Photosynthesis Research 39: 175-181, 1994. © 1994KluwerAcademicPublishers.Printedin the Netherlands. Regular paper

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus Yosepha Shahak 1, Christof Klughammer2, Ulrich Schreiber 2, Etana Padan 3, Inge Herrman 4 & Giinter Hauska 4 l lnstitute of Horticulture, The Volcani Center, ARO, Bet-Dagan, Israel; 2 Julius-von-Sachs-Institut far Biowissenschaften mit Botanischem Garten, Universitiit Wiirzburg, Germany; 3 Division of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, Israel; 4 Lehrstuhl far Zellbiologie und Pflanzenphysiologie, Universitiit Regensburg, Germany Received 23 July 1993; acceptedin revisedform 29 October 1993

Key words: sulfide-quinone reductase (SQR), Oscillatoria limnetica, Chlorobium limicola, electron transport, LED array spectrophotometer, anoxygenic photosynthesis Abstract

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for VmaX of 300 and 10/xmoles reduced/mg bacteriochlorophyll a. h, and for K m of 5 and 3/zM were estimated, for decyl-ubiquinone- and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bcl-complex via the ubiquinone pool.

Abbreviations: BChl a - bacteriochlorophyll a; D A D - diaminodurene; d e c y l - U Q - decyl-ubiquinone; LED - light emitting diode; NQNO - 2-n-nonyl-4-hydroxyquinoline-N-oxide; -PQ-1 - plastoquinone 1; SQR - sulfide-quinone reductase (E.C. 1.8.5.'.); UQ - ubiquinone 10; Qc - the quinone reduction site on the cytochrome b6f/bcl, complex (also termed Qi or Qr or Qn); Qs - the quinone reduction site on SQR; Q z - quinol oxidation site on the b6f/bc 1, complex (also termed Qo or Qp)

Introduction

Sulfide can induce anoxygenic photosynthesis in the otherwise oxygenic cyanobacterium Oscillatoria limnetica (Padan 1979). The inducible factor was characterized as sulfide-quinone reductase (SQR), which was isolated as a single 57kDa potypeptide, and most recently was shown to be a flavoprotein (Shahak et al. 1987, Arieli et al. 1991, Arieli, Shahak, Taglicht, Hauska and Padan, in preparation). Constitutive sulfide photooxidation by anoxygenic photo-

synthetic bacteria is well known, and has been extensively reviewed by D.C. Brune (Brune 1989). The sufide oxidizing entity in these organisms has been considered to be a flavocytochrome c (Kusai and Yamanaka 1973). However, not all sulfide oxidizing species contain this component and alternatives have been discussed (Brune 1989). Oxidation of sulfide by quinone has first been described for Rhodobacter sulfidophilus (Brune and Triiper 1986, Brune 1989), and more recently for the green sulfur bacterium Chlorobium limicola (Shahak et al. 1992). Thus,

176 it is not clear whether SQR or flavocytochrome c or both are responsible for sulfide oxidation in these photosynthetic bacteria. Rhodospirillaceae were little studied with respect to sulfide oxidation and formely even grouped as 'Athiorhodaceae'. However, they have recently been shown to be able to grow on sulfide (Brune 1989). Rhodobacter capsulatus utilizes and tolerates sulfide up to 2 mM (Hansen and van Germerden 1972). Here we demonstrate SQR-activity in chromatophores of Rb. capsulatus, and show that sulfide oxidation is connected to the reduction of the cytochrome bcl-complex via the ubiquinone pool. Preliminary reports on the comparison of SQR-activity in Rb. capsulatus and other systems have been published recently (Shahak et al, 1992a,b). The significance of this discovery is beyond the realization of the widespread occurrence of SQR-activity amongst photosynthetic prokaryotes. Since Rb. capsulatus is genetically well characterized it will permit the application of a molecular biology approach to the study of SQR.

Materials and methods

Rb. capsulatus was obtained from the DSM G6ttingen, Germany. The cells were grown, chromatophores were prepared and their bacteriochlorophyll a-content was measured as described (Baccarini-Melandri and Melandri 1972). Photosynthetic membranes from O. limnetica and Chlorobrium were prepared according to Arieli et al. (1991) and Shahak et al. (1992), respectively. SQR-activity was measured as before (Arieli et al. 1991, Shahak et al. 1992) under nitrogen atmosphere, with decyl-UQ (Sigma) as the electron acceptor. The dual-wavelength mode of an Aminco DW2 spectrophotometer (280 minus 300 nm), served for monitoring UQ-reduction, using a millimolar differential extinction coefficient of 15 (Morton 1965). The reaction mixture contained 50mM glycylglycine, pH7.4, chromatophores equivalent to 5/xg BChl a/ml, 20/~M decyl-UQ, and was briefly flushed with nitrogen. The reaction was started by the addition of sulfide to 40/xM. Cytochrome changes

were recored under nitrogen with the LED-array spectrophotometer previously described in detail (Klughammer et al. 1990). This instrument allows simultaneous recordings at 16 different wavelengths in the range from 530 to 600 nm, with a maximal time resolutions of 1 ms/point (Fig. 3). The changes were deconvoluted to cytochrome c- and b- kinetics in the following way: a cytochrome c-spectrum was obtained by the difference spectrum induced by the addition of 25/xM DAD (Merck; recrystallized). Subsequently full reduction of cytochromes was achieved by the addition of 30/xM dithionite (Fig. 2). The dithionite induced difference spectrum contained combinations of cytochrome c and b. The cytochrome b-model spectrum was obtained by subtracting an adjusted extent of the DAD-induced change. A curve fitting routine (Klughammer et al. 1990) optimally fitted the two components, peaking at 551 and 560 nm, as Lorentz functions with appropriate half widths and peak heights, together with a drift correcting parabola to the observed changes. The result of this fitting is shown in Fig. 2. Differential millimolar extinction coefficients of 20 were assumed for both components. More accurate values for the complex complement of cytochromes b and c in Rb. capsulatus (Boyer et al. 1981) did not significantly change the results. The reaction mixture resembled the one for SQR-activity described above, except that chromatophores were present at 30 ~g BChl a/ml and decyl-UQ was omitted. The reaction was started by the addition of 13/zM sulfide. Aurachin C (Oettmeier et al. 1990) was kindly provided by Dr G.H. H6fle, Braunschweig. Other inhibitors were obtained from Sigma.

Results and discussion

Reduction of ubiquinone by sulfide Decyl-UQ is rapidly reduced by sulfide in chromatophores of Rb. capsulatus. Under aerobic conditions reduction is transient only and decylU Q H 2 is reoxidized after exhaustion of sulfide (not shown). In buffer flushed with nitrogen the extent of reduction levels off at about 2/3 of the total, and runs to completion in a second step,

177 after residual oxygen has been exhausted (Fig. 1, trace a). These two step characteristics are abolished by the presence of glucose/glucose oxidase/catalase (trace b), or by extensive flushing with nitrogen (not shown). The reaction in the absence of membranes, or with heated membranes (trace c) was negligible. From the initial slopes of trace a or b a rate of about 80 p, moles U Q reduced/mg BChl a - h was calculated. This rate can be increased at higher concentrations of the substrates, but the background rate also increases, as has been observed even more dramatically with SQR of Chlorobium (Shahak et al. 1992). A Km-value of about 5/zM for sulfide was determined, which is close to the value reported for half maximal growth (Brune 1989, Hansen and Van Germerden 1972). A Vmax of approximately 250/.~moles UQ reduced/mg BChl a. h was obtained from the double reciprocal plot of rates versus sulfide concentrations. The addition of valinomycin and nigericin to 1/~M each, plus 20 mM KCI did not stimulate

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the reduction rate. Plastoquinone-1 was about half as active as decyl-UQ, and the activity obtained with duroquinone was too low to be measured accurately. The optimum pH of the reaction was 7.4, with half maximal rates at pHs of 5 and 8.2. The reaction rate was almost insensitive to ionic strength (90% at 0.4M NaC1). The sensitivities of SQRs to quinone analog inhibitors (Von Jagow and Link 1986) are listed in Table 1. SQR-activity in Rb. capsulatus was significantly less sensitive to aurachin C and N Q N O (Table 1) than in O. limnetica or in Ch. limicola (Shahak et al. 1992b). Stigmatellin was the most potent inhibitor in Chlorobium but less efficient in the other two systems. Antimycin and myxothiazol were somewhat less efficient in Rb. capsulatus than in Chlorobium and were inactive in Oscillatoria. Rotenone had a weak inhibitory effect on the enzyme of Rb. capsulatus. The pIs0-value of 4.60 compares with 6.96 for the rotenone inhibition of reduction of decyl-UQ by NADH-dehydrogenase (unpublished and see also Ragan 1976). As shown previously for SQR of Chlorobium (Shahak et al. 1992), and found in O. limnetica (Arieli et al., in preparation), KCN also inhibits sulfide oxidation in Rb. capsulatus with similar pIs0 values (Table 1). KCN is known to be a potent inhibitor (/~M range) of sulfide oxidation by flavo-cytochrome c in photosynthetic bacteria

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Table 1. The efficiency of inhibitors of SQR in membranes of Rb. capsulatus, 0. limnetica and Chl. limicola. The rates were measured as described in 'Methods and materials', and previously (Arieli et al. 1991, Shahak et al. 1992). The quinone substrate was decyI-UQ for Rb. capsulatus, while PQ-1 for O. limnetica and Chl. limicola, pIs0-values give the negative logarithms of the inhibitor concentrations for 50% inhibition; 'none' indicates no inhibition, ' - ' stands for not determined. The KCN-inhibition in Oscillatoria was tested on the solubilized enzyme (Arieli et al., in preparation) Inhibitor

Fig. 1. Reduction of ubiquinone by sulfide in chromatophores of Rb. capsulatus. For (a) the reaction was measured under air, after briefly flushing the buffer with nitrogen, as otherwise described under 'Materials and methods'. In (b) 20 mM glucose, 1 unit glucose oxidase (Boehringer) and about 20 units of catalase (Sigma) were additionally present. Chromatophores eqivalent to 5/zg BChl a/ml were present. In (c) they were kept in boiling water for 10 min before the experiment.

Aurachin C NQNO Stigmatellin Antimycin A Myxothiazol Rotenone KCN

pls0

Rb. capsulatus

O. limnetica

Chl. limicola

6.64 5.62 5.60 4.30 4.37 4.60 3.92

7.31 6.85 5.17 none none none 4.92

7.92 6.12 8.30 6.02 5.22 4.0

178 (Kusai and Yamanaka 1973, Brune 1989) and a moderate inhibitor of flavo-enzymes (Massey and Ghisla 1983). Accordingly, the O. limnetica SQR has been recently found to be a flavoenzyme (Arieli et al., in preparation). The inhibition of SQR in Rb. capsuIatus by KCN was transient, the rates recovering after sulfide addition. This recovery was more pronounced at higher sulfide concentrations, suggesting a replacement of cyanide by sulfide at the active site. However, a double reciprocal plot of the initial rates versus sulfide concentration in the presence of 0.1mM KCN indicated a stronger effect o n Vma x (decrease from about 250 to 100/.~moles UQ reduced/mg BChl a. h) than on K m (increase from about 5 to 8/xM sulfide), resembling non-competitive behaviour. Presently we are attempting to purify SQR of Rb. capsulatus, which can be solubilized from chromatophores by cholate (Shahak et al. 1992b).

chrome c, or b to BChl a of about 1/200 can be estimated for our chromatophore prepartion. The absorbance response traces at the 16 wavelenghts to the addition of sulfide to the chromatophore suspension (under nitrogen) are shown in Fig. 3A. The actual spectral changes at the 5 time-intervals (symbols, see Fig. 3A) and the computer fit (lines) are shown in Fig. 3B. The deconvoluted responses of cytochromes c and b are shown in Fig. 4A. Sulfide rapidly reduces all the cytochrome c, but only sluggishly reduces part of cytochrome b in the given time period. The rate and extent of cytochrome reduction by sulfide was not changed by either aerobic, or completely anaerobic conditions (in

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Fig. 3. Response of the 16 LEDs to the addition of sulfide (A) and sulfide-induced spectral changes of the cytochromes (B). The middle of 5 time-intervals are indicated by different symbols, and correspond to - 5 , 1, 5, 20 and 40 s after the addition of sulfide. The reaction mixture contained 30 # g BChl a/ml and is described under 'Materials and methods'. The lines in (B) give the spectra fitted by the computer as explained under 'Materials and methods'.

179 the presence of glucose, glucose oxidase and catalase), but the illumination by saturating red light reversed the cytochrome c-reduction by about 60% (not shown). The addition of valinomycin, nigericin and KC1 did not stimulate the reaction. These results indicate that either the SQR reaction of Rb. capsulatus is not coupled to proton movement or that the membranes are leaky to protons, or that the SQR is the rate limiting step. Stigmatellin (1/zM) drastically inhibited cytochrome c-reduction, leaving cytochrome b-reduction unaffected (Fig. 4B). Myxothiazol (2/xM) acted similarly on cytochrome c-reduction, but stimulated the reduction of cytochrome b (Table 2). Both compounds are similarly efficient inhibitors of the ubiquinol oxidizing site (Qz, see Fig. 6) of the cytochrome bcl-complex (Von Jagow and Link 1986, Robertson et al. 1990), but stigmatellin is the better inhibitor of SQR (Table 1). Therefore, it is feasible that in the presence of myxothiazol, but not stigmatellin, the ubiquinol pool becomes reduced enough to reduce cytochrome b via the quinone reduction site Qc (Von Jagow and Link 1986 and Fig. 6). Antimycin A (2 ~M) less drastically inhibited cytochrome c-reduction, but dramatically stimulated the reduction of cytochrome b (Fig. 4C, Fig. 6 and Table 2). Aurachin C (2/zM) acted similar to antimycin A, but stimulated cytochrome b-reduction less (Table 2). Both inhibitors block the Qc-site of the cytochrome bc~complex very efficiently (Von Jagow and Link 1986, Oettmeier et al. 1990), but aurachin C also inhibits SQR (Table 1). The accelerated reduction of cytochrome b in these two cases represents the pathway via the Qz-site (Von Jagow

Table 2. Inhibition of sulfide-cytochrome reduction by various inhibitors. The rates of reduction in the absence of inhibitors (100%) was 616 and about 0.2/~moles/mg BChl a . h for cytochrome c and b, respectively.

Inhibitor

Control Stigmatellin Myxothiazol AntimycinA AurachinC

Concentration

1 ~M 2 ~M 2 ~M 2~M

Rate (%) cyt c

cyt b

100 6 8 27 26

100 100 270 2400 I100

and Link 1986 and Fig. 6). The extent of this accelerated reduction amounts to about 60% of total cytochrome b present, and we conclude that these comprise both the low- and the highpotential hemes, of cytochrome b of the bc~complex (Von Jagow and Link 1986, Hauska et al. 1983). NQNO acted like antimycin A (not shown). The control rate of cytochrome c-reduction by sulfide (Fig. 4A) was much lower than that of decyl-UQ (about 7 compared to up to 300~equivalents of electron/mg BChl a . h ) . This is only partially caused by the lower sulfide concentration applied (13/xM compared to 40/zM, respectively). Titration with sulfide yielded a Vmax of only 10/~moles cytochrome c reduced/ mg BChl a - h and a Km-valuc of about 3/xM in the double reciprocal plot (Fig. 5). The difference between the two apparent rates can be ascribed to the different quinone species involved: exogenous decyl-UQ in the case of the SQR assay and the endogenous UQ pool in the cytochrome reduction reaction. Other reasons for the relatively slow rate could be a stronger rate limitation by the endogenous pool of UQ-10 than by externally added decyl-UQ, as is observed for UQ-reduction in mitochondrial membranes (Schatz 1967). Alternatively, not all electrons reach cytochromes c in the branched electron transport system of Rb. capsulatus (Baccarini-Melandri and Zannoni 1978). In Fig. 4D we show that cytochrome c-reduction by sulfide is sensitive to cyanide. As with SQR-activity (Table 1), the inhibition was partially transient, leading to a lag in the onset of the reaction. KCN had no effect on cytochrome reduction when added after sulfide. Three ways of cytochrome reduction by sulfide can be considered: direct chemical reduction; indirect chemical reduction via UQ, and enzymatic reduction by SQR via UQ. The sensitivity of the reaction to inhibitors of the cytochrome bcl-complex excludes the first possiblity, while the sensitivity to KCN excludes the second alternative. Indeed, as expected for an enzymatic reaction, sulfide dependent cytochrome reduction shows saturation kinetics with sulfide (Fig. 5). It may be concluded that in Rb. capsulatus SQR catalyzes electron flow from sulfide into the ubiquinone pool, which eventually reduces the

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cytochrome bcl-complex as schematically illustrated in Fig. 6.

Acknowledgements This work was supported by the Basic Research Foundation administered by the Israel Academy

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Fig. 6. Scheme of SQR dependent electron transport in chromatophores of Rb. capsulatus. FI, flavin; Q,, the quinone-binding site on SQR; RC, reaction center; FeS, c l and c2 are the Rieske protein and cytochromes c 1 and c 2, respectively. b L and b H are the low and the high potential heroes of cytochrome b, respectively, The sites of inhibition of the various inhibitors used in this study are indicated. Parentheses indicate low potency.

of Sciences and Humanities (to YS) and the Deutsche Forschungsgemeinschaft (SFB 176, to ChK and US). The German-Israeli Foundation for Scientific Research and Development (GIF to EP and GH), and The Moshe Shilo Center for Marine Biogeochemistry are acknowledged for supporting I. Herrmann's visit to Israel.

181 References Arieli B, Padan E and Shahak Y (1991) Sulfide induced sulfide-quinone reductase activity in thylakoids of Oscillatoria limnetica. J Biol Chem 266:104-111 Baccarini-Melandri A and Melandri BA (1972) Partial resolution of the photophosphorylating system of Rhodopseudornonas capsulatus. Methods Enzymol 23:556-561 Baccarini-Melandri A and Zannoni D (1978) Photosynthetic and respiratory electron flow in the dual functional membrane of facultative photosynthetic bacteria. J Bioenerg Biomembr 10:109-138 Boyer JR, Meinhardt SW, Tierney GV and Crofts AR (1981) Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodobacter capsulatus and Rhodobacter sphaeroides. Biochim Biophys Acta 635: 167186 Brune DC (1989) Sulfur oxidation by phototrophic bacteria. Biochim Biophys Acta 975:189-221 Brune DC and Tr/iper HG (1986) Noncyclic electron transport in chromatophores from photolithotrophically grown Rhodobacter sulfidophilus. Arch Microbiol 145:295-301 Hansen TA and Van Germerden H (1972) Sulfide utilization by purple nonsulfur bacteria. Arch Mikrobiol 86:49-56 Hauska G, Hurt E, Gabellini H and Lockau W (1983) Comparative aspects of quinol-cytochrome c/plastocyanin oxidoreductases. Biochim Biophys Acta 726:97-133 Klughammer C, Kolbowski J and Schreiber U (1990) LED array spectrophotometer for time resolved difference spectra in the 530-600 nm wave length region. Photosynth Res 25:317-327 Kusai K and Yamanaka T (1973) The oxidation mechanisms of thiosulphate and sulphate in Chlorobium thiosulphatophilum: Role of cytochrome c-551 and cytochrome c-553. Biochim Biophys Acta 325:304-314 Massey V and Ghisla S (1983) The mechanism of action of flavoprotein-catalyzed reactions. In: Sund H and Ulrich V (eds) Biological Oxidations, pp 114-139. Springer-Verlag, Berlin

Morton RA (1965) Spectroscopy of quinones and related substances In: Morton RA (ed) Biochemistry of Quinones, pp 23--64. Academic Press, London Oettmeier W, Dostatni R, Majewski C, Hrfle G, Fecker T, Kunze B and Reichenback H (1990) The aurachins, naturally occurring inhibitors of photosynthetic electron flow through Photosystem II and the cytochrome brfcomplex. Z Naturforsch 45C: 311-328 Padan E (1979) Facultative anoxygenic photosynthesis in cyanobacteria. Ann Rev Plant Physiol 30:27-40 Ragan CI (1976) The stucture and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria. Biochem J 154:295 Robertson DE, Daldal F and Dutton PL (1990) Mutants of ubiquinol-cytochrome c 2 oxidoreductase resistant to Qosite inhibitors: Consequences for ubiquinone and ubiquinol affinity and catalysis. Biochemistry 29:11249-11260 Schatz G (1967) The measurement of oxidative phosphorylation in the NADH-cytochrome b segment of the mitochondria respiratory chain. Methods Enzymol 10:30-33 Shahak Y, Arieli B, Binder B and Padan E (1987) Sulfidedependent photosynthetic electron flow coupled to proton translocation in thylakoids of the cyanobacterium Oscillatoria limnetica. Arch Biochem Biophys 259:605-615 Shahak Y, Arieli B, Padan E and Hauska G (1992) Sulfide quinone reductase (SQR) in Chlorobium. FEBS Lett 299: 127-130 Shahak Y, Arieli B, Hauska G, Herrmann I and Padan E (1992a) Isolation of sulfide-quinone reductase (SQR) from prokaryotes. Phyton 32:131-135 Shahak Y, Hauska G, Herrmann I, Arieli B, Taglicht D and Padan E (1992b) Sulfide quinone reductase (SQR) drives anoxygenic photosynthesis in prokaryotes. In: Murata N (ed) Research in Photosynthesis, Vol II, pp 483-486. Kluwer Academic Publishers, Dordrecht, The Netherlands Von Jagow G and Link TA (1986) Use of specific inhibitors on the mitochondrial bc complex. Methods Enzymol 126: 253-271

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus.

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrati...
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