Separation o f the two photochemical system s of photosynthesis 355
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Wessels, J . S. C. 1958 Biochim. biophys. Acta, 29, 113.
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The role of benzoquinones in the electron transport system
By A. Tr e b s t
Orgctnisch-chemisches Institut, Technische Hochschule, Miinchen
During the past few years two new classes of benzoquinones have been added to the list of biologically active quinone compounds, namely the ubiquinones and the plastoquinones. Consideration has also been given to the possible functions of the vitamins tocopherol and vitamin K in electron-transport systems (Slater 1959; Martius i960); but contradictory views have not so far been reconciled.
The importance of ubiquinones was quickly recognized and indeed it was their participation in oxidative phosphorylation which led to their discovery (Ciba Foundation Symposium i960). In accordance with the value of their redox potential they link the succinate and the N A D H oxidation systems to the cyto
chrome chain (Green 1962). At the time when identification of the ubiquinones was proceeding, a different benzoquinone was found in leaves (Lester & Crane 1959). Its structure proved to be dimethyl-solanosyl-benzoquinone (Trenner,
23 Vol. 157. B.
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Arison, Erickson, Shunk, Wolf & Folkers, 1959) as compared with the dimethoxy- substitution of the ubiquinones.
0
A
JA
0CHS- / X CHS c h3o- |/ \ —CH3 c h3
CH3-J J
—[CH2—CH==C—CH2]9—H| c h3o—!\ / —[CH2—CH=C—CH2]n—H
y
Y
0 plastoquinone
011
ubiquinone
Plastoquinone is localized in chloroplasts, there replacing the vitamin K, which cannot be detected in appreciable amounts in chloroplasts (Crane 1959; Bishop
1959). The presence of closely related benzoquinones in mitochondria and in chloroplast grana emphasizes the similarities of these two subcellular particles in respect of both structure and function. Both particles catalyze electron transport coupled to A TP-formation.
The reversible oxidation and reduction of plastoquinone during photosynthesis has been shown by Crane, Ehrlich & Kegel (1960) and by Redfeam & Friend (1961).
A more direct experiment to prove its participation in photosynthetic reactions was provided by Bishop (1959, i960), who demonstrated th a t the reduction of ferricyanide by illuminated chloroplasts depends on the presence of plastoquinone.
More recently Krogmann (1961) found th at cyclic photophosphorylation in petrol- ether-extracted chloroplasts with phenazine methosulphate ( ) as co-factor was enhanced by the addition of plastoquinone. The participation of plastoquinone in photosynthesis is thus certain, but it remains to place it in the sequence of oxido-reduction reactions which lead to photosynthesis.
The photochemical reactions, so far described in isolated chloroplasts, may be classified into four types:
(1) The Hill-reaction, th a t is the reduction of electron-acceptors accompanied by oxygen-evolution. This type can be subdivided according to the electron- acceptor reduced, which may be (a) ferricyanide (Hill & Searisbrick 1940), (6) benzoquinones (Warburg & Liittgens 1946), and (c) N A D P (San Pietro &
Lang 1956). These three photoreduction-reactions are inhibited by 3-(3,4-diehloro-
phenyl)-l,l dimethyl urea (DCMU) (Wessels & van der Veen 1956) and o-phenan- throline (Warburg & Luttgens 1946), and are coupled to the formation of one
molecule of A T P for every two electrons transferred (Amon, Whatley & Allen 1958).
(2) Cyclic photophosphorylation independent of the presence of oxygen (Arnon 1959). Here it is only possible to measure A T P formation, and a co-factor must be added to the isolated grana to promote the reaction. The most commonly used are P M S and vitamin K. This reaction is not inhibited by either DC M U or o-phenanthroline.
(3) Aerobic cyclic photophosphorylation (Jagendorf & Forti 1961; Amon, Losada, Whatley, Tsujimoto, Hall & Horton 1961). In this reaction A T P - formation is dependent on the presence of oxygen (Hill & Walker 1959; Warburg,
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The role o f benzoquinones in the electron transport system 357 Krippahl, Gewitz & Volker 1959; Nakamoto, Krogmann & Vennesland 1959), and numerous compounds such as quinones and dyes are stimulatory. The reaction is similar to type (1), in which a reduced electron-acceptor is continuously re
oxidized by oxygen, permitting its reduction to be repetitive.
(4) Photo-oxidation of hydroquinones. This reaction is not coupled to phos
phorylation and forms th a t p art of the reaction-sequence in type (3), which oxidizes the reduced electron-acceptor (Trebst & Eck 19616). Reactions (1) and (4) together give rise to reaction-type (3).
Reaction (4) which is formally the reversal of reaction (1), may be in fact such a reversal as will be discussed later. Since hydrogen peroxide is formed during reaction (4), it is possible to measure the rate of reaction (3) concurrently by determining the amount of H 20 2 formed as well as the amount of A T P . Reaction- types (3) and (4) do not necessarily occur in nature.
To get some more information as to the position of plastoquinone in the electron transport chain, we have studied the properties of chloroplast grana freed from plastoquinone and carotenes by extraction with petrol-ether {PE). To this end, isolated chloroplast-grana were lyophilized and quickly extracted with P E under nitrogen and illuminated in the usual way. Some of the reactions, mentioned above, were studied using these grana-preparations and their activation by plastoquinone examined. The reduction of ferricyanide, benzoquinones and N A D P was measured by the absorption-changes at 400 m/u- and 340 m/u. respectively. Formation of H 20 2, which accumulates in reaction (3) when KCN is added to inhibit the endo
genous catalase (Trebst & Eck 19616) was estimated by reaction with titanyl- sulphonate, measuring the extinction at 395 m/x.
Ta b l e 5. In a c t iv a t io noft h ef e r r ic y a n id er e d u c t io n s y s t e mb y e x t r a c t io n
OF LYOPHILIZED CHLOROPLAST GRANA {LCG) WITH PETROL-ETHER {PE)
/nmoles ferricyanide
reduced in fu rth er tre a tm e n t of 15 m in in
lyophilized grana light
none 9-8
extracted w ith P E 0 1
ex tracted w ith P E and e x tra c t added back
4-5
As Bishop (1958) has shown, extraction of lyophilized grana with P E leads to inactivation of the ferricyanide reduction system, but activity is restored by addition of the PE extract (in our experiments to about 50 %, see table 5). When the ferricyanide system is compared with other Hill-reagents, as is shown in table 6, it is found th at the reduction of benzoquinones is not necessarily impaired by conditions which inactivate reduction of ferricyanide.
If benzoquinones are reduced in the absence of plastoquinone, it would be expected th at they would reactivate the ferricyanide system. This proved to be the case, as is shown in table 7. In these experiments catalytic amounts of the
23-2
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benzoquinones were added to the ferricyanide system which had been inactivated by P E extraction of chloroplasts.
We have tested a larger number of quinones than is shown in table 7, and all are not equally active. The important character seems to be the redox potential;
the more negative the potential, the more active the quinone in promoting
Ta b l e 6. Re d u c t io n o f f e r r ic y a n id e a n d o f b e n z o q u in o n e s b y
CHLOROPLAST GRANA AFTER EXTRACTION WITH PE, IN NITROGEN /nmoles electrons
transferred in
acceptor added 15 m in in light
10 /nmoles ferricyanide 0-8
5 /nmoles 2,3-dim ethoxybenzoquinone 5-2 5 /nmoles 2,3-dim ethoxytoluquinone 5-6 5 /nmoles 2,3-dim ethylbenzoquinone 5-6
Ta b l e 7. Re a c t iv a t io n o f t h e f e r r ic y a n id e s y s t e m o f P E e x t r a c t e d
CHLOROPLAST GRANA BY ADDITION OF BENZOQUINONES, IN NITROGEN /nmoles ferricyanide
reduced in additions to 10 /nmoles ferricyanide 15 m in in light
none 0*6
0*3 /nmole dichlorophenol-indophenol 0*7
0*3 /nmole 2,3-dim ethoxybenzoquinone 1*7
0*3 /nmole 2,3-dimethoxybenzoquinone-5-propionie acid 2*2 0*3 /nmole 2-m ethylbenzoquinone-5-butyric acid 2*4
0*3 /nmole trim ethylbenzoquinone 2*5
Ta b l e 8. Re a c t iv a t io n o f t h e f e r r ic y a n id e s y s t e m of P E e x t r a c t e d
CHLOROPLAST GRANA BY ADDITION OF QUINONES, IN NITROGEN /nmoles
ferricyanide reduced in additions to 10 /nmoles ferricyanide 15 m in in light
none 1*0
0*3/nmole/?-carotene 0*7
0*3/nmole p hytol 1*1
0*3 /nmole plastoquinone-45 3*7
0*3 /nmole plastoquinone-20 3*0
0*3 /nmole vitam in K x 2*0
0*3 /nmole ubiquinone-50 0*6
0*3 /nmole ubiquinone-30 0*6
0*3 /nmole a-tocopherol 2*1
0*3 /nmole a-tocopherylquinone 3*6
reduction. Dichlorophenol-indophenol ( DG)does not reactivate as might be expected, indeed Bishop (1959, i960) has already shown th at its reduction is
plastoquinone-dependent.
The effect of redox potential is altered in the case of benzoquinones with an isoprenoid side chain. Only methyl-substituted benzoquinones are active. The
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methoxybenzoquinones—th a t is the ubiquinones show very little activity, vitamin K j in agreement with Bishop’s finding (1959, i960) also has some activity as does a-tocopherol, probably due to its oxidation to the tocopherylquinone.
The a-tocopherylquinone is a trim ethyl benzoquinone with twenty carbon atoms in the side chain and is as active as plastoquinone. The relationship of plasto- quinone to the tocopherylquinones, which might be classified as plastoquinones were it not for the saturated side chain, becomes even more apparent in a second plastoquinone found in chestnut leaves. Identification is not yet complete, but it has probably two methyl groups and tw enty carbon atoms in the side chain (thus resembling y-tocopherol). This quinone also reactivates the ferricyanide system. A number of theories of the reaction mechanism of the reversible reduction of benzoquinones with an isoprenoid side chain, are based on the supposition th a t it proceeds by the chroman ring (Slater 1959; Ciba Foundation Symposium i960), as found in the tocopherols, ubichromenols and solanochromenols (Rowland 1958). Vishniac (1962) recently found tritium activity in the plastoquinone, after illuminating chloroplasts in tritiated water; an observation which also suggests th a t plastoquinone cycling involves a chroman structure.
Ta b l e 9. Re d u c t io n o f N A D P a n d o f f e r r ic y a n id e b y P E e x t r a c t e d
CHLOROPLAST GRANA, IN NITROGEN
mmoles electrons transferred in 15 m in in light b y
<---i— --- ---v e x tracted g ran a ex tracted gran a + dialyzed acceptor added e x tracted grana + P E e x tra c t P E e x tra c t
5 pm oles N A D P — 3-2 3-6
10 jumoles ferricyanide 0-7 1-9 0*2
Since N A D P is considered as the naturally occurring electron acceptor (Hill reagent), it was of particular interest to ascertain the effect of plastoquinone on N A D P reduction. Table 9 summarizes the results obtained. Reduction of N A D P by chloroplast grana is known to require an additional water soluble enzyme or enzyme system (San Pietro & Lang 1956), which is removed during the preparation of the grana. We added this enzyme by supplying some of the chloroplast extract {CE). As seen in table 9 added CE reactivates the ferricyanide system, but this effect is removed by subsequent dialysis. We have not examined this dialyzable factor extensively. In addition to being water soluble it is petrol-ether insoluble and heat stable; properties which might suggest a flavin. I f P E extracted grana, which do not reduce ferricyanide appreciably, are supplied with N A D P and CE, N A D P H is formed and oxygen is evolved.
Conclusions from the experiments discussed so far are, th at reduction of ferricyanide by chloroplast grana is easily abolished by extraction with PE, but can be restored by adding naturally occurring methyl-substituted benzoquinones.
The reduction of benzoquinones and N A D P is not prevented by P E extraction and so these reactions by-pass plastoquinone. The position of the plastoquinone in the reaction sequence of electron acceptors, according to these results, is just The role o f benzoquinones in the electron transport system 359
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after a hypothetical compound Z which can reduce either plastoquinone or (since the reduction of ferricyanide is coupled to A T P formation, the electron
flow must go by Z).
This position of the plastoquinone in the sequence is difficult to reconcile with the results of Krogmann (1961), who believes th a t plastoquinone is involved in cyclic phosphorylation, and with the experiments of W itt, Muller & Rumberg (1961), which suggest th at plastoquinone might be the compound which links the two light reactions recently shown to be involved in oxygen evolution. With this difficulty in mind a more thorough extraction of the grana with P E was made and
Ta b l e 10. Fe r r ic y a n id e r e d u c t io n, N A D P r e d u c t io n a n d H 20 2 fo r m a tio n
BY PE EXTRACTED CHLOROPLAST GRANA AS AFFECTED BY QUINONES AND BY PLASTOQUINONE
In air an d 0*2 /xmoles ^-carotene added to each vessel.
/xmoles electrons tra n s ferred in 15 m in in light
( _A \
tim es ex tracted
additions to ex tracted grana 2 5
1 . 10 /xmoles ferricyanide 0-7 0-5
2. 10 /xmoles ferricyanide+ 0*2 /xmoles plastoquinone 2-5 1*9
3. 5 /xmoles N A D P 2-8 1*6
4. 5 /xmoles N A D P + 0 2 /xmole plastoquinone 3 0 2*2
5. 5 /xmoles i\L4DP + 0-1 /xmole D0 /xmoles ascorbate1 2-9 2*3
6. 0-2 /xmole 2,3-dim ethylbenzoquinone-butyric acid 1-9 0*9
+ 10"3mKCN
7. 0-2 /xmole 2,3-dim ethylbenzoquinone-butyric a c id + 1 0 _3M KCN + 0-2 /xmoles plastoquinone
2-5 2*1
8. 0-2 /xmole chlorogenic a c id + 10~3m KCN 0-4 0*4
9. 0-2 /xmole chlorogenic acid-|- 1 0 _8m KCN + 0-2 /xmole plastoquinone
2-0 2*1
Measurements w ere: in treatm en ts 1 and 2 fall in extinction a t 400 m/x, 3, 4, 5 rise in extinction a t 340 m/x, 6, 7, 8, 9 extinction a t 395 m/x after reaction w ith titan y l sulphate.
led to the discovery of a second possible role for plastoquinone. The experiments, described so far, were done by carefully extracting grana twice with PE which removes about 70 % of the plastoquinone originally present. Extraction of grana five times with P E leads to quite a different picture. As shown in table 10, extrac
tion of grana twice with P E results in an impairment of the capacity to reduce ferricyanide and also chlorogenic acid in a system of reaction-type (3), as measured by the H 20 2 formation occurring by photo-oxidation. N A D P and quinone reduction, the latter also measured by H 20 2 formation, however, still proceed.
Addition of plastoquinone affects only ferricyanide reduction and the chlorogenic acid system. Extraction with P E five times, prevents reduction of all four accep
tors, and addition of plastoquinone now activates all four reactions (table 10).
The role of plastoquinone now emerging, is therefore twofold. The main function, at a position in the electron transport chain where it is at a rather low concentra
tion and from which it is difficult to extract it completely, is in catalyzing all
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photochemical reactions, which lead to the evolution of oxygen. This is in accord
ance with results of experiments of W itt et al. (1961), and places plastoquinone in the electron-transport chain as the electron acceptor of the second light reaction.
We showed recently, th a t the reduced co-factor in anaerobic cyclic photophos
phorylation reacts with a compound with a redox potential of zero (Trebst & Eck 1961 a). I t now seems justifiable to assume th a t this compound is the plastoquinone which can either accept electrons during a second light reaction associated with a Hill reaction, or during the first light reaction in a cyclic electron flow system involving the added co-factor. This hypothesis would also be consistent with the results of Krogmann (1961). This main function of plastoquinone is depicted in the following scheme as position one.
The role o f benzoquinones in the electron transport system 361
°. ^
V
chlorophyll a
cytochrome / ADP
vitamin K 3 plastoquinone PMS
(position 1)
plastoquinone (position 2)
z ---
benzoquinones
/ /
ferricyanide
The second role for plastoquinone (position 2) is a t the hypothetical position
of compound Z, where it is coupled to the first light reaction. At this location plastoquinone is a t a higher concentration and is also more easily completely
extracted. I t may be by-passed in this position by N A D P and low redox potential quinones, but it is necessary for the reduction of ferricyanide, dichlorophenol- indophenol, chlorogenic acid and possibly for other compounds with high positive potentials. Experiments with mitochondria have suggested, th at only a small portion of the ubiquinones present actually take part in the electron transport in respiration (Chance i960; Redfearn 1962). The second suggested position of plastoquinone might also be only a storage site at which it is reduced in light, but not rapidly turned over, although there might be a role for plastoquinone at this site too. We do not yet know which is the natural co-factor of cyclic photophos
phorylation. I t is conceivable, th a t it is plastoquinone, if so all th a t is required is to establish contact between the two plastoquinone sites, which for some unknown reason are disconnected during isolation of the chloroplast, so making it necessary to add an artificial co-factor to bridge the gap. Some support for this idea comes from the observation th a t substituted benzoquinones are excellent co-factors of photophosphorylation, though only in air. They share this property with a number of o-hydroquinones, like chlorogenic acid or Dopamin, which are also found in nature.
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Another aspect of the role of benzoquinones in photosynthesis is the re-oxidation of hydroquinones. In anaerobic cyclic photophosphorylation the reduced co-factor is re-oxidized by an oxidized cytochrome chain (Amon 1959) and, as already discussed, probably via plastoquinone before oxygen is evolved. The re-oxidation of the reduced co-factor in aerobic cyclic photophosphorylation is effected by oxygen, and the enzyme responsible might well be a phenoloxidase (Warburg et al.
1959). If this were so the reaction should proceed in the dark. However, oxygen uptake in the dark, with a number of hydroquinones as substrate, particularly those with positive redox potentials, is too slow to account for the rapid turnover in photophosphorylation. For this and other reasons we came to the conclusion
Ta b l e 11. Cyclic p h o t o p h o sp h o r y l a t io n i n a ir i n c h lo r o pla st g r a n a
juxnoles A T P form ed in
co-factor added 15 m in in light
0-2 jumole vitam in K 3 3 4
0-2 fjanole 2,3-dim ethylbenzoquinone 0-6 0-2 jtmiole 2,3-dim ethylbenzoquinone-butyric acid 4-6
O'2 jumole trim ethylbenzoquinone 2*2
0*2 nmole duroquinone 4-6
0-2 /xmole chlorogenic acid 3*4
0-2 ^mole D opam in 5*1
Ta b l e 12. Ox id a t io n o f c h lo r o g e n ic a c id b y c h lo r o pla st g r a n a b e f o r e
AND AFTER DISRUPTION BY A DETERGENT ( )
jLiatoms oxygen tak en up
____________ A___________ ■
f \
w ithout w ith additions to 5 /mmoles chlorogenic acid detergent detergent
none 15 m in light 1-6 4-8
10-3M KCN 15 m in light 6-4 1-7
10-3 m K C N + 1 0 ~ 6 DCM U 15 m in light 0 —.
none 15 m in dark 0 5-3
10-3m KCN 15 m in dark 0-2 0-2
10-4m DCM U 15 m in dark — - 5-1
th a t re-oxidation of hydroquinones occurs by way of a photo-oxidation, with H 20 2 as the primary product. In the absence of inhibitors, the H 20 2 is decomposed by the endogenous catalase of the grana (Trebst & Eck 19616). This photo-oxidation
is inhibited by DCMU and o-phenanthroline, as shown in table 12. These com
pounds are known as specific inhibitors of the oxygen-evolution system in the Hill reaction (Warburg & Luttgens 1946; Wessels & van der Veen 1956), so th at it may be th a t this photo-oxidation is a direct reversal of the process of oxygen evolution. A detailed mechanism cannot be proposed at present, but as a specula
tion there might be a peroxide which is either decomposed to oxygen (which might be an explanation of the oxygen capacity of Chlorella (Warburg, Krippahl, Schroder & Buchholz 1954) or transfers its peroxide oxygen to a hydroquinone to form a quinolperoxide which in turn is split into H 20 2 and quinone. The purely
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speculative scheme below attem pts to set out a possible reaction sequence, p art of which has been proposed by Forti & Jagendorf (1961):
H 20 + X + 7 -> X H 2+ 7 0 7 0 + £ 0 2 -> 7 0 2
7 0 2 -> 7 + 0 2 or 7 0 2 + Z H 2 -> 7 + Z H 2- 0 2
x h2- o2 ->x +h2o2
The role o f benzoquinones in the electron transport system 363
O
This photo-oxidation depends on the structural integrity of the grana, as is also shown in table 12. Breaking the structure by treatm ent with a detergent, like B R IJ , abolishes the photo-oxidation of hydroquinones. This distinguishes it from the photo-oxidation of ascorbic acid (Wessels 1955), which as a non-enzymatic photo-oxidation is enhanced by treatm ent with a detergent (Hinkson & Vernon 1959). In disrupted grana the hydroquinones are oxidized in the dark, a reaction which is inhibited by KCN. Breaking the structure of the chloroplast grana, liberates a polyphenoloxidase with specificity towards o-hydroquinones (Trebst &
Wagner 1962). Latent phenoloxidases have been described before (Kenten 1957, 1958) and one might speculate, whether the phenoloxidase activity of a leaf is attributable to damaged chloroplasts. A role for this latent copper enzyme in photosynthesis cannot be suggested as yet.
The experiments described herein were done in collaboration with Herbert Eck.
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Vishniac, W . 1962 Vth International Congress of Biochemistry, Moscow 1961 (in th e Press).
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W arburg, O. & L uttgens, W . 1946 Biokhimiya, 11, 303.
Wessels, J . S. C. 1955 Rec. Trav. chim. Pays-Bas, 74, 832.
Wessels, J . S. C. & v an der Veen, R . 1956 Biochim. biophys. Acta,19, 548.
W itt, H . T., Muller, A. & R um berg, B. 1961 Angew. Chem. 73, 507.
Discussion
E. R. Redfearn and J . Friend. Department of Biochemistry, University of Liverpool and Department of Botany, University of Hull.
Several speakers in this Discussion have referred to the possible role of plasto- quinone or koflerquinone (2,3-dimethyl-5-solanesyl-l,4-benzoquinone) in the photo
synthetic electron transport system. We should like to present the results of some experiments which we have carried out in an attem pt to elucidate the function of plastoquinone.
Chloroplasts were isolated from sugar-beet or spinach leaves. In the first series of experiments the oxidation-reduction reactions of endogenous plastoquinone were studied (Redfearn & Friend 1961, 1962; Friend & Redfearn 1962). The results of these experiments may be summarized as follows:
(1) Illumination of freshly isolated chloroplasts resulted in approximately 20 % reduction of the endogenous plastoquinone. Crane, Ehrlich & Kegel (i960) have reported 80 % reduction.
(2) This light-catalyzed reduction was inhibited by o-phenanthroline and 3-(3,4-dichlorophenyl)-l,l-dimethylurea ( Addition of ascorbic acid and 2,6-dichlorophenol-indophenol stimulated the photoreduction of plastoquinone and this reaction was not affected by either of the two inhibitors.
(3) Endogenous plastoquinone was reduced by N A D P H in the dark or by N A D P plus chloroplast extract (containing photosynthetic pyridinenucleotide reductase) in the light.
(4) Hydroxylamine hydrochloride, potassium cyanide and sodium azide stimu
lated the photoreduction of plastoquinone but ammonia was inhibitory.
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In a second series of experiments the effects of extraction of the endogenous plastoquinone with organic solvents were studied. Chloroplasts were extracted with light petroleum without prior lyophilization and photolytic activity measured by the potentiometric determination of the reduction of potassium ferricyanide.
I t was shown th a t extraction resulted in a loss of the Hill-reaction activity, th a t the loss could be correlated with the amount of plastoquinone removed, and th a t activity could be restored by adding plastoquinone to the extracted chloroplasts, thus confirming the earlier findings of Bishop (1959). In addition to plasto
quinone, however, it was found th a t a number of other quinones would also re
activate extracted preparations and some appeared to be more effective than plastoquinone. Thus trimethyl-1,4-benzoquinone and 2-methoxy-6-propyl-l,4- benzoquinone gave greater reactivations th an an equimolar concentration of plastoquinone. Furthermore, these quinones, including plastoquinone, also stimulated the rate of ferricyanide reduction, although quantitatively to a smaller extent, in unextracted chloroplasts. Of the quinones tested related to the ubi
quinone (coenzyme Q) series, aurantiogliocladin and ubiquinone-5 were effective in the activation and reactivation reactions but ubiquinone-25 was inhibitory.
In the naphthoquinone series, 2-methyl-1,4-naphthoquinone (menadione) was active but vitamin K 2-5, K 2-10 and K 2-25 were inhibitory.
The experiments on the endogenous plastoquinone suggest th a t it acts at a point after the light reaction concerned in the photolysis of water. This is in agreement with the main site of action proposed in the schemes of Duysens, W itt, Wessels and Trebst presented in this Discussion. The extraction-reactivation experiments have indicated th a t a wide range of quinones may restore ferricyanide reduction in extracted chloroplasts and it is possible th a t the oxidation-reduction potential may be a more im portant factor than structural specificity in this respect.
However, Dr Trebst’s interesting scheme with plastoquinone acting a t two sites in the isolated chloroplast may mean th a t reactivation is a more complex process.
Thus, ferricyanide reduction may be mediated specifically by plastoquinone at one site and non-specifically by other quinones having the proper oxidation- reduction potential at the other.
Re f e r e n c e s (Redfearn & Friend)
Bishop, N. I. 1959 Proc. Nat. Acad. Sci., Wash. 45, 1696.
Crane, F . L., Ehrlich, B. & Kegel, L. P . i960 Biochem. Res. Com. 3, 37.
F riend, J . & R edfearn, E . R . 1962 Biochem. J . 82, 13p. R edfeam , E . R . & F riend, J . 1961 Nature, Bond. 191, 806.
R edfeam , E . R . & Friend, J . 1962 Phytochemistry 1, 147.
L. W. Mapson. Low Temperature Research Station, Cambridge.
We have obtained evidence th a t suggests th a t ascorbic acid may act as an electron donor and dehydroascorbic acid (DHA) or more probably monodehydroascorbic acid as an electron acceptor in the photosynthetic process. When intact straw
berry leaves are illuminated there is a photo-oxidation of ascorbic acid, the rapidity of the reaction depending on the intensity of the illumination. This photo-oxidation
The role o f benzoquinones in the electron transport system 365
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leads to an increase in the steady-state level of in the leaf, and on removal of light this steady-state level falls to the value previously recorded in the darkened leaf. The photo-oxidation of ascorbic acid is suppressed on poisoning the leaf with cyanide, and at the same time the steady-state level of D H A is enhanced in the darkened leaf. W ith such poisoned leaves light stimulates the reduction of D H A, and the rate again depends on the intensity of illumination.
Both photo-oxidation and photoreduction are abolished in leaves poisoned with 3-4-dichlorophenyl-l,l-dimethylurea, hydroxyalamine or o-phenanthroline.
An evaluation of the respective rates of photo-oxidation and photoreduction, suggest th a t the increase in the steady-state level of D H A consequent upon illumination, is the result of an equilibrium between the opposing reactions of photo-oxidation and photoreduction.
The significance of these results in relation to the photosynthetic mechanism remains to be determined. One possibility is th a t ascorbic acid may act as an electron donor and the oxidized form as electron acceptor, positioned as the intermediate A in the sequence of reactions postulated by Losada, Whatley &
Amon (1961).
Re f e r e n c e (Mapson)
Losada, M., W hatley, F . R . & A m on, D. I. 1961 Nature, Lond. 190, 606.
The production of glycollate during photosynthesis in Chlorella
By C. P . Wh it t in g h a m a n d G. G. Pr it c h a r d
Botany Department, Queen Mary College, London University
Blackman (1905) deduced from his study of the effect of external factors on the rate of photosynthesis th a t photosynthesis must include both photochemical and thermochemical processes. Later it appeared probable th a t the fixation of carbon dioxide was a thermochemical process driven in the direction of the forma
tion of reduced carbon compounds by reaction with some product of the photo
chemical process. Work with radioactive carbon isotopes has confirmed this viewpoint.
Calvin and his colleagues (Bassham & Calvin 1957) utilized the radioactive isotope of carbon, carbon-14.
In the first type of experiment the percentage radioactivity in any one compound as a fraction of the total activity fixed was measured. By extrapolation it was possible to estimate which compound would have 100% of the radioactivity immediately after the addition of tracer and was therefore the first intermediate formed. I t was found to be phosphoglyceric acid (PGA). From the shape of the time course of the percentage activity for individual compounds it was possible
