Apr 26, 1990 - potent inhibitor of both light and dark reactions, work- ... binding site or the & site) or the plastoquinone pool. The analogy to other anoxygenic ...
THEJOURNAL OF BIOLOGICALCHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 266, No. 1, Issue of January 5, ~ p 104-,lll, : 1991 rmted m U.S.A.
Sulfide-induced Sulfide-Quinone Reductase Activity in Thylakoids of Oscillatoria limnetica* (Received for publication, April 26, 1990)
Boaz ArieliS,Etana Padan$, andYosepha Shahakg From the $Division of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904, Jerusalem and the §Biochemistry Department, Weizmann Institute of Science, 76100 Rehovot, Israel
Sulfide-dependent partial electron-transport reacSeveral cyanobacteria are capable of shifting from oxygenic, tions were studied in thylakoids isolated from cells of plant-type photosynthesis to anoxygenic, bacterial type phothe cyanobacterium Oscillatoria limnetica, which had tosynthesis (1-3). This ability to perform both types of phobeen induced to performsulfide-driven anoxygenic tosynthesis by the same organism is unique. Anoxygenic photosynthesis. It was found that these thylakoids have photosynthesis was extensively studied in the cyanobacterium the capacity to catalyze electron transfer, from sulfide Oscillatoria limnetica. It was found that, while sulfide comto externally added quinones, in the dark. Assay conditions were developed to measure the reaction either pletely inhibits the PSI1’-dependent oxygenic reaction (4), it as quinone-dependent sulfide oxidation (colorimetri- is the ultimate electron donor to PSI-dependent anoxygenic cally) or as sulfide-dependent quinone reduction (by photosynthesis (5,6).The electrons accepted from sulfide can U V dual-wavelength spectrophotometry). The main be used for either C02 fixation (6-8), H2 evolution (9), or N2 features of this reaction are as follows, (i) It is exclu- fixation (lo), depending onthe growth and physiological sively catalyzed by thylakoids of sulfide-induced cells. conditions. The pathway of electrons, from sulfide to ferreNoninduced thylakoids lack this reaction. (ii) Plasto- doxin, is probably common to all three reactions (11). The shift from oxygenic to anoxygenic photosynthesis ocquinone-1 or -2are equally good substrates. Ubiquinone- 1and duroquinone yield somewhat slower rates. curs in the presence of sulfide under light and anaerobic (iii) The apparent K,,, for plastoquinone-1 was 32 WM conditions. It requires an adaptation period of2-3 h, and and for sulfide about 4 MM. Maximal rates (at 25 “C) depends on the synthesis of new proteins (8).Inhibitor studies were about 75 $molof quinone reduced per mgof of induced cells revealed the inhibition of the anoxygenic chlorophyll. h. (iv) The reaction was not affected by reaction by DBMIB and DNP-INT, butnot by DCMU. Thus, extensive washes of the membranes. (v) Unlikesulfide- sulfide donation siteis probably located beyond QB, but before, dependent NADP photoreduction activity of these thyor at thecytochrome bd complex (3, 12). lakoids, which is sensitive toall the specific inhibitors Sulfide-dependent electron transport has also been obof the cytochrome b6f complex, the new dark reaction exhibited differential sensitivityto these inhibitors. 2- served in cell-free systems prepared from 0. limnetica cells n-Nonyl-4-hydroxyquinoline-N-oxide wasthe most after induction periods of2.5 h (3, 13) or 48 h (14, 15). In potent inhibitorof both light and dark reactions, work- these preparations sulfide reacts at two distinct siteswith the ing at submicromolar concentrations. 5-n-Undecyl-6- electron-transport system. The one which is considered to be hydroxy-4,7-dioxobenzothiazolealso inhibited the two the physiologically important site is inducible by, and has a reactions to a similar extent, but at 10 times higher high affinity to, sulfide (apparent K,,, = 20-40mM). The concentrationsthan 2-n-nonyl-4-hydroxyquinoline- reaction to NADP, whichinvolves this site is sensitive to N-oxide. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzo-inhibitors of the bsf complex (DNP-INT, stigmatellin, NQNO quinone, 2-iodo-6-isopropyl-3-methyl-2’,4,4’-trini-(13) and BAL and DBMIB (14, 15)), is coupled to proton trodiphenyl ether, and stigmatellin had no effect on pumping (13), and is not lost by washing these membranes the dark reaction at concentrations sufficient to fully (13). Therefore, we have suggested that theinducible factorb) inhibit the light reaction from sulfide. We propose that which enables anoxygenic electron transport is a membranal the sulfide-induced factor which enables the use of protein, interacting at the PQ- bsf site. sulfide as the electrondonor for anoxygenic photosynIn thiswork we report on a novel electron transport reaction thesis in Oecillatria limnetica is a membrane-bound sulfide-quinone reductase. Its siteof interaction issug- from sulfide to externally added quinones, which is catalyzed gested to be either thecytochrome be (at the QEquinone exclusively by induced 0.limnetica thylakoids, in the dark. binding site or the & site) or the plastoquinone pool. This sulfide-quinone oxidoreduction reaction is most probably The analogy to other anoxygenic photosynthetic sys- the initial step in sulfide-dependent anoxygenic photosynthesis. tems is discussed. The abbreviations used PSII, photosystem
* This work was supported by the German-Israeli Foundation for Scientific Research and Development (GIF), Grant I-91-118.9/88 (to E.P. and Y. S.) and the Israeli Ministry of Science and Development, the National Council for Research and Development (to B. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore behereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
II; PSI, photosystem
I; Fd, ferredoxin; FNR, Fd-NADP reductase; SQR, sulfide-quinone reductase; bH (bL),high (low) potential cytochrome bs heme; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DNP-INT, 2iodo-6-isopropyl-3-methyl-2’,4,4’-trinitrodiphenyl ether; DCMU, 3(3’4’-dichlorophenyl)-l,l-dimethylurea; HQNO, 2-heptyl-4-hydroxyquinoline-N-oxide; NQN0,2-n-nonyl-4-hydroxyquinoline-N-oxide; UHDBT, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole; Hepes, 4(2-hydroxymethyl)-l-piperazineethanesulfonic acid; pE, microEinstein; PQ, plastoquinone; UQ, ubiquinone; DQ, duroquinone.
104
Sulfide-Quinone Reductase and Anoxygenic Photosynthesis EXPERIMENTALPROCEDURES
CyanobacteriulGrowth and Induction-0. limnetica wasgrown photoautotrophically in the aerobic growth medium (3) in 10-liter batches, in a Microferm Fermentor (New Brunswick Scientific Co., Edison, NJ). Growth conditions were: 37 "C, 180 rpm stirring rate, 3 liter/min air bubbling rate and illumination by 2 Natur-escentlamps (each 200 pE r n-'s-'). The cultures of the cyanobacteria were kept axenic and routinely checked microscopically, and by streaking on rich media plates. For induction of anoxygenic photosynthesis, cells were harvested during the logarithmic growth phase, washed, and resuspended in the anaerobic growth medium (3) supplemented with 25 mM Hepes-Na, pH 7.6, to a final concentration of3-8pgof chlorophyll/ml. The suspension was bubbled with Nz for 20 min, the bottle (0.5-3 liters) stoppered, and 10 p~ DCMU, 3.5 mM NazS (pH 7-8), and 2.5 mM NaHC03 injected. The mixture was stirred and illuminated (cool white fluorescent lamp, 150 pE m-' s-') a t 35 "C,for 3 h. Noninduced cells were treated similarly, but the Nz bubbling, DCMU, and NazS were omitted. Thylakoid Preparation-After the induction, or control treatments, cells were washed twice with the anaerobic medium supplemented with 25 mM Hepes-Na, pH 7.6, and thenonce with a freshly prepared spheroplast buffer containing: 20 mM Hepes-Na, pH 7.9,0.3 mM EDTA-Na, 5 mM NaCl, 5 mM KCl, 5 mM MgClz, 0.5 M glycine betaine, and 0.1 M sucrose. The cells were resuspended in the same buffer, containing also bovine serum albumin (1mg/ml) and lysozyme (2 mg/ml), to a final concentration of about 100 pg of chlorophyll/ ml, and incubated for 20-25 min at 35 "C. The cells having partially digested cellwallswere then gently centrifuged (70 s, 1500 X g), resuspended in an ice-cold buffer containing 10 mM Hepes-K, pH 7.4, 10 mM KCl, 10 mM MgCl,, and 0.3 M sucrose, and centrifuged again as above. The pellet was resuspended in the same buffer to yield 0.5-1.0 mg of chlorophyll/ml and transferred to a conical 15-ml plastic test tube. After 15 min of flashing with Nz at 0 "C, the cells were gently sonicated (typically four times, 5 s each, at the lowest power) with a BransonSonicator, model S-100 equipped with a micro tip probe. The sonicate was diluted 4-fold and centrifuged (5 min, 1700 X g), and the pellet was resuspended in the same buffer to a minimal volume. Glycerol was added to yield 14%(v/v) and the thylakoid preparation was either immediately used or frozen under liquid nitrogen and stored a t -70 "C. Frozen preparations retained their activity for at least 7 months. This procedure differs from the one previously described (13) mainly in shortening the lysozyme step, and the addition of the gentle sonication and washing steps. Thylakoid preparations obtained by the modified procedure yielded higher (25-50 pmol NADP reduced/mg of chlorophyll. h) and more reproducible electron-transport rates. Electron-Transport Reactions-All spectroscopic assays were measured in an Aminco dual wavelength spectrophotometer, model DW2a, equipped with a thermostated cuvette holder, stirring unit, and an optional side-illumination device. The reaction mixture (2.3 ml) was kept anaerobic under Nz atmosphere, at 25 "C, in a stoppered cuvette. NADP photoreduction was measured essentially as described elsewhere (12, 13). Reaction mixture contained in 2.3 ml: 10 mM HepesNa, pH 7.4, 10 mM MgCl,, 10 mM KC1, 0.75 mM NADP, 3.5 p~ spinach ferredoxin, 0.025 unit/ml spinach FNR, and chloroplasts containing 10 pg of chlorophyll/ml. The reduction of externally added quinones was initially monitored by changes in UV-absorption spectra. Data were stored and analyzed using an Apple I1 computer interfaced with the Aminco spectrophotometer. Spectra (typically between 240 and 340 nm) were taken in the dual mode of the Aminco, with 320 nm as the reference wavelength, to reduce a relatively high background absorption and scattered light. Spectra were taken before and after the addition of a particular quinone, and atdifferent time intervals after the addition of sulfide. The accumulation of each spectrum took 100 s. Continuous quinone reduction was monitored without the computer system, using again the dual-mode of the Aminco. A couple of wavelengths was chosen for each quinone to monitor maximal absorption difference between the reduced and oxidized forms, as described under "Results." The reaction mixture routinely contained 10 mM Hepes-K, pH 7.4, 10 mM M&l,, 10 mM KCl, 29 p M quinone 100 p M NaZS, and thylakoids containing 10 pg of chlorophyll/ml. Sulfide oxidation was measured colorimetrically. Reaction mixture was as above, except for the addition 0.3 M sucrose, and the higher chlorophyll concentration (50 pgof chlorophyll/ml). 4.5-ml glass
105
bottles were used, sealed with thick (1 cm) black rubber stoppers (Bellco Glass, Inc., Vineland, NJ). Final liquid volume was 2.4ml and gas volume was0.7 ml. The bottles were flushed for 15 min with ) injected and the bottles incubated for Nz, then sulfide (100 p ~ was several minutes with a magnetic stirrer in a water bath (at 25 or 35 "C). The reaction was started by injection of 100 p~ quinone. 50100-pl samples were withdrawn and fixed with 0.45 ml of 2% zinc acetate. Sulfide was determined by the methylene-blue method (16). CO, Fixation-Sulfide-dependent COz fixation was assayed under anaerobic atmosphere. The reaction conditions were the same as the induction conditions described above, except for the inclusion of ["C] NaHC03 (14 mCi/mol), the different chlorophyll concentration (0.51.0 pg of chlorophyll/ml), and the reaction vessel (12-ml stoppered tubes). The reaction was initiated by illumination. Aliquots (0.3 ml) were withdrawn for 30 min, with a gas-tight syringe at different time intervals, into 5 mlof ice-cold trichloroacetic acid (5%, w/v) and passed through Whatman GF/C filters. The filters were washed twice with 5 ml of cold trichloroacetic acid (5%).Radioactivity was determined by a scintillation counter. Materials-Lysozyme, Hepes, bovine serum albumin, chloramphenicol, and spinach FNRand Fd were purchased from Sigma;NazS from British Drug House, duroquinone from either (K&K Chemicals, ICN Biomedicals Inc., New York, or Aldrich and antimycin A from either Sigma or Boehringer Mannheim (both used). PQ-1 was a generous gift from Dr. G. Hauska, Regensburg University, PQ-2 from Dr. G. Hind, Brookhaven National Laboratory, and UQ-1 from Drs. G. B. Brubacher and R. Salkeld, Hoffmann-La Roche. Stock solutions of quinones were prepared in ethanol (spectroscopic grade). Concentrations were determined spectroscopically, using the following millimolar extinction coefficients: plastoquinone, 19.0 at 255 nm; ubiquinone, 15.1 at 275 nm, and duroquinone, 18.2 at 267 nm (17). The following quinone-analog inhibitors were also generous gifts: stigmatellin from Dr. G. H. Hofle, Braunschweig; DBMIB and DNP-INT from Dr. A. Trebst, Ruhr University, Bochum; and NQNO from Dr. B. Trumpower, Dartmouth Medical School, Hanover. Fresh stock solutions were made before each set of experiments, and the concentrations determined specteroscopically, according to Von Jagow and Link (18) and Hauska et al. (19). RESULTS
Thylakoids of Sulfide-induced 0. limnetica CellsCatalyze Quinone-dependent Sulfide Oxidation in the Dark-Washed thylakoids of 0. limnetica retain the induced capacity to use sulfide as an electron donor for NADP photoreduction, in a pathway which includes the cytochrome bd complex, photosystem I, and externally added ferredoxin (13). In order to better define the inducible factor, we have looked for an assay that will require the least possible components of the electrontransport system. Considering the possibility that the factor might mediate electron transfer to PQ (either directly or via the cytochrome complex), we have tested the effect of externally added quinones on sulfide oxidation. As illustrated in Fig. 1, induced membranes can indeed catalyze a duroquinonedependent sulfide oxidation. The reaction occurs in the dark, at a rate which is &fold faster than thenoncatalytic reaction (Fig. lA)and depends on the addition of duroquinone (Fig. 1B). The rate of DQ-dependent sulfide oxidation (after subtraction of the noncatalyzed rates) ranged between 11 ( f 3 ) and 28 ( f l )pmol of sulfide oxidized per mg of chlorophyll. h, in different preparations. The rates of thisdark reaction correlated with the rates of sulfide-dependent NADP photoreduction (10-30 (kO.1) pmol of DQ reduced per mg of chlorophyll. h). Quinone-dependent sulfide oxidation, catalyzed by induced membranes, was also observed with UQ-1 and PQ1. The catalyzed Na2S + PQ-1 reaction was 2.4 times faster than Na2S + DQ, under similar conditions (not shown). Sulfide-dependent Quinone Reduction-Since the inducible dark oxidation of sulfide depends on the addition of quinone, it is suggested that quinone is the electron acceptor of the reaction. Quinones have typical absorption spectra in the UV range, with distinct peaks for the reduced and oxidized species (17) (see also Fig. 2C). We have thus attempted to measure
Sulfide-Quinone Reductase and Anoxygenic Photosynthesis
106
FIG. 1. Duroquinone-dependent sulfide oxidation. Thylakoids (50 pg of chlorophyll/ml) ofinducedor noninduced 0. lirnnetica cells were incubated under anaerobic conditions in an assay medium containing: 10 mM Hepes-K, pH 7. 4, 10 mM MgClz, 10 mM KCl, 0.3 M sucrose, and 100 p~ NazS. Reaction was started after about 3 min by injection of 100 p M duroquinone (A) or equivalent amount of ethanol (B).Buffer indicates a no membrane control. Samples were taken at the indicated times for colorimetric sulfide determination.
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FIG. 2. Spectroscopic determination of sulfide-dependent plastoquinone reduction. A, induced thylakoids (10 pg of chlorophyll/ml) were incubated under anaerobicconditions in the assay medium (without sucrose). Spectrum ( I ) was taken before further additions; (2) after the injection of 29 p~ PQ-2, and (3) taken 3 min after sequential injection of 100 p~ Na,S. Spectra were taken in the dual mode of the Aminco spectrophotometer, with 320 nm as the fixed reference wavelength. B , digital subtraction of the indicated spectra. C, spectra of oxidized and borohydride-reducedPQ-2 (43 PM) in 95% ethanol.
the dark reaction by monitoring the reduction of the externally added quinones. To reduce a rather high background (equivalent to about 1 OD) due to light scattering and also the absorption of membranal components in theUV, we have used dual wavelength spectrometry. The reference wavelength was chosen to be 320 nm, since at this range ( f 1 0 nm) absorption was minimal and no apparent spectral changes observed under the experimental conditions used. Thus, in the spectra shown in Figs. 2-4 the absorption at 320 nm was continuously subtracted from the absorption at thechanging wavelengths, using the “Dual” mode of the Aminco spectrophotometer. Fig. 2A illustrates spectra which were taken with induced membranes, placed in a stoppered cuvette under anaerobic conditions. Spectrum ( I ) was monitored before, while spectrum ( 2 ) was after the addition of PQ-2 (29 p ~ ) .Then 100 pM Na2S was injected, and after 3 more min spectrum ( 3 ) taken. Digital subtraction of spectrum ( I ) from ( 2 ) or (3) yields spectra which are typical to oxidized and reduced PQ (Fig. 2B). Fig. 2C shows, for comparison, the spectra of oxidized and borohydride-reduced PQ-2 in ethanol. The results thus show that PQ-2 is reduced to plastoquinol in the presence of sulfide and induced membranes. Fig. 3 shows a timecourse of sulfide-dependent PQ-2 spectral changes which occur in the presence of induced membranes (Fig. 3A), as compared with noninduced membranes (Fig. 3B). In thisfigure the spectra of membranes plus PQ-2 (equivalent to spectrum ( 2 ) in Fig. 2.4) were subtracted from the spectra taken at the indicated time after the addition of , reflecting sulfide ( i e . equivalent to Fig. 2A spectrum ( 3 ) ) thus “reduced minus oxidized” difference spectra. After the addition of sulfide there was a gradual decrease in the oxidized
PQ-2 species (indicated by the negative peak around 260 nm), concomitant with an increased positive peak of the reduced species, around 290 nm. The reaction was completed within 3 min, due to the limiting amount of PQ-2. Further addition of PQ-2 to induced membranes can restore the spectral changes (not shown). No such changes couldbe detected in the presence of noninduced membranes, even after 6-min incubation with sulfide (Fig. 3B). This indicates that sulfide-dependent PQ-2 reduction is specifically catalyzed by induced membranes. It further demonstratesthat under our experimental conditions, the rate of the chemical reduction of PQ-2 is negligible. The positive shoulder which is apparent in the low wavelength range (below 250 nm) originates from absorption by reduced sulfide, and is not dependent on the presence of quinone or membranes (not shown). It becomes smaller upon sulfide oxidation (compare this part in the spectra of Fig. 3, A with
B). Sulfide-dependent reduction of quinones other than PQ-2 were also assayed. Fig. 4 shows the difference spectra obtained with UQ-1 and duroquinone. In both cases the difference spectra (plus-minus sulfide) fit well with their typical reduced minus oxidized difference spectra. The disturbance of the quinone spectra by sulfide absorption is also apparent in Fig. 4. Based on the above difference spectra, we have chosen a couple of wavelengths for each quinone, which will give the largest absorption change upon reduction, and have further used these couples to continuously monitor the reaction. Typical traces areshown in Fig. 5. As can be seen, the reaction is linear for about 2 min; it has a high signal to noise ratio, and the rateof the catalyzed reaction clearly exceeds the rate
107
Sulfide-Quinone Reductase and Anoxygenic Photosynthesis
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obtained with noninduced membranes, or without membranes. Table I compares the rateof the sulfide-dependent reaction using different quinone acceptor. The calculations were based on extinction coefficients which were empirically determined by titration of each quinone under the reaction conditions, namely in the presence of membranes and thereaction buffer. The coefficients thus determined were (for 1 mM):16.9 for PQ-1 or PQ-2 (at 292-265 nm), 17.8 for UQ-1 (at 300-280 nm), and 18.0 for duroquinone (at 295-272 nm). The highest rates of sulfide-dependent quinone reduction were obtained with plastoquinone 1or 2. Using 29 p~ quinone and 100 p~ Na2S, the reaction rate was 55 pmol of quinone
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reduced per mg of chlorophyll. h. The catalyzed reduction of UQ-1 was slower by 6-fold, while duroquinone reduction by 33-fold. The calculated reduction rates of PQ-1 and UQ-1 were in good agreement with the rates determined by the analogous sulfide oxidation measurement. However, the rates of the Na2S + DQ reaction, as determined spectroscopically were 5-10-foldsmaller than those determined colorimetrically (compare Fig. 1 and Table I). Thus, duroquinone seems to be a rather good electron acceptor for the dark,partial reaction, but for a reason which is not clear yet, its spectral changes are masked. The dependence of the dark reaction on the donor and the acceptor concentrations is shown in Fig. 6. The rate of qui-
108
Sulfide-Quinone Reductase and Anoxygenic Photosynthesis
A . Induced thylakoids
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TABLE I Sulfide-dependent quinone reduction catalyzed by thylakoids of 0. lirnnetica in the dark Reaction conditions were the same as for Fig. 5, except for varying the quinone acceptors as indicated, and using the appropriate wavelength coude for each auinone. as described in the text. Rate of quinone reduction Quinone
Induced
Noninduced membranes pmol/mg Chl. h
0.5
PQ-1 PQ-2 UQ-1
DQ
57.7 56.6 10.8 2.2
2.8 1.1 0.5
none reduction measured in thepresence of noninduced membranes was much smaller than with induced membranes, throughout the tested concentrationrange of PQ-1 andNa2S. After subtraction of the noninduced rates, the apparent K,,, values for the catalyzed reaction were 4.4 p~ for Na2S and32 p~ for PQ-1. These indicate high affinity of the induced reaction for bothsubstrates. The maximal rates observed under saturating substrates ranged between 50 and 80 (k5%) pmol of quinone reduced per mg of chlorophyll. h in different membrane preparations. Effect of Chloramphenicol onthe Induction of the New Dark Reaction-The induction of sulfide-dependent anoxygenic photosynthesis in whole 0. limnetica cells had been shown to be inhibited by chloramphenicol, indicating the dependence of the anoxygenic reaction on newly synthesized proteins (8). In the experiments summarized in Table 11,we tested the effect of chloramphenicol, present during the induction period, on the dark sulfide-quinone reductase activity of thylakoids isolated after this treatment. The rates of sulfidedependent CO, fixation by the cells, as assayed prior to their lysis for thylakoid preparation,are given in the table for comparison. Clearly, the chloramphenicol treatment, which prevented anoxygenic CO, fixation by the cells (Table 11, left column), also inhibited to a great extent the ability of the related thylakoids to catalyze the sulfide-quinone reaction.
The remaining rate of the dark reaction, after subtraction of the noncatalyzed rate(as determined by the noninduced control), was only 6% of the reaction rate performed by thylakodis of cells induced in the absence of chloramphenicol (Table 11, right column). The nature of the remaining slow rate, inthe chloramphenicol pretreated thylakoids, is not clear yet. Inhibitor Sensitivity of the Dark Reaction-In order to define what part of the photosynthetic electron transport system is involved in the dark reaction from sulfide to quinones, we have studied the effect of several wellknown inhibitors. Table I11 compares the inhibitor sensitivity of the two sulfide-dependent reactions as studied in the same membrane preparations. DCMU had no effect on either the light or dark reaction, suggesting that components of PSI1 are not involved in both reactions (Table 111).Two potent inhibitors of mitochondrial bc complex, weretested myxothiazol, which does not affect chloroplast bdcomplex (18-20), and antimycin A, which in chloroplasts inhibits cyclic electron transport, but not the b$ complex directly (18-20). Neither affected the sulfide-dependent reactions. Surprisingly, the cytochrome bdinhibitors, which all inhibit the NazS + NADP photoreduction in induced 0. limnetica thylakoids (Table 111) (13,14), had adifferential effect on the dark reaction. Two groups could be distinguished. One group includes NQNO and UHDBTwhich inhibited both reactions, each to a similar extent. NQNO, though, was 10 times more efficient than UHDBT, as judged by the concentration dependence. The second group (DNP-INT, DBMIB, and stigmatellin) inhibitedwell the light reaction, but had no, or very little, effect on the dark electron transport reaction. DBMIB and DNP-INThad no effect at concentrations up to about 10 p ~ which , were beyond the concentration required for maximal inhibition of the NazS+ NADP photoreaction. At higher ) caused only 35% inhibition concentration (20 p ~ DNP-INT of the dark quinone reduction (not shown). Stigmatellin inhibited the dark reaction, but at a concentration range, 20fold higher, than that required for the inhibition of the light reaction (Table 111). These minor inhibitor effects were not enhanced even when the PQ-1 concentration was lowered 4fold (not shown). These results may indicate that the poor inhibition is not due to competition by excess of the quinone acceptor. DISCUSSION
The results presented in this paper demonstrate a novel electron transport reaction from sulfide to externally added quinones. The reaction is catalyzed by photosynthetic membranes of 0. limnetica in the dark. It can be measured both by following the oxidation of the donor, as well as the reduction of the acceptor. Quinone-dependent sulfide oxidation was assayed colorimetrically (Fig. I), while sulfide-dependent quinone reduction was detected by UV dual-wavelength spectroscopy. Assay conditions were defined to enable a reliable measurement of quinone-specific absorption changes on top of the relatively high nonspecific absorption and scattering background (Figs. 2-5). We suggest here that thedark sulfide-quinone oxidoreduction is a partial reaction related to the first step of sulfidedependent anoxygenic electron transport to NADP. Several points argue that the dark reaction is part of the light dependent electron transport from sulfide. (i) Both reactions are catalyzed exclusively bymembranes of sulfide-induced 0. limnetica cells. Noninduced membranes neither catalyze the light-dependent Na2S + NADP electron transport which is coupled to proton translocation (13), nor do they catalyze the
Sulfide-Quinone Reductase and Anoxygenic Photosynthesis
109
FIG. 6. Dependence of PQ-1 reduction on sulfide and PQ-1 concentrations. Reaction conditions were the same as in Table I, except for varying the PQ-1 (A) or Na2S ( B ) concentrations. Either induced or noninduced membranes were used, as indicated. Sulfide concentration in A was 100 pM. 0
100
200
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I
[ Sulfide 1, pM
TABLE I1 lease of loosely bound components on the donor and acceptor Effect of chloramphenicol pretreatment on sulfide-dependent quinoneside of PSI ( i e . plastocyanin FNR, Fd). The dark quinone reduction as compared with COZ fixation reduction, which does not involve this part of the electron Aerobically grown 0. limnetica cells were harvested and treated in transport system, is even less sensitive to washing treatments. three different ways: (i) sulfide-induction, for 3 h as described under “Experimental Procedures;” (ii) sulfide induction in the presence of It is unaffected by treatments which are known to wash out chloramphenicol (68 pg/ml); (iii) aerobic incubation for 3 h without peripheral proteins of thylakoids (e.g. EDTA treatment, not sulfide and DCMU. After pretreatment, cells were washed twice in shown). Thus, theinducible factor involved in both reactions anaerobic growth medium supplemented with 25 mM Hepes-Na, pH is probably a membranal component. (iii) The apparent K,,, 7.6. Cell samples were assayed for sulfide-dependent COZ fixation in for sulfide in both reactions is in the micromolar range (Fig. the light, as described under “Experimental Procedures.” The rest of the cells were used for thylakoid preparation. The thylakoids were 6) (13,14). As previously shown, there is an additional sulfide assayed for sulfide-dependent PQ-1 reduction in the dark, as de- donation site for NADP photoreduction in cell-free systems, scribed in Fig. 5,but with 41 p M PQ-l. Values represent the statistical with apparent K,,, in the millimolar range (13, 14). This site, averaee of three to six different exDeriments. which is not inducible, is located on the immediate donor side CO, fixation in whole PQ-1 reduction in thylakoids cells pmollmg Chl. h
Cell pretreatment
Induced Induced chloramphenicol Noninduced
+
35.3 0.3 1.7
* 3.7
* 0.2 * 1.1
85.6 k 9.4 9.0 3.7 4.4 0.4
*
*
TABLE111 Inhibitor sensitivity of sulfide-&pendent electron transport in induced 0. limnetica thylakoids Sulfide-dependent quinone reduction, with either 20 p~ PQ-1 or 29 p M DQ (as indicated) was measured in the dark, as in Table I. Sulfide-dependent NADP photoreduction was measured as described under “Experimental Procedures.” A concentration dependence of the effect of each inhibitor was done. The numbers presented in the table, indicate the inhibitor concentration required for 50% inhibition.
Inhibitor DQ
Quinone darkreduction
NADP photoreduction
PQ-1 PM
NQNO 0.30 UHDBT Stigmatellin 8.00 DNP-INT DBMIB Myxothiazol Antimycin A DCMU
0.14 1.45 6.70 None None None
None None
None
0.15 1.15 0.38 1.35 2.40 None None None
dark quinone reduction (Fig. 1, 3-5). Thus, both reactions depend on an inducible factor. (ii) The capacity to utilize sulfide is not lost upon washing the membranes. Some loss of Na2S + NADP photoreduction which progressively occurs upon repetitive washes (13) probably reflects the partial re-
of PSI. As expected, no indications were found for the participation of such a low affinity site in the quinone reduction (not shown). (iv) The apparent V,,, for Na2S + PQ-1 oxidoreduction ranged between 50 and 80 pmol of quinone reduced per mg of chlorophyll. h in different membrane preparations (e.g. Fig. 6), measured at 25 “C. A rate of 135 pmol/ mg chlorophyll. h (i.e. 270 peqe-/mg chlorophyll. h) was measured at 35 “C, under saturating PQ-1 and NazSconcentrations (not shown). The rate of sulfide-dependent COz fixation in whole cells, after an induction period of 3 h,was reported to be around 50 pmol of COz fixed per mg of chlorophyll-h (= 200 peq e-/mg of chlorophyl1.h) at 35 “C (21). The similarity of the rates supports the involvement of the partialdark reaction in the overall anoxygenic photosynthesis. With the membrane preparations used in the present work rates of 30-50 pmol of NADP photoreduced per mg of chlorophyll. h were obtained (at 25 “C). These are about half the rates of NazS + PQ-1 which are measured in the same preparations. The spinach Fd and FNR added to the NADP reaction mixture might not fully substitute for the native components, thus accounting for the lower rates of this reaction. (v) Specific inhibitors of the cytochrome bd complex inhibit light-dependent electron transport from sulfide, in induced whole cells as well as induced membrane preparations (3, 13, 15). Some of them also inhibit the SQR activity in these membranes (Table 111), suggesting that in both cases sulfide donates electrons to the PQ-bd part of the electron transport system. Although the above arguments supportthe linkage between the dark sulfide-quinone reduction and the anoxygenic photosynthesis, amore direct proof forthis linkage is still missing. Such a proof might be provided in the future if an SQR factor is isolated, having the ability to induce anoxygenic photosynthesis in noninduced membranes. As a first step toward this aim, we have now obtained results indicating that the SQR
110
Sulfide-Quinone Reductase and Anoxygenic Photosynthesis
activity can be solubilized out of induced thylakoids in an active form.' As mentioned above, the lack of SQR activity in noninduced thylakoids indicates the inducible nature of the SQR factor. Chloramphenicol, aprotein-synthesisinhibitor,has been shown to inhibit the induction of anoxygenic photosynthesis in 0. limnetica cells (8) (see also Table 11). The inhibitory effect of chloramphenicol on the induction of SQR activity observed in the present work (Table 11) suggests that this activity depends on a newly synthesized protein(s). Furthermore, the recently solubilized activity is found to be sensitive to heat and protease treatments,' suggesting that the SQR factor itself is aprotein. What component of the PQ-bGf segment of the electron transport chain does the SQR interact with? Several models have beensuggested for the mechanism of action of the cytochrome bGf complex (see Refs. 19, 20, 22-25 for review). The one which is more accepted, though with different modifications and variations, is the Q cycle model (26). The model assumes two distinct quinone binding sites on cytochrome b, designated Q. and Qc (or Q,, and Qi, or Qn and Q,, according to other nomenclatures). The Qz site is located close to the lumen side of the thylakoid membrane, near the bL heme, and is probably shared with the Rieske protein (27). Qe is located close to the stromal side of the membrane, near the bH. Reduced PQ preferentially binds to the Qz site, while the oxidized species binds to the Qc site. The two sites differ in their sensitivity to quinone-analog inhibitors. NQNO at submicromolar concentrations, inhibits PQ reduction by b H at the Qc site (25). At concentrations above WM, it also inhibits be reduction (28). HQNO was shown to increase the midpoint potentials of the two bs hemes, with bH being significantly more sensitive (29). In the Oscillatoria system described here, NQNO was found to be the most potent inhibitor of the dark sulfide-quinone reduction, working at submicromolar concentrations (Table 111). It is thus suggested that this partial electron transport reaction occurs via the Qc site. In contrast, Qz inhibitors (with the exception of UHDBT) wereverypoor inhibitors, if at all, of the SQR reaction. DBMIB and DNP-INT had essentially no effect, suggesting that the dark reaction does not directly involve QZ.Stigmatellin inhibited the reaction, but at concentrations 20 times higher than those inhibiting the light reaction of Na2S + NADP, in the same system (Table 111). Stigmatellin is a Qz inhibitor (30,31) which also induces a red shift of the a band of cytochrome b6 (31, 32). Thus, its inhibitory effect on SQR activity might reflect an indirect effect via cytochrome bg. Based on the differential effect of Qe and Qz inhibitors, it is possible that SQR interacts with the bGf complex at the level of bH - Qc. The model, schematically described in Fig. 7A, suggests that SQR is bound to cytochrome bs near the bH heme, and participates inthe binding domain of the oxidized PQ. In the light, sulfide electrons are being transferred to the PQ pool by SQR, via Qe. Reduced PQ then binds to the QZ site, and electron transfer to PSI proceeds, ending in NADP. Therefore, the light reaction is sensitive to both Qc and Qz inhibitors. The dark reaction, on the other hand, involves only the Qc siteand is thus essentially insensitive to Qz inhibitors. The effects of NQNO, DBMIB, DNP-INT, and stigmatellin all fit this view. UHDBT is an exception, being equally effectiveas aninhibitor of both light an dark reactions from sulfide (Table 111). UHDBT is a typical Q. inhibitor of bGf and bcl complexes (18, 33). In bcl complexes, it can also bindto the Qc site,but with a lower affinity then to Qz (reviewed in Ref. 18). If the model of Fig. 7A is correct, it
* B. Arieli, Y. Shahak, and E. Padan, unpublished results.
DNP-INT UHDBT
Oext
NADP
DNP-INT IIHDET
FIG. 7. Proposed schemes for possible sites of interaction of the sulfide-quinone reductase with the electron transport system. A , direct interaction of SQR with the Cytochrome complex. B, independentinteraction of SQR with the PQ pool. The scheme includes the sulfide-quinone reductase (SQR);externally added quinone ( Q ext); cytochrome bg high (bH) and low (&) potential hemes; subunit IV of the cytochrome complex ( I V ) ; Rieske protein ( R ) ; cytochrome f (f ) ; plastocyanin (PC); and photosystem I (PSI). PC might be substituted by cytochrome c663.Q. is a hypothetical sulfidereducible quinone binding site on the SQR. For further details, see under "Discussion."
means that in our system UHDBT binds equally well to Qe and Qz. The detailed molecular mechanism of electron transport coupled to proton translocation in bGf/bc, complexes is not solved yet. The model in Fig. 7 is based on a simplified Q cycle model,which includes the recycling of one electron back to PQ via bs. It should be stated, though, that our proposal is not restricted to one version of the cycle oranother. In particular, the question has arose recently to what extent is the turnover of cytochrome b6 obligatory in photosynthetic electron transport (e.g. Ref. 34). Sulfide- and light-dependent spectral changes have recently been reported to occur in 0. limnetica thylakoids, which might indicate the involvement of cytochrome bs in the anoxygenic reaction (15). Qc is not the only possible direct electron acceptor from SQR. Alternative candidates are the bH heme and the PQ pool. In the first case, the differential inhibitor sensitivity will still reflect the Qe and Qz sites. However, if the PQpool is the initial electron acceptor, it will implythat thequinone analog inhibitors affect the SQR directly and that their differential effect is unrelated to their specific interaction with the bsf complex. In thatcase, SQR is expected to be an independent protein, or protein complex, which interacts with sulfide on one hand and with the quinone pool on the other hand. The latter requires that it should have a PQ binding site, which we tentatively name Q., as schematically illustrated in Fig. 7B. The differential effect of the inhibitors on SQR will reflect their interaction with the Qs site. Like 0. limnetica, many photosynthetic bacteria utilize the oxidation of sulfide to elemental sulfur asthe source of electrons for COz photoassimilation. In several cases, a flavocytochrome c was shown to catalyze electron transfer from sulfide to C-type cytochromes (see Ref. 35 for review). An
Sulfide-Quinone Anoxygenic Reductase Photosynthesis and alternative, or parallel, pathway from sulfide to ubiquinone or menaquinone has recently been suggested to occur in these bacteria (36). Chromatophores of Rhodobacter sulfidophilus catalyze the reduction of the endogenous UQ pool in thedark (37). Antimycin A and HQNO inhibit sulfide- (and light-) dependent electron transport in Chlorobium species (38, 39). Our results in the present work strongly suggest that PQ is involved in sulfide-dependent anoxygenic photosynthesis in the cyanobacterium 0. limnetica. In plants andcyanobacteria, the PQ-bdsegmentmost commonly accepts electrons from PSII. However, there are alternative sources whichfeed electrons intothis segment. (i) Cyclic electron transport was suggested to be mediated by FNR (40) or by an unknown factor called ferredoxin-quinone reductase (FQR) (41), both feeding electrons to PQ. (ii) An unknown factor (G) has been predicted to mediate electron input from stromal reducing compounds to thebsf complex in Chlorella (42). (iii) A respiratory NADH dehydrogenase was found to donate electrons to the PQ-bd of Chlamydomonas reinhardii (43). (iv) In heterocysts of Anabaena, NADPH, NADH, and H2 all feed electrons into this part of the chain (44-46). Dark electron transfer from H2 to external quinones in this system was reported to be HQNO (but not DBMIB) sensitive (45), thus resembling the SQR reaction in 0. limnetica. (v) In bundle sheath chloroplasts of C4plants, electrons from malate dehydrogenase are also being fed into the PQ-bd (47). We hypothesize the existence of a family of electron carriers which mediate electron transfer from different physiological donors to the PQ-bd . These carriers are predicted to have a common site which interacts with the PQor bs, and a different site which interacts with their specific donor. Acknowledgments-We thank Dr. G. Hauska (Regensburg University, Federal Republic of Germany) for stimulating discussions. A. Auerbach and M. Mamistvalov of the Weizmann Institute Scientific Services are gratefully acknowledged for constructing the dataacquisition and analysis software connected to theAminco spectrophotometer. REFERENCES 1. Padan, E. (1979) in Advances in Microbial Ecology (Alexander, M.,ed) Vol. 3, pp. 1-48, Plenum Press, New York 2. Padan, E. (1979)Annu. Rev. Plant Physiol. 30, 27-40 3. Belkin, S., Shahak, Y., and Padan, E. (1987) Methods Enzymol. 167,380-386 4. Oren, A., Padan, E., and Malkin, S. (1979) Biochim. Biophys. Acta 546, 270-279 5. Oren, A., Padan, E., and Avron, M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2152-2156 6. Cohen, Y., Padan, E., and Shilo, M. (1975) J. Bacteriol. 1 2 3 , 858-861 7. Cohen, Y., Jorgensen, B.B., Padan, E., and Shilo, M. (1975) Nature 257,489-492 8. Oren, A., and Padan, E. (1978) J. Bacteriol. 133,558-563 9. Belkin, S., and Padan, E. (1978) FEBS Lett. 94, 291-294 10. Belkin, S., Arieli, B., and Padan, E. (1982) Isr. J. Bot. 31, 199200 11. Belkin, S., and Padan, E. (1983) J. Gen. Microbiol. 129, 30913098 12. Belkin, S., Siderer, Y., Shahak, Y., Arieli,B., and Padan, E. (1984) Biochim. Biophys. Acta 766,563-569
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