Localization of the reaction site of cytochrome 552 in chloroplasts from Euglena gracilis

ARCHIVES

OF BIOCHEMISTRY

AND

Localization

BIOPHYSICS

164, 127-135 (1974)

of the Reaction Chloroplasts

Cytochrome

Content

Site of Cytochrome

from Euglena

and Photooxidation Preparations’,

G. F. WILDNER Lehrstuhl

fiir

Biochemie

der Pflanzen,

AND

Abt. Biologie, Received

gracilis in Different

Chloroplast

2

G. HAUSKA

Ruhr-Universittit March

552 in

Bochum, 463 Bochum,

West Germany

16, 1974

Isolated Euglena chloroplasts retain up to 50% of cytochrome 552 on a chlorophyll basis compared to the content of cells. Cytochrome 563 is found in equal amount in chloroplasts and cells. The amount of cytochrome 552 retained depends on the isolation procedure of chloroplasts. Cytochrome 552 can be further liberated from chloroplasts by mechanical treatment or incubation with detergent. It is concluded that cytochrome 552 is not tightly bound in the membrane but rather trapped in the thylakoids of the chloroplasts. In photosynthetic electron flow, cytochrome 552 is functioning as donor for photosystem I, mediating electron flow from cytochrome 558 to P,,, under our conditions. Antimycin A stimulates the photooxidation of cytochrome 552 and of cytochrome 558. The rates of electron flow from water to NADP+ and of cyclic photophosphorylation mediated by phenazine methosulfate correlate with the content of endogenous cytochrome 552 in the chloroplasts. External readdition of cytochrome 552 to deficient chloroplasts causes reconstitution of NADP’ reduction but not of cyclic photophosphorylation. Mechanical treatment or other means of fragmentation of chloroplasts results in the exposure of originally buried reaction sites for external cytochrome 552.

Chloroplasts of Euglena grucilis cells contain a c-type cytochrome with an (Yband at 552 nm, and two b-type cytochromes with a-bands at 558 and 563 nm. Cytochrome 552 is a soluble protein and is well characterized, whereas the other two are membrane bound and many of their properties remain to be elucidated. Cytochrome 552 in Euglena cells was discovered by Davenport and Hill (1); it has been purified and further characterized by Perini et al. (2) and its role as a redox carrier analog to cytochrome f from higher plants, linking photosystem II to photosystern I has been established by Olson and ‘This is paper I in a series. Paper II is the next article in this issue (Arch. Biochem. Biophys. 164, 136-144, 1974). 2Dedicated to Prof. Otto Hoffmann-Ostenhof on occasion of his 60th birthday. 127 Copyrigtlt All riphts

0 1974 by Academic Press, of repr,durti,m in HOG form

Inc. reserved.

Smillie (3). Contrary to cytochrome f from higher plants, which remains bound to the thylakoid membrane during isolation of the chloroplasts, most of the cytochrome 552 after disruption of Euglena cells is found in the supernatant, and only a minor part is sedimenting with the chloroplast fraction. This leaves us with two possibilities for the compartmentation of this cytochrome in the cell. It either is localized in the cytoplasm and also in the chloroplasts, or it is associated with chloroplasts only but is liberated during mechanical treatment. The latter would result in a chloroplast preparation deficient in cytochrome 552. Katoh and San Pietro supported the latter view showing that electron flow from water to NADP+ and other low-potential electron acceptors showed a strict dependence on readdition of cytochrome 552 to a

128

WILDNER

AND HAUSKA

Cytochmme 552 was purified according to Perini chloroplast preparation obtained after French press treatment of the cells (4). et al. (2) from supematants obtained after freezing Shneyour and Avron, using a milder pres- and thawing of cells. Ferredoxin from spinach (8) as well as from Euglena sure device for breaking the cells (Yeda press), reported on a chloroplast prepara- (9) was isolated by the standard procedure. Plastocyanin was purified from spinach chloroplasts action which retained good rates of photosyn- cording to Anderson and McCarty (10). thetic electron flow without readdition of Antimycin A and valinomycin were obtained from cytochrome 552 (5). The question remains Sigma and Calbiochem, respectively. whether this reflected a higher content of endogenous cytochrome 552. Assays In this paper we correlate cytochrome Chlorophyll was measured according to Amon (11). 552 content to NADP+ reduction and cyclic Fractions containing cells were sonicated in 2-ml photophosphorylation in chloroplast prep- suspensions four times for 15 set with the microtip of a arations obtained after these two different Branson sonifier, model S 75, before chlorophyll pressure-cell treatments of Euglena cells. estimation and recording difference spectra (reduced minus oxidized). The difference spectra were meaIn addition, we studied the photooxidation of endogenous as well as of externally sured in an Aminco DW-2UV/Vis spectrophotometer operated in the split-beam mode. The different added cytochrome 552 and its interaction Euglena fractions or cell sonicates were diluted with with cytochrome 558 after earlier investigathe buffer described above to 35 pg chlorophyll per ml, tions by Ikegami et al. (6). and filled into two 3-ml fluorescence cuvettes, one A subsequent paper will present more serving as reference. detailed evidence for the location of the The spectra were recorded at a slit with of 2-nm reaction site of cytochrome 552 obtained by handpass and a scanning speed of 2 nm per sec. The reconstitution experiments and the use of base line was adjusted and was recorded for every a specific antibody. fraction before additions and whenever the same METHODS

Preparations

and Materials

Cells of Euglenn gracilis, strain z, were cultivated in the medium described by Bijger and San Pietro (7) under photoautotrophic conditions at 23°C. The cultures were gassed with 5% CO, (v/v) in air and illuminated with white incandescent light of 150 lux intensity. The cells were harvested in the logarithmic growth phase, were washed once with buffer containing 40 mM Tricine-NaOH,3 pH 7.8, 0.33 M mannitol, and 5 mM MgCl, and were finally suspended in the same buffer at about 10 g wet wt per 100 ml of buffer. For isolation of chloroplasts, cells were broken by a single pressure-cell treatment. Either the Yeda press (Yeda Research and Development Co., Ltd., Rehovoth, Israel) was employed at 650 psi of air and at a flow rate of 2 ml per minute (Method A), or a Sorvall Ribi RF-l cell fractionator (French press) was used at 6000 psi (Method B). In both cases the homogenate was centrifuged for 1 min at 500g for sedimentation of cells and cell fragments. The top portion of the supematant was decanted, avoiding carry-over of cells, and was spun 5 min at 1OOOgto sediment the chloroplasts, which were subsequently suspended in the buffer described above. BAbbreviations: Tricine, (N-methyl)trishydroxymethylglycine; Hepes, N-2hydroxyethylpiperazineN-2-ethanesulfonate.

additions had been made to the sample and the reference cuvette. Redox compounds were added in solid form. Further details are given in Fig. 1 and Table I. Photooxidation of cytochromes and plastocyanin was monitored in the Aminco DW-2UV/Vis spectrophotometer operated in the dual-wavelength mode, modified by illumination from the side. The actinic light from a 100-W halogen lamp passed through 7 cm of water, a red glass filter (Schott RG 665) an IR-filter (Schott KG l), and a shutter, and was guided by fiberglass optics (Streppel, Dhiinn, Germany) to the side of the cuvette. The light intensity at the place of the cuvette was 1.2 x lo5 ergs per cm2 and sec. The photomultiplier was shielded from scattered light by 0.4 cm of a saturated copper sulfate solution and a BG18 filter from Schott. All measurements were carried out at room temperature. The reaction mixture and further details are given in the legends to the figures and tables. NADP+ reduction was measured in a Zeiss spectrophotometer, model PMQII, similarly modified for cross illumination, using the same filters and the shutter in the actinic light path as described above. A second monochromator, model M20, instead of filters, was employed to shield the phototube. The actinic light had an intensity of 4 x 10’ ergs per cm2 and set at the place of the cuvette. The assay mixture of 1 ml contained: Hepes-NaOH 50 mM, pH 6.5, 50 mM NaCl, 5 rnrw MgCl,, 0.25 mM NADP+, 10.~~ fer-

CYTOCHROMES

IN EUGLENA

WI-Homogenate

-

552

WI

nm

C”@=+~~

~-oo

FIG. 1. Reduced-oxidized difference spectra of cytochromes in a cell homogenate and in chloroplasts from Euglena gracilis. The measurement of the difference spectra is described under Methods. Chloroplasts were isolated by Method B. The labels of the traces indicate: O-O, difference spectrum of the cuvettes before additions; O-F, difference spectrum after addition of ferricyanide to the reference cuvette; A-F, difference spectrum after addition of ferricyanide to both cuvettes and of excess ascorbate to the sample cuvette; D-A, difference spectrum after subsequent addition of excess ascorbate also to the reference cuvette and of dithionite to the sample cuvette. The spectra F-F, A-A and D-D corresponded to O-O (not shown).

which is seen in the spectra D-A, is present in almost equal amounts. Taking a millimolar extinction coefficient of 20 for the reduced minus oxidized form for cytochromes in first approximation, we can calculate a chlorophyll/cytochrome ratio of 150 for cytochrome 563 and of 50 and 200 for cytochrome 552 in the homogenate and the chloroplasts, respectively. The A-F spectra reveal the third component, i.e., cytochrome 558. In chloroplasts this accounts for the dominant peak at 558 nm, while in the cell homogenate a shoulder is seen in this region, which is already present in the O-F spectrum of the homogenate, but not in the corresponding spectrum for chloroplasts. A shift of the half-reduction potential of cytochrome 558 to lower values after mechanical perturbation of the chloroplast membrane might be considered. Such a behavior would correspond to similar observations with cytochrome b,,, from higher plants (14). The amount of cytochrome 558 present in chloroplasts correTABLE

RESULTS

Disruption of Euglena cells results in the liberation of cytochrome 552 into the soluble fraction. In accordance the difference spectra (O-F) in Fig. 1 show that on a chlorophyll basis only about 25% of this cytochrome is found in the chloroplast fraction compared to the total cell homogenate. Cytochrome 563, on the other hand,

I

LIBERATIONOFCHLOROPHYLL AND CYTOCHROME~~~ BY PRESSURE CELL TREATMENTOF Euglenagr~cilis~ Fraction

redoxin and chloroplasts equivalent to 10 wg chlorophyll. Ferredoxin-NADP+ reductase was not added because it did not increase the forward reaction but increased the back reaction in the dark. Cyclic photophosphorylation was measured according to McCarty and Racker (12). The reaction mixture contained in 1 ml: 50 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, 5 mM MgCl,, 3 mM ADP, 2 mM phosphate containing lo6 cpm s2P, 0.05 mM phenazine methosulfate, 5 mM ascorbate, 0.02 mM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, 1 mg defatted bovine serum albumin, and chloroplasts equivalent to 30 pg chlorophyll. The recommended addition of hexokinase and glucose (13) did not increase the observed rates.

129

GRACILIS

Method A Cell homogenate Cell sediment Cell supernatant Chloroplasts Supernatant Method B Cell homogenate Cell sediment Cell supernatant Chloroplasts Supernatant

Chlororg; c

cytochrome 552 (9%)

Chlorophylll cytochrome 552

100 90 7 5 2

100 92 10 3 8

50 49 38 84 12

100 59 38 27 10

100 54 43 7 30

45 49 40 174 15

n The disruption of Euglena cells by the Yeda press (Method A) or the Ribi cell fractionator (Method B) and subsequent fractionation by centrifugation are described under Methods, as well as the measurement of chlorophyll and the assay for the cytochrome 552 content by measurement of reducel-oxidized difference spectra (reduced: no addition, oxidized: ferricyanide added). The ratio of chlorophyll to cytochrome 552 is given on a molar basis.

130

WILDNER

AND HAUSKA

sponds to that of cytochrome 563 assuming equal extinction coefficients. Neither of the cytochromes was found in the supernatant of the sedimented chloroplasts and appear to be bound to the chloroplast membrane. The contribution of mitochondrial and cytoplasmic b-type cytochromes to the spectra of the Euglena cell homogenate, therefore, seemed to be small, which is also obvious from comparison of the D-A spectra. As already pointed out, we face two possibilities for the compartmentation of endogenous cytochrome 552, In favor of a location inside the chloroplasts are the data presented in Table I. There it is shown that milder treatment for breaking the cells yields chloroplasts with a higher content in cytochrome 552. This clearly suggests that chloroplasts are affected during cell disruption, the harsher the treatment, i.e., the higher the pressure applied, the greater the effect. We can see from Table I that only 7% of chlorophyll is found in the supernatant of the first spin after Yeda press treatment (Method A) compared to 38% after passage of the cell through the Ribi fractionator (Method B). This percentage is underestimating the degree of cell disruption, because a large portion of chloroplasts might have become trapped in the fluffy sediment of the cell debris. However, we could not attain 50-90s disruption which has been reported (6) even counting Euglena cells before and after Yeda press treatment under the microscope. Following the second column of Table I we observe that most of the liberated cytochrome 552 is found in soluble form in the supernatant of the second spin, but a portion is retained in the chloroplasts which is about twice as high after the milder treatment by Method A. The chlorophyll/cytochrome 552 ratio increases from 50 to 84 for Method A, and from 45 to 174 for Method B comparing cells with chloroplasts. It is important to note that even after the harsher treatment by Method B, this ratio in the cell sediment containing the debris is the same as in the starting cell homogenate, indicating that cytochrome 552 is not liberated from broken cells without release of chloroplasts. Altogether,

cytochrome 552 seems to be associated closely with chloroplasts. The serological identity of cytochrome 552 still bound to chloroplasts and solubilized cytochrome will be proved in the subsequent paper. A correlation of cytochrome 552 content and photosynthetic activity may be seen in Table II. Indeed higher rates of electron flow from water to NADP+ and of phenazine methosulfate-mediated cyclic photophosphorylation are observed in the chloroplast preparation with more endogenous cytochrome 552. However, readdition of cytochrome 552 had no better reconstitutive effect on the more deficient chloroplast preparation in the case of NADP+ reduction, and had no effect at all in the case of cyclic phosphorylation. Also, the stimulation of NADP+ reduction by external cytochrome 552 was not paralleled by an increase of the very low noncyclic phosphorylation rate (data not shown). We conclude from these results that endogenous cytochrome 552 functions at a different site as re-added external cytochrome 552 as will be substantiated in the subsequent paper. These findings are reminiscent of results obtained in reconstitution experiments with plastocyanin and spinach chloroplast membranes (15, 16). It should be considered that, as in spinach chloroplasts, mechanical treatment or action of detergents in addition to liberation of components like plastocyanin or cytochrome 552 from chloroplasts might cause structural changes (16). Such membrane scrambling (17) could shift internal oxidation sites, such as the one for cytochrome 552, to the outside, accessible from the suspending medium. This possibility is verified by the experiments in Fig. 2 where it is shown that the rate of photooxidation of external cytochrome 552, i.e., the number of oxidation sites, increases by sonication of Euglena chloroplasts or even more in the presence of cholate. Photooxidation of spinach plastocyanin is also shown, demonstrating that cytochrome 552 and plastocyanin can mutually replace each other in spinach chloroplasts (18) and in Euglena chloroplasts. The analogous experiment with spinach chloroplasts (class II) showed no photooxidation at all unless

IN EUGLENA

CYTOCHROMES TABLE

II

CORRELATION OF CYTOCHROME 552 CONTENT WITH NADP REDUCTION AND CYCLIC PHOSFHORYLATIONIN Euglena CHLOROPLASTSISOLATED AFTER DIFFERENT PRESSURECELL TREATMENTS’ Addition of 552

F&I;;;;

r”,=JrcP tion

Method

A

-

1.7pM 6.8~~ MethodB

-

1.7/.&M 6.8PM

84 -

174

-

Cyclic photophosphorylation

29

83

56 100

78

5

75 25

45 87

25 22

131

GRACILIS

the spectra of the insert. Calculated from the cytochrome spectra obtained from redox difference spectra (Fig. 1) 30-40’S of cytochrome 552 and about 60% of cytochrome 558 are photooxidized under our conditions. Nevertheless, the cytochrome 552 pool seems to participate in electron transport to photosystem I in both chloroplast preparations. In additional experiments we studied the effect of antimycin A on the light-induced change (Fig. 4). Antimycin A increases the extent of the photooxidation of both cyto-

“The procedures for the isolation of chloroplasts and the assays are described under Methods. Other details are found in Table I. 552 stands for Cytochrome 552. The rates for NADP reduction and ATP formation in cyclic photophosphorylation are given in amoles NADP, or ATP formed per mg chlorophyll per hr.

chloroplasts were passed through the pressure cell, were sonicated, or treated with detergents. In analogy we suggest that the observed rate of photooxidation in Euglena chloroplasts before sonication or additions of cholate results from perturbation of the membrane during pressure-cell treatment, occurring already at the low pressures in the Yeda press. Taking a closer look at Table II it is surprising that chloroplasts prepared by Method B still contain cytochrome 552 but are almost inactive in NADP+ reduction. Possibly this remainder of the endogenous cytochrome is not participating in electron flow at all. We tried to sort this out by measuring the photooxidation of endogenous cytochromes in Euglena chloroplasts. Figure 3 presents typical traces for the photooxidation and dark reduction of cytochrome 552 and cytochrome 558 (the latter was measured at 560 nm to avoid overlapping from cytochrome 552). The traces show that much less cytochrome 552 is photooxidized in chloroplasts isolated by Method B than by Method A in accordance with the lower content in this cytochrome (Fig. 1 and Table I). The extent of photooxidation of cytochrome 558 is the same in both preparations. This is also seen from

-2Osec FIG. 2. Photooxidation of externally added cytochrome 552 and spinach plastocyanin by Euglena chloroplasts at different degrees of fragmentation. The spectroscopic measurement of photooxidation is described under Methods. The assay mixture of 3 ml in a fluorescence cuvette contained: 50 rnM TricineNaOH, pH 8.0,50 mM NaCl, 5 mM MgCl,, 2 x 1O-5 M 3-(3,4-dichlorophenyl)-l, l-dimethylurea, lo-’ M anthraquinone-2-sulfonate. Chloroplasts equivalent to only 20 pg chlorophyll were added to minimize the contribution of endogenous cytochromes. Cytochrome 552 was added to 0.7 PM in cases b, c, and d, and spinach plastocyanin was added to 1.4 PM in the cases f, g, and h. Cytochrome photooxidation was measured at 552 nm with 540 nm as reference, plastocyanin photooxidation at 580 nm with 520 nm as reference. Traces a and e correspond to the chloroplast preparation (Method A) without addition, traces b and f show the change after addition of cytochrome 552 and plastocyanin, respectively, traces c and g show the corresponding change in chloroplasts sonicated in the assay medium four times for 15 set (see Methods); traces d and h depict the changes after addition of cholate to 1% before the assay. Upward arrows reflect “light on,” downward arrows “light off.”

132

WILDNER AND HAUSKA Method A

Method 0

550

550 hnm

FIG. 3. Photooxidation of cytochromes 552 and 558 in Euglena chloroplasts isolated after different pressure cell treatments. The measurements, the assay mixture, and other details are described under Methods and in the legend for Fig. 2. Method A stands for chloroplasts isolated after Yeda press treatment, Method B for chloroplasts isolated after passage through the Ribi cell fractionator. The insert compares the spectra of cytochrome photooxidation in chloroplasts isolated by the two procedures. (X-X) Method A, (O---O) Method B.

chromes, but the most drastic effect is the increase in the rate of cytochrome 558 photooxidation. This is also seen from the two inserts in Fig. 4 which show the spectra of photooxidation in the whole cytochrome c and b region for the initial fast phase and for the total change. Thus, antimycin seems to inhibit the reduction of cytochrome 558 in photosynthetic electron flow, possibly by blocking cytochrome 563 in agreement with the electron transport scheme proposed by Ikegami et al. (7). It was of interest to study the photooxidation of cytochrome 558 in a system of Euglena chloroplasts even more deficient in cytochrome 552. This was achieved by treating the chloroplast membranes with cholate, liberating endogenous cytochrome 552. As seen from Fig. 5, increasing amounts of cholate in the assay mixture abolish the fast photooxidation of cytochrome 552 and drastically decrease the rate of cytochrome 558 photooxidation. Re-addition of cytochrome 552 in about the 5-fold amount of endogenous cytochrome 552 in the presence of cholate partially restores the photooxidation of cytochrome 556. In addition a fast photooxidation of cytochrome 552 is observed again, which has a rapid back reaction in the dark on our time scale, reflecting probably the direct reaction of cytochrome 552 with ascorbate, present in the assay mixture. The rela-

tively slower back reaction of cytochrome 552 before addition of cholate is another indication that endogenous cytochrome 552 is not directly accessible. Reconstitution of cytochrome 558 photooxidation in the presence of cholate can also be achieved with plastocyanin. In this case, a small change is restored in the 552-nm region, which exhibits quite different kinetics to the change at 560 nm. It appears as if plastocyanin can mediate not only between cytochrome 558 and photosystem I but also between solubilized cytochrome 552 and photosystem I. DISCUSSION

The results in this paper demonstrate that cytochrome 552 in Euglena cells is localized in chloroplasts in agreement with its function in photosynthetic electron transport (3, 4, 6). Contrary to former results of Katoh and San Pietro (4) we were able to obtain chloroplasts from Euglena which retained up to 50% of cytochrome 552 on a chlorophyll basis, present in the cell before disruption by French press treatment. The amount decreased with the pressure, and varied with the pressure device applied (Table I). In contradiction to a statement by Ikegami et al. (6), cytochrome 552 can be easily further liberated from chloroplasts by sonication or detergent treatment. We, therefore, sug-

CYTOCHROMES

IN EUGLENA

,002 00 P +2Gz

,:550

560q

wlthxlt

AA

550 With

560 PA

FIG. 4. Photooxidation of cytochromes 552 and 558 in Euglena chloroplasts and the effect of antimycin A. The spectroscopic measurement is described under Methods. The basic assay mixture is given in the legend for Fig. 2. In addition, it contained 0.3 mM ascorbate (to accelerate dark reduction and allow for repeated measurements), 3 mM NH&l, and 5 PM valinomycin (to avoid contribution from electrochromic pigment shifts) and 5 PM antimycin A where indicated. Chloroplasts (Method A) were added equivalent to 100 pg chlorophyll. Photooxidation of cytochromes 552 and 558 was followed at 552 nm and 560 nm, respectively, with the reference set at 540 nm. The inserts show the spectra of the change with respect to 540 nm before and after addition of antimycin A (AA). The total change (x-x) and the initial fast phase (2 set) (04) are shown. Upward arrows reflect “light on,” downward arrows “light off.”

gest that this hydrophilic protein is trapped within the thylakoid system rather than bound to the membrane proper. We think that there is no tightly bound cytochrome 552 in EugEena chloroplasts (6) different from the soluble form. “Tightly bound” cytochrome f from other algae should be reconsidered in this respect (19, 20). Our data in Table I show that the debris retained as much endogenous cytochrome 552 as the intact cell, which suggests that during pressure-cell treatment cytochrome 552 is not lost from cells unless chloroplasts are released. This differs from results of analogous experiments regarding cytochrome cz in photosynthetic bacteria (18).

133

GRACILIS

There it was found that the debris was deficient in cytochrome c2 compared to intact cells, and in addition, preparation of spheroplasts resulted in liberation of almost all the cytochrome. The conclusion was drawn that cytochrome cz is located in the periplasmic space in continuum with the inner space of the chromatophores. Of course, this difference finds an explanation in the different organization of an eukaryotic and a prokaryotic cell. It is more feasible to compare the latter with intact chloroplasts (class I), the periplasmic space corresponding to the stroma. Unfortunately, intact chloroplasts are not yet available from Euglena gracilis. The content of endogenous cytochrome 552 varied with the different methods for isolating chloroplasts while the content of cytochrome 563 remained constant. Cytochrome 552 retained in chloroplasts is participating in photosynthetic electron flow 552 - ‘30

560 - 5LO

05% cholate l 1.3pM 552:

0.5% cholate + 1.3yM plastocyanin.

+

Vkmim 20

FIG. 5. Effect of cholate and of subsequent addition of cytochrome 552 or spinach plastocyanin on the photooxidation of cytochromes in Euglena chloroplasts. Details of the measurement and the assay mixture are given under Methods and in the legends to the preceding Figures.

WILDNER

AND HAUSKA

since it is oxidized during illumination. Supporting a schema of Ikegami et al. for electron flow in Euglena chloroplasts (6), our results indicate that external cytochrome 552 is able to mediate electron flow between cytochrome 558 and photosystem I in chloroplasts treated with cholate (Fig. 5). This needs not to reflect the situation in the intact cell. The photooxidation of endogenous cytochrome 552 and especially of cytochrome 558 was stimulated by antimycin A. According to the scheme of Ikegami et al. the action of this compound could be on cytochrome 563 consistent with the inhibitory action on b-type cytochromes in mitochondrial (see Ref. 22 for a review) and bacterial (23) respiration. In bacterial photosynthetic electron transport antimycin A greatly stimulates the photooxidation of cytochrome c, simultaneously increasing the reduction of b-type cytochromes (23). On the other hand, so far no effect of antimycin A on photooxidation of cytochromes in chloroplasts of higher plants could be found (own experiments and personal communication of H. BBhme), but the inhibition of cyclic photophosphorylation, mediated by ferredoxin, by antimycin was interpreted as an action on cytochrome b, (24). Altogether, the photosynthetic electron transport system of Euglena chloroplasts seems to differ significantly from the system of higher plant chloroplasts and has some similarities with the bacterial system. It is an important question whether this statement could be extended to other eukaryotic algae. In one respect it can: algae have soluble c-type cytochromes functional in photosynthetic electron flow, while the ctype cytochrome of chloroplasts of higher plants, cytochrome f, is membrane bound. It seems a hasty classification to call all c-type cytochromes in chloroplasts cytochrome f, which indeed led to wrong interpretations of experiments. Cytochrome 552 from Euglena has been studied in spinach chloroplasts as a soluble substitute for cytochrome f, which at that time could not be purified (18). In fact, however, it behaves as a substitute for plastocyanin as donor for P,,,. Isolated homologous cytochrome f is poorly photooxidized by P,,,

unless plastocyanin is added (25). Vice versa, plastocyanin can substitute for external cytochrome 552 in Euglena chloroplasts as shown in this paper, indicating that cytochrome 552 might be the physiological electron donor for P,,, in Euglena, as is plastocyanin in spinach. In that context it is of interest to note, that no plastocyanin could be detected so far in Euglena. However, other algal chloroplasts contain plastocyanin in addition to a soluble c-type cytochrome (20, 26). It could be demonstrated in our experiments that NADP+ reduction from water and PMS-mediated cyclic photophosphorylation are higher in those chloroplasts which retained more endogenous cytochrome 552, indicating its participation in these reactions. An inconsistency, however, was observed regarding reconstitution experiments. Re-addition of cytochrome 552 to deficient chloroplasts stimulated NADP+ reduction but had no effect on cyclic photophosphorylation. We interpret this result as evidence for two sites of action for cytochrome 552. One is trapped in the membrane system and only endogenous cytochrome 552 is reacting with it, the other is accessible for cytochrome 552 added back to the assay mixture. The possibility exists that both reaction sites reflect the same component, the external site being artificial and resulting from disorientation of photosystem I in the membrane during mechanical treatment. This is supported by the finding that external reaction sites are increased by further fragmentation of chloroplasts (Fig. 2). Additional experiments on this aspect will be presented and discussed in the subsequent paper (27). ACKNOWLEDGMENTS We are indebted to Prof. Dr. A. Trebst for encouragement and interest. The technical assistance of Miss A. Hellmich and Miss C. Axt is gratefully acknowledged. The work was supported by Deutsche Forschungsgemeinschaft. REFERENCES 1. DAVENPORT, H. E., AND HILL, R. (1954) Proc. Roy. Sot. Ser. B 139, 327-336. 2. PERINI, F., SCHIFF, J. A., AND KAMEN, M.D. (‘1964) Biochim. Biophys. Acta 88, 74-90.

CYTOCHROMES

IN EUGLENA GRACILIS

3. OLSON, J. M., AND SMILLIF, R. M. (1963) Photosynthetic Mechanisms in Green Plant, Nat. Acad. Sci-Nat. Res. Council Publ. 1145,56-65. 4. KATOH, S., AND SAN PIETRO, A. (1967) Arch. Biochem. Biophys. 118, 466496. 5. SHNEYOUR, A., AND AVRON, M. (1970) Fed. Eur. Biochem. Sot. Lett. 8, 164-166. 6. IKEGAMI, I., KATOH, S., AND TAKAMIYA, A. (1970) Plant Cell Physiol. 11, 777-791. 7. BANGER,P., AND SAN PIETRO, A. (1967) Z. Pflanzenphysiol. 58, 70-75. 8. TAGAWA, K., AND ARNON, D. I. (1962) Nature (London) 195,5377541. 9. BBGER, P. (1969) Z. Pflanrenphysiol. 61,447-461. 10. ANDERSON, M. M., AND MCCARTY, R. E. (1969) Biochim. Biophys. Acta 189, 193-206. 11. ARNON, D. I. (1949) Plant Physiol. 24, 1-15. 12. MCCARTY, R. E., AND RACKET E. (1967) J. Biol.

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(1971) Methods Enzymol. 23, 327-344. 15. HAUSKA, G. A., MCCARTY, R. E., BERZBORN, R. J., AND RACKER, E. (1971) J. Biol. Chem. 246,

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Proc. Nat. Acad. Sci. USA 66.928-935. 18. ELSTNER, E., PISTORIUS, E., BBGER, P., AND TREBST, A. (1968) Planta 79, 146-161. 19. BIGGINS, J. (1967) Plant Physiol. 42, 1447-1456. 20. POWLS, R., WONG, J., AND BISHOP, N. (1969) Biochim. Biophys. Acta 180,490-499. 21. PRINCE, R., HAUSKA, G., AND CROWS, A. R. Biohem. J. (in press). 22. SLATER, E. C. (1973) Biochim. Biophys. Acta 301, 129-154. 23. NISHIMURA, M. (1963). Biochim. Biophys. Acta 66, 17-21. 24. TAGAWA, K., TSUJIMOTO, H. Y., AND ARNON, D. I. (1963) Nature (London) 199, 1247-1252. 25. NELSON, N., AND RACKER, E. (1972) J. Biol. Chem. 247, 3848-3853. 26. GORMAN, D. S., AND LEVINE, R. P. (1966) Proc. Nat. Acad. Sci. USA 54, 166551669. 27. WILDNER, G. F., AND HAUSKA, G. A. (1974) Arch. Biochem. Biophys. 164, 136-144.