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Ascorbate-supported NADP photoreduction by heated Euglena chloroplasts
ARCHIVES
OF
BIOCHEMISTRY
AND
122, 144-152 (1967)
BIOPHYSICS
Ascorbate-Supported
NADP Euglena
SAKAE Charles
KATOH2
F. Kettering Received
Photoreduction
Chloroplasts’
AND
ANTHONY
Research Laboratory, March
by Heated
SAN PIETRO
Yellow Springs,
29, 1967; accepted
May
Ohio 46.387
8, 1967
The effect of heating of Euglena chloroplasts on various photooxidation-reduction activities has been studied. When the chloroplasts were heated for 5 minutes at 40” the Hill activity was abolished completely, but there was no significant inhibition of NADP photoreduction with reduced Euglena cytochrome-552 as electron donor. Interestingly, the addition of ascorbate restored the ability of the heated chloroplasts to photoreduce NADP as well as did the ascorbate-DPIP couple. However, the ascorbate-supported NADP photoreduction exhibited an intermediate sensitivity toward treatment of the chloroplasts at higher temperatures or for a longer duration of incubation, that is, lower than for the normal Hill reaction but higher than for the ascorbate-DPIP system. In addition, the ascorbate-supported NADP photoreduction by heated chloroplasts was sensitive to various poisons of the oxygen evolution system, but the ascorbate-DPIP system was not. A possible mechanism to account for heat inactivation of the Hill reaction and reactivation by ascorbate is presented.
The mechanism of oxygen evolution is the least understood facet of photosynthesis even though the production of oxygen during photosynthesis is a well-documented phenomenon (1,2). One explanation may be the very unstable nature of the oxygen evolution system toward physical and chemical modification of chloroplasts. A number of these treatments of chloroplasts will inactivate efiectively and specifically the oxygen evolution system but will leave unchanged many of the other reactions of the photosynthetic electron transport system. The treatment of chloroplasts at mild temperature is one such specific treatment. The Hill activity is abolished easily by heating chloroplasts at relatively low temperatures (3, 4). Hinkson and Vernon (5) found that ‘Contribution No. 284 of the Charles F. Kettering Research Laboratory. This research was supported in part by a research grant (GM10129) from the National Institutes of Health, United States Public Health Service. * Present address: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo, Japan.
incubation of chloroplasts at 51’ for 10 minutes resulted in complete abolition of the Hill activity with DPIP as oxidant. In contrast, indigo carmine photoreduction with ascorbate and DPIP as electron donor and ascorbate photooxidation in the presence of DPIP were reduced only partially. The photoreduction of ferredoxin by mildly heated chloroplasts at the expense of reduced DPIP was described also by Whatley et al. (6). In the present study, the effect of mild heat treatment of Eugkna chloroplasts on the Hill reaction and the photoreduction of NADP with artificial electron donors was investigated. Evidence is presented which indicates that the mechanism of heat inactivation of the Hill reaction is significantly different from the mechanism of inhibition caused by various poisons which are specific inhibitors of the oxygen evolution system. MATERIALS
AND
METHODS
The general experimental procedures employed were described in the preceding papers (7, 8). Heat treatment of chloroplast,s was carried out 144
.
ASCORBATE-SUPPORTED
NADP
in a small test tube containing chloroplasts suspended in the preparation medium (0.4 M sucrose, 0.01 M XaCl, and 0.05 M phosphate buffer, pH 6.5) in a water bath of the desired temperature in the dark. Special care was taken to avoid itlhomogeneous heating of the chloroplasts. Toward this end, incubation of 0.5 ml of a chloroplast suspension containing about 250 pg of chlorophyll with gentle and colrtillrmrls slirring was found to be satisfactory and was used throughout. Incubation of either a larger volume of the ehloroplast, suspension or a more dense suspension of chloroplasts tended to result in an iuhomogclleous prepsratioll, t,herefore, a less selective inhibitinn of the Hill reaction. RESULTS
Kinetics of NADP photoreduction and cytochrome-552 photooxidation-reduction. The
time course of light-induced reduction of ;“\‘ADP and oxidation-reduction of cytochrome-&Q by Euglena chloroplasts which were heated at 45” for 5 minutes is shown in Fig. 1. The absorbance changes at 340 rnp for KADP and at 552 rnp for cytochrome552 were measured with separate but identical reaction mixtures. As reported previously (7), the unheated chloroplasts reduced XADP at a substantial rat,e provided ferredoxin and cytochrome-552 were present (Fig. 1, A). A correlatjive absorbance change ascribable to cyt’ochrome-552 which occurred during XADP photoreduct,ion was also described (S). After heating, Euglena chloroplasts were incapable of catalyzing the Hill reaction with SADP as oxidant and showed only a slight initial increase of absorbance at 340 rnp (Fig. lB, upper curve). The corresponding absorbance change at 552 rnp, which is indicative of an enhanced photooxidation of reduced cytochrome-552, is shown as the lower (dashed) curve of Fig. 1B. Similar absorbance changes were observed previously with chloroplasts poisoned with atrazine (S). It therefore appears that the heat treat,ment of b’uqlena chloroplasts resulted in a preferential destruction of only the oxygen-evolution system (photosystem 2). In contrast, the long wavelength system (photosystem 1) n-as unaffected and, as indicated by a short-lived, initial increase in absorbance at 340 m,u, catalyzed the photoreduction of
145
PHOTOREDUCTION
N6 I,
D o-
0
I
2
3
0
I
2
3
MINUTES
absorbance I?IG. 1. Time course of light-induced changes at 340 rnp and 552 mnMwith heat,ed chloroplasts. Each reaction mixture contained 50 mM phosphate buffer, pH 6.0, 10 m&l NaCl, 0.86 mnr NADP, a saturating amount of spinach ferredoxin, 21 PM Ezcglena cytochrome-552 and Euglena chloroplasts equivalent to 14.2 pg chlorophyll. Final volume, 0.7 ml. Cytochrome-552 was in the reduced form when added except for curves D, in which oxidized cytochrome was added. Unt,reated chloroplasts were used in A and heated chloroplasts ilt B to G. A and B, no addition; C, 50 MM atrazine; D, no addition; E, 14 mu neutralized ascorbate; F, 14 IIIM ascorbate plus 50 PM atrazine; and G, 14 mM ascorbate, 20 MM DPIP, and 50 PM atrazinc. (-), 340 rnw; (-), 552 rnp.
KADP with reduced cytochrome-552 as the electron donor. This conclusion is supported by the observation that the addition of atrazine to the heated chloroplasts had no significant effect on the absorbance changes at 340 and 552 rnF (Fig. 1, C). Furthermore, no absorbance changes were noted when the reaction mixture contained oxidized rat.her than reduced cytochrome-552 (Fig. 1, D). Of special interest was the finding that ascorbate alone could support a substantial rate of NADP photoreduction (Fig. 1, E). This was not the case with the atrazinepoisoned chloroplasts, where ascorbate served only poorly as an electron donor for the photoreduction of NADP (S; Fig. 1, 17). In addition, the ascorbat’e-supported NADP photoreduction by the heated chloroplasts n-as susceptible t.o the poison, although complete inhibition could not be obtained with a
146
KATOH
AND
concentration of the poison which inhibited completely the Hill reaction with untreated chloroplasts (Fig. 1, F). This is in marked contrast to NADP photoreduction with ascorbate and DPIP as electron donor which occurred in the presence of the poison (Fig. 1, G). AS would be expected, there was no appreciable absorbance change at 552 mp in the presence of ascorbate (Fig. 1, E-G) under the conditions employed. Effect of temperature. The Hill reaction with ferricyanide and NADP, as well as NADP photoreduction in the presence of ascorbate or ascorbate and DPIP, were measured using chloroplasts which had been heated for 5 minutes at various temperatures (Fig. 2). It was found that the reactions exhibited markedly different sensitivities toward heat treatment of the chloroplasts. The Hill reaction with ferricyanide was most heat, labile and the activity was lost completely at 40”. The Hill reaction with NADP was somewhat complicated by apparent inactivation during the reaction, The initial rate of NADP photoreduction was moderately sensitive to the heat treatment, and at 40” corresponded to about one-half of the original activity. When the rate of the reaction measured after 2 minutes of illumination was plotted against temperature, the resultant curve was similar to that for ferricyanide reduction except for a residual temperatureinsensitive activity of about 10%. The photoreduction of NADP supported by reduced cytochrome-552 as the electron donor, which became marked concurrent with inactivation of the oxygen evolution system, would account for the apparent temperatureinsensitivity of the initial rate and some of the rate measured 2 minutes after illumination. When the phot,oreduction of NADP was carried out in the presence of ascorbate, the activity was much less affected by heating than the regular Hill reaction (Fig. 2, lower curves). With chloroplasts heated at 40” which exhibited no or very low Hill activity, ascorbate restored the activity to about 70 % of the original activity. Further, there was no significant difference between the initial rate of NADP photoreduction and the rate measured after 2 minutes of illumination.
SAN PIETRO
HILL
O -‘>30
40 TEMPERATURE
REACTION
I 50
60
t-2)
FIG. 2. Effect of temperature on the Hill referricyanide and DPIP and NADP photoreduction with ascorbate and ascorbate plus DPIP. The chloroplasts were heated for 5 minutes at each temperature. Ferricyanide photoreduction was determined with a Clark electrode obtained from the Yellow Springs Instrument Co. The reaction mixture, in a final volume of 4.9 ml, contained 50 mM citrate-phosphate buffer, pH 5.0,lO mM NaCl, 1 mM potassium ferricyanide, and chloroplasts equivalent to 63 pg chlorophyll. NADP photoreduction was determined as described in Fig. 1, and the chloroplast content was equivalent to 13 rg of chlorophyll. Upper curves: (M), ferricyanide Hill reaction; (0, A), initial and a-minute rate, respectively, of the NADP Hill reaction. Lower curves: (IJ), initial and 2-/M minute rate, respectively, of NADP photoreduction in the presence of 14 mM ascorbate; (A, A) initial and a-minute rate, respectively, of NADP photoreduction with 14 mM ascorbate, 20 /IM DPIP, and 50 mM atrazine.
action with
The photoreduction of NADP with ascorbate and DPIP was most resistant to the heat treatment. The activit,y was unaffected, or increased slightly, at temperatures up to 40”, and a t’emperature slightly above .50” was required to decrease the activity to onehalf. In this case also, the initial rate and the rate measured after 2 minutes of illumination were the same. Effect of time of heating at 40” on NADP
ASCORBATE-SUPPORTED
NADP
photoreduction. The Hill reaction with ferricyanide and that with NADP determined 2 minutes after illumination were very sensitive to heat treat’ment of chloroplasts at 40” and were lost completely after 3 minutes of heating (l?ig. 3). Again, the initial rate of the NADP Hill reaction was less sensitive than the rate measured after 2 minutes of illumination. The presence of ascorbate increased greatly t’he activity for NADP photoreduction by the heated chloroplast’s. A further increase was obtained by the subsequent addition of DPIP. E$ect of temperature on Cytochrome-552 photooxidation-leduction. The photooxidation and photoreduction of cytochrome-552 represent partial reactions of the P\‘ADP Hill react,ion and are associat,ed wit,h photosysterns 1 and 2, respectively (8). Figure 4 shows the effect of temperature on these two reactions. The photoreduction of cytochrome-552 leas as sensit,ive to heat,ing as the Hill reacbion with ferricyanide and reflects the heat sensitivity of photosystem 2. On the other hand, t,he photooxidat’ion of
HILL
NADP
REACTION
PHOTOREDUCTION
I
I OO
2
I TIME
OF
3 INCWXTION
4 (MIN)
5
FIG. 3. Effect of time of heating on the Hill reaction with ferricyanide and NADP and NADP photoredllction in the presence of ascorbate and ascorbate plus DPIP. Reaction conditions were as described in Fig. 2. Temperature of heating, 40”.
147
PHOTOREDUCTION
P,
0'L, 0
I
' 30
50 TE%RATURE
60
(“C)
FIG. 4. Photooxidation and photoreduction of cytochrome-552 by chloroplasts heated for 5 minutes at various temperatures. Photooxidation of reduced cytochrome-552 was determined wit,h a reaction mixture of 0.7 ml which contained 50 mM phosphate buffer, pH 6.0, 1 mM methyl vioand chlorologen, 53 pM reduced cytochrome-552, plasts equivalent to 6 pg of chlorophyll. The reaction mixture employed for photoreduction contained 50 mM phosphate buffer, pH 6.0, 106 PM oxidized cytochrome-552, 1 mM NaCl, and chloroplasts equivalent to 18 rg of chlorophyll in a final volume of 2 ml. Photoreduction of cytochrome was carried out anaerobically.
reduced cytochrome-,552, catalyzed by photosystem 1, was markedly resistant’ to heating. At t’he lower temperatures, there was either no effect or a slight increase in activity; at’ temperat’ures above 50”, the activity decreased. Reactim cmditions for ascorbate-suppoffed NADP photoreduction by heated chloroplasts. The preceding observations indicate that the ascorbate-supported NADP photoreduction by heated chloroplasts is significantly different from NADP photoreduction using either water or the ascorbate-DPIP couple as electron donor. The reaction conditions for the ascorbate-supported NADP photoreduction mere elucidated wit,h Euglena chloroplasts which were heat’ed for 5 minutes at 45”. Figure 5 shows the effect of ascorbate concentration on the rate of NADP photoreduct,ion. Since t,he reaction was linear with time (see Fig. 1, E), only the initial rate of reducGon
is plotted
against
ascorbate
con-
148
KATOH
AND
OO24 ASCORBATE
( pmoles)
FIG. 5. Dependence of NADP photoreduction by heated chloroplasts on ascorbate concentration. Reaction conditions were as described in Fig. 1, E and G, except that the concentration of ascorbate was varied as indicated.
centration. The activity observed without ascorbate represents NADP photoreduction coupled to the oxidation of reduced cytochrome-552. It was shown previously that the photooxidation of reduced cytochrome552 contributed to the absorbance increase at 340 rnp (8). The observation that the rate of NADP photoreduction decreased at low ascorbate concentrat8ion means that this amount of ascorbate maintains cytochrome552 in the reduced form but cannot support NADP photoreduction at an efficient rate. Aftjer this initial decrease, the rate of NADP reduction increased with increasing concentration of ascorbate and reached a constant rate which was independent of ascorbat,e concentration. A similar dependence on ascorbate concentration was noted for NADP photo reduction wit,h the ascorbate-DPIP couple (Fig. 5), The data presented in Fig. 6 shows clearly that cytochrome-552 is essential for the ascorbate-supported NADP photoreduction by heated chloroplasts. There was no NADP photbreduction in the absence of the cytochrome; at low cytochrome concentrations, the rate was related linearly to the cytochrome concentration. At higher cyt’ochrome concentrations, the rate was maximal and independent of the cytochrome concentration. The concentration of cytochrome required for maximal activity was similar to that for the NADP Hill reaction, i.e., about 10 mpmoles per cuvet,te (7).
SAN
PIETRO
The pH-activity curves for NADP photoreduction are shown in Fig. 7. The ascorbatesupported NADP photoreduction exhibited maximal activity at a pH slightly higher than 6. The addition of DPIP resulted in a slight shift of the curve to a higher pH. These curves are again comparable with those for the NADP Hill reaction. Sensitivity toward various poisons. A variety of inhibitors such as DCMU inhibit the Hill reaction at very low concentration (9). Certain reactions which are thought not to require the oxygen evolution system were inhibited at high concentrations of the poisons. For example, Asahi and Jagendorf (10) reported that 1O-4 M CMU inhibited PMScatalyzed photo-phosphorylation. It was important, therefore, t’o compare the sensitivity of the ascorbate-supported NADP photoreduction toward various poisons with that of the Hill reaction or the photoreduction of NADP with the ascorbate-DPIP couple as the electron donor. Figure 8 shows the effect of atrazine concentration on the Hill reaction of untreated chloroplasts nith ferricyanide and NADP, as well as on the NADP photoreduction by heated chloroplasts with either ascorbate or ascorbate and DPIP as electron donor. The percentage inhibition is defined as (one-rate with inhibitor/rate without inhibitor) X 100 and is plotted against the logarithm of the molar concentration of the poison. The Hill reaction with ferricyanide and
30 IO 20 CYTOCHROME 552 (mpmoles)
40
FIG. 6. Dependence of NADP photoreduction by heated chloroplasts on reduced cytochrome-552 concentration. Reaction conditions were as described in Fig. 1, E and G, except the concentration of reduced cytochrome-552 was varied as indicated.
149
ASCORBATE-SUPPORTED NADP PHOTOREDUCTIOK
r’ 601!
.4 DP’P n-. . \ . . Y ’ . \\ . .
\ .-. 1 OL-----J 5
7
6
8
9
PH FIG. 7. PII-dependency of ?r’ADP photoreduction by heated chloroplasts. Reaction couditions were as described in Fig. 1, E and G, except t,hat the pH was varied by using 50 rnbr citrate-phosphate or phosphate buffer. ( q ), no addition (2.minute rate); (U), ascorbate; (A, ascorbate plus DPIP.
SADP with untreated chloroplasts was inhibited significantly by low concentrations of atrazine. Also, the ascorbate-supported photoreduction of KADP by heated chloroplasts exhibitled a sensitivity comparable to that of the Hill react,ion, although inhibit,ion was less complete at higher concentratjions of the poison. In contrast, NADP photoreduct’ion with ascorbate and DPIP by heated chloroplasts was much less sensitive t,o the poison, and only about 30 % inhibition was obt,ained at, the highest concentration of t,he poison tested. Similar experiments were carried out with several other inhibitors. For comparison, the data are presented both as t’he concentrat8ion required for 50% inhibition (&J and the maximum inhibit’ion (MI), obtained nith the highest, concentration of inhibitor tested (Table I). The value of 150 for t’he various reactions varied with the nat,ure of the inhibitor. DCJIU was the most pot’ent inhibitor, followed by atrazine and simazine, whereas o-phenanthroline and hydroxylamine were effective only at a high concentration. It is apparent, however, t,hat the 150values for a given inhibitor were essentially the same
for the Hill reaction with ferricyanide and NADP with unt’reated chloroplasts or the ascorbate-supported NADP photoreducion with heated chloroplast’s. On the other hand, each reaction exhibited a charact’eristic Rlr value regardless of t.he nature of t’he poison employed. Complet)e inhibition was observed for the ferricyanide Hill reaction, but t,he NADP Hill reaction was inhibited about, 90% at, t,he highest concentration tested. Furthermore, a significant portion (about 30 %) of t)he ascorbaksupported NADP phot,oreduction \vas always resistant t,o the poisons. In contrast, the inhibition of SADP photoreduction \\-ith ascorbate and DPIP never exceeded 50% of the original activky with any inhibitor tested. Replacement of ascorbate by cysteke. As shown in Table II cysteine could replace ascorbate in supporting NADP photoreduction by heated chloroplast,s. Although the concentration of cysteine required to give the maximal rate of SADl’ photoreduction \vas approximatjely twice that of ascorbate, t,he maximal velocity \vas about the same with the two reduct’ants. The presence of cytochrome-552 was necessary also for the cysteine-supported SADP photoreduction, and the react’ion was inhibit,ed by poisons such as DCnlU. The inhibition with DCbIU
ATRAZINE
(-Log
[Ml)
FIG. 8. Effect of atrazine on the Hill reaction with ferricyanide and NADP and NADP photoreduction by heated chloroplasts with ascorbate and ascorbate plus DPIP. Reaction conditions were as described in Figs. 1 and 2. (U), ferricyanide Hill reaction; (O), NADP Hill reaction; (0), NADP photoreduction with ascorbate; (A), NADP photoreduction with ascorbate and DPIP.
150
KATOH
AND
SAN
PIETRO
TABLE I EFFECT OF INHIBITORS ON THE HILL REACTION WITH UNTREATED ChLortoPL.ksrs NADP PHOTOREDUCTION WITH HEATED CHLOROPLASTS Hill reaction Inhibitor
Ferricyanide 150 (Ed)
DCMU o-Phenanthroline Simazine Hydroxylamine CCCP Atrazine
9.5 9 7.8 4 2.3
TABLE
10-e x 105 x 10-e x RI--* x 10-b X 106
160(aa)
loo 100 90 100 100 90
9.5 9 7.8 4 3
II
CYSTEINE-SUPPORTED BY HEATED
NADP
Reaction conditions except that ascorbate cysteine.
were as described in Fig. 1 was replaced by 20 mM
PHOTOREDUCTION CHLOROPLASTS
Additions
None Cysteine Cysteine
f
low7 M DCMU
CySteine CySteine
+ f
1om6 M DCMU 1o-5 M DCMU
Cysteine + 105 M DCMU + 20 /.&MDPIP (1In the absence was only 4.
of cytochrome-552,
NADP photoreduction NADP
MI (%)
pmoles NADPH/mg chl./hour
0 82a 56 18
12 81
the rate
was alleviated by the further addition of DPIP. These observations indicate that cysteine acts in a manner similar to ascorbate in supporting NADP photoreduction by heated chloroplasts. DISCUSSION
It is clear that when Euglena chloroplasts are heated at mild temperature (40” for 5 minutes), the oxygen evolution system is inhibited preferentially and the Hill activity is lost completely. The heated chloroplasts can still catalyze the photoreduction of NADP with reduced cytochrome-552 or, in in the presence of cytochrome-552, with reduced DPIP as electron donor. A similar specific inhibition of the oxygen evolution system can be achieved with a number of inhibitors. These include hydrox-
AND
10-C x 10-S x 106 x lo-4 x 106 x 10-G
As&at;
Ascorbate MI (%)
80 90 90 90 93 90
Is0 (Ml
lo-6 9.8 X 103 9 x lo-” 10-z 4 X 105 3 X 1O-6
MI (%I
75 75 75 68 63 77
+
Iso (M) MI (%)
L -
20 24 22 7 10 32
ylamine, o-phenanthroline, CMU, DCMU, atrazine, simazine, BIMU, etc. (9, 11-13). Regardless of their divergent chemical structure, they bring about a similar inhibition of the photosynthetic process. Any reaction which involves photosystem 2 is always inhibited by these poisons. Kishimura FLal. (13), and recently Gingras and Lemasson (14), studied the effect of the poisons on the action spectrum and the kinetics of the Hill reaction with spinach chloroplasts and Chlorella cells, respectively. Their results indicate that the poisons inhibited a reaction associated closely with photosystem 2. A similar inference can be drawn from the studies of fluorescence and delayed light emission of photosynthetic systems (15, 16). Duysens and Sweers (17) proposed that the poisons inhibit on the reductive side of photosystem 2 from their experiments on the two lighteffect on fluorescence. More recently, Murata et al. (18) reached a similar conclusion from a study of the kinetics of fluorescence. In contrast, the poisons are without effect on react’ions catalyzed solely by photosystem 1; for example, photoreductive COZ fixation by adapted algae (19), photoreduction of NADP or low potential acceptors lvith reduced DPIP as electron donor (20), photooxidation of cytochrome f in viva and in vitro (21,22), andphotophosphorylation with P&IS as cofactor (23). Our results indicate that the inactivation of the oxygen evolution system by heat involves a mechanism different than that of inhibition by the poisons described above. Vernon and Zaugg (20) showed that the inhibition of SADP photoreduction by poisons
ASCORBATE-SUPPORTED
XADP
PHOTOREDUCTION
151
photosystem 2. The intermediate sensitivit’y of the ascorbate-supported SADP photoreduction could mean there is another moderately heat-labile st,ep between [OJ and cyt’ochrome-552. It is highly likely that the inhibitory site s>srem 2 ascnrbate UPIPHl system 1 C)Stel”c TM PDH z of the poisons in the ascorbate-supported SADP photoreduction system is the same FIG. 9. Proposed mechanism of heat iuactias that in the regular Hill reaction sytem. vation of Euglena chloroplasts. In fact, the agreement between t’he 150values could be alleviated by providing ascorbate for the two reactions is remarkable when one and DPIP as the electron donor couple. The considers the diverse chemical nature of the presence of ascorbate alone was much less poisons tested. The poisons could inhibit’ a react’ion between photosystem 2 and cyt,oeffective in restoration of the act’ivity with chrome-.552 as suggested by Duysens and poisoned chloroplasts (S). On t’he contrary, ascorbate alone supported a high rate of Saeers (17) and Murata et al. (IS). In t,he presence of the poisons, therefore, electron NADP photoreduction with the heat-treated Euglena chloroplast’s. Moreover, this ascor- transfer t’o cyt~ochromeZi2 from ascorbate, bate-supported NADP photoreduct’ion was as from water, via [O,] is blocked. Incomplete inhibition of the ascorbate-supported X;ADP inhibited by t,he poisons. These observaGons form the basis for the photoreduct’ion with high concentration of the poisons might reflect the ability of ascormechanism of heat-inact,ivation of the Hill bate to reduce endogenous electron carriers reaction proposed in Fig. 9. The electron other t>han [O,] and bevond t’he sit,e of inhitransfer system of the Hill reaction requires bition, i.e., in the proximity of cytochrometwo photosystems, phot’osystems 1 and 2 according to the terminology of Duysens and 552. In fact, a slow but continual photoAmesz (21). The result’ant’ of photjosystem 2 reduction of KADP by the poisoned chloroplasts was observed previously (S). is the formation of a weak reductant, [RJ, In the above hypothesis, the role of ascnrand a strong oxidant, [OJ, at, t’he expense of light energy absorbed bv t’he shorter wave- bate is that of an electron donor for SADP photoreduction. An alternat,ive is that nscorlength pigment system. The weak reductant, bat’e simply reactivates the oxygen evolution [R,], reduces ferricyanide directly or indisyst’em of the heated chloroplasts. This imrectly or, in the presence of cytochrome-552 plies that the actual electron donor for provides electrons (or hydrogens) to photoIYADP photoreduction is water and oxvgen system 1 via a series of oxidation-reduction reactions. At the same t’ime, the strong oxi- is evolved during the reaction. Det,ermination of oxygen evolution during the reaction dant, [O,], could oxidize water to produce was diflicuh because of the rapid, light-inmolecular oxygen. This latter reaction might duced oxygen consumption n-hich occurred in involve a very heatlabile st)ep which is in activated preferentially by treat’ment of the presence of nscorbate. However, the folchloroplasts at mild temperatures. In the lowing considerations make this possibility heat#ed chloroplasts, photosystem 2 could unlikely. First, ascorbate must be present in the still be relat,ively intact’ but, seemingly nonreacCon mixt,ure during the react,ion since functional and unable to produce cont’inually [RJ because the necessary removal or reduc- preincubation of the heat,ed chloroplasts wit,h ascorbate did not restore the activity, that tion of [OJ by n-at’er is no longer possible. However, the [OJ can react n-it’h subst,ances is, when the activity n-as measured subsemore reducing than wat’er such as ascorbate quently in the absence of ascorbate. Second, or cysteine. Thus, ascorbate or cysteine could the presence of ascorbate during the heat support a continued production of [R2] by treatment of the chloroplasts did not facilitating removal of [OJ. In other words, preserve the Hill reaction when the activity ascorbate or cysteine can serve in place of of the chloroplasts was measured after water as the ultimate electron donor for removal of the ascorbate by washing the
152
KATOH
AND
chloroplasts by centrifugation. These chloroplasts still showed, however, the DCMUsensitive NADP photoreduction upon readdition of ascorbate to the reaction system. Third, there is evidence which indicates that ascorbate is actually oxidized by the chloroplasts in a stoichiometric manner during an analogous reaction. Marre et al. (24) observed an anaerobic photooxidation of ascorbate by chloroplast fragments in the presence of an XADPH-trapping system. They found that the amount of ascorbate consumed and the amount of reduced glutathione formed were comparable. Ikeda (25) showed that ascorbate could support oxygen consumpt’ion in the light in the presence of menadione or FhIN but in the absence of DPIP. In the reaction, 2 moles of oxygen were consumed per mole of ascorbate, and the oxygen uptake was inhibited by o-phenanthroline. This indicates that the light-induced, ascorbatedependent oxygen consumption studied by Ikeda is another expression of the react’ion described in the present paper. Chiba and Ikayama (26) confirmed the inhibitory effect of o-phenanthroline on t’he photooxidative consumption of ascorbate by chloroplasts and proposed that ascorbate provided electrons to photosyst’em 2. Trebst et al. (27) also noted that t’he photooxidation of several hydroquinones by chloroplast,s was sensitive to DCMU. They showed further that acorbate photooxidation in the presence of a low redox potential quinone was accompanied by photophosphorylation and peroxide formation in a stoichiometric manner. Ascorbate-dependent photophosphorylation was also described by Forti and Jagendorf (28) and recently by Jacobi (29). They suggested that ascorbat’e reacted with the electron transfer system at a site related to the oxygen evolution system. REFERENCES 1. INMAN, 0. L., Cold Spring Harbor Symp. &ant. Biol. 3, 184 (1935). 2. HILL, R., Proc. Roy. Sot. (London), Ser. B 137, 192 (1939). 3. WARBURG, O., AND LUTTGENS, W., Biochimia 11, 303 (1946). 4. HOLT, A. S., .~ND FRENCH, C. S., Arch. Biothem. 9, 25 (1946).
SAN
PIETRO
5. HINKSON, J. W., AND VERNON, L. P., Plant Physiol. 34, 268 (1959). 6. WHATLEY, F. R., TAGAWA, K., AND ARNON, D. I., Proc. Matl. Acad. Sci. U.S. 49, 266 (1967). 7. KATOH, S., -END SAN PIETRO, A., Arch. Biothem. Biophys. 118, 488 (1967): 8. KATOH, S., IND SUN PIETRO, A., Arch. Biothem. Biophys. 121, 211 (1967). 9. WESSELS, J. S. C., AND V-4~ DER VEEN, R., Biochim. Biophys. Acta 19, 548 (1956). 10. ASAHI, T., BND J~GENDORF, A. T., Arch. Biothem. Biophys. 100, 531 (1963). 11. MORELAND, D. E., GENTNER, W. A., HILTON, J. L., .4ND HILL, K. L., Plant Physiol. 34, 432 (1959). 12. GOOD, N. E., Plant Physiol. 36, 788 (1961). 13. NISIIIMUR.~, M., SAKURAE, H., END T.IKaMIYa, A., Biochim. Biophys. Acta 79, 241, (1964). 14. GINGRAS, G., AND LEM.ISSON, C., Biochim. Biophys. Acta 109, 67 (1965). 15. SWEETER, P. B., TODD, C. W., .IND HERSH, R. T., Biochim. Biophys. Acta 61, 509 (1961). 16. BERTSCH, W. F., DAVIDSON, J. B., AND AZZI, J. R., in “Photosynthetic Mechanisms of Green Plants” (B. Kok and A. T. Jagendorf, eds.), p. 701. Natl. Acad. Sci.-Natl. Res. Council (1963). 17. DUYSENS, L. N. M., -&ND SWEERS, H. E., “Studies on Microalgae and Photosynthetic Bacteria,” p. 353. Jap. Sot. Plant Physiol. (1963). 18. MuR.~T.~, N., NISHIMURA, M., AND TAKAMIYA, A., Biochim. Biophys. Acta 120,23 (1963). 19. BISHOP, N. I., Biochim. Biophys. Acta 27, 205 (1958). 20. VERNON, L. P., AND ZAUGG, W. S., J. Biol. Chem. 236, 2728 (1960). 31 DUYSENS, L. N. M., AND AMES~, J., Biochim. __. Biophys. Acta 64, 243 (1962). 22. NIEMAN, R. H., AND VENNESLSND, B., Plant Physiol. 34, 255 (1959). 23. JAGENDORF, A. T., .IND MSRGULIES, M., Arch. Biochem. Biophys. 90, 184 (1960). 24. MARRE, E., ARRIGONI, O., AND ROSSI, G., Biochim. Biophys. Acta 36, 56 (1959). 25. IKED~, S., Mem. Res. Inst. Food Sci. Kyofo Univ. 18, 57 (1959). 26. CHIB~, Y., AND OK~Y.~MA, S., Plant Cell Physiol. 3, 379 (1962). 27. TREBST, A., ECK, H., AND WAGNER, S., il, “Photosynthetic Mechanisms of Green Plants” (B. Kok and A. T. Jagendorf, eds.), p. 174. Natl. Acad. Sci.-Natl. Res. Council (1963). 28. FORTI, G., AND JSGENDORF, A. T., Biochim. Biophys. Acta 64, 322 (1961). 29. JACOBI, G., 2. Naturforsch. 19b, 470 (1964).
OF
BIOCHEMISTRY
AND
122, 144-152 (1967)
BIOPHYSICS
Ascorbate-Supported
NADP Euglena
SAKAE Charles
KATOH2
F. Kettering Received
Photoreduction
Chloroplasts’
AND
ANTHONY
Research Laboratory, March
by Heated
SAN PIETRO
Yellow Springs,
29, 1967; accepted
May
Ohio 46.387
8, 1967
The effect of heating of Euglena chloroplasts on various photooxidation-reduction activities has been studied. When the chloroplasts were heated for 5 minutes at 40” the Hill activity was abolished completely, but there was no significant inhibition of NADP photoreduction with reduced Euglena cytochrome-552 as electron donor. Interestingly, the addition of ascorbate restored the ability of the heated chloroplasts to photoreduce NADP as well as did the ascorbate-DPIP couple. However, the ascorbate-supported NADP photoreduction exhibited an intermediate sensitivity toward treatment of the chloroplasts at higher temperatures or for a longer duration of incubation, that is, lower than for the normal Hill reaction but higher than for the ascorbate-DPIP system. In addition, the ascorbate-supported NADP photoreduction by heated chloroplasts was sensitive to various poisons of the oxygen evolution system, but the ascorbate-DPIP system was not. A possible mechanism to account for heat inactivation of the Hill reaction and reactivation by ascorbate is presented.
The mechanism of oxygen evolution is the least understood facet of photosynthesis even though the production of oxygen during photosynthesis is a well-documented phenomenon (1,2). One explanation may be the very unstable nature of the oxygen evolution system toward physical and chemical modification of chloroplasts. A number of these treatments of chloroplasts will inactivate efiectively and specifically the oxygen evolution system but will leave unchanged many of the other reactions of the photosynthetic electron transport system. The treatment of chloroplasts at mild temperature is one such specific treatment. The Hill activity is abolished easily by heating chloroplasts at relatively low temperatures (3, 4). Hinkson and Vernon (5) found that ‘Contribution No. 284 of the Charles F. Kettering Research Laboratory. This research was supported in part by a research grant (GM10129) from the National Institutes of Health, United States Public Health Service. * Present address: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo, Japan.
incubation of chloroplasts at 51’ for 10 minutes resulted in complete abolition of the Hill activity with DPIP as oxidant. In contrast, indigo carmine photoreduction with ascorbate and DPIP as electron donor and ascorbate photooxidation in the presence of DPIP were reduced only partially. The photoreduction of ferredoxin by mildly heated chloroplasts at the expense of reduced DPIP was described also by Whatley et al. (6). In the present study, the effect of mild heat treatment of Eugkna chloroplasts on the Hill reaction and the photoreduction of NADP with artificial electron donors was investigated. Evidence is presented which indicates that the mechanism of heat inactivation of the Hill reaction is significantly different from the mechanism of inhibition caused by various poisons which are specific inhibitors of the oxygen evolution system. MATERIALS
AND
METHODS
The general experimental procedures employed were described in the preceding papers (7, 8). Heat treatment of chloroplast,s was carried out 144
.
ASCORBATE-SUPPORTED
NADP
in a small test tube containing chloroplasts suspended in the preparation medium (0.4 M sucrose, 0.01 M XaCl, and 0.05 M phosphate buffer, pH 6.5) in a water bath of the desired temperature in the dark. Special care was taken to avoid itlhomogeneous heating of the chloroplasts. Toward this end, incubation of 0.5 ml of a chloroplast suspension containing about 250 pg of chlorophyll with gentle and colrtillrmrls slirring was found to be satisfactory and was used throughout. Incubation of either a larger volume of the ehloroplast, suspension or a more dense suspension of chloroplasts tended to result in an iuhomogclleous prepsratioll, t,herefore, a less selective inhibitinn of the Hill reaction. RESULTS
Kinetics of NADP photoreduction and cytochrome-552 photooxidation-reduction. The
time course of light-induced reduction of ;“\‘ADP and oxidation-reduction of cytochrome-&Q by Euglena chloroplasts which were heated at 45” for 5 minutes is shown in Fig. 1. The absorbance changes at 340 rnp for KADP and at 552 rnp for cytochrome552 were measured with separate but identical reaction mixtures. As reported previously (7), the unheated chloroplasts reduced XADP at a substantial rat,e provided ferredoxin and cytochrome-552 were present (Fig. 1, A). A correlatjive absorbance change ascribable to cyt’ochrome-552 which occurred during XADP photoreduct,ion was also described (S). After heating, Euglena chloroplasts were incapable of catalyzing the Hill reaction with SADP as oxidant and showed only a slight initial increase of absorbance at 340 rnp (Fig. lB, upper curve). The corresponding absorbance change at 552 rnp, which is indicative of an enhanced photooxidation of reduced cytochrome-552, is shown as the lower (dashed) curve of Fig. 1B. Similar absorbance changes were observed previously with chloroplasts poisoned with atrazine (S). It therefore appears that the heat treat,ment of b’uqlena chloroplasts resulted in a preferential destruction of only the oxygen-evolution system (photosystem 2). In contrast, the long wavelength system (photosystem 1) n-as unaffected and, as indicated by a short-lived, initial increase in absorbance at 340 m,u, catalyzed the photoreduction of
145
PHOTOREDUCTION
N6 I,
D o-
0
I
2
3
0
I
2
3
MINUTES
absorbance I?IG. 1. Time course of light-induced changes at 340 rnp and 552 mnMwith heat,ed chloroplasts. Each reaction mixture contained 50 mM phosphate buffer, pH 6.0, 10 m&l NaCl, 0.86 mnr NADP, a saturating amount of spinach ferredoxin, 21 PM Ezcglena cytochrome-552 and Euglena chloroplasts equivalent to 14.2 pg chlorophyll. Final volume, 0.7 ml. Cytochrome-552 was in the reduced form when added except for curves D, in which oxidized cytochrome was added. Unt,reated chloroplasts were used in A and heated chloroplasts ilt B to G. A and B, no addition; C, 50 MM atrazine; D, no addition; E, 14 mu neutralized ascorbate; F, 14 IIIM ascorbate plus 50 PM atrazine; and G, 14 mM ascorbate, 20 MM DPIP, and 50 PM atrazinc. (-), 340 rnw; (-), 552 rnp.
KADP with reduced cytochrome-552 as the electron donor. This conclusion is supported by the observation that the addition of atrazine to the heated chloroplasts had no significant effect on the absorbance changes at 340 and 552 rnF (Fig. 1, C). Furthermore, no absorbance changes were noted when the reaction mixture contained oxidized rat.her than reduced cytochrome-552 (Fig. 1, D). Of special interest was the finding that ascorbate alone could support a substantial rate of NADP photoreduction (Fig. 1, E). This was not the case with the atrazinepoisoned chloroplasts, where ascorbate served only poorly as an electron donor for the photoreduction of NADP (S; Fig. 1, 17). In addition, the ascorbat’e-supported NADP photoreduction by the heated chloroplasts n-as susceptible t.o the poison, although complete inhibition could not be obtained with a
146
KATOH
AND
concentration of the poison which inhibited completely the Hill reaction with untreated chloroplasts (Fig. 1, F). This is in marked contrast to NADP photoreduction with ascorbate and DPIP as electron donor which occurred in the presence of the poison (Fig. 1, G). AS would be expected, there was no appreciable absorbance change at 552 mp in the presence of ascorbate (Fig. 1, E-G) under the conditions employed. Effect of temperature. The Hill reaction with ferricyanide and NADP, as well as NADP photoreduction in the presence of ascorbate or ascorbate and DPIP, were measured using chloroplasts which had been heated for 5 minutes at various temperatures (Fig. 2). It was found that the reactions exhibited markedly different sensitivities toward heat treatment of the chloroplasts. The Hill reaction with ferricyanide was most heat, labile and the activity was lost completely at 40”. The Hill reaction with NADP was somewhat complicated by apparent inactivation during the reaction, The initial rate of NADP photoreduction was moderately sensitive to the heat treatment, and at 40” corresponded to about one-half of the original activity. When the rate of the reaction measured after 2 minutes of illumination was plotted against temperature, the resultant curve was similar to that for ferricyanide reduction except for a residual temperatureinsensitive activity of about 10%. The photoreduction of NADP supported by reduced cytochrome-552 as the electron donor, which became marked concurrent with inactivation of the oxygen evolution system, would account for the apparent temperatureinsensitivity of the initial rate and some of the rate measured 2 minutes after illumination. When the phot,oreduction of NADP was carried out in the presence of ascorbate, the activity was much less affected by heating than the regular Hill reaction (Fig. 2, lower curves). With chloroplasts heated at 40” which exhibited no or very low Hill activity, ascorbate restored the activity to about 70 % of the original activity. Further, there was no significant difference between the initial rate of NADP photoreduction and the rate measured after 2 minutes of illumination.
SAN PIETRO
HILL
O -‘>30
40 TEMPERATURE
REACTION
I 50
60
t-2)
FIG. 2. Effect of temperature on the Hill referricyanide and DPIP and NADP photoreduction with ascorbate and ascorbate plus DPIP. The chloroplasts were heated for 5 minutes at each temperature. Ferricyanide photoreduction was determined with a Clark electrode obtained from the Yellow Springs Instrument Co. The reaction mixture, in a final volume of 4.9 ml, contained 50 mM citrate-phosphate buffer, pH 5.0,lO mM NaCl, 1 mM potassium ferricyanide, and chloroplasts equivalent to 63 pg chlorophyll. NADP photoreduction was determined as described in Fig. 1, and the chloroplast content was equivalent to 13 rg of chlorophyll. Upper curves: (M), ferricyanide Hill reaction; (0, A), initial and a-minute rate, respectively, of the NADP Hill reaction. Lower curves: (IJ), initial and 2-/M minute rate, respectively, of NADP photoreduction in the presence of 14 mM ascorbate; (A, A) initial and a-minute rate, respectively, of NADP photoreduction with 14 mM ascorbate, 20 /IM DPIP, and 50 mM atrazine.
action with
The photoreduction of NADP with ascorbate and DPIP was most resistant to the heat treatment. The activit,y was unaffected, or increased slightly, at temperatures up to 40”, and a t’emperature slightly above .50” was required to decrease the activity to onehalf. In this case also, the initial rate and the rate measured after 2 minutes of illumination were the same. Effect of time of heating at 40” on NADP
ASCORBATE-SUPPORTED
NADP
photoreduction. The Hill reaction with ferricyanide and that with NADP determined 2 minutes after illumination were very sensitive to heat treat’ment of chloroplasts at 40” and were lost completely after 3 minutes of heating (l?ig. 3). Again, the initial rate of the NADP Hill reaction was less sensitive than the rate measured after 2 minutes of illumination. The presence of ascorbate increased greatly t’he activity for NADP photoreduction by the heated chloroplast’s. A further increase was obtained by the subsequent addition of DPIP. E$ect of temperature on Cytochrome-552 photooxidation-leduction. The photooxidation and photoreduction of cytochrome-552 represent partial reactions of the P\‘ADP Hill react,ion and are associat,ed wit,h photosysterns 1 and 2, respectively (8). Figure 4 shows the effect of temperature on these two reactions. The photoreduction of cytochrome-552 leas as sensit,ive to heat,ing as the Hill reacbion with ferricyanide and reflects the heat sensitivity of photosystem 2. On the other hand, t,he photooxidat’ion of
HILL
NADP
REACTION
PHOTOREDUCTION
I
I OO
2
I TIME
OF
3 INCWXTION
4 (MIN)
5
FIG. 3. Effect of time of heating on the Hill reaction with ferricyanide and NADP and NADP photoredllction in the presence of ascorbate and ascorbate plus DPIP. Reaction conditions were as described in Fig. 2. Temperature of heating, 40”.
147
PHOTOREDUCTION
P,
0'L, 0
I
' 30
50 TE%RATURE
60
(“C)
FIG. 4. Photooxidation and photoreduction of cytochrome-552 by chloroplasts heated for 5 minutes at various temperatures. Photooxidation of reduced cytochrome-552 was determined wit,h a reaction mixture of 0.7 ml which contained 50 mM phosphate buffer, pH 6.0, 1 mM methyl vioand chlorologen, 53 pM reduced cytochrome-552, plasts equivalent to 6 pg of chlorophyll. The reaction mixture employed for photoreduction contained 50 mM phosphate buffer, pH 6.0, 106 PM oxidized cytochrome-552, 1 mM NaCl, and chloroplasts equivalent to 18 rg of chlorophyll in a final volume of 2 ml. Photoreduction of cytochrome was carried out anaerobically.
reduced cytochrome-,552, catalyzed by photosystem 1, was markedly resistant’ to heating. At t’he lower temperatures, there was either no effect or a slight increase in activity; at’ temperat’ures above 50”, the activity decreased. Reactim cmditions for ascorbate-suppoffed NADP photoreduction by heated chloroplasts. The preceding observations indicate that the ascorbate-supported NADP photoreduction by heated chloroplasts is significantly different from NADP photoreduction using either water or the ascorbate-DPIP couple as electron donor. The reaction conditions for the ascorbate-supported NADP photoreduction mere elucidated wit,h Euglena chloroplasts which were heat’ed for 5 minutes at 45”. Figure 5 shows the effect of ascorbate concentration on the rate of NADP photoreduct,ion. Since t,he reaction was linear with time (see Fig. 1, E), only the initial rate of reducGon
is plotted
against
ascorbate
con-
148
KATOH
AND
OO24 ASCORBATE
( pmoles)
FIG. 5. Dependence of NADP photoreduction by heated chloroplasts on ascorbate concentration. Reaction conditions were as described in Fig. 1, E and G, except that the concentration of ascorbate was varied as indicated.
centration. The activity observed without ascorbate represents NADP photoreduction coupled to the oxidation of reduced cytochrome-552. It was shown previously that the photooxidation of reduced cytochrome552 contributed to the absorbance increase at 340 rnp (8). The observation that the rate of NADP photoreduction decreased at low ascorbate concentrat8ion means that this amount of ascorbate maintains cytochrome552 in the reduced form but cannot support NADP photoreduction at an efficient rate. Aftjer this initial decrease, the rate of NADP reduction increased with increasing concentration of ascorbate and reached a constant rate which was independent of ascorbat,e concentration. A similar dependence on ascorbate concentration was noted for NADP photo reduction wit,h the ascorbate-DPIP couple (Fig. 5), The data presented in Fig. 6 shows clearly that cytochrome-552 is essential for the ascorbate-supported NADP photoreduction by heated chloroplasts. There was no NADP photbreduction in the absence of the cytochrome; at low cytochrome concentrations, the rate was related linearly to the cytochrome concentration. At higher cyt’ochrome concentrations, the rate was maximal and independent of the cytochrome concentration. The concentration of cytochrome required for maximal activity was similar to that for the NADP Hill reaction, i.e., about 10 mpmoles per cuvet,te (7).
SAN
PIETRO
The pH-activity curves for NADP photoreduction are shown in Fig. 7. The ascorbatesupported NADP photoreduction exhibited maximal activity at a pH slightly higher than 6. The addition of DPIP resulted in a slight shift of the curve to a higher pH. These curves are again comparable with those for the NADP Hill reaction. Sensitivity toward various poisons. A variety of inhibitors such as DCMU inhibit the Hill reaction at very low concentration (9). Certain reactions which are thought not to require the oxygen evolution system were inhibited at high concentrations of the poisons. For example, Asahi and Jagendorf (10) reported that 1O-4 M CMU inhibited PMScatalyzed photo-phosphorylation. It was important, therefore, t’o compare the sensitivity of the ascorbate-supported NADP photoreduction toward various poisons with that of the Hill reaction or the photoreduction of NADP with the ascorbate-DPIP couple as the electron donor. Figure 8 shows the effect of atrazine concentration on the Hill reaction of untreated chloroplasts nith ferricyanide and NADP, as well as on the NADP photoreduction by heated chloroplasts with either ascorbate or ascorbate and DPIP as electron donor. The percentage inhibition is defined as (one-rate with inhibitor/rate without inhibitor) X 100 and is plotted against the logarithm of the molar concentration of the poison. The Hill reaction with ferricyanide and
30 IO 20 CYTOCHROME 552 (mpmoles)
40
FIG. 6. Dependence of NADP photoreduction by heated chloroplasts on reduced cytochrome-552 concentration. Reaction conditions were as described in Fig. 1, E and G, except the concentration of reduced cytochrome-552 was varied as indicated.
149
ASCORBATE-SUPPORTED NADP PHOTOREDUCTIOK
r’ 601!
.4 DP’P n-. . \ . . Y ’ . \\ . .
\ .-. 1 OL-----J 5
7
6
8
9
PH FIG. 7. PII-dependency of ?r’ADP photoreduction by heated chloroplasts. Reaction couditions were as described in Fig. 1, E and G, except t,hat the pH was varied by using 50 rnbr citrate-phosphate or phosphate buffer. ( q ), no addition (2.minute rate); (U), ascorbate; (A, ascorbate plus DPIP.
SADP with untreated chloroplasts was inhibited significantly by low concentrations of atrazine. Also, the ascorbate-supported photoreduction of KADP by heated chloroplasts exhibitled a sensitivity comparable to that of the Hill react,ion, although inhibit,ion was less complete at higher concentratjions of the poison. In contrast, NADP photoreduct’ion with ascorbate and DPIP by heated chloroplasts was much less sensitive t,o the poison, and only about 30 % inhibition was obt,ained at, the highest concentration of t,he poison tested. Similar experiments were carried out with several other inhibitors. For comparison, the data are presented both as t’he concentrat8ion required for 50% inhibition (&J and the maximum inhibit’ion (MI), obtained nith the highest, concentration of inhibitor tested (Table I). The value of 150 for t’he various reactions varied with the nat,ure of the inhibitor. DCJIU was the most pot’ent inhibitor, followed by atrazine and simazine, whereas o-phenanthroline and hydroxylamine were effective only at a high concentration. It is apparent, however, t,hat the 150values for a given inhibitor were essentially the same
for the Hill reaction with ferricyanide and NADP with unt’reated chloroplasts or the ascorbate-supported NADP photoreducion with heated chloroplast’s. On the other hand, each reaction exhibited a charact’eristic Rlr value regardless of t.he nature of t’he poison employed. Complet)e inhibition was observed for the ferricyanide Hill reaction, but t,he NADP Hill reaction was inhibited about, 90% at, t,he highest concentration tested. Furthermore, a significant portion (about 30 %) of t)he ascorbaksupported NADP phot,oreduction \vas always resistant t,o the poisons. In contrast, the inhibition of SADP photoreduction \\-ith ascorbate and DPIP never exceeded 50% of the original activky with any inhibitor tested. Replacement of ascorbate by cysteke. As shown in Table II cysteine could replace ascorbate in supporting NADP photoreduction by heated chloroplast,s. Although the concentration of cysteine required to give the maximal rate of SADl’ photoreduction \vas approximatjely twice that of ascorbate, t,he maximal velocity \vas about the same with the two reduct’ants. The presence of cytochrome-552 was necessary also for the cysteine-supported SADP photoreduction, and the react’ion was inhibit,ed by poisons such as DCnlU. The inhibition with DCbIU
ATRAZINE
(-Log
[Ml)
FIG. 8. Effect of atrazine on the Hill reaction with ferricyanide and NADP and NADP photoreduction by heated chloroplasts with ascorbate and ascorbate plus DPIP. Reaction conditions were as described in Figs. 1 and 2. (U), ferricyanide Hill reaction; (O), NADP Hill reaction; (0), NADP photoreduction with ascorbate; (A), NADP photoreduction with ascorbate and DPIP.
150
KATOH
AND
SAN
PIETRO
TABLE I EFFECT OF INHIBITORS ON THE HILL REACTION WITH UNTREATED ChLortoPL.ksrs NADP PHOTOREDUCTION WITH HEATED CHLOROPLASTS Hill reaction Inhibitor
Ferricyanide 150 (Ed)
DCMU o-Phenanthroline Simazine Hydroxylamine CCCP Atrazine
9.5 9 7.8 4 2.3
TABLE
10-e x 105 x 10-e x RI--* x 10-b X 106
160(aa)
loo 100 90 100 100 90
9.5 9 7.8 4 3
II
CYSTEINE-SUPPORTED BY HEATED
NADP
Reaction conditions except that ascorbate cysteine.
were as described in Fig. 1 was replaced by 20 mM
PHOTOREDUCTION CHLOROPLASTS
Additions
None Cysteine Cysteine
f
low7 M DCMU
CySteine CySteine
+ f
1om6 M DCMU 1o-5 M DCMU
Cysteine + 105 M DCMU + 20 /.&MDPIP (1In the absence was only 4.
of cytochrome-552,
NADP photoreduction NADP
MI (%)
pmoles NADPH/mg chl./hour
0 82a 56 18
12 81
the rate
was alleviated by the further addition of DPIP. These observations indicate that cysteine acts in a manner similar to ascorbate in supporting NADP photoreduction by heated chloroplasts. DISCUSSION
It is clear that when Euglena chloroplasts are heated at mild temperature (40” for 5 minutes), the oxygen evolution system is inhibited preferentially and the Hill activity is lost completely. The heated chloroplasts can still catalyze the photoreduction of NADP with reduced cytochrome-552 or, in in the presence of cytochrome-552, with reduced DPIP as electron donor. A similar specific inhibition of the oxygen evolution system can be achieved with a number of inhibitors. These include hydrox-
AND
10-C x 10-S x 106 x lo-4 x 106 x 10-G
As&at;
Ascorbate MI (%)
80 90 90 90 93 90
Is0 (Ml
lo-6 9.8 X 103 9 x lo-” 10-z 4 X 105 3 X 1O-6
MI (%I
75 75 75 68 63 77
+
Iso (M) MI (%)
L -
20 24 22 7 10 32
ylamine, o-phenanthroline, CMU, DCMU, atrazine, simazine, BIMU, etc. (9, 11-13). Regardless of their divergent chemical structure, they bring about a similar inhibition of the photosynthetic process. Any reaction which involves photosystem 2 is always inhibited by these poisons. Kishimura FLal. (13), and recently Gingras and Lemasson (14), studied the effect of the poisons on the action spectrum and the kinetics of the Hill reaction with spinach chloroplasts and Chlorella cells, respectively. Their results indicate that the poisons inhibited a reaction associated closely with photosystem 2. A similar inference can be drawn from the studies of fluorescence and delayed light emission of photosynthetic systems (15, 16). Duysens and Sweers (17) proposed that the poisons inhibit on the reductive side of photosystem 2 from their experiments on the two lighteffect on fluorescence. More recently, Murata et al. (18) reached a similar conclusion from a study of the kinetics of fluorescence. In contrast, the poisons are without effect on react’ions catalyzed solely by photosystem 1; for example, photoreductive COZ fixation by adapted algae (19), photoreduction of NADP or low potential acceptors lvith reduced DPIP as electron donor (20), photooxidation of cytochrome f in viva and in vitro (21,22), andphotophosphorylation with P&IS as cofactor (23). Our results indicate that the inactivation of the oxygen evolution system by heat involves a mechanism different than that of inhibition by the poisons described above. Vernon and Zaugg (20) showed that the inhibition of SADP photoreduction by poisons
ASCORBATE-SUPPORTED
XADP
PHOTOREDUCTION
151
photosystem 2. The intermediate sensitivit’y of the ascorbate-supported SADP photoreduction could mean there is another moderately heat-labile st,ep between [OJ and cyt’ochrome-552. It is highly likely that the inhibitory site s>srem 2 ascnrbate UPIPHl system 1 C)Stel”c TM PDH z of the poisons in the ascorbate-supported SADP photoreduction system is the same FIG. 9. Proposed mechanism of heat iuactias that in the regular Hill reaction sytem. vation of Euglena chloroplasts. In fact, the agreement between t’he 150values could be alleviated by providing ascorbate for the two reactions is remarkable when one and DPIP as the electron donor couple. The considers the diverse chemical nature of the presence of ascorbate alone was much less poisons tested. The poisons could inhibit’ a react’ion between photosystem 2 and cyt,oeffective in restoration of the act’ivity with chrome-.552 as suggested by Duysens and poisoned chloroplasts (S). On t’he contrary, ascorbate alone supported a high rate of Saeers (17) and Murata et al. (IS). In t,he presence of the poisons, therefore, electron NADP photoreduction with the heat-treated Euglena chloroplast’s. Moreover, this ascor- transfer t’o cyt~ochromeZi2 from ascorbate, bate-supported NADP photoreduct’ion was as from water, via [O,] is blocked. Incomplete inhibition of the ascorbate-supported X;ADP inhibited by t,he poisons. These observaGons form the basis for the photoreduct’ion with high concentration of the poisons might reflect the ability of ascormechanism of heat-inact,ivation of the Hill bate to reduce endogenous electron carriers reaction proposed in Fig. 9. The electron other t>han [O,] and bevond t’he sit,e of inhitransfer system of the Hill reaction requires bition, i.e., in the proximity of cytochrometwo photosystems, phot’osystems 1 and 2 according to the terminology of Duysens and 552. In fact, a slow but continual photoAmesz (21). The result’ant’ of photjosystem 2 reduction of KADP by the poisoned chloroplasts was observed previously (S). is the formation of a weak reductant, [RJ, In the above hypothesis, the role of ascnrand a strong oxidant, [OJ, at, t’he expense of light energy absorbed bv t’he shorter wave- bate is that of an electron donor for SADP photoreduction. An alternat,ive is that nscorlength pigment system. The weak reductant, bat’e simply reactivates the oxygen evolution [R,], reduces ferricyanide directly or indisyst’em of the heated chloroplasts. This imrectly or, in the presence of cytochrome-552 plies that the actual electron donor for provides electrons (or hydrogens) to photoIYADP photoreduction is water and oxvgen system 1 via a series of oxidation-reduction reactions. At the same t’ime, the strong oxi- is evolved during the reaction. Det,ermination of oxygen evolution during the reaction dant, [O,], could oxidize water to produce was diflicuh because of the rapid, light-inmolecular oxygen. This latter reaction might duced oxygen consumption n-hich occurred in involve a very heatlabile st)ep which is in activated preferentially by treat’ment of the presence of nscorbate. However, the folchloroplasts at mild temperatures. In the lowing considerations make this possibility heat#ed chloroplasts, photosystem 2 could unlikely. First, ascorbate must be present in the still be relat,ively intact’ but, seemingly nonreacCon mixt,ure during the react,ion since functional and unable to produce cont’inually [RJ because the necessary removal or reduc- preincubation of the heat,ed chloroplasts wit,h ascorbate did not restore the activity, that tion of [OJ by n-at’er is no longer possible. However, the [OJ can react n-it’h subst,ances is, when the activity n-as measured subsemore reducing than wat’er such as ascorbate quently in the absence of ascorbate. Second, or cysteine. Thus, ascorbate or cysteine could the presence of ascorbate during the heat support a continued production of [R2] by treatment of the chloroplasts did not facilitating removal of [OJ. In other words, preserve the Hill reaction when the activity ascorbate or cysteine can serve in place of of the chloroplasts was measured after water as the ultimate electron donor for removal of the ascorbate by washing the
152
KATOH
AND
chloroplasts by centrifugation. These chloroplasts still showed, however, the DCMUsensitive NADP photoreduction upon readdition of ascorbate to the reaction system. Third, there is evidence which indicates that ascorbate is actually oxidized by the chloroplasts in a stoichiometric manner during an analogous reaction. Marre et al. (24) observed an anaerobic photooxidation of ascorbate by chloroplast fragments in the presence of an XADPH-trapping system. They found that the amount of ascorbate consumed and the amount of reduced glutathione formed were comparable. Ikeda (25) showed that ascorbate could support oxygen consumpt’ion in the light in the presence of menadione or FhIN but in the absence of DPIP. In the reaction, 2 moles of oxygen were consumed per mole of ascorbate, and the oxygen uptake was inhibited by o-phenanthroline. This indicates that the light-induced, ascorbatedependent oxygen consumption studied by Ikeda is another expression of the react’ion described in the present paper. Chiba and Ikayama (26) confirmed the inhibitory effect of o-phenanthroline on t’he photooxidative consumption of ascorbate by chloroplasts and proposed that ascorbate provided electrons to photosyst’em 2. Trebst et al. (27) also noted that t’he photooxidation of several hydroquinones by chloroplast,s was sensitive to DCMU. They showed further that acorbate photooxidation in the presence of a low redox potential quinone was accompanied by photophosphorylation and peroxide formation in a stoichiometric manner. Ascorbate-dependent photophosphorylation was also described by Forti and Jagendorf (28) and recently by Jacobi (29). They suggested that ascorbat’e reacted with the electron transfer system at a site related to the oxygen evolution system. REFERENCES 1. INMAN, 0. L., Cold Spring Harbor Symp. &ant. Biol. 3, 184 (1935). 2. HILL, R., Proc. Roy. Sot. (London), Ser. B 137, 192 (1939). 3. WARBURG, O., AND LUTTGENS, W., Biochimia 11, 303 (1946). 4. HOLT, A. S., .~ND FRENCH, C. S., Arch. Biothem. 9, 25 (1946).
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