almost free of chlorophyll. Part of this solution was heated to 100°C. for about 1 min. The suspended chloroplasts (0.28 mg. chlorophyll per vessel) were illuminated in the presence of catalase and alcohol, with or without the addition of the leaf extract, or with potassium ferricyanide. Oxygen was reduced slowly in the absence of leaf extract but rapidly in its presence. The catalytic factors from the leaves were not destroyed when the extract was heated. It is noteworthy, however, that the oxygen consumption only attained maximum velocity after some minutes of illumination. bonus-henricus
3. The Stimulation of Oxygen Reduction by Treatment Preparations with Quinone (Figs. 2-4)
of Chloroplasf
Mehler showed that spinach chloroplasts reduced oxygen more rapidly after they had reduced quinone (2). Our preparations from spinach and Chenopodium bonus-henricus did not show this effect (Fig. 3, vessels 1 and 2). However, the quinone stimulation was very marked with chard preparations (Fig. 2, vessels 1 and 2). Moreover, a chlorophyll-free portion of the quinone-treated chard suspensions (obtained by centrifu-
PHOTOCHEMICAL
OXYGEN
10
0
REDUCTION.
359
II
20 MINUTES
FIG. 1. Chenopodium
hen&us and
The effect of leaf extracts on the reduction of oxygen by chloroplasts of bonus-henricus. Each vessel contained chloroplasts from C. bonus(0.28 mg. chlorophyll) suspendedinphosphatebuffer, pH 7.2, withcatalase
ethanol.
Vessel
1 contained
in
addition
13.0
mg.
potassium
ferricganide;
vessel 2, no addenda; vessel 3, 1.5 ml. leaf extract; and vessel 4, 1.5 ml. boiled leaf extract. Total volume of liquid in each vessel, 3.0 ml. Temperature, 20°C. gation of the reaction mixture from vessel 1, Fig. 2) was effective in stimulating the reduction of oxygen by chloroplasts from Chenopodium bonus-henricus (Fig. 3, vessel 4). Since it was also possible to show that, systems catalyzing the reduction of oxygen by ascorbic acid are sometimes formed when quinone-treated chloroplasts are illuminated (Fig. 4), it seemslikely that autoxidizable substances capable of oxidizing either the chloroplast system or ascorbic acid are photochemically produced
from quinone derivatives and substancespresent in chard leaves.
4. The Stimulation of Oxygen Reduction by Nanganous Ions (Figs. 2 and 3) Mehler also reported that manganous ions accelerated the reduction of oxygen by illuminated chloroplasts. This observation has been confirmed and extended to preparations from chard and Chenopodium
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FIG. 2. The effect of quinone and manganous chloride on the reduction of oxygen by illuminated chard chloroplasts. Each vessel contained chloroplasts from young chard plants suspended in tris(hydroxymethyl)aminomethane-maleic buffer, pH 6.7, with catalase and ethanol. Vessel 1 contained in addition 0.5 mg. quinone; vessel 2, no addenda; and vessel 3, manganous chloride (6.0 X lo-4 M). Total volume of liquid in each vessel, 3.0 ml. Temperature, 20°C.
bonus-hen&us (Fig. 2, vessel 3; and Fig. 3, vessel 3). The increased oxygen uptake must have been due to hydrogen peroxide formation since it did not occur when the peroxide-trapping catalase and ethanol were omitted (Fig. 3, vessel 5). The manganous-ion effect has not been further investigated. 5. The Stimulation of Oxygen Reduction by Chloroplast ReagentsWhose ReducedForms are A&oxidizable (Table II) A large number of substances readily undergo reversible reduction when treated with appropriate reagents. The reduced forms of many of these react spontaneously with molecular oxygen to give hydrogen peroxide and the reoxidized substances. Such autoxidizable materials should catalyze the reduction of oxygen by chloroplasts if they function as chloroplast reagents, and if they fail to act catalytically it is probable
PHOTOCHEMICAL
I
1 IO
OXYGEN
I 20 MINUTES
REDUCTION.
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1 30
I 40
FIG. 3. The effect of quinone and manganous chloride on the reduction of oxygen by chloroplasts of Chenopodium bonus-henricus. Each vessel contained chloroplasts of C. bonus-henricus suspended in tris-maleic buffer, pH 7.2. All the vessels except vessel 5 contained catalase and ethanol. Vessel 1 contained in addition 0.5 mg. quinone; vessel 2, no addenda; vessel 3, manganous chloride (6.0 X lo-* M); vessel 4, 1.0 ml. of a chlorophyll-free portion of the reaction mixture from vessel 1, Fig. 2; and vessel 5, manganous chloride (6.0 X IO-’ M) but no catalase and ethanol.
that they are not reduced by the chloroplasts. Thus Horowitz (6), using the isotope method described in the previous communication (4), found that benzylviologen caused a fivefold increase in t’he rate of reduction of oxygen by spinach chloroplasts. The effect of various other additions have been investigated, and the results of the survey are presented in Table II. Many substances stimulated oxygen uptake, some at very low concentrations. With most chloroplasts, flavines saturated the syst’em (caused oxygen to be reduced at the same rate as quinone or ferricyanide) when about 1O-6 M, but, surprisingly, the chloroplast,s of Chenopodium bonus-henricus scarcely responded to riboflavine or its derivatives. Ant,hocyanins, janus green, and litmus were all effective catalysts. Methylviologen and benzylviologen were less act,ive and did not saturate the system at any concentration. Methylene blue, pyocyanin, and antho xanthines were inactive. The pyridine nucleotides (DPK and TPN) are not themselves autoxidizable nor did they stimulate the reduction of oxygen by the chloroplast preparations. However, DPK was st,ill inactive
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FIG.
4. The production of a catalyst of ascorbic acid oxidation by the illumination of quinone-treated chard chloroplasts. The vessel contained chard chloroplasts (0.2 mg. chlorophyll) suspended in phosphate buffer, pH 6.5. Quinone (0.5 mg.) and then ascorbic acid (3.5 mg.) were added in the dark. Illumination by fluorescent lights, about 1.50 ft.-candles. Temperature, 20.9”C.
in the presence of a system which mediates its oxidation oxygen (diaphorase and methylene blue).
by molecular
DISCUSSION It has been found that the reduction of molecular oxygen by chloroplast preparations is not simply dependent upon the activity of the preparations as measured by oxygen output with other hydrogen acceptors. Photochemically active preparations with no detectable ability to reduce oxygen have been obtained. When certain additions were made to these preparations, it was found that the rate of oxygen reduction became equal to the rate of reduction of conventional chloroplast reagents. In some cases it was demonstrated that the substance responsible for the increased capacity to reduce oxygen was itself reduced by illuminated chloroplasts and reoxidized in air. It seems probable, therefore, that most of the reduction of oxygen by chloroplasts is indirect in the sense that intermediate hydrogen transport systems are required, systems which are not required for the reduction of chloroplast reagents such as quinone or ferricyanide. Many plant products constitute appropriate autoxidizable oxidation-reduction systems, and it has been shown that some of these can couple the chloroplast reaction with oxygen re-
PHOTOCHEMICAL
The Egects of Various
OXYGEN
REDUCTIOK.
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TABLE II Substances on the Rate of Reduction by Illuminated Chloroplasts
of
Oxygen
=
Active
catalysts
of oxygen
reduction
Ribollavine Riboflavine-5-phosphate @MN) Triacetylriboflavine-5-phosphate Flavine-adenine-dinucleotide @‘AD)
Anthocyaninsa (delphinidin) (cyanin) Janus greena Litmus Manganous ion p-Benzoquinoneh only)
Inefficient oxygen
catalysts reduction
of
Inactive
Benzyl viologen Methyl viologen
Anthoxanthines (quercetin) (apigenin) Thionine, hiethylene
blue
Diphosphopyridine-nucleotide (DPN) DPN + diaphorase -+ methylene blue Triphosphopyridine-nucleotide (TPN) Pyocyanin p-Benzoquinone Hydroquinone Ferric ion Cupric ion Flavines (with C. bonus-henricus only)
(with chard
I L
a Unstable; catalytic substance not definitely known. * Associated with the photoproduction of oxidation catalysts?
duction. However, the endogenous systems mediating the reaction of chloroplasts with molecular oxygen have not been identified. The lag in oxygen uptake which is often encountered at the beginning of a period of illumination (see most of the figures) suggests that some of these “endogenous” intermediates may be produced by photochemical reactions. Apparently an enhanced photoproduction of oxidation catalysts occurs when quinone-treated chard chloroplasts are illuminated. Although much of the ability of chloroplasts to reduce oxygen is assoiiated with the presence of soluble autoxidizable substances, some preparations continue to reduce oxygen slowly even after repeated washings. Moreover, treatments which have not yet been related to t,he formation of autoxidizable hydrogen transport systems, for instance the addition of manganous ions, stimulate the reduction of oxygen. It is
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conceivable, therefore, that certain conditions may bring about the “exposure” of reactive sites not ordinarily available to oxygen. Since it is difficult to distinguish between true catalysis and such noncatalytic activations, the possibility that oxygen may occassionally react directly with the photolysis reduction product [H] cannot be excluded. However, there is no unequivocal evidence that oxygen is thus directly reduced photochemically in living plants (4). Therefore we might suppose that the frequently observed inactivity of the isolated chloroplasts toward molecular oxygen represents the state of the photochemical system as it exists in the cell. But if during the process of isolation the chloroplasts lost a component which normally prevents the “shortcircuiting” of the reducing system by moIecular oxygen, the resulting preparation might reduce oxygen directly. Analogous reductions of oxygen by light-activated pigment systems are well known, and the discovery of such reactions in chloroplasts could make a considerable contribution to our knowledge of the photochemistry of the chloroplast reaction. The reduction of autoxidizable chloroplast reagents may be conveniently followed by utilizing Mehler’s method of measuring oxygen reduction. The great disadvantage of studying chloroplast reactions by this indirect method is the element of ambiguity introduced by the fact that the proposed mechanisms may sometimes by questioned; it is well to recognize that some addenda, for instance quinone and manganous ions, stimulate oxygen reduction in an unknown manner. However, conclusions derived from observations of oxygen reduction have been confirmed in several instances by experiments in which the reduction of the supposed chloroplast reagent was measured in the absence of oxygen. Under sufficiently anaerobic conditions, benzylviologen and FAD were reduced, the latter almost completely, while pyocyanin was not reduced at all. Details of these experiments will be published in another paper. Under the same conditions methylene blue, which does not enhance the Mehler reaction, was also slowly but completely reduced. This apparent discrepancy with regard to methylene blue perhaps reflects the great difference in the sensitivities of the optical and manometric techniques employed, since both the reduction and the reoxidation of the dye were slow. Also, feeble anaerobic reductions are relatively difficult to interpret because they may be indirect. The failure of DPN to stimulate a Mehler reaction, even in the presence of a specific oxidase, may mean that this substance was also slowly (and perhaps indirectly) reduced. Thus
PHOTOCHEMICAL
OXYGEN
REDUCTION.
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the participation of the pyridine nucleotides in chloroplast reactions, reported by Vishniac and Ochoa and by others (7, 8) is probably less immediate than the participation of the flavine nucleotides. Since the flavines are very active in mediating oxygen reduction and are rapidly reduced in VCKUOby illuminated chloroplasts, they certainly may be added to the list of biologically important substances which have been found to react with the isolated photochemical system of photosynthetic organisms. However, the authors do not wish to imply that the observed reductions of the flavine nucleotides in vitro constitute evidence that similar reactions in viva play a part in the photosynthetic process. The fact that potentially active chloroplast preparations from Chenopodium bonus-henricus apparently do not react with either class of coenzyme suggests that the known coenzymes may not be directly concerned in normal chloroplast reductions. ACKNOWLEDGMENTS The authors are grateful to Dr. Allan Brown for his helpful criticism of this paper. The riboflavine derivatives used in these studies were kindly supplied by the late Dr. S. M. H. Christie. This research was supported by the Agricultural Research Council of Great Brit,ain.
SUMMARY
1. The reduction of oxygen by illuminated chloroplast preparations represents a reaction which corresponds t’o t,he reduction of other oxidants such as quinone but is probably dependent on additional hydrogen transport systems. Chloroplast preparations which reduce oxygen very slowly, if at all, have been obtained from the leaves of all five of t,he plant speciestested. 2. Many substances whose reduced forms are reoxidized by molecular oxygen stimulate the reduction of oxygen by chloroplasts. Some of t,hese substancesare normal components of leaves. Unidentified, soluble, heatstable factors from leaves of Chenopodiumbonus-henricuscatalyze oxygen reduction. 3. Usually the reduction of oxygen associated with endogenous oxidation catalysts only begins after several minutes of illumination. Moreover, the illumination of quinone-treated chard chloroplasts causesthe formation of substances capable of catalyzing the oxidation of either ascorbic acid or the chloroplast system by molecular oxygen. It is therefore suggested that much of the ability of chloroplasts to reduce oxygen depends on the photoproduction of autoxidizable hydrogen acceptors.
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4. If the reduction of oxygen measures the reduction of autoxidizable chloroplast reagents, it may be concluded that the flavines are rapidly reduced by most chloroplasts. Reduction of diphosphopyridine nucleotide (DPN) could not be detected by this method although diaphorase and methylene blue were added in order to catalyze the reaction with molecular oxygen. REFERENCES 1. MEHLER, A. H., Arch. Biochem. and Biophys. 33, 65 (1951). 2. MEHLER, A. H., Arch. Biochem. and Biophys. 34, 339 (1951). 3. MEHLER, A. H., AND BROWN, A. H., Arch. Biochem. and Biophys. 38,365 (1952). 4. BROWN, A. H., AND GOOD, N., Arch. Biochem. and Biophys. 67,340 (1955). 5. KEILIN, D., AND HARTREE, E. F., Biochem. J. 39, 148 (1945). 6. HOROWITZ, L., Dissertation, University of Minnesota, 1952. 7. VISHNIAC, W., AND OCHOA, S., J. Biol. Chem. 196, 75 (1952). 8. ARNON, D. I., Nature 187, 1008 (1951).