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The role of C-type cytochrome in the Hill reaction with Euglena chloroplasts
ARCHIVES
OF
BIOCHEMISTRY
AND
The Role of C-Type
BIOPHYSICS
488-496 (1967)
118,
Cytochrome
in The Hill Reaction
with
Euglena
Chloroplasts’ SAKAE
KATOH
Charles F. Kettering
AND
ANTHONY
Research Laboratory,
Yellow
SAN PIETRO Springs,
Ohio 45387
Received July 19, 1966 Euglena chloroplasts catalyze the Hill reaction with ferricyanide or dichlorophenol indophenol (DPIP) but not with nicotinamide adenine dinucleotide phosphate (NADP), methyl viologen or horse-heart cytochrome c. The latter compounds can serve as Hill oxidants with Euglena chloroplasts provided Euglena cytochrome-552 is included in the reaction mixture. This c-type cytochrome is solubilized during preparation of the algal chloroplasts. It acts in a catalytic fashion and appears to function in the electron transport chain which interconnects the two photosystems of photosynthesis. Euglena cytochrome-552 is most effective in restoration of NADP photoreduction activity; some restoration is observed with another algal c-type cytochrome, Porphyra tenera cytochrome-553. In contrast, restoration was not observed with either Euglena cytochrome-556 or horse-heart cytochrome c.
The occurrence of cytochromes in various algae and the extraction and partial purification of c-type cytochrome from the red alga, Porphyra tenera, were described first by Yakushiji (1). The involvement of cytochromes in the photosynthetic process was suggested by Hill and Scarisbrick (a), who found a c-type cytochrome, cytochrome f, in the photosynthetic apparatus of higher plants. Davenport and Hill (3) noted the presence of cytochrome(s) with an absorption maximum at 552-553 rnp in an acetone powder of several algae, including Euglena, which they believed was a composite of the absorption maxima of cytochromes c and f. Katoh (4) extended these observations and found,that a soluble c-type cytochrome was ubiquitous among green, brown, red, and blue-green algae. Although many of the chemical and physical properties of this cytochrome were similar to those of mito1 Contribution No. 257 of the Charles F. Kettering Research Laboratory. Supported in part by Grant GM-10129 from the National Institutes of Health, United States Public Health Service.
chondrial cytochrome c (5), its oxidationreduction potential was above 300 mV (4). It was localized in the photosynthetic apparatus (6) indicating that, like cytochrome f in higher plants, the cytochrome was functional in photosynthesis rather than in respiratory metabolism. In accord with this assumption was the finding that a chlorophyll-containing preparation from P. tenera catalyzed a rapid and cyanide-insensitive photooxidation of P. tenera cytochrome-553; whereas, the dark oxidation of the cytochrome by the algal extract was insignificant (5). The presence of a similar cytochrome in green cells of Euglena was first observed by Nishimura (7). This study, and the subsequent studies of Wolken and Gross (S), Smillie (9), and Perini et al. (lo), revealed that the cytochrome was absent from either dark-grown Euglena or chlorophylless mutants and appeared concomitant with the light-induced formation of chlorophyll. Additional evidence for the role of algal cytochrome(s) in photosynthesis comes from the spectrophotometric studies of the light488
PHOTORE,4CTIONS
OF EUGLENa4
induced oxidation-reduction of cytochromes in algae. Duysens (11) observed that illumination of the red alga, Porph~rirliurn cruentu?n, caused oxidation of a c-type cytochrome. Duysens and Amesz (12) showed further t’hat light absorbed by chlorophyll a (690 mp) was much more effcctivc for oxidation of a c-type cytochrome t’han was light absorbed by phycoerythrin (560 mp). Reduction of the cytochrome oxidized by 690-rnp light occurred on illumination with 560-rnp light. Thus, they suggested that, the cytochrome was an electron c:arrier in the electron-transfer system connect’ing the two photosystems of photosynthesis, i.e., Photosystems I and II according to their terminology (12). A similar response of cytochrome-552 in Euglena cells to light of different, wavelength was described by Olson and Smillie (13) : far-red light. was effective for cytochrome oxidation; whereas, light of shorter wavelength was effective for oxidation, and subsequent reduction, of the cytochrome. The results described in this paper relate to t,hc role of algal c-type cytochrome in the photochemical activities of algal chloroplasts. Euglena chloroplasts, devoid of cytochrome-552, were employed and t’he effect of the cytochrome on the photooxidation-reduction system of the preparations was studied. MATERIALS
AND
METHODS
Growth of Euglenn. E. yracilis, strain Z, WAS allt,otrophically or hetrotrophically as grown described by Evans and San Piet.ro (14). Preparation of chloroplasfs. Cells from 2 liters of an autotrophic culture, grown for 3 or 4 days, were harvested by cent,rifugation and washed with 0.05.nz phosphate buffer, pH 7. The washed cells were suspended in 50 ml of 0.05.M phosphate, pH 7, containing 0.4+1 sucrose and 0.01.~ NaCl, and disrupted with a French press at 500 psi. The broken cell suspension was centrifuged at 1OOg for 5 minutes and then 270g for 5 minutes to remove whole cells and large cell fragments, and the chloroplasts were collected by centjrifugation of the sllpernatant, fraction at 1OOOgfor 10 minutes. The chloroplast,s were washed once with, and finally suspended in, the above medium. Microscopic inspection showed that the chloroplast preparation consisted mainly of whole chloroplasts of fairly uniform size and some chloroplast
CHLOROPLASTS
4s9
fragments but was completely free from unbroken cells and larger cell fragments. Prepuration of redoz profeins. Euglena cyt ochrome-552 and cytochrome-556 were prepared according to the method of Perini el al. (15). Cytochrome-552 was extracted from light-grown green cells and purified by repeated column chromatography using DEdE cellulose. Heterotrophic cells, grown in the dark, were rlsed as the source of cyt ochrome-55G, which was partially ptlrified by a single passage t.hrorlgh a cnrboxymetjhyl cellulose col~mm. P. lenet~s rytochrome553 was prepared as described previously (16) from dried algal thalli obtained from a local market Ferrcdoxill and plast ocyanin were prepared from spinach leaves (17, 18). Transhgdrogenase (ferredoxill-NAIlI’ reductase) was kindly provided by 1)r. 1). I,. Keister (19). Jssaq of a&&es. In general, Hill reactions were carried 011t, in a crivet,te (1 X 1 X 4 rm) placed in a Berkman spectrophotometer (Model DB) as described by Zallgg (20) with certain modifications. The light, sollrce was a Unitron lamp which provided, through a microsrope Corning glass filter (No. 2103), a light intensity of abollt 3.7 X 106 ergs/set. cm2. An appropriate glass filter was inserted between the cuvette and phototj~~be to prevent scattered light, from reaching the phototlrbe. Xach reaction mist iire contained in a final volllme of 2 ml: 100 @moles of buffer, 20 pmoles of N&l, rhloroplasts, and the electron acceptor. Phosphate butier, pH 6, was used for NADP (0.4 pmole) and cytochrome c (horse heart, 0.12 pmole) photoreduction and the reaction mixt,ure rolltained all exress of spinach ferredoxin. For the redllction of fcrricyatnide (2 pmolcs) or DPIP (0.1 pmole), citrate-phosphitt e buffer, pH 5, was llsed. In some experiments, the final volume of the reaction mixtllre was decreased to 0.7 ml and a smaller cllvette (0.5 X 1.0 X 4 cm) was used. The ronrcntration of t,he various components was decreased proportionately. Corrections were applied for the changes in the absorption spectrum of I>PIP at. different pH’s. Additional components are indirated for the respective experiments. The photoreclrlctioll of NAI)P with the ascorbate-DPIP corlple was carried olltj anaerobically in a Thlmberg-type cuvette. Prodllction or uptake of oxygen was measured losing a Clark elect.rodc in a semiclosed vessel which was illuminated, t.hrough 20 cm of water and 5 cm of a CUSO~ solution, by a 1030-w turlgsten lamp. The light, intensity was approximately 4000 ft candles. tiarh react,ion mixture contained in a final volume of 4.8 ml: 250 j.moles of cit,rat.ephosphate buf’fer, pH 5; 50 pmoles of NaCl; chlo-
490
KATOH
AND
roplasts; and 0.4 pmole of DPIP or 5 pmoles of ferricyanide. To determine oxygen uptake according to the method of Mehler (21)) 4 mg of catalase and 1.1 mmoles of ethanol was added. Chlorophyll was determined by the method of Arnon (22).
SAN PIETRO
2
.6
RESULTS
Photoreduction activity of Euglena chloroplasts. The photoreduction activity of EugZena chloroplasts with several oxidants are presented in Table I. Both DPIP and ferricyanide were efficient electron acceptors for the Hill reaction system of Eugkna chloroplasts. In contrast, the photoreduction of NADP and cytochrome c was very low even in the presence of excess ferredoxin. The photoreduction of methyl viologen was insignificant as measured by the photochemical uptake of oxygen by Euglena chloroplasts in the presence of catalase and ethanol (21). It seemed, therefore, that Euglena chloroplasts could photoreduce only electron acceptors with a rather high oxidationreduction potential but not those with low potentials. Cytochrome c is included in the TABLE
I
PHOTOREDUCTION ACTIVITY OF Euglena CHLOROPLASTS WITH VARIOUS OXIDANTP Electron Acceptor
DPIP Ferricyanide NADP NADP plus ascorbate Cytochrome c Methyl viologen
and DPIP
146 237 12 9 6 4b
a Photoreductions were measured spectrophoas described in the section on tometrically, Methods, and the reaction mixture contained Euglena chloroplasts equivalent to 48 pg of chlorophyll. Where indicated, 15 pmoles of ascorbate, neutralized with NaOH, and 0.04 pmole of DPIP were added. b Determined polarographically with an oxygen electrode. The reaction mixture contained phosphate buffer, pH 6, Euglena chloroplasts equiva-
lent to 68 pg. of chlorophyll,
10 pmoles of methyl
viologen, catalase and ethanol. The rate is presented as pmoles of oxygen consumed per milligram chlorophyll per hour.
ON
I
0
I
I
tCyt.552 I
2 3 TIME (MINI
-NADP I
4
5
FIG. l.Photoreductionof NADPin the presence and absence of cytochrome-552. The change in absorbance at 340 rnp was measured with a Beckman Model DB spectrophotometer. Reaction mixture of 2 ml contained chloroplasts equivalent to 37 rg of chlorophyll and, where indicated, 0.033 pmole of reduced cytochrome-552. Other
experimental Methods
conditions were as described in the
section.
“low” redox potential group because its reduction by chloroplasts requires an intermediary electron carrier such as ferredoxin (23, 24). It appeared, therefore, that either Euglena chloroplasts lack the activity associated with Photosystem I or that interaction of the two photosystems in these chloroplasts is prevented. That is, some component of the electron-transport system which interconnects the two photosystems was removed during preparation of the chloroplasts. The results presented below demonstrate that the latter explanation is the correct one. E$ect of cytochrome-552 on Hill reaction with NADP. Smillie (9) and Perini et al. (10) found that cytochrome-552 was located originally in the chloroplasts but was very easily solubilized during preparation of chloroplasts in an aqueous medium. This observation was confirmed in the present study. The difference spectrum (reduced minus oxidized) of an aqueous suspension of an acetone powder of Euglena chloroplasts showed an absorption maximum around 560 rnp, indicative of a b-type cytochrome, but no appreciable absorbance indicative of cytochrome-552. It is clear, therefore, that the inability of Euglena
PHOTOREACTIONS TABLE REACTION
II
COMPONENTS FOR PHOTOREDUCTION~
Reaction system
Complete system minus cytochrome-552 minus ferredoxin plus transhydrogenase plus DCMU
OF EUGLENA
NADP
Rate of NADP Reduction rmolesjmg chl.hr
117 9 0 106 10*
5 The complete system contained 100 rmoles of phosphate, pH 6.0, 0.4 pmole of NADP, a saturating amount of spinach ferredoxin, 0.04 pmole of reduced Euglena cytochrome-552, and chloroplasts equivalent to 24 ug of chlorophyll. Where indicated, 0.004 pmole of DCMU or 45 units (as NADPH: DPIP oxidoreductase) of spinach transhydrogenase (19) were added. * Initial rate: see text.
chloroplasts to photoreduce low-potential substances (Table I) could be related to the loss of cytochrome-552 from the electrontransport system of the chloroplasts. If such were the case, the addition of cytochrome-552 to the chloroplasts should restore the Hill activity with NADP as the oxidant. The data presented in Fig. 1 indicate clearly that this is the case. The rate of NADP photoreduction by Euglena chloroplasts was enhanced markedly on addition of cytochrome-552, and became comparable to that of DPIP or ferricyanide photoreduction. In the presence of cytochrome552, the rate of reduction of NADP was constant during four minutes of illumination; whereas, the low endogenous rate of photoreduction of NADP decreased rapidly. There was no appreciable change in absorbance at 340 rnp in the absence of NADP but in the presence of cytochrome-552. In contrast to NADP photoreduction, the addition of cytochrome-552 had no significant effect on the rate of oxygen consumption by chloroplasts with ferricyanide as oxidant. In the case of the DPIPHill reaction, however, the rate of reduction of DPIP, measured spectrophotometrically was stimulated somewhat when cytochrome552 was present. Reaction components for NADP photo-
CHLOROPLASTS
491
reduction. The data presented in Table II show that the photoreduction of NADP by Euglena chloroplasts requires both ferredoxin and cytochrome-552. The addition of ferredoxin-NADP reductase (transhydrogenase) has no effect or was slightly inhibitory. Presumably, the Euglena chloroplasts contain the reductase in sufficient concentration. Further addition of the reductase to the reaction system was inhibitory probably because of an acceleration of the rate of reoxidation of NADPH (25). In the presence of 10-5-~ DCMU, which completely inhibited the Hill reaction with ferricyanide and DPIP, some slight photoreduction of NADP occurred initially, but the rate dropped to zero within a few minutes. This sensitivity to DCMU indicates that the cytochrome-552 activated NADP photoreduction is a Hill reaction. In the presence of DCMU, the amount of NADP reduced appears to be related to the amount of cytochrome-552 present. Since the cytochrome-552 is present in the reduced form, it can serve as the electron donor for NADP reduction. Details of the inhibitory effect of DCiWJ on NADP photoreduction will be described elsewhere. It was also observed that, on addition of cytochrome-552, Euglena chloroplasts evolve oxygen with NADP as oxidant at a rate 1
%
a z
4 E i
0
-I
0
IO
20 CYTOCHROME 552 CONC.(pM)
30
FIG. 2. EffectofEuglenacytochrome-552concen. lration on photoreduction of NADP. Reaction conditions were as described in Fig. 1, except that the concentration of cytochrome-552 was varied.
492
KATOH
comparable to that photometrically.
determined
AND
spectro-
Concentration of cytochrome-552. The relationship between the rate of NADP photoreduction and the concentration of cytochrome-552 is shown in Fig. 2. The rate of reduction was optimal when the concentration of cytochrome-552 was approximately 15-20 pM. The optimal ooncentration of cyt’ochrome-552 seems rather high and was equivalent to the chlorophyll concentration (20.5 PM). However, in the presence of 18.5 PM cyt,ochrome-552, that is, 37 mpmoles in 2 ml, 240 mpmoles of WADP were reduced in four minutes of illumination (Fig. 1). In contjrast, t’he amount of NADP reduced in the absence of cytochrome-552, was only 29 mpmoles, thereby indicating t,he catalytic function of the cytochrome in NADP photoreduction (Fig. 1). E$ect of pH. The pH dependency of the Hill activity of Euglena chloroplasts is shown in Fig. 3. The photoreduction of I
A
/
4
I
0 DPIP n
5
SAN PIETRO
0’ 0
I I I 1000 2000 LIGHT INTENSITY (foot candles)
FIG. 4. Effect of light intensity on photoreduction of ferricyanide and NADP. Each reaction mixture of 2 ml, containing chloroplasts equivalent to 42 pg of chlorophyll, was illuminated with a 200-W tungsten lamp through a wat,er layer of 7 cm thickness. Light intensity was varied by placing the lamp at different distances from the reaction mixture. The absorbance at 420 rn* for ferricyanide, and at 340 rnp for NADP, photoreduction was determined before and after illumination of 2 minlltes. For NADP photoreduction, 0.033 pmole of cytochrome-552 was added to each reaction mixture.
Fe+++cy
6
7
5
6
7
8
PH FIG. 3. PH-dependency of photoreduction of ferricyanide and NADP. The buffers employed were citrate-phosphate (for pH’s below 5.5) and phosphate (for pH’s higher than 5.6). Hill activities with ferricyanide and DPIP (A) were determined polarographically; NADP photoreduction (B) was determined spectrophotometrically in the 2 ml system (see section on Methods) in the presence of 0.033 pmole of reduced cytochrome-552. Chloroplasts equivalent to 68 pg of chlorophyll were used for ferricyanide and DPIP photoreduction and equivalent to 27 rg of chlorophyll for NADP photoreduction. Other reaction conditions were as described in Methods.
ferricyanide and DPIP were optimal at an acidic pH, around 5-5.5. The activities decreased sharply when the pH was either increased or decreased. The rate of NADP photoreduction in the presence of cytochrome-552 was opt’imal around pH 6.4. Light intensity. The effect of light intensity on the rate of the Hill reaction with ferricyanide and NADP was measured (Fig. 4). It is seen that the rates of both react’ions were almost the same at low light intensity but t,he rate of ferricyanide reduction was much higher than that of NADP reduction at saturating light int,ensity. SpeciJicity of cytochrome. Wolken and Gross (8) and Perini et al. (15) found that Euglena cells contained, besides cytochrome552, another soluble cytochrome showing an absorption maximum at 556-558 rnp. Because of its presence in heterotrophically grown cells (8, 15) and its location on particles smaller than chloroplasts (15), cytochrome-556 was presumed to have no specific role in the photosynthetic process.
PHOTOREACTIONS TABLE SPECIFICITY
OF EUGLENA
III
OF CYTOCHROME IN REACTIVATION OF NADP PHOTOREDUCTION~ Final Concentration w
Cytochrome
None Euglena cytochrome-552 Euglena cytochrome-552 Euglena cytochrome-556 Euglena cytochrome-556 P. tenera cytochrome-553 P. lenera cytochrome-553 Horse-heart cytochrome
c
4.6 15.4 4.5 15.5 4.4 14.6 15.0
%Gf
Reduction
5 146 187 2 4 50 90 0
a The chloroplast content was equivalent to 5.3 pg of chlorophyll and the volume of the reaction mixture was 0.7 ml.
As shown in Table III, cytochrome-556 was completely inactive in restoring NADP photoreduction activity of Euglena chloroplasts in contrast to the restoration observed with cytochrome-552. Horse-heart cytochrome c was also without effect on NADP reduction; whereas, another algal cytochrome of the c-type, P. tenera cytochrome-553 (4) was capable, although to a lesser extent, of restoring the activity. Effect of cytochrome-552 on methyl viologen photoreduction. Mehler found that illuminated spinach chloroplasts could use molecular oxygen as a Hill reagent (21). This reaction is markedly stimulated by autoxidizable Hill reagents, such as methyl viologen, which accelerate the reaction between a primary reductant and oxygen. Using Euglena chloroplasts, the effect of cytochrome-552 on methyl viologen photoreduction was studied (Table IV). The endogenous rate of oxygen uptake by the Euglena chloroplasts was negligible and was not affected by the addition of methyl viologen (Table IV, Expts. 1 and 2). On addition of only cytochrome-552, however, a substantial oxygen uptake was observed in the light (Table IV, Expt. 3). Apparently, EugZena chloroplasts contained a significant amount of an endogenous, autoxidizable substance which serves as a Hill reagent in the presence of cytochrome-
493
CHLOROPLASTS
552. That the chloroplasts were also capable of photoreducing methyl viologen was indicated by the additional stimulation of the cytochrome-552 supported endogenous oxygen uptake by inclusion of methyl viologen (Table IV, Expt. 4). This latter reaction is inhibited by DCMU (Table IV, Expt. 5). Effect of cytochlwne-552 on cytochrome-c photoreduction. The effect of cytochrome552 on cytochrome-c phot’oreduction by Euglena chloroplasts is shown in Table V. The observation that cytochrome-552 alone could support a significant photoreduction of cytochrome c, without addition of ferredoxin, suggested that t,he cyt,ochrome c was reduced directly by cytochrome-552 which in turn was reduced by PhotosystBem II. An alternative explanation is that an endogenous Hill oxidant, the presence of which was indicated in the previous section, served as the electron carrier between the reductnnt’ produced by Photosystem I and cytochrome c. As shown in the last column of Table V, ferredoxin, stimulated markedly the rate of cytochrome-c photoreduction in the presence of cytochrome-552. As expected, ferredoxin was without effccat in t,he absence of cytochrome-552. TABLE EFFECT
Expt.
IT’
OF CYTOCHROME-552 ON METHYL VIOLOGEN PHOTOREDUCTION~
Additions
None Methyl viologeu Euglena Cytochrome-552 Methyl viologen and Euglena cytochrome-552 Methyl yiologen, Euglena cyto. chrome-552 and DCMU
oxygen Consumption m;$.s,/w
0 0 23 39 8
a Oxygen uptake was measured with a Clark electrode. Each reaction mixture contained chloroplasts equivalent to 56 rg of chlorophyll, 4 mg of catalase and 1.1 mmoles of ethanol in a final volume of 4.9 ml. Where indicated 0.055 rmole of reduced Euglena cytochrome-552, 10 pmoles of methyl viologen and 0.05 pmole of DCMU was added.
494
KATOH TABLE
EFFECT
OF
Euglena
CYTOCHROME
..~
V CYTOCHROME-5%
ON
c PHOTORED~CTION~
of Cytochrome c Reduction LI moles/mg chl.hr
Rate Amount of Cytochrome-552
AND
minus ferredoxin -
plus ferredoxin
WZpPde
2 33 84 81
0 18 39 71
0.0
2.2 5.5 11.0
a Reduction of horse heart cytochrome c was measured spectrophotometrically at 550 mr. The chloroplast content was equivalent to 11 rg of chlorophyll and the volume of the reaction mixture was 0.7 ml. TABLE EFFECT
OF
Euglena
PHOTOREDUCTION OPLASTS
ON NADP
DCMU-INHIBITED
WITH
ASCORBATE-DPIP
Electron
Donor
CHLOR-
OR
ASCOI~-
BATE-TMPD” Rate of NADP Reduction pmoles/mg chl.hr minus cyt-552
None Ascorbate Ascorbate Ascorbate Ascorbate Ascorbate
+ + + +
PIETRO
redox component between Photosystems I and II. It can be seen (Table VI) that ascorbate plus either 6 X IO-“-M DPIP or 1.1 X 10-4-~ TXIPD did not, support NADP photoreduction in the absence of cytochrome-552. However, in the presence of cytochrome-552 and the couple, the rate of NADP reduction was comparable to that of the Hill reaction system. It is concluded that cytochrome-552 is an essential component for electron transfer from the electron-donor couple to NADP with Euglena chloroplasts. An increase in concentration of DPIP or TMPD of lo-fold resulted in slight increase in the endogenous rate, but a significant inhibition of t’he rate with cytochrome-552.
VI
CYTOCHROME-552 BY
SAN
0.04 pmole DPIP 0.4 rmole DPIP 0.077 rmole TMPD 0.77 pmole TMPD
0 0 3 9 3 6
PlUS
cyt-552
21* 21* 71 33 116 33
Q Reactions were followed spectrophotometritally; each reaction mixture contained 0.02 pmole of DCMU, chloroplasts equivalent to 20 pg of chlorophyll and 15pmoles of ascorbate in a final volume of 2 ml. Other experimental conditions were as described in t,he Methods section. * Initial rate of reduction: see Table II.
E$ect of cytochrome-552 on NADP photoreduction by ascorbate-DPIP or ascorbateTMPD. Inhibition of NADP photoreduction by DCMU could be overcome by the addition of DPIP or TMPD in combination with an excess of ascorbate (26-28). The effect of DPIP or TMPD on this photoreduction was of special interest because these artificial electron donor systems replace the oxygen evolving system (Photosystem II) by reacting with some
DISCUSSION
Euglena chloroplasts can photoreduce both ferricyanide and DPIP, but are inactive in NADP, methyl viologen, and cytochrome-c photoreduction. A similar finding was reported by Kok and Datko (29) for chloroplasts from a Scenedesmus mutant (No. 8) isolated by Bishop (30). Whereas substances, such as ferricyanide and benzoquinone, having a redox potential higher than 0.19 V, served as Hill oxidants with good quantum efficiency, low-potential substances were reduced with a poor yield. The inability of these chloroplasts to photoreduce low redox potential substances was due to the absence of Photosystem I, since other photoreactions characteristic of Photosystem I were also missing from the mutant (29, 30). In contrast, the results obtained with Euglena chloroplasts are due to the absence of cytochrome-552 from the electrontransfer chain of the chloroplasts. In fact, cytochrome-552 was so readily solubilized that all our attempts thus far to obtain containing chloroplasts cytoEuglena chrome-552 have been unsuccessful. On readdition of cytochrome-552, however, the photoreduction of NADP, methyl viologen, and cytochrome c by chloroplasts was restored. These results are consistent with the Hill and Bendall scheme for photosynthesis (31), which consists of two photo-
PHOTOREACTIONS
OF EUGLENA
reactions connected by an electron-transfer chain involving cytochrome(s). They are also in good agreement with the observation of Olson and Smillie concerning the lightinduced oxidation-reduction of cytochrome5.32 in Euglena cells (13). Witt el al. (32) indicated that the entry of electrons from reduced DPIP could take place at several sites along the electrontransfer chain of the chloroplasts. Gorman and Levine (33) have proposed that reduced DPIP functions at a site between cytochrome f and Photosystem I because a Chlamydomonas mutant, lacking cytochrome f was still capable of photoreducing NADP with ascorbate and DPIP. Based on spectrophotometric observations of cytochrome-j oxidation-reduction in spinach chloroplasts, Avron and Chance (34) concluded that cytochrome f is not involved in the electron transfer from reduced DPIP to NADP. The present results are inconsist’ent with those proposals. It has been shown that cytochrome-552 is essential for KADP photoreduction by the ascorbateDPIP or ascorbate-TJIPD couple. Thus, reduced DPIP or reduced TMPD could not react directly wit’h Photosystem I at a significant rate but must int’eract with Photosyst,em I via cyt.ochrome-552 or another redox component between cytochrome552 and Photosystem II. Whether this discrepancy about the role of cytochrome in NADP photoreduction with reduced DPIP is due to the different organisms conditions employed, or experimental adopted, must await, further experiment,ation. It is interesting that ascorbate alone was rather inefficient in supporting NADP photoreduction with the DCMU-inhibited chloroplasts in spite of its ability to reduce readiIy cytochrome-552 and nonenzymatically. This implies that the main point of entry of electrons from reduced DPIP or TMPD might not be cytochrome-552 but between cytochrome-552 and photosystem II. Presumably, cytochrome-552 would react preferentially with the oxidized and reduced components in the elect’rontransfer system of the chloroplasts rather than the redox substances added exog-
CHLOROPLASTS
495
enously. This assumption would also explain the effect of ferredoxin on the photoreduction of cytochrome c. Presumably, cytochrome-552 reduced by Photosystem II could react directly with cytochrome c, thereby creating a short circuit for cytochrome-c photoreduction involving only Photosystem II. However, the rate of photoreduction of cytochrome c, in the presence of cytochrome-552, was stimulated further by ferredoxin. This indicates that cytochrome-552 is still functioning, at least in part, as an electron carrier between the two photosystems. The photochemical activities of Euglena chloroplasts described herein resemble, in many respects, those of sonicated spinach chloroplasts described previously (35, 36). Both preparations possessed the capacity to catalyze the Hill reaction using ferricyanide or DPIP as oxidant but were inactive in NADP photoreduction. Each lost a redox protein: plastocyanin in the case of sonicated spinach chloroplnets; and cytochrome-552 in the case of Euglena chloroplasts. Readdition of the protein to the appropriate chloroplast preparation effectively restored NADP photoreduction activity supported by the Hill reaction system or the arcorbate-DPIP system. It seems, therefore, that both proteins are involved in the NADP photoreduction system of photosynthesis. In agreement with this finding, Gorman and Levine found that chloroplasts from two Chlamyclomonas mutants, one devoid of cytochrome f and the other devoid of plastocyanin, were incapable of photoreducing NADP through the Hill reaction system (33). Further studies concerning the role and the location in the electron-transfer system of cytochrome f and plastocyanin are in progress. REFERENCES 1. YAKUSHIJI, E. Acta Phytochim. 8, 325 (1935). 2. HILL, R., AND SCARISBRICK, R., New Phytologist 60, 98 (1951). 3. DAVENPORT, H. E., AND HILL, R., Proc. Roy. Sot. (London) B 139, 327 (1954). 4. KATOH, S., J. Biochem. 46, 629 (1959). 5. KATOH, S., Plant Cell Physiol. 1, 29 (1959). 6. KATOH, S., Plant Cell Physiol. 1, 91 (1960). 7. NISHIMURA, M., J. Biochem. 46, 219 (1959).
496
KATOH
AND
8. WOLKEN, J. J., AND GROSS, J. A., J. Protozoology 10, 189 (1963). 9. SMILLIE, R. M., Can. J. Botany 41, 123 (1963). 10. PERINI, F., SCHIFF, J. A., AND KAMEN, M. D., Biochim. Biophys. Acta 88, 91 (1964). 11. DUYSENS, L. N. M., Science 121, 210 (1955). 12. DUYSENS, L. N. M., AND AYESZ, J., Biochim. Biophys. Acta 64, 249 (1962). 13. OLSON, J., AND SMILLIE, R. M., in “Photosynthetic Mechanisms of Green Plants” (B. Kok and A. T. Jagendorf, Eds.), p. 56. National Academy of Sciences-National Research Council, Wash., D. C. (1963). 14. EVANS, W. R., AND SAN PIETRO, A., Arch. Biochem. Biophys. 113, 236 (1966). 15. PERINI, F., KAMEN, M. D., AND SCHIFF, J. A., B&him. Biophys. Acta 88, 74 (1964). 16. KATOH, S., Nature 186, 138 (1960). 17. KATOH, S., SHIRATORI, I., AND TAKAMIYA, A., J. Biochem. 61, 32 (1962). 18. SAN PIETRO, A., AND LANG, H. M., J. Biol. Chem. 231, 211 (1958). 19. KEISTER, D. L., SAN PIETRO, A., AND STOLZENBACH, F. E., J. Biol. Chem. 236, 2989 (1960). 20. ZAUGG, W. S., Proc. Natl. Acad. Sci. U.S. 60, 100 (1963). 21. MEHLER, A. H., Arch. Biochem. Biophys. 33, 65 (1951). 22. ARNON, D. I., Plant Physiol. 24, 1 (1959). 23. DAVENPORT, H. E., Biochem. J. 77, 471 (1960).
SAN PIETRO 24. KEISTER, D. L., AND SAN PIETRO, A., Arch. Biochem. Biophys. 103,45 (1963). 25. LAZZARINI, R. A., AND SAN PIETRO, A., Biochim. Biophys. Acta 62, 417 (1962). 26. VERNON, L. P., AND ZAUGG, W. S., J. Biol. Chem. 236, 2728 (1960). 27. TREBST, A., AND SISTORIUS, E., 2. Naturforsch. 2OB, 143 (1965). 28. WESSELS, J. S. C., Biochim. Biophys. Acta 79, 640 (1964). 29. KOK, B., AND DATKO, E. A., Plant Physiol. 40, 1171 (1965). 30. BISHOP, N. I., Record Chem. Prog. 26, 181 (1964). 31. HILL, R., AND BENDALL, F., Nature 186, 136 (1960). 32. WITT, H. T., RUMBERG, B. SCHMIDT-MENDE, P., SIGGEL, U., SKERRA, B., VATER, J., AND WEIKARD, J., Angewandte Chemie 4, 799 (1965). 33. GORMAN, D. S., AND LEVINE, R. P., Proc. N&Z. Acad. Sci, U.S. 64, 1665 (1965). 34. AVRON, M., AND CHANCE, B., in ‘Currents in Photosynthesis,” p. 455. Ad. Donker, Rotterdam, The Netherlands (1966). 35. KATOH, S., AND TAKAMIYA, A., Biochim. Biophys. Acta 99, 156 (1965). 36. KATOH, S., AND SAN PIETRO, A., in “Proceedings of the International Symposium on the Biochemistry of Copper,” p. 407, Academic Press, New York (1966).
OF
BIOCHEMISTRY
AND
The Role of C-Type
BIOPHYSICS
488-496 (1967)
118,
Cytochrome
in The Hill Reaction
with
Euglena
Chloroplasts’ SAKAE
KATOH
Charles F. Kettering
AND
ANTHONY
Research Laboratory,
Yellow
SAN PIETRO Springs,
Ohio 45387
Received July 19, 1966 Euglena chloroplasts catalyze the Hill reaction with ferricyanide or dichlorophenol indophenol (DPIP) but not with nicotinamide adenine dinucleotide phosphate (NADP), methyl viologen or horse-heart cytochrome c. The latter compounds can serve as Hill oxidants with Euglena chloroplasts provided Euglena cytochrome-552 is included in the reaction mixture. This c-type cytochrome is solubilized during preparation of the algal chloroplasts. It acts in a catalytic fashion and appears to function in the electron transport chain which interconnects the two photosystems of photosynthesis. Euglena cytochrome-552 is most effective in restoration of NADP photoreduction activity; some restoration is observed with another algal c-type cytochrome, Porphyra tenera cytochrome-553. In contrast, restoration was not observed with either Euglena cytochrome-556 or horse-heart cytochrome c.
The occurrence of cytochromes in various algae and the extraction and partial purification of c-type cytochrome from the red alga, Porphyra tenera, were described first by Yakushiji (1). The involvement of cytochromes in the photosynthetic process was suggested by Hill and Scarisbrick (a), who found a c-type cytochrome, cytochrome f, in the photosynthetic apparatus of higher plants. Davenport and Hill (3) noted the presence of cytochrome(s) with an absorption maximum at 552-553 rnp in an acetone powder of several algae, including Euglena, which they believed was a composite of the absorption maxima of cytochromes c and f. Katoh (4) extended these observations and found,that a soluble c-type cytochrome was ubiquitous among green, brown, red, and blue-green algae. Although many of the chemical and physical properties of this cytochrome were similar to those of mito1 Contribution No. 257 of the Charles F. Kettering Research Laboratory. Supported in part by Grant GM-10129 from the National Institutes of Health, United States Public Health Service.
chondrial cytochrome c (5), its oxidationreduction potential was above 300 mV (4). It was localized in the photosynthetic apparatus (6) indicating that, like cytochrome f in higher plants, the cytochrome was functional in photosynthesis rather than in respiratory metabolism. In accord with this assumption was the finding that a chlorophyll-containing preparation from P. tenera catalyzed a rapid and cyanide-insensitive photooxidation of P. tenera cytochrome-553; whereas, the dark oxidation of the cytochrome by the algal extract was insignificant (5). The presence of a similar cytochrome in green cells of Euglena was first observed by Nishimura (7). This study, and the subsequent studies of Wolken and Gross (S), Smillie (9), and Perini et al. (lo), revealed that the cytochrome was absent from either dark-grown Euglena or chlorophylless mutants and appeared concomitant with the light-induced formation of chlorophyll. Additional evidence for the role of algal cytochrome(s) in photosynthesis comes from the spectrophotometric studies of the light488
PHOTORE,4CTIONS
OF EUGLENa4
induced oxidation-reduction of cytochromes in algae. Duysens (11) observed that illumination of the red alga, Porph~rirliurn cruentu?n, caused oxidation of a c-type cytochrome. Duysens and Amesz (12) showed further t’hat light absorbed by chlorophyll a (690 mp) was much more effcctivc for oxidation of a c-type cytochrome t’han was light absorbed by phycoerythrin (560 mp). Reduction of the cytochrome oxidized by 690-rnp light occurred on illumination with 560-rnp light. Thus, they suggested that, the cytochrome was an electron c:arrier in the electron-transfer system connect’ing the two photosystems of photosynthesis, i.e., Photosystems I and II according to their terminology (12). A similar response of cytochrome-552 in Euglena cells to light of different, wavelength was described by Olson and Smillie (13) : far-red light. was effective for cytochrome oxidation; whereas, light of shorter wavelength was effective for oxidation, and subsequent reduction, of the cytochrome. The results described in this paper relate to t,hc role of algal c-type cytochrome in the photochemical activities of algal chloroplasts. Euglena chloroplasts, devoid of cytochrome-552, were employed and t’he effect of the cytochrome on the photooxidation-reduction system of the preparations was studied. MATERIALS
AND
METHODS
Growth of Euglenn. E. yracilis, strain Z, WAS allt,otrophically or hetrotrophically as grown described by Evans and San Piet.ro (14). Preparation of chloroplasfs. Cells from 2 liters of an autotrophic culture, grown for 3 or 4 days, were harvested by cent,rifugation and washed with 0.05.nz phosphate buffer, pH 7. The washed cells were suspended in 50 ml of 0.05.M phosphate, pH 7, containing 0.4+1 sucrose and 0.01.~ NaCl, and disrupted with a French press at 500 psi. The broken cell suspension was centrifuged at 1OOg for 5 minutes and then 270g for 5 minutes to remove whole cells and large cell fragments, and the chloroplasts were collected by centjrifugation of the sllpernatant, fraction at 1OOOgfor 10 minutes. The chloroplast,s were washed once with, and finally suspended in, the above medium. Microscopic inspection showed that the chloroplast preparation consisted mainly of whole chloroplasts of fairly uniform size and some chloroplast
CHLOROPLASTS
4s9
fragments but was completely free from unbroken cells and larger cell fragments. Prepuration of redoz profeins. Euglena cyt ochrome-552 and cytochrome-556 were prepared according to the method of Perini el al. (15). Cytochrome-552 was extracted from light-grown green cells and purified by repeated column chromatography using DEdE cellulose. Heterotrophic cells, grown in the dark, were rlsed as the source of cyt ochrome-55G, which was partially ptlrified by a single passage t.hrorlgh a cnrboxymetjhyl cellulose col~mm. P. lenet~s rytochrome553 was prepared as described previously (16) from dried algal thalli obtained from a local market Ferrcdoxill and plast ocyanin were prepared from spinach leaves (17, 18). Transhgdrogenase (ferredoxill-NAIlI’ reductase) was kindly provided by 1)r. 1). I,. Keister (19). Jssaq of a&&es. In general, Hill reactions were carried 011t, in a crivet,te (1 X 1 X 4 rm) placed in a Berkman spectrophotometer (Model DB) as described by Zallgg (20) with certain modifications. The light, sollrce was a Unitron lamp which provided, through a microsrope Corning glass filter (No. 2103), a light intensity of abollt 3.7 X 106 ergs/set. cm2. An appropriate glass filter was inserted between the cuvette and phototj~~be to prevent scattered light, from reaching the phototlrbe. Xach reaction mist iire contained in a final volllme of 2 ml: 100 @moles of buffer, 20 pmoles of N&l, rhloroplasts, and the electron acceptor. Phosphate butier, pH 6, was used for NADP (0.4 pmole) and cytochrome c (horse heart, 0.12 pmole) photoreduction and the reaction mixt,ure rolltained all exress of spinach ferredoxin. For the redllction of fcrricyatnide (2 pmolcs) or DPIP (0.1 pmole), citrate-phosphitt e buffer, pH 5, was llsed. In some experiments, the final volume of the reaction mixtllre was decreased to 0.7 ml and a smaller cllvette (0.5 X 1.0 X 4 cm) was used. The ronrcntration of t,he various components was decreased proportionately. Corrections were applied for the changes in the absorption spectrum of I>PIP at. different pH’s. Additional components are indirated for the respective experiments. The photoreclrlctioll of NAI)P with the ascorbate-DPIP corlple was carried olltj anaerobically in a Thlmberg-type cuvette. Prodllction or uptake of oxygen was measured losing a Clark elect.rodc in a semiclosed vessel which was illuminated, t.hrough 20 cm of water and 5 cm of a CUSO~ solution, by a 1030-w turlgsten lamp. The light, intensity was approximately 4000 ft candles. tiarh react,ion mixture contained in a final volume of 4.8 ml: 250 j.moles of cit,rat.ephosphate buf’fer, pH 5; 50 pmoles of NaCl; chlo-
490
KATOH
AND
roplasts; and 0.4 pmole of DPIP or 5 pmoles of ferricyanide. To determine oxygen uptake according to the method of Mehler (21)) 4 mg of catalase and 1.1 mmoles of ethanol was added. Chlorophyll was determined by the method of Arnon (22).
SAN PIETRO
2
.6
RESULTS
Photoreduction activity of Euglena chloroplasts. The photoreduction activity of EugZena chloroplasts with several oxidants are presented in Table I. Both DPIP and ferricyanide were efficient electron acceptors for the Hill reaction system of Eugkna chloroplasts. In contrast, the photoreduction of NADP and cytochrome c was very low even in the presence of excess ferredoxin. The photoreduction of methyl viologen was insignificant as measured by the photochemical uptake of oxygen by Euglena chloroplasts in the presence of catalase and ethanol (21). It seemed, therefore, that Euglena chloroplasts could photoreduce only electron acceptors with a rather high oxidationreduction potential but not those with low potentials. Cytochrome c is included in the TABLE
I
PHOTOREDUCTION ACTIVITY OF Euglena CHLOROPLASTS WITH VARIOUS OXIDANTP Electron Acceptor
DPIP Ferricyanide NADP NADP plus ascorbate Cytochrome c Methyl viologen
and DPIP
146 237 12 9 6 4b
a Photoreductions were measured spectrophoas described in the section on tometrically, Methods, and the reaction mixture contained Euglena chloroplasts equivalent to 48 pg of chlorophyll. Where indicated, 15 pmoles of ascorbate, neutralized with NaOH, and 0.04 pmole of DPIP were added. b Determined polarographically with an oxygen electrode. The reaction mixture contained phosphate buffer, pH 6, Euglena chloroplasts equiva-
lent to 68 pg. of chlorophyll,
10 pmoles of methyl
viologen, catalase and ethanol. The rate is presented as pmoles of oxygen consumed per milligram chlorophyll per hour.
ON
I
0
I
I
tCyt.552 I
2 3 TIME (MINI
-NADP I
4
5
FIG. l.Photoreductionof NADPin the presence and absence of cytochrome-552. The change in absorbance at 340 rnp was measured with a Beckman Model DB spectrophotometer. Reaction mixture of 2 ml contained chloroplasts equivalent to 37 rg of chlorophyll and, where indicated, 0.033 pmole of reduced cytochrome-552. Other
experimental Methods
conditions were as described in the
section.
“low” redox potential group because its reduction by chloroplasts requires an intermediary electron carrier such as ferredoxin (23, 24). It appeared, therefore, that either Euglena chloroplasts lack the activity associated with Photosystem I or that interaction of the two photosystems in these chloroplasts is prevented. That is, some component of the electron-transport system which interconnects the two photosystems was removed during preparation of the chloroplasts. The results presented below demonstrate that the latter explanation is the correct one. E$ect of cytochrome-552 on Hill reaction with NADP. Smillie (9) and Perini et al. (10) found that cytochrome-552 was located originally in the chloroplasts but was very easily solubilized during preparation of chloroplasts in an aqueous medium. This observation was confirmed in the present study. The difference spectrum (reduced minus oxidized) of an aqueous suspension of an acetone powder of Euglena chloroplasts showed an absorption maximum around 560 rnp, indicative of a b-type cytochrome, but no appreciable absorbance indicative of cytochrome-552. It is clear, therefore, that the inability of Euglena
PHOTOREACTIONS TABLE REACTION
II
COMPONENTS FOR PHOTOREDUCTION~
Reaction system
Complete system minus cytochrome-552 minus ferredoxin plus transhydrogenase plus DCMU
OF EUGLENA
NADP
Rate of NADP Reduction rmolesjmg chl.hr
117 9 0 106 10*
5 The complete system contained 100 rmoles of phosphate, pH 6.0, 0.4 pmole of NADP, a saturating amount of spinach ferredoxin, 0.04 pmole of reduced Euglena cytochrome-552, and chloroplasts equivalent to 24 ug of chlorophyll. Where indicated, 0.004 pmole of DCMU or 45 units (as NADPH: DPIP oxidoreductase) of spinach transhydrogenase (19) were added. * Initial rate: see text.
chloroplasts to photoreduce low-potential substances (Table I) could be related to the loss of cytochrome-552 from the electrontransport system of the chloroplasts. If such were the case, the addition of cytochrome-552 to the chloroplasts should restore the Hill activity with NADP as the oxidant. The data presented in Fig. 1 indicate clearly that this is the case. The rate of NADP photoreduction by Euglena chloroplasts was enhanced markedly on addition of cytochrome-552, and became comparable to that of DPIP or ferricyanide photoreduction. In the presence of cytochrome552, the rate of reduction of NADP was constant during four minutes of illumination; whereas, the low endogenous rate of photoreduction of NADP decreased rapidly. There was no appreciable change in absorbance at 340 rnp in the absence of NADP but in the presence of cytochrome-552. In contrast to NADP photoreduction, the addition of cytochrome-552 had no significant effect on the rate of oxygen consumption by chloroplasts with ferricyanide as oxidant. In the case of the DPIPHill reaction, however, the rate of reduction of DPIP, measured spectrophotometrically was stimulated somewhat when cytochrome552 was present. Reaction components for NADP photo-
CHLOROPLASTS
491
reduction. The data presented in Table II show that the photoreduction of NADP by Euglena chloroplasts requires both ferredoxin and cytochrome-552. The addition of ferredoxin-NADP reductase (transhydrogenase) has no effect or was slightly inhibitory. Presumably, the Euglena chloroplasts contain the reductase in sufficient concentration. Further addition of the reductase to the reaction system was inhibitory probably because of an acceleration of the rate of reoxidation of NADPH (25). In the presence of 10-5-~ DCMU, which completely inhibited the Hill reaction with ferricyanide and DPIP, some slight photoreduction of NADP occurred initially, but the rate dropped to zero within a few minutes. This sensitivity to DCMU indicates that the cytochrome-552 activated NADP photoreduction is a Hill reaction. In the presence of DCMU, the amount of NADP reduced appears to be related to the amount of cytochrome-552 present. Since the cytochrome-552 is present in the reduced form, it can serve as the electron donor for NADP reduction. Details of the inhibitory effect of DCiWJ on NADP photoreduction will be described elsewhere. It was also observed that, on addition of cytochrome-552, Euglena chloroplasts evolve oxygen with NADP as oxidant at a rate 1
%
a z
4 E i
0
-I
0
IO
20 CYTOCHROME 552 CONC.(pM)
30
FIG. 2. EffectofEuglenacytochrome-552concen. lration on photoreduction of NADP. Reaction conditions were as described in Fig. 1, except that the concentration of cytochrome-552 was varied.
492
KATOH
comparable to that photometrically.
determined
AND
spectro-
Concentration of cytochrome-552. The relationship between the rate of NADP photoreduction and the concentration of cytochrome-552 is shown in Fig. 2. The rate of reduction was optimal when the concentration of cytochrome-552 was approximately 15-20 pM. The optimal ooncentration of cyt’ochrome-552 seems rather high and was equivalent to the chlorophyll concentration (20.5 PM). However, in the presence of 18.5 PM cyt,ochrome-552, that is, 37 mpmoles in 2 ml, 240 mpmoles of WADP were reduced in four minutes of illumination (Fig. 1). In contjrast, t’he amount of NADP reduced in the absence of cytochrome-552, was only 29 mpmoles, thereby indicating t,he catalytic function of the cytochrome in NADP photoreduction (Fig. 1). E$ect of pH. The pH dependency of the Hill activity of Euglena chloroplasts is shown in Fig. 3. The photoreduction of I
A
/
4
I
0 DPIP n
5
SAN PIETRO
0’ 0
I I I 1000 2000 LIGHT INTENSITY (foot candles)
FIG. 4. Effect of light intensity on photoreduction of ferricyanide and NADP. Each reaction mixture of 2 ml, containing chloroplasts equivalent to 42 pg of chlorophyll, was illuminated with a 200-W tungsten lamp through a wat,er layer of 7 cm thickness. Light intensity was varied by placing the lamp at different distances from the reaction mixture. The absorbance at 420 rn* for ferricyanide, and at 340 rnp for NADP, photoreduction was determined before and after illumination of 2 minlltes. For NADP photoreduction, 0.033 pmole of cytochrome-552 was added to each reaction mixture.
Fe+++cy
6
7
5
6
7
8
PH FIG. 3. PH-dependency of photoreduction of ferricyanide and NADP. The buffers employed were citrate-phosphate (for pH’s below 5.5) and phosphate (for pH’s higher than 5.6). Hill activities with ferricyanide and DPIP (A) were determined polarographically; NADP photoreduction (B) was determined spectrophotometrically in the 2 ml system (see section on Methods) in the presence of 0.033 pmole of reduced cytochrome-552. Chloroplasts equivalent to 68 pg of chlorophyll were used for ferricyanide and DPIP photoreduction and equivalent to 27 rg of chlorophyll for NADP photoreduction. Other reaction conditions were as described in Methods.
ferricyanide and DPIP were optimal at an acidic pH, around 5-5.5. The activities decreased sharply when the pH was either increased or decreased. The rate of NADP photoreduction in the presence of cytochrome-552 was opt’imal around pH 6.4. Light intensity. The effect of light intensity on the rate of the Hill reaction with ferricyanide and NADP was measured (Fig. 4). It is seen that the rates of both react’ions were almost the same at low light intensity but t,he rate of ferricyanide reduction was much higher than that of NADP reduction at saturating light int,ensity. SpeciJicity of cytochrome. Wolken and Gross (8) and Perini et al. (15) found that Euglena cells contained, besides cytochrome552, another soluble cytochrome showing an absorption maximum at 556-558 rnp. Because of its presence in heterotrophically grown cells (8, 15) and its location on particles smaller than chloroplasts (15), cytochrome-556 was presumed to have no specific role in the photosynthetic process.
PHOTOREACTIONS TABLE SPECIFICITY
OF EUGLENA
III
OF CYTOCHROME IN REACTIVATION OF NADP PHOTOREDUCTION~ Final Concentration w
Cytochrome
None Euglena cytochrome-552 Euglena cytochrome-552 Euglena cytochrome-556 Euglena cytochrome-556 P. tenera cytochrome-553 P. lenera cytochrome-553 Horse-heart cytochrome
c
4.6 15.4 4.5 15.5 4.4 14.6 15.0
%Gf
Reduction
5 146 187 2 4 50 90 0
a The chloroplast content was equivalent to 5.3 pg of chlorophyll and the volume of the reaction mixture was 0.7 ml.
As shown in Table III, cytochrome-556 was completely inactive in restoring NADP photoreduction activity of Euglena chloroplasts in contrast to the restoration observed with cytochrome-552. Horse-heart cytochrome c was also without effect on NADP reduction; whereas, another algal cytochrome of the c-type, P. tenera cytochrome-553 (4) was capable, although to a lesser extent, of restoring the activity. Effect of cytochrome-552 on methyl viologen photoreduction. Mehler found that illuminated spinach chloroplasts could use molecular oxygen as a Hill reagent (21). This reaction is markedly stimulated by autoxidizable Hill reagents, such as methyl viologen, which accelerate the reaction between a primary reductant and oxygen. Using Euglena chloroplasts, the effect of cytochrome-552 on methyl viologen photoreduction was studied (Table IV). The endogenous rate of oxygen uptake by the Euglena chloroplasts was negligible and was not affected by the addition of methyl viologen (Table IV, Expts. 1 and 2). On addition of only cytochrome-552, however, a substantial oxygen uptake was observed in the light (Table IV, Expt. 3). Apparently, EugZena chloroplasts contained a significant amount of an endogenous, autoxidizable substance which serves as a Hill reagent in the presence of cytochrome-
493
CHLOROPLASTS
552. That the chloroplasts were also capable of photoreducing methyl viologen was indicated by the additional stimulation of the cytochrome-552 supported endogenous oxygen uptake by inclusion of methyl viologen (Table IV, Expt. 4). This latter reaction is inhibited by DCMU (Table IV, Expt. 5). Effect of cytochlwne-552 on cytochrome-c photoreduction. The effect of cytochrome552 on cytochrome-c phot’oreduction by Euglena chloroplasts is shown in Table V. The observation that cytochrome-552 alone could support a significant photoreduction of cytochrome c, without addition of ferredoxin, suggested that t,he cyt,ochrome c was reduced directly by cytochrome-552 which in turn was reduced by PhotosystBem II. An alternative explanation is that an endogenous Hill oxidant, the presence of which was indicated in the previous section, served as the electron carrier between the reductnnt’ produced by Photosystem I and cytochrome c. As shown in the last column of Table V, ferredoxin, stimulated markedly the rate of cytochrome-c photoreduction in the presence of cytochrome-552. As expected, ferredoxin was without effccat in t,he absence of cytochrome-552. TABLE EFFECT
Expt.
IT’
OF CYTOCHROME-552 ON METHYL VIOLOGEN PHOTOREDUCTION~
Additions
None Methyl viologeu Euglena Cytochrome-552 Methyl viologen and Euglena cytochrome-552 Methyl yiologen, Euglena cyto. chrome-552 and DCMU
oxygen Consumption m;$.s,/w
0 0 23 39 8
a Oxygen uptake was measured with a Clark electrode. Each reaction mixture contained chloroplasts equivalent to 56 rg of chlorophyll, 4 mg of catalase and 1.1 mmoles of ethanol in a final volume of 4.9 ml. Where indicated 0.055 rmole of reduced Euglena cytochrome-552, 10 pmoles of methyl viologen and 0.05 pmole of DCMU was added.
494
KATOH TABLE
EFFECT
OF
Euglena
CYTOCHROME
..~
V CYTOCHROME-5%
ON
c PHOTORED~CTION~
of Cytochrome c Reduction LI moles/mg chl.hr
Rate Amount of Cytochrome-552
AND
minus ferredoxin -
plus ferredoxin
WZpPde
2 33 84 81
0 18 39 71
0.0
2.2 5.5 11.0
a Reduction of horse heart cytochrome c was measured spectrophotometrically at 550 mr. The chloroplast content was equivalent to 11 rg of chlorophyll and the volume of the reaction mixture was 0.7 ml. TABLE EFFECT
OF
Euglena
PHOTOREDUCTION OPLASTS
ON NADP
DCMU-INHIBITED
WITH
ASCORBATE-DPIP
Electron
Donor
CHLOR-
OR
ASCOI~-
BATE-TMPD” Rate of NADP Reduction pmoles/mg chl.hr minus cyt-552
None Ascorbate Ascorbate Ascorbate Ascorbate Ascorbate
+ + + +
PIETRO
redox component between Photosystems I and II. It can be seen (Table VI) that ascorbate plus either 6 X IO-“-M DPIP or 1.1 X 10-4-~ TXIPD did not, support NADP photoreduction in the absence of cytochrome-552. However, in the presence of cytochrome-552 and the couple, the rate of NADP reduction was comparable to that of the Hill reaction system. It is concluded that cytochrome-552 is an essential component for electron transfer from the electron-donor couple to NADP with Euglena chloroplasts. An increase in concentration of DPIP or TMPD of lo-fold resulted in slight increase in the endogenous rate, but a significant inhibition of t’he rate with cytochrome-552.
VI
CYTOCHROME-552 BY
SAN
0.04 pmole DPIP 0.4 rmole DPIP 0.077 rmole TMPD 0.77 pmole TMPD
0 0 3 9 3 6
PlUS
cyt-552
21* 21* 71 33 116 33
Q Reactions were followed spectrophotometritally; each reaction mixture contained 0.02 pmole of DCMU, chloroplasts equivalent to 20 pg of chlorophyll and 15pmoles of ascorbate in a final volume of 2 ml. Other experimental conditions were as described in t,he Methods section. * Initial rate of reduction: see Table II.
E$ect of cytochrome-552 on NADP photoreduction by ascorbate-DPIP or ascorbateTMPD. Inhibition of NADP photoreduction by DCMU could be overcome by the addition of DPIP or TMPD in combination with an excess of ascorbate (26-28). The effect of DPIP or TMPD on this photoreduction was of special interest because these artificial electron donor systems replace the oxygen evolving system (Photosystem II) by reacting with some
DISCUSSION
Euglena chloroplasts can photoreduce both ferricyanide and DPIP, but are inactive in NADP, methyl viologen, and cytochrome-c photoreduction. A similar finding was reported by Kok and Datko (29) for chloroplasts from a Scenedesmus mutant (No. 8) isolated by Bishop (30). Whereas substances, such as ferricyanide and benzoquinone, having a redox potential higher than 0.19 V, served as Hill oxidants with good quantum efficiency, low-potential substances were reduced with a poor yield. The inability of these chloroplasts to photoreduce low redox potential substances was due to the absence of Photosystem I, since other photoreactions characteristic of Photosystem I were also missing from the mutant (29, 30). In contrast, the results obtained with Euglena chloroplasts are due to the absence of cytochrome-552 from the electrontransfer chain of the chloroplasts. In fact, cytochrome-552 was so readily solubilized that all our attempts thus far to obtain containing chloroplasts cytoEuglena chrome-552 have been unsuccessful. On readdition of cytochrome-552, however, the photoreduction of NADP, methyl viologen, and cytochrome c by chloroplasts was restored. These results are consistent with the Hill and Bendall scheme for photosynthesis (31), which consists of two photo-
PHOTOREACTIONS
OF EUGLENA
reactions connected by an electron-transfer chain involving cytochrome(s). They are also in good agreement with the observation of Olson and Smillie concerning the lightinduced oxidation-reduction of cytochrome5.32 in Euglena cells (13). Witt el al. (32) indicated that the entry of electrons from reduced DPIP could take place at several sites along the electrontransfer chain of the chloroplasts. Gorman and Levine (33) have proposed that reduced DPIP functions at a site between cytochrome f and Photosystem I because a Chlamydomonas mutant, lacking cytochrome f was still capable of photoreducing NADP with ascorbate and DPIP. Based on spectrophotometric observations of cytochrome-j oxidation-reduction in spinach chloroplasts, Avron and Chance (34) concluded that cytochrome f is not involved in the electron transfer from reduced DPIP to NADP. The present results are inconsist’ent with those proposals. It has been shown that cytochrome-552 is essential for KADP photoreduction by the ascorbateDPIP or ascorbate-TJIPD couple. Thus, reduced DPIP or reduced TMPD could not react directly wit’h Photosystem I at a significant rate but must int’eract with Photosyst,em I via cyt.ochrome-552 or another redox component between cytochrome552 and Photosystem II. Whether this discrepancy about the role of cytochrome in NADP photoreduction with reduced DPIP is due to the different organisms conditions employed, or experimental adopted, must await, further experiment,ation. It is interesting that ascorbate alone was rather inefficient in supporting NADP photoreduction with the DCMU-inhibited chloroplasts in spite of its ability to reduce readiIy cytochrome-552 and nonenzymatically. This implies that the main point of entry of electrons from reduced DPIP or TMPD might not be cytochrome-552 but between cytochrome-552 and photosystem II. Presumably, cytochrome-552 would react preferentially with the oxidized and reduced components in the elect’rontransfer system of the chloroplasts rather than the redox substances added exog-
CHLOROPLASTS
495
enously. This assumption would also explain the effect of ferredoxin on the photoreduction of cytochrome c. Presumably, cytochrome-552 reduced by Photosystem II could react directly with cytochrome c, thereby creating a short circuit for cytochrome-c photoreduction involving only Photosystem II. However, the rate of photoreduction of cytochrome c, in the presence of cytochrome-552, was stimulated further by ferredoxin. This indicates that cytochrome-552 is still functioning, at least in part, as an electron carrier between the two photosystems. The photochemical activities of Euglena chloroplasts described herein resemble, in many respects, those of sonicated spinach chloroplasts described previously (35, 36). Both preparations possessed the capacity to catalyze the Hill reaction using ferricyanide or DPIP as oxidant but were inactive in NADP photoreduction. Each lost a redox protein: plastocyanin in the case of sonicated spinach chloroplnets; and cytochrome-552 in the case of Euglena chloroplasts. Readdition of the protein to the appropriate chloroplast preparation effectively restored NADP photoreduction activity supported by the Hill reaction system or the arcorbate-DPIP system. It seems, therefore, that both proteins are involved in the NADP photoreduction system of photosynthesis. In agreement with this finding, Gorman and Levine found that chloroplasts from two Chlamyclomonas mutants, one devoid of cytochrome f and the other devoid of plastocyanin, were incapable of photoreducing NADP through the Hill reaction system (33). Further studies concerning the role and the location in the electron-transfer system of cytochrome f and plastocyanin are in progress. REFERENCES 1. YAKUSHIJI, E. Acta Phytochim. 8, 325 (1935). 2. HILL, R., AND SCARISBRICK, R., New Phytologist 60, 98 (1951). 3. DAVENPORT, H. E., AND HILL, R., Proc. Roy. Sot. (London) B 139, 327 (1954). 4. KATOH, S., J. Biochem. 46, 629 (1959). 5. KATOH, S., Plant Cell Physiol. 1, 29 (1959). 6. KATOH, S., Plant Cell Physiol. 1, 91 (1960). 7. NISHIMURA, M., J. Biochem. 46, 219 (1959).
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