Abundance, Subunit Composition, Redox Properties, and Catalytic ...

Vol. 175, No. 6

JOURNAL OF BACrERIOLOGY, Mar. 1993, p. 1629-1636 0021-9193/93/061629-08$02.00/0 Copyright © 1993, American Society for Microbiology

Abundance, Subunit Composition, Redox Properties, and Catalytic Activity of the Cytochrome be1 Complex from Alkaliphilic and Halophilic, Photosynthetic Members of the Family Ectothiorhodospiraceae TINA LEGUIJT,l PIETER W. ENGELS,1 WIM CRIELAARD,1 SIMON P. J. ALBRACHT,2 AND KLAAS J. HELLINGWERFl* E. C. Slater Institute for Biochemical and Microbiological Research, University ofAmsterdam, Department of Microbiology, 1 Nieuwe Achtergracht 127, 1018 WS Amsterdam, and the Department of Biochemistry,2 Plantage Muidergracht 12, 1018 TVAmsterdam, The Netherlands Received 13 October 1992/Accepted 11 January 1993

Ubiquinol-cytochrome c oxidoreductase (cytochrome bej) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy. The gy values of the Rieske [2Fe-2S] clusters observed in membranes of E. mobifis and E. halophila were 1.895 and 1.910, respectively. In E. mobilis membranes, the cytochrome be1 complex was present in a stoichiometry of approximately 0.2 per reaction center. This complex was isolated and characterized. It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; E. = 116 mV), cytochrome cl (Em = 341 mV), and a Rieske iron-sulfur cluster. The absorbance spectrum of cytochrome bL displayed an asymmetric a-band with a maximum at 564 nm and a shoulder at 559 nm. The a bands of cytochrome bH and cytochrome cl peaked at 559.5 and 553 nm, respectively. These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome cl, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively. No evidence for the presence of a fourth subunit was obtained. Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced (mol of cytochrome cj)-1 s-1. The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc complexes. from quinol to a c-type cytochrome (see references 22 and 45 for reviews). They generally consist of three polypeptides, which contain four redoxactive prosthetic groups: the Rieske iron-sulfur protein contains a single [2Fe-2S] cluster, cytochrome c1 contains a single, covalently bound heme c, and cytochrome b contains two noncovalently bound protohemes. For Rhodobacter sphaeroides, a fourth subunit, lacking prosthetic groups, has been reported (see references 22, 45, and 54 for reviews). Although many purple bacteria contain a cytochrome bc1 complex, it is not universal. An alternative ubiquinol-cytochrome c2 oxidoreductase has been proposed to be present in some strains of Rhodobacter capsulatus (3). Furthermore, in the BChl b-containing members of the Ectothiorhodospiraceae family, only low-potential soluble cytochromes were detected (43, 44), which seems to be inconsistent with a function in quinone oxidation via a cytochrome bc1 complex. In addition to this, no cytochrome c2 has been detected in the BChl a-containing members of the Ectothiorhodospiraceae family (25, 28, 33). In Ectothiorhodospira mobilis, a linear, light-driven electron transport chain transfers electrons from S2- to NAD' (probably via reversed electron transfer [27]). Such a linear chain may (partly) substitute a cyclic electron transfer chain in the conversion of light energy into a proton gradient. Ubiquinol-cytochrome c oxidoreductase activity of a cytochrome bc1 complex is essential at low and neutral pH, while at higher pH values, the reaction between quinol and cytochrome c proceeds spontaneously at high rates (e.g., see

The Ectothiorhodospiraceae family consists of a single genus of gram-negative, photosynthetic purple sulfur bacteria that grow anaerobically (46). For optimal growth, they require an alkaline pH (except for the recently described Ectothiorhodospira marismortui [36]) and high salinity. Elemental sulfur is deposited outside the cells when sulfide is used as an electron donor (21). The Ectothiorhodospiraceae family can be divided into two groups that contain either bacteriochlorophyll (BChl) a or b. These groups also display physiological differences; only the former group is able to grow photoautotrophically with reduced sulfur compounds as electron donors (see reference 6 for a review). These characteristics of the Ectothiorhodospiraceae family give rise to questions about the composition of their electron transport chain and about the generation and maintenance of ion gradients across their cytoplasmic membrane. In purple bacteria, a cyclic electron transfer chain is supposed to be primarily responsible for the conversion of light energy into a transmembrane proton gradient (e.g., see reference 37). Because most characterizations of this cyclic electron transfer chain have been performed on representatives of the a or ,B branches of the purple bacteria (41), little information on members of the y branch, the purple sulfur bacteria, is available. Cytochrome bc1 complexes of photosynthetic bacteria are membrane-spanning complexes which catalyze electron flow * Corresponding author. Electronic mail address: mond.sara.nl.

a4l7hell@dia1629

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LEGUIJT ET AL.

reference 35). Considering the high pH for optimal growth of the Ectothiorhodospiraceae family (and therefore the presumably high pH of the periplasm), in combination with the apparent absence of membrane-bound cytochrome b (7, 25, 28) and the light-driven, linear electron transport chain observed in E. mobilis (27), the question of whether or not a cytochrome be complex is present in these organisms arises. In this communication, we report on the identification and characterization of a cytochrome be1 complex in three BChl a-containing Ectothiorhodospira species. MATERIALS AND METHODS Materials. Polyclonal antibodies, raised against R sphaeroides cytochrome c1 (18) were a gift from C.-A. Yu (Oklahoma State University, Stillwater) and polyclonal antibodies raised against the R. sphaeroides Rieske iron-sulfur protein (40) were a gift from R. A. Niederman (Rutgers University, New Brunswick, N.J.). The inhibitor 2-heptyl4-hydroxyquinoline-N-oxide (HQNO) was a gift from J. A. Berden (University of Amsterdam, The Netherlands). All other materials were obtained from commercial sources; the ubiquinones were purchased from Sigma (St. Louis, Mo.). Growth conditions and isolation of intracellular membranes. E. mobilis BN9903, Ectothiorhodospira halophila BN9630, Ectothiorhodospira shaposhnikovii M3 (49), and R. sphaeroides 2.4.1 were grown as reported previously (28, 29). After harvesting, cells were washed twice with buffer (29) and stored at -200C. Intracellular membranes (chromatophores) were isolated as reported (28) and resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 8% [wt/vol] glycine betaine, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride). (Note that the intracellular membranes isolated from the Ectothiorhodospiraceae family do not form closed vesicles, but rather have the form of thylakoid stacks. They are termed chromatophores for convenience only.) For electron paramagnetic resonance (EPR) measurements, the manganese content of the media was reduced to minimize EPR signals originating from Mn2+, and chromatophores of E. mobilis and E. halophila were washed (four times) with a solution containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride and 0.5 (E. mobilis) or 2.2 M (E. halophila) NaCl. Isolation of the cytochrome bc, complex. The E. mobilis cytochrome be1 complex was isolated according to a combination of reported procedures (1, 9, 15). All steps were performed at 4°C. Chromatophores were solubilized with n-dodecyl ,B-D-maltoside (dodecyl maltoside) at the optimal ratio of 1.1 g/g of protein for 1 h under continuous stirring. The mixture was centrifuged at 120,000 x g for 1.5 h; the supernatant was collected, while the pellet was reextracted under the same conditions. The combined supernatants were layered on linear sucrose gradients (20 to 35% [wt/vol] sucrose and 0.1% [wt/vol] dodecyl maltoside in buffer A with omission of phenylmethylsulfonyl fluoride) and centrifuged at 200,000 x g for 65 h. This long centrifugation time was essential to obtain a clear separation between the membraneassociated c-type cytochromes and the cytochrome bc1 complex and allowed an estimation of the total amount of cytochrome b present in the chromatophores (see below). Fractions with cytochrome bc1 complex were recovered from the gradients, analyzed spectrophotometrically, pooled, and kept on ice until further use within 5 days. Otherwise, the samples were diluted with 50% (vol/vol) glycerol and stored at -20°C. For some experiments, the

J. BA=rRIOL.

cytochrome be1 complex was concentrated with Centricon-10 concentrators (Amicon, Danvers, Mass.). Preceding concentration, the samples were dialyzed briefly (35 min) against 50 mM Tris-HCl (pH 8.0) to remove most of the sucrose and diluted in buffer A with 0.1% (wt/vol) dodecyl maltoside. Spectral analyses. Absorbance measurements were performed as reported previously (28). Chemical redox titrations were carried out anaerobically at 20'C with a thermostated multipurpose 1.8-ml cuvette (23) equipped with a platinum electrode and an Ag-AgCl reference electrode. Cleaning of the platinum electrode and calibration of the setup was performed as reported (28). The following redox mediators were used: 50 jxM 1,4-benzoquinone, 50 jiM hydroquinone, 50 p.M UQo (2,3-dimethoxy-5-methyl-1,4benzoquinone), 10 p.M N-methylphenazonium methosulfate, 10 p.M N-ethyldibenzopyrazine ethyl sulfate, 50 tLM tetramethyl-p-benzoquinone, 50 p.M 2-methyl-1,4-naphthoquinone, 50 p.M 2-OH-1,4-naphthoquinone, 10 p.M anthraquinone-1,5-disulfonate, 10 p.M anthraquinone-2-sulfonate, 1 mM NADH, 1 mM NADPH, 50 p.M 1,1'-dibenzyl-4,4'bipyridylium dichloride, and 50 JuM 1,1'-dimethyl-4,4'-bipyridylium dichloride. Both reductive and oxidative titrations were performed (28). At each value of the ambient redox potential (Eh), six spectra between 500 and 600 nm were recorded. These spectra were averaged and corrected for baseline drift, and the areas under the curves were calculated. Extinction coefficients used for cytochromes b and c1 were 25.6 mM-1 * cm-' at 562 - 577 nm (4) and 20 mM-1 cm-1 at 553 - 542 nm (26), respectively. Quinol-cytochrome c oxidoreductase activity was measured at room temperature (approximately 20°C) as described (15). Routinely, the reaction was started by adding

Q2H2 (2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol)

from an ethanolic stock solution. Where indicated, QoH2 (ethanolic stock solution), Q6H2 (2,3-dimethoxy-5-methyl-6-

[all-trans]famesylfarnesyl-1,4-benzoquinol), or Q10H2 [2,3dimethoxy-5-methyl-6-(prenyl)10-1,4-benzoquinol] was used instead; the latter two ubiquinols were added from stock solutions in Triton X-100 (20). Nonenzymatic reaction rates were determined in the absence of enzyme. Quinones were reduced as described (2) and stored at -70°C. EPR measurements were performed with a Bruker ECS 106 EPR spectrometer operating at a field-modulation frequency of 100 kHz and equipped with an Oxford Instruments ESR 900 cryostat with an ITC4 temperature controller. The microwave frequency and magnetic field values were taken from the instrument without further correction. Analytical procedures. Protein concentrations were measured as described (5). BChl a concentrations were determined as reported (8). Reaction center (RC) concentrations were calculated from the light-induced absorption difference at 605 - 540 nm by using an extinction coefficient of 29.8 mM-1 * cm-' (14) in the presence of 300 ,uM 2,3,5,6-tetramethyl-phenylenediamine to obtain fully reduced RCs. Protoheme was determined according to the method of Takaichi and Morita (42) after extraction of chromatophores (three times) with a 20-fold volume of acetone-methanol (7:2, vol/vol) to remove all pigment. Preceding the extraction, the chromatophores were washed extensively with a high-salinity buffer (matching the salinity of the growth medium) to remove soluble cytochromes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), heme staining, and Western blotting (immunoblotting) were performed as described (28).

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bc, COMPLEX FROM ECTOTHIORHODOSPIRA SPECIES

A

0.02

1

B 2

3

1

2

3

4

9766-

a) 0 0 co

1631

4331-

0.01

Co

V0

i-

D

21140.040

- FeS -

L

500

FIG. 2. Immunodetection of proteins in chromatophores by us520

540

560

580

600

Wavelength (nm) FIG. 1. Redox difference spectra of pyridine hemochromes prepared from chromatophores of E. halophila (..... ), E. mobilis ( ), and E. shajposhnikovii (-- -). Acetone-HCl extracts were prepared as describeed in Materials and Methods. Reduction was performed by adding,,a saturating amount of sodium dithionite.

l

ing polyclonal antibodies against the Rieske iron-sulfur protein (A)

and cytochrome cl (B) of R. sphaeroides. Proteins resolved on SDS-PAGE (15% polyacrylamide) gels were transferred to nitrocellulose filters as described in Materials and Methods. Lanes 1 to 3 of panels A and B contain chromatophores of the following organisms: 1, R. sphaeroides; 2, E. halophila; and 3, E. shaposhnikovii. Lane 4 of panel B contains E. mobilis chromatophores. The amount of protein applied was 10 ,ug in lane 1 and 40 pg in lanes 2 to 4. The positions of cytochrome cl and the Rieske iron-sulfur protein are indicated by l and FeS, respectively. The positions of the molecular mass standards (in kilodaltons) are indicated in the left margin.

RESULTS AND DISCUSSION Distribution of the cytochrome bc, complex among BChl a-containing members of the Ectothiorhodospiraceae family. Despite the apparent absence of cytochrome b in the intracellular membranes of some BChl a-containing Ectothiorhodospira species (7, 25, 28), its presence could be obscured in redox difference spectra by membrane-associated c-type cytochromes, which are abundantly present in these samples (e.g., see reference 28). Therefore, a procedure which includes separation of protoheme (heme b) and heme c (42) was applied. The spectra resulting from this analysis, as applied to chromatophores of three Ectothiorhodospira species, E. halophila, E. mobilis, and E. shaposhnikovii, are presented in Fig. 1. After extraction of the membranes with acidic acetone, protoheme was observed to be present in the supernatant fractions of all three species, as judged by the characteristic absorption maximum at 555 or 556 nm. Because the amounts of cytochrome b, as determined with this procedure, were highly irreproducible, these spectra can only be regarded as a qualitative indication for the presence of cytochrome b. Because the membranes were extensively washed with high-salinity buffer prior to protoheme extraction, these data suggest that the observed protoheme is the prosthetic group of a membrane-bound component, rather than of a soluble component trapped during chromatophore preparation. To investigate whether the observed protoheme is a component of a cytochrome bc, complex, Western blots were made with polyclonal antibodies raised against subunits of the R sphaeroides cytochrome bc, complex (Fig. 2). As a control, we show the cross-reaction in chromatophores of R. sphaeroides between the Rieske iron-sulfur protein and cytochrome c1 with a polyclonal antiserum primarily raised against the Rieske protein (Fig. 2A, lane 1). Membranes of both E. halophila and E. shaposhnikovii displayed a clear cross-reaction with a polypeptide about 21 kDa in size, indicating the presence of a Rieske iron-sulfur protein (Fig. 2A, lanes 2 and 3). Chromatophores of R. sphaeroides treated with antibodies raised primarily against cytochrome c1 displayed several cross-reacting bands (Fig. 2B, lane 1), suggesting that the antibodies reacted with all cytochrome bc, subunits. In the

chromatophores of E. halophila (lane 2), E. shaposhnikovii (lane 3), and E. mobilis (lane 4), a clear cross-reaction with a peptide about 30 kDa in size was observed, indicating the presence of cytochrome c1. The observed band at about 66 kDa (Fig. 2) was presumably due to aggregation (24, 40). The weaker-staining bands at about 23 and 25 kDa in R. sphaeroides chromatophores (Fig. 2B) were probably degradation products (17, 53). To confirm that the Ectothiorhodospira species investigated indeed contain a cytochrome bc, complex, EPR spectroscopy of their membranes was performed. Low-temperature EPR spectra of ascorbate-treated chromatophores of both E. mobilis and E. halophila (Fig. 3) show a rather sharp line around g = 1.90 (g ), which is indicative for the presence of a Rieske [2Fe-'S] cluster (see reference 45 for a review). The gy values observed in the spectra of E. mobilis (Fig. 3A) and E. halophila (Fig. 3B and C) were 1.895 and 1.910, respectively. This latter value is slightly higher than the one reported for the Rieske iron-sulfur cluster in R. sphaeroides chromatophores (11, 31). In both spectra, theg, signals (expected at g = 1.76 to 1.81) could not be resolved from the background noise because of reduction of the Q pool (12, 13, 31). Interfering signals from radicals and high-potential iron-sulfur proteins (34) prevented detection of the g, lines (at g = 2.02 to 2.03). Thus, EPR spectroscopy supports the conclusion that the investigated alkaliphilic Ectothiorhodospira species do contain a cytochrome bc, complex. Purification of the E. mobilis cytochrome be1 complex. By using solubilization of chromatophores with dodecyl maltoside and subsequent sucrose-gradient centrifugation, we were able to purify the cytochrome bc, complex from E. mobilis membranes (Fig. 4). Only minor contamination with carotenoids was observed in the spectrum of this sample. The low absorbance in the wavelength region between 480 and 600 nm relative to the Soret absorbance of oxidized cytochrome near 422.5 nm, indicates a successful separation of most of the BChl and carotenoids from the cytochrome bc, complex (Table 1). The hydroquinone-reduced minus ferricyanide-oxidized difference spectrum (Fig. 4, inset) shows the absorption maximum (A553) of the a band of

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J. BACTERIOL.

TABLE 1. Prosthetic group content of chromatophores and the purified cytochrome bc, complex from E. mobilis Prosthetic group content"

(nmol/mg of protein) of:

Prosthetic group

Chromatophores

Cytochrome cl

Cytochrome b BChl a RC

C

I..

0.34 100.9 1.0

Cytochrome bcl 5.8 10.4 0.9 _F

aThe amounts of cytochromes b and c were calculated on basis of redox difference spectra. For chromatophores, the total amount of cytochrome b was estimated by summation of the amount of cytochrome b of the sucrosegradient fractions after solubilization and sucrose-gradient centrifugation of the membranes (see Materials and Methods). b ND, not determined. c _, not detectable.

.I

2.1

1.9 1.8 G - VALUE

2.0

1.7

FIG. 3. Low-temperature EPR spectra of the [2Fe-2S] cluster of the cytochrome bc, complexes from E. mobilis (A) and E. halophila (B and C). Spectrum C is a magnification (18.5 times) of a part of spectrum B. Chromatophores (approximately 30 mg/ml of protein) were incubated with 40 mM ascorbate, 25 pM cytochrome c, and 3 ILM TMPD (NNNN'-tetra-methyl-p-phenylenediamine) for 10 min at room temperature and frozen in liquid N2. EPR conditions: modulation frequency (u), 9.44 GHz; modulation amplitude, 1.27 mT; microwave power, 20 mW; temperature, 36 K. The G valve is defined as 0.7145 * v/gauss.

reduced cytochrome cl (24, 50, 51). The dithionite-reduced hydroquinone-reduced difference spectrum (Fig. 4, inset) reveals the absorption band, with a maximum at 560.5 nm, of the a band of reduced cytochrome b (24, 50, 51). There was no evidence in this latter spectrum for a shoulder at 553 nm, demonstrating the successful separation of membrane-associated low-potential cytochrome c, which is abundantly present in E. mobilis chromatophores (28). minus

1.0

0.10

0.5

0.05

0 0

a

D

NDb

0 m .0

0.0

400

0.00

450

500

550

600

650

Wavelength (nm) FIG. 4. Absolute and redox difference spectra of the E. mobilis cytochrome bc, complex. The solid line with the peak at 422.5 represents the absolute spectrum of the complex. The inset shows the hydroquinone-reduced minus ferricyanide-oxidized ( ) and dithionite-reduced minus hydroquinone-reduced (-- -) difference spectra. The complex (equivalent to 1.2 F.M cytochrome cl) was dissolved in a mixture of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, and 0.01% (wt/vol) dodecyl maltoside.

Table 1 shows that the E. mobilis cytochrome bc, complex contains 1.8 mol of cytochrome b per mol of cytochrome c1, which is comparable to values of 1 to 2 mol of cytochrome b per mol of cytochrome cl, as reported for bc, complexes isolated from other photosynthetic bacteria (1, 24, 30, 50, 51). On the basis of this b/c, ratio and the assumption that all heme b observed in E. mobilis chromatophores belonged to a bc, complex, a ratio of about 0.2 mol of cytochrome bc, complex per mol of RC could be calculated (Table 1). This ratio is comparable to the stoichiometry of 0.1 to 0.2 reported for Rhodospirillum mbrum (49), but it is significantly below the ratio of 0.5 to 0.7 cytochrome bcl per RC described for R sphaeroides and K capsulatus (38, 39, 47). This low stoichiometry in E. mobilis cells did not significantly increase when the cells were grown at higher light intensities, as was reported for Rhodopseudomonas viridis and R capsulatus (9, 16). This low and constant bc,/RC ratio may be due to the presence of a linear, light-driven electron transfer chain in E. mobilis, transferring electrons from S2via the RCs to NAD', thereby bypassing the cytochrome bc1 complex (27). Further studies on the nature of the components involved in light-driven electron transport in the BChl a-containing members of the Ectothiorhodospiraceae family may clarify this issue. Composition of the E. mobifis cytochrome bce complex. Although so far no spectral indications for the presence of two different types of heme b (Fig. 4) have been obtained, a redox titration of the E. mobilis cytochrome bc, complex clearly revealed two different b hemes with Em values (at pH 7.5) of -142 mV (standard deviation = 6.3 mV) and 116 mV (standard deviation = 4.3 mV), i.e., cytochrome bL and bH, respectively (Fig. 5A). The reduced minus oxidized difference spectrum of cytochrome bL displayed an asymmetric a band with an absorption maximum at 564 and a shoulder at 559 nm, as was also reported for cytochrome bL from R. rubrum (17). Cytochrome bH displayed the characteristic symmetric a band with an absorption maximum at 559.5 nm (1, 15, 17). The titration of c-type cytochromes (Fig. SB), revealed a single component with Em = 341 mnV (standard deviation = 1.5 mV). The Em values obtained for cytochrome bL and cytochrome c1 are comparable to those reported for other photosynthetic bacteria, particularly R. rubrum, while the value obtained for cytochrome bH is higher (1, 9, 15, 17, 24). It has been reported for bacterial, as well as for chloroplast cytochrome bc/bf complexes, that the Em values are pH dependent (see reference 20 for a review). Because the

CYTOCHROME

VOL. 175, 1993

bc,

100

1

80 -

43-

ID

60

m1-

ID ._

116

0)

14-

20

A\ 0

-400

-300

-200

-100 0 Eh (mV)

100

200

300

100 B

80 -

-

60

3

-b0 - C.,-

FIG. 6. Peptide composition of the E. mobilis cytochrome be1 complex. Electrophoresis of the complex on a 15% polyacrylamide gel and Western blotting were performed as described in Materials and Methods. Lanes: 1, cytochrome bc1 complex stained for proteins with Coomassie brilliant blue; 2, immunodetection with polyclonal antibodies raised against the R. sphaeroides Rieske ironsulfur protein; 3, immunodetection with polyclonal antibodies raised against R sphaeroides cytochrome cl. The amount of complex applied to each lane was equivalent to 60 pmol of cytochrome cl. The positions of cytochromes b and cl and the Rieske iron-sulfur protein are indicated by f, and FeS, respectively. The positions of the molecular mass standards (in kilodaltons) are indicated in the left margin. b,

0 -

2

1633

- FeS~~~~21-

~~~

116

40 cc

-s~

COMPLEX FROM ECTOTHIORHODOSPIRA SPECIES

40

carOD

obtained for the presence of a fourth as reported for R. sphaeroides (52). As in Fig. 2, cross-reaction in the higher-molecular-weight region of the gel was observed, which again was presumably due to aggregation. Ubiquinol-cytochrome c oxidoreductase activity. The pH optimum of ubiquinol-cytochrome c oxidoreductase activity 50). No evidence

20

was

subunit (about 15 kDa),

0

200

300

400

500

Eh (mV)

FIG. 5. Redox titration of the cytoc :hromes present in the E.

mobilis cytochrome bc1 complex. (A) Fc cytochromes bH and bL, the data were plotted to give the best fit to the Nernst equation for two n = 1 components. (B) For cytochrc)me cl, the data were fitted to a single n = 1 component. The titrrations were performed as described in Materials and Methods. Thie mixture contained cyto)r

of the E. mobilis

bc

complex is shown in Fig.

7.

With Q2H2

(15) and cytochrome c from Saccharomyces cerevisiae, the purified complex displayed a pH optimum at pH 8, which is

higher than the optima observed for R viridis (pH 6.8 [9]) and R. sphaeroides (pH 7 to 7.5 [15]). This is possibly a

chrome bc1 complex equivalent to 2 ,uM cytochrome cl, 50 mM Tris-HCI (pH 7.5), 100 mM NaCi, 1 mM MgCl2, and 0.01% (wt/vol) dodecyl maltoside plus several redox mediators, which are de-

50

scribed in Materials and Methods.

0

Go

determination of the Em values was performed at pH 7.5, whereas the optimal pH for growth (which presumably equals the pH of the periplasm) of Ectothiorhodospira species is about pH 9, it is possible that under these physiological conditions, the span in Em values between cytochromes bH and bL is smaller and therefore comparable to those of other photosynthetic bacteria. The polypeptide composition of the purified E. mobilis cytochrome be1 complex was determined by SDS-PAGE followed by staining for protein or Western blotting with antibodies against the R sphaeroides cytochrome bc1 complex (Fig. 6). Three major polypeptides with apparent molecular masses of 43, 30, and 21 kDa were observed in lane 1 after staining for protein with Coomassie brilliant blue. From the cross-reaction with antibodies raised against either the R sphaeroides Rieske iron-sulfur protein (lane 2) or cytochrome cl (lane 3), the 30- and 21-kDa components could be identified as cytochrome cl and the Rieske ironsulfur protein, respectively. Heme staining revealed only the former polypeptide. The third component (43 kDa) is therefore most probably cytochrome b. Its apparent molecular weight is comparable to the ones observed for cytochrome be1 complexes from other photosynthetic bacteria (1, 15, 32,

_

40 30

._

0

. >00 co -D

8d_ E

:

08

0

20

s

10

* 0

-

L-o

0

3

4

5

-

*

MIl l

1

-

6

I

8

7

9

0

10

11

12

pH

FIG. 7. pH dependence of the oxidoreductase activity of the E. mobilis cytochrome (cyt) be1 complex. The assay was performed as described in Materials and Methods. The assay mixture contained 15 mM MES [2-(N-morpholino)ethanesulfonic acid], 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 15 mM glycyl glycine, 1 mM EDTA, 50 ,uM cytochrome c from S. cerevisiae, and 28 nM cytochrome be1 complex, as measured on the basis of cytochrome cl. The reaction was started by the addition of 150 'PM Q2H2 (final concentration). The activities have been corrected for the uncatalyzed reaction, which is indicated by the dashed curve.

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reflection of the alkaliphilic nature of the Ectothiorhodospiraceae family. The uncatalyzed rate of quinol oxidation becomes more prominent at a higher pH (20), requiring increasing corrections in this assay at higher pH values. Variation of electron donors and acceptors clearly showed that Q2H2 as electron donor and equine cytochrome c as acceptor yielded maximal catalytic activity of the E. mobilis cytochrome be1 complex. The turnover rate was 57 mol of cytochrome c reduced (mol of cytochrome c1)-l s-1. This is comparable to values reported for R rubrum (24) and R. vinidis (9), but is lower than the rate of about 300 s5 reported for R. sphaeroides (1). The effects of specific inhibitors on the cytochrome bc1 complex are presented as follows (assay conditions were as in Fig. 7 at pH 7.5). In the presence of 23 ,M myxothiazol, an inhibitor known to act at the quinol oxidation (Qz) site of the complex, 50% of the activity was inhibited (value was obtained by determination of the percentage of inhibition at a range of myxothiazol concentrations between 100 nM and 100 jxM). Addition of 100 ,uM stigmatellin, also an inhibitor of the Qz site, showed no effect at all. The maximal inhibition obtained after addition of myxothiazol was 96% (at 100 ,uM of the inhibitor). In the presence of 100 ,uM concentrations of the Qc inhibitors HQNO and antimycin A, only 29 and 41%, respectively, of the activity was inhibited. This low degree of inhibition of the activity by these inhibitors was not dependent on the nature of the ubiquinol used as electron donor, because the same values were observed when either Q6H2 or Q1oH2 was used. The lack of sensitivity of the E. mobilis cytochrome bc1 complex for inhibitors contrasts with results reported for other photosynthetic bacteria, but is comparable to the situation observed for cytochrome b J complexes from chloroplasts (see reference 19 for a review). This low degree of sensitivity might indicate a disturbance of the catalytic site due to loss of a fourth subunit. However, in view of the fact that the isolated complex shows a high turnover rate, combined with the inhibitor resistance of the complex in the membrane, it is more likely that this inhibitor resistance is due to an intrinsic property of the E. mobilis be complex rather than to loss of a fourth subunit. The suggestion that the resistance of cytochrome b f complexes to antimycin A and HQNO, inhibitors known to act at the Qc site, and myxothiazol, a Qz site inhibitor (see reference 19 for a review), may be due to some amino acid substitutions in cytochrome b6 (10) has attained firm support. All known mutations leading to inhibitor resistance of bacterial cytochrome bc1 complexes indeed map in the gene encoding for cytochrome b (see references 45 and 54 for reviews). Comparison of the sequences of the genes coding for the subunits of the E. mobilis bc1 complex with those reported for various inhibitor-resistant mutants, combined with EPR spectroscopy in the presence of inhibitors, will therefore be essential to establish the basis of the inhibitor resistance of the E. mobilis cytochrome bc1 complex. By using the data described in this paper, in combination with results presented earlier (28, 29), a preliminary description of the cyclic electron transport chain in the BChl a-containing members of the Ectothiorhodospiraceae family can be provided. Like many purple bacteria, these alkaliphilic and halophilic bacteria contain a three-subunit RC and a presumably three-subunit quinol-cytochrome c oxidoreductase. This latter complex of E. mobilis has a slightly increased pH optimum and a strikingly low sensitivity towards inhibitors. Insight into the energy-transducing mechanisms of the

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BChl a-containing members of the Ectothiorhodospiraceae family will be important for elucidation of the mechanism of the generation and maintenance of ion gradients across their cytoplasmic membrane. This is the subject of our current investigations. ACKNOWLEDGMENTS We gratefully acknowledge C.-A. Yu and R. A. Niederman for kindly providing us with antibodies. We also thank J. A. Berden for his kind gift of inhibitor. S.P.J.A. is indebted to the Dutch Organization for the Advancement of Pure Research (NWO) for grants (supplied via the Dutch Foundation for Chemical Research [SON]) which enabled the purchase of the EPR spectrometer.

1.

2. 3.

4. 5. 6.

7.

8. 9. 10.

11.

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