Natural Resistance to Inhibitors of the Ubiquinol Cytochrome c Oxidoreductase of Rubrivivax gelatinosus: Sequence and Functional Analysis of the Cytochrome bc1 Complex

Biochemical analyses of Rubrivivax gelatinosus membranes haverevealed that the cytochrome bc₁ complex is highly resistantto classical inhibitors including myxothiazol, stigmatellin,and antimycin. This is the first report of a strain exhibitingresistance to inhibitors of both catalytic Q₀ and Q_i sites.Because the resistance to cytochrome bc₁ inhibitors is primarilyrelated to the cytochrome b primary structure, the petABC operonencoding the subunits of the cytochrome bc₁ complex of Rubrivivaxgelatinosus was sequenced. In addition to homologies to thecorresponding proteins from other organisms, the deduced aminoacid sequence of the cytochrome b polypeptide shows (i) an E303Vsubstitution in the highly conserved PEWY loop involved in quinol/stigmatellinbinding, (ii) other substitutions that could be involved inresistance to cytochrome bc₁ inhibitors, and (iii) 14 residuesinstead of 13 between the histidines in helix IV that likelyserve as the second axial ligand to the b_H and b_L hemes, respectively.These characteristics imply different functional propertiesof the cytochrome bc₁ complex of this bacterium. The consequencesof these structural features for the resistance to inhibitorsand for the properties of R. gelatinosus cytochrome bc₁ arediscussed with reference to the structure and function of thecytochrome bc₁ complexes from other organisms.

INTRODUCTION

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Introduction

Ubihydroquinone cytochrome c oxidoreductase (cyt bc₁ complex)is an integral membrane complex involved in energy transductionin a wide range of organisms. Anoxygenic photosynthetic bacteriacontain a cyt bc₁ complex, which serves for both light-drivenelectron transfer and dark respiration. For Rhodobacter sphaeroidesand Rhodobacter capsulatus this enzyme catalyzes electron transferfrom ubiquinol to soluble ferricytochrome c coupled to protontranslocation across the membrane, thus generating an electrochemicalgradient used for ATP synthesis. Oxygenic photosynthetic organismscontain a similar complex, i.e., cyt b_6f, which functions asa plastoquinol plastocyanin oxidoreductase (12). Structuraland functional similarities have been reported for both typesof cyt bc (3, 9, 41). The cyt bc complex was found to be madeup of at least three subunits: cyt b (or b₆), containing thetwo b-type hemes of low (b_L) and high (b_H) redox potentials,cyt c₁ (or f) containing a c-type heme, and the Rieske protein(ISP) containing the [2Fe-2S] cluster. In addition to subunitscontaining the prosthetic groups and cofactors, the mitochondrialcyt bc₁ complex contains other subunits (4, 29, 30, 46, 47).

Photosynthesis and aerobic respiration are affected by inhibitors.Some of these molecules are specific to the cyt bc complex.In fact, in many species the ubiquinol-cyt c oxidoreductaseactivity was found to be sensitive to quinone analogs such asmyxothiazol, stigmatellin, antimycin, hydroxyquinoline N-oxide(HQNO), and diuron. Extensive kinetic and genetic studies (7,13, 16, 17, 19, 28) as well as recent structural data availablefrom X-ray crystallography of the mitochondrial cyt bc₁ complex(4, 29, 30, 47) in the presence of these inhibitors have allowedbetter knowledge of the properties of the two sites of quinonebinding, the Q₀ and the Q_i sites (3). The Q₀ pocket, which isthe site of oxidation of quinol, is located toward the periplasmicside of the membrane and is essentially composed of cyt b residuesclose to the heme b_L and of the extrinsic domain of the Rieskeprotein. It is the site of two concerted electron transfers,one from quinol through ISP and cyt c₁ to the external electronacceptor and the other through hemes b_L and b_H to the reducingsite of quinone Q_i, located near the cytoplasmic side of themembrane close to heme b_H. Much experimental data support theidea that this bifurcated electron flow through cyt bc₁ is controlledby the movement of the periplasmic domain of the Rieske proteincontaining the [Fe-S] prosthetic group (4, 17, 18, 47). ThisQ cycle results in the oxidation of two molecules of ubiquinolwith the release of four H⁺s at the Q₀ site and the reductionof one molecule of quinone with uptake of two H⁺s at the Q_isite. Myxothiazol and stigmatellin bind to the Q₀ pocket, displacingubiquinol, and thus block the electron pathway to ferricytochromec and to heme b_L. Antimycin, HQNO, and diuron block the reductionof quinone at the Q_i site.

Rubrivivax gelatinosus, a facultative phototrophic nonsulfurbacterium belonging to the ß subclass of purple bacteria,can grow either aerobically, deriving its energy from aerobicrespiration, or photosynthetically by using the cyclic electrontransport chain. In this paper we report the study of naturalresistance to electron transport inhibitors myxothiazol, stigmatellin,and antimycin in this purple bacterium. The level of the resistanceof the R. gelatinosus cyt bc₁ complex to these inhibitors wasdetermined by in vitro enzymatic activity and compared to thoseobserved for Rhodobacter sphaeroides, Blastochloris viridis,and Allochromatium vinosum. To explain the observed phenotypes,the sequence of the petABC operon was determined and comparedto known pet sequences and to a compilation of substitutionslocated in the cyt b subunit of the bacterial and mitochondrialcyt bc₁ complexes associated with sensitivity or resistanceto inhibitors (7, 13, 21, 22, 26). The primary sequence comparisonbetween different photosynthetic bacteria revealed the presenceof substitutions in R. gelatinosus and in A. vinosum petB (cytb) and suggested that the resistance to inhibitors of both speciesmay be attributed to substitution of these residues in the quinonebinding sites. The identified substitutions in the cyt b sequencemay account for the unusual properties of both Q₀ and Q_i sitesin the R. gelatinosus cyt bc₁ complex.

MATERIALS AND METHODS

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Materials and Methods

Chemicals. Decylubiquinone, horse heart cyt c, and antimycin were purchasedfrom Sigma, myxothiazol was from Boehringer, and stigmatellinwas from Fluka.

DBH₂ preparation. The reduction of the 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone(decylubiquinol; DBH₂) was done as described previously (38).The concentration of DBH₂ in ethanol was determined by measuringthe absorbance using an extinction coefficient ( ${varepsilon}$ ) at 290 nmof 4 mM^-1 cm^-1.

Bacterial strains and growth media. R. gelatinosus wild-type strain S1 (45), Rhodobacter sphaeroides,and B. viridis were grown anaerobically in light in a malategrowth medium (1). R. gelatinosus mutant strain S ${Delta}$ C2 (36) wasgrown semiaerobically in the dark in the same medium supplementedwith kanamycin (50 µg/ml). A. vinosum strains D and DSM180T(German collection) were grown under reducing photoheterotrophicconditions as described previously (33).

Membrane preparation. Membranes were prepared by cell disruption with a French pressin the presence of 0.1 M sodium phosphate buffer, pH 7.4, containing1 mM phenylmethylsulfonyl fluoride, followed by differentialultracentrifugation as previously described (1). Subsequently,the Rhodobacter sphaeroides membranes were resuspended in thesame buffer and R. gelatinosus, B. viridis, and A. vinosum membraneswere resuspended in 0.1 M Tris-HCl buffer, pH 8. Protein concentrationwas determined by using the Pierce BCA protein assay reagentmethod with bovine serum albumin as the reference standard.

Isolation of soluble electron carriers of R. gelatinosus. It has been shown that the main periplasmic redox proteins inR. gelatinosus are cyt c₈ and the high-potential iron protein(HiPIP) and that, in cells grown under semiaerobic dark conditions,the amount of cyt c₈ is much larger than that in cells grownunder photosynthetic conditions (34). Therefore, we purifiedthis cytochrome from the R. gelatinosus S ${Delta}$ C2 strain grown inthe dark. The cyt c₈ was recovered from the supernatant of cellextracts after ultracentrifugation and purified by precipitationwith ammonium sulfate (45% saturation). The precipitate wasdialyzed against 10 mM MES (morpholineethanesulfonic acid)-NaOHbuffer, pH 6, and subsequently adsorbed onto a carboxymethyl(CM)-Sepharose ion exchange resin equilibrated with the samebuffer. The cytochrome was eluted at 25 mM NaCl. The HiPIP wasprepared by a method resembling that of Osyczka et al. (35).It was recovered from the supernatant of cell extracts and purifiedfrom the fraction precipitating at 100% saturation of ammoniumsulfate, which was then dialyzed against 10 mM MES-NaOH buffer,pH 6. Subsequently the dialyzed fraction was adsorbed on CM-Sepharoseequilibrated with the same buffer. The HiPIP was eluted at 50mM NaCl, and the fractions with absorbance ratios (A₂₈₀/A₃₈₈)of 3 to 4 were pooled and concentrated. The sample was furtherpurified by gel filtration on a Sephadex G-50 column equilibratedwith 50 mM Tris-HCl, pH 8, and fractions with A₂₈₀/A₃₈₈ valuesof 2 to 2.2 were pooled.

Before the cyt bc₁ activities were measured, horse heart cytc, cyt c₈, and the HiPIP were oxidized with potassium ferricyanide.The excess oxidant was then removed on a Sephadex PD-10 column.

Spectrophotometric measurements. The absolute and differential absorption spectra of differentsamples were recorded with a Cary 2300 absorption spectrometer.Ubiquinol cyt c, cyt c₈, and HiPIP oxidoreductase activitieswere measured with a DW-Aminco Chance spectrometer in the dual-wavelengthmode. The membranes were suspended in a 3-ml stirred cell containing4 mM KCN (to prevent cyt c oxidase activity), and oxidized cytochromesor HiPIPs were then added. The mixture in the presence or absenceof inhibitors was stirred for 5 min in the dark, and the activitymeasurements were initiated by the addition of DBH₂.

The kinetics of the enzymatic reduction of oxidized cyt c (cytc_ox) and cyt c_8ox were monitored as an increase in the valueof A₅₅₀ - A₅₄₀ (isobestic point) in the dark. From the initialrate of absorption increase (V_i), the amount of reduced cytc per minute was quantified by using a value for the differentialextinction coefficient ( ${Delta}$ ${varepsilon}$ ) between 550 and 540 nm of 19 mM^-1cm^-1 (23)_. Specific activities were determined as micromolesof cyt c reduced per nanomole of cyt bc₁ per hour as describedpreviously (25). When the HiPIP was used as the electron acceptor,its reduction was monitored as the decrease of A₄₈₃ - A₆₀₀ witha ${Delta}$ ${varepsilon}$ of 7 mM^-1 cm^-1. Under our experimental conditions the nonenzymaticreduction of ferricytochrome c or the HiPIP by DBH₂ was notdetected.

Estimation of the cyt bc₁ concentration in the membrane fraction. The amount of cyt bc₁ complex was estimated by determining thecyt b concentration at 562 nm from the absorption differencespectra for the membrane suspension (dithionite minus ascorbate); ${Delta}$ ${varepsilon}$ [562 to 575 nm] = 28 mM^-1 cm^-1) (11). Alternatively, the amountof cyt b was estimated by the pyridine hemochrome method (5)for B. viridis, R. gelatinosus, and A. vinosum.

Molecular biology techniques. Standard methods were performed, if not otherwise indicated,as described by Sambrook et al. (39). Plasmids were purifiedwith the Bio-Rad Quantum Prep plasmid kit. DNA was treated withrestriction enzymes and other nucleic acid-modifying enzymesaccording to the manufacturers' specifications. DNA fragmentswere analyzed on agarose gels, and restriction fragments werepurified with the Geneclean kit (Bio 101, Inc.). PCRs were carriedout by using genomic or plasmid templates in a 50-µl reactionmixture containing the DNA, the PCR buffer, 200 µM (each)deoxynucleoside triphosphate, 1 µM (each) primer, 5% dimethylsulfoxide, and 2.5 U of Taq DNA polymerase. Twenty cycles wereperformed in a Hybaid thermal cycler; each cycle consisted of30 s at 92°C, 40 s at 60°C, and 40 s at 72°C. Theprimers used to clone the DNA fragment of R. gelatinosus containingthe pet genes were 5'-CGGTCGAGCACATCATGC-3' and 5'-CACCGCGCGCAGCATCGA-3'.The primers used to clone the A. vinosum 0.4-kb fragment ofthe petB gene (5'-CGCTGGAGCGACAGGCTCCG-3' and 5'-CGGTCGCCCACTTGAAGATC-3')were based on the previously published sequence (10). DNA sequencingwas performed with an ABI 373 automatic DNA sequencer.

Nucleotide sequence accession number. The sequences reported in this paper were deposited in GenBankunder accession no. AF380164.

RESULTS

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Results

Effects of inhibitors on decyl quinol oxidoreductase activity of R. gelatinosus membranes. Most of the cyt bc₁ reductase activity measurements for R. gelatinosuswere carried out with horse heart cyt c as the electron acceptorto allow a comparison with the cyt bc₁ activities from Rhodobactersphaeroides, B. viridis, and A. vinosum. Nevertheless, in severalexperiments physiological carriers cyt c₈ and HiPIP were usedas substrates.

Figure 1 shows the time course for reduction of cyt c by DBH₂catalyzed by R. gelatinosus membranes in the presence and absenceof specific inhibitors of the cyt bc₁ complex acting eitheron the Q₀ site (myxothiazol and stigmatellin) or on the Q_i site(antimycin). The reduction of cyt c by cyt bc₁ displays monoexponentialdecay kinetics with an average rate constant (k) of 0.046 s^-1;values of k in the presence and absence of inhibitors were similar.Furthermore, the activities were measured with different cytbc₁ concentrations in the presence of different amounts of eachinhibitor (Table 1). From inspection of Fig. 1 and Table 1 itis obvious that the R. gelatinosus cyt bc₁ complex is resistantto the three inhibitors. A weak decrease in the activity inthe presence of a very high concentration (0.15 mM) of myxothiazolwas observed, namely, a maximum of 50% inhibition of cyt bc₁activity was reached (not shown).

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FIG. 1. Decyl ubiquinol horse heart cyt c oxidoreductase activity of R. gelatinosus membranes in the presence and absence of specific inhibitors of the cyt bc₁ complex. Membranes were suspended at 0.66 mg of protein/ml in 0.1 M Tris-HCl buffer, pH 8, containing 4 mM KCN and 20 µM horse heart cyt c. The reaction was started by the addition of 30 µM DBH₂. The time course of activity follows a monoexponential decay with time constants as follows: without inhibitor (•), 0.046 s^-1; with 25 µM antimycin ( ${circ}$ ), 0.046 s^-1, with 25 µM myxothiazol (_*), 0.04 s^-1; with 10 µM stigmatellin ( ${blacktriangleup}$ ), 0.04 s^-1. (Inset) Initial linear rates of absorption increase (derived from the larger graph) from which specific activities (A) were determined as described in Materials and Methods. •, A = 36; ${circ}$ , A = 32; _*, A = 30; ${blacktriangleup}$ , A = 30.

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TABLE 1. Measurements of cyt bc₁ activity in the membranes isolated from different species^a

When R. gelatinosus cyt bc₁ activity in the presence of itsphysiological electron acceptors cyt c₈ and HiPIP was determined,a higher turnover of the enzyme was obtained. For cyt c₈, theactivity increased by a factor of 2 to 3 relative to that forequine cyt c, whereas, with the very reactive HiPIP, the activitywas 30 times higher than that for cyt c reductase measured underthe same experimental conditions (not shown). As with cyt c,cyt c₈ and the HiPIP reductase were not affected by the inhibitors(data not shown). These results demonstrate that the cyt bc₁complex from R. gelatinosus is resistant to both Q₀ and Q_i siteinhibitors, i.e., stigmatellin, myxothiazol, and antimycin,irrespective of its external electron acceptor.

Comparison of decyl quinol cyt c oxidoreductase activities of Rhodobacter sphaeroides and B. viridis. For reference, we measured the decyl quinol cyt c oxidoreductaseactivity in Rhodobacter sphaeroides and B. viridis membranesunder the same conditions. A high sensitivity to the inhibitorsin both species has already been demonstrated (15, 25, 44).B. viridis and R. gelatinosus membranes contain much less cytbc₁ than Rhodobacter sphaeroides membranes (Table 1). However,the small amount of cyt bc₁ in these species was compensatedfor by its high enzymatic turnover. In the presence of the inhibitors,enzymatic activity was completely abolished in Rhodobacter sphaeroides(Fig. 2) and was strongly reduced in B. viridis to only 10 to20% residual activity (Fig. 3). Table 1 summarizes the cyt creductase activities measured with different cyt bc₁ concentrationsin the presence of different amounts of each inhibitor.

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FIG. 2. cyt bc₁ activity of Rhodobacter sphaeroides chromatophores in the presence and absence of the inhibitors. The chromatophores were suspended at 0.24 mg of protein/ml in 0.1 M sodium phosphate buffer, pH 7.5, containing 4 mM KCN and 20 µM horse heart cyt c. The reaction was started with 20 µM DBH₂. Additions and conditions were as in Fig. 1. •, specific activity (A) = 14; ${circ}$ , A = 0.28; _*, A = 0.54; ${blacktriangleup}$ , A = 0.42.

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FIG. 3. cyt bc₁ activity of B. viridis chromatophores in the presence and absence of inhibitors. The chromatophores were suspended at 0.66 mg/ml in 0.1 M Tris buffer, pH 8, containing 20 µM cyt c; 30 µM DBH₂ was added to start the reaction. •, specific activity (A) = 248; ${circ}$ , A = 27; ${blacktriangleup}$ , A = 34; _*, A = 13.7.

We conclude that Rhodobacter sphaeroides and B. viridis aresensitive to the inhibitors as already reported (15, 25, 44)and confirm that the R. gelatinosus cyt bc₁ complex is resistantto myxothiazol, stigmatellin, and antimycin.

Because the resistance or sensitivity to the cyt bc₁ inhibitorsis primarily related to the cyt b primary structure, we thenanalyzed the cyt b sequence from R. gelatinosus and comparedit to the sequences of those from other species.

Sequence of the petABC operon from R. gelatinosus. A genomic R. gelatinosus DNA library (37) was screened by PCRfor the presence of the petB gene encoding cyt b. A plasmidcontaining an approximately 7-kb DNA fragment was isolated,and a 2.3-kb EcoRI fragment containing three open reading frames(ORFs) was subcloned and sequenced. BLASTX (2) sequence similaritysearches against the nonredundant protein database suggestedthat the ORFs are homologues of the petA, petB, and petC genes,which encode the Rieske Fe-S protein (ISP), cyt b, and cyt c₁,respectively. The DNA fragment contains the entire sequenceof petA and petB and the 5' end of petC. The three genes havethe same transcriptional orientation, with no putative promotersequences in the intergenic region, suggesting that they mightconstitute an operon.

cyt b amino acid sequence analyses. The petB gene consists of 1,272 bp and encodes a polypeptideof 423 amino acids with a molecular mass of 48 kDa. PetB hassignificant similarity to the cyt b polypeptides from Pseudomonasaeruginosa (65% identity and 76% similarity), Neisseria meningitidis(63% identity and 74% similarity), and PetB from A. vinosum(61% identity and 74% similarity). Lower matches to the PetBpolypeptides from Rhodobacter sphaeroides (42% identity and59% similarity), B. viridis (41% identity and 58% similarity),and Rhodobacter capsulatus (40% identity and 57% similarity)were seen. According to TMpred and SOSUI transmembrane regiondetection programs, PetB possesses eight hydrophobic transmembranehelices, in agreement with the published structure of the mitochondrialcyt bc₁ complex (46). In addition, according to the helicalwheel analysis (SOSUI), an extra amphipathic helix could beexposed on the periplasmic side of the membrane linking helicesIII and IV. The alignment of the deduced amino acid sequenceof cyt b from R. gelatinosus with homologues from bacteria andSaccharomyces cerevisiae (not shown) revealed three interestingfeatures. (i) Whereas the spacing (13 residues) between thehistidines supposed to serve as axial ligands to the hemes isconserved in all cyt b sequences in helix II, there are 14 residuesbetween the heme binding histidines in helix IV in R. gelatinosus(Fig. 4A), as already observed in cyt b₆ (41) and in the cytb subunits of the ß, ${gamma}$ , and ${varepsilon}$ subdivisions of proteobacteria,with the exception of A. vinosum (41). (ii) The highly conservedPEWY motif found in hundreds of cyt b sequences from differentorganisms is changed to PVWY in R. gelatinosus and A. vinosum(Fig. 4B). The glutamic acid in the PEWY loop is involved ininhibitor binding at the Q₀ site, as shown by the structureof the chicken cyt bc₁ cocrystallized with stigmatellin (47).The replacement of glutamic acid 303 with the hydrophobic valinemight then prevent the binding of stigmatellin to cyt b (seeDiscussion). (iii) Several substitutions of residues localizedin the regions presumed to be involved in the Q₀ binding sitewere identified in PetB sequences from R. gelatinosus, i.e.,V151L, T163Q, T166I(V), E303V, L307T, T145E, and F331G. In A.vinosum only the first five substitutions are conserved (Fig.4B). The possible consequences of these changes for the propertiesof the Q₀ pocket are discussed below. Mutations that conferresistance to antimycin at the Q_i site were also reported. Acomparison to the Rhodobacter sequence identified substitutionsthat might be responsible for resistance to antimycin in R.gelatinosus in the putative Q_i site. In particular, we focuson the I49F and the A55M substitutions (Fig. 4C); both aminoacids are directly involved in antimycin resistance in Rhodobacter(7). Also, the I254T substitution is located in a critical regioninvolved in antimycin resistance, as shown by the differentmutations mapped in this region (7, 14).

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FIG. 4. Partial amino acid sequence comparison of the deduced petB gene products (cyt b) of R. gelatinosus (R. gel), A. vinosum (A. vin), and Rhodobacter sphaeroides (R. sph). _*, identical residues; :, similar residues (from the Clustal W program). (A) Comparison of the helix IV sequences showing the two histidines that bind the hemes (shaded) and the extra residue (boldface and underlined) in the R. gelatinosus sequence. (B) Amino acid sequence comparison of the putative Q₀ pockets. ${blacktriangledown}$ , most-relevant residues, shown to be involved in sensitivity or resistance to cyt bc₁ inhibitors stigmatellin and myxothiazol in other organisms (7) and likely to be responsible for the resistance to Q₀ pocket inhibitors in R. gelatinosus and A. vinosum. (C) Amino acid sequence comparison of the putative Q_i pockets. ${triangledown}$ , substitutions which might be responsible for the resistance to the Q_i site inhibitor, antimycin.

Inhibitor effects on A. vinosum cyt bc₁ activity. The sequence homologies between the cyt b primary sequencesof R. gelatinosus and A. vinosum, especially in the area ofthe Q₀ site (E303V substitution), suggest that the cyt bc₁ fromA. vinosum would be resistant to at least stigmatellin. To testthis hypothesis, we measured the cyt bc₁ reductase activityof A. vinosum membranes in the presence of the three inhibitors(Table 1). As for R. gelatinosus, the A. vinosum cyt bc₁ activitywas not inhibited by the addition of antimycin, myxothiazol,and stigmatellin. This resistance of A. vinosum strain D toinhibitors was confirmed with the A. vinosum DSM180T collectionstrain.

Concerning the resistance to myxothiazol and antimycin, ourresults are different from the previous ones obtained with A.vinosum membranes (6, 11, 44). In the work of Tan et al. (44)cyt c reductase activity was measurable only after a treatmentwith dodecyl maltoside detergent. This implies that the quinonebinding site could be masked in situ. In our system, cyt c reductaseactivity on intact chromatophores was measured, showing thatquinone has access to the cyt bc₁ complex. However, to checkwhether the inhibitors have access to the binding sites, wepreincubated chromatophores with the detergent as describedpreviously (44). Detergent treatment did not allow the complexto regain sensitivity, suggesting that resistance is an intrinsicproperty of the complex.

In the cyt b sequence of A. vinosum strain D the E303V substitutionin the PEWY motif was reported by Chen et al. (10). To checkif the PVWY motif was present in the A. vinosum strain D usedin our work, a 0.4-kb DNA fragment of the region flanking thismotif in the petB gene was cloned. The DNA sequence confirmedthe presence of the GTC valine codon.

DISCUSSION

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Discussion

In the present work we have studied the cyt bc₁ complex fromR. gelatinosus by measuring the decylquinol oxidoreductase activityof membranes with an artificial electron acceptor, the horseheart ferricytochrome c, and with its physiological electroncarriers, namely, cyt c₈ and HiPIP, in the presence of threedifferent inhibitors.

It is conspicuous that the rate of reduction of the HiPIP isincreased by a factor of 20 to 30 relative to the rate of cytc reduction. The role of HiPIP as an efficient electron mediatorbetween the cyt bc₁ complex and the reaction center (RC) haspreviously been suggested by the experiments of Schoepp et al.(40). The high reactivity of the cyt bc₁ complex with HiPIPcould be explained by the presence of a specific docking siteon the cyt c₁, as is present in the mitochondrial cyt c₁ subunit,shown to contain a binding site for cyt c (43, 47).

The specific inhibitors of the Q₀ and of the Q_i sites stronglyreduce the enzymatic activity of the cyt bc₁ complex in bothRhodobacter sphaeroides and B. viridis, as has already beenshown in the literature. In contrast, for R. gelatinosus membranes,this activity was insensitive to these inhibitors, irrespectiveof the nature of the final electron acceptor. Flash-inducedelectron transfer experiments carried out by Shoepp et al. (40)on R. gelatinosus whole cells have shown that the rereductionrate of the photo-oxidized RC was not affected by myxothiazolor antimycin. On the contrary, stigmatellin appreciably slowedthe light-induced electron flow through the cyt bc₁ complex.It has been shown that stigmatellin is also an inhibitor ofthe photochemical RC Q_B site in B. viridis (32). Therefore itis very likely that, in these light-induced experiments, stigmatellinacts as an inhibitor of the RC by binding to the Q_B site. Inthis way it would prevent the regeneration of the ubiquinolpool necessary for cyt bc₁ turnover, decreasing the electrontransfer rate.

A series of constructed mutations as well as spontaneous substitutionsin the cyt b subunit of bacterial and mitochondrial cyt bc₁are available (7, 13, 21, 22, 28). These mutations have beenshown to confer either hypersensitivity or resistance to thecyt bc₁-specific inhibitors. Amino acids associated with resistancewere mapped essentially in the quinone binding domains. To explainthe natural resistance of R. gelatinosus to these inhibitors,we studied the petB gene sequence, encoding cyt b. The comparisonof the primary structures of R. gelatinosus and A. vinosum cytb polypeptides with those of Rhodobacter species and yeast allowedthe identification of substitutions which may contribute toresistance. Among them, we identified a substitution which hasbeen reported to induce resistance to both myxothiazol and stigmatellin,T163Q, and those inducing resistance only to stigmatellin, i.e.,T166I(V) and E303V (7). The resistance-associated amino acidsintroduce different residue volumes, polarities, or chargesinto the Q₀ binding site. According to structural data (13),the accommodation of the inhibitor inside the Q₀ pocket is accompaniedby a series of conformational changes, and we presume that mutationsof some residues may prevent access of the inhibitor to, orits binding in, the pocket. Stigmatellin is bound in the distaldomain of the Q₀ pocket by two H bonds, one from His161 (ISP)and the other from Glu303 (cyt b) of the highly conserved PEWYloop. In R. gelatinosus and A. vinosum the replacement of glutamicacid 303 with a hydrophobic valine might prevent stigmatellinH bonding to cyt b and hence the inhibitor might not be retainedin the site. Nevertheless, Thiobacillus ferroxidans PetB containsa PPWY loop (instead of PEWY), and stigmatellin could bind inthe proximity of heme b_L, as demonstrated by the red shift ofits ${alpha}$ band (24). Yet, like valine, the hydrophobic proline isunable to form an H bond with the inhibitor. Thus, an H bondprovided by the cyt b subunit is not always required to bindstigmatellin, and the whole structure of the Q₀ site thereforeshould be taken into account when assessing binding.

In the R. gelatinosus Q₀ pocket, other substitutions were identified(V151L, T145E, L307T, and F331G) (Fig. 4B). Their proximityto the binding domains of the inhibitors may also explain theobserved resistance.

Similarly, we propose that the resistance of the R. gelatinosuscyt bc₁ complex to myxothiazol may be explained by the factthat some of the substituted residues are involved in inhibitorbinding at the Q₀ site. The weak sensitivity to myxothiazoldetected suggests that its binding site in the Q₀ pocket differsfrom that of stigmatellin, as already shown by the crystallographicdata for the mitochondrial cyt bc₁ complex cocrystallized withdifferent inhibitors (13).

Crystallographic and mutagenesis data have highlighted the structuraland functional role of the glutamic acid in the PEWY loop inthe cyt bc₁ and cyt b_6f complexes in the rate of ubiquinol (QH₂)oxidation (7, 16, 48). It was reported that this rate was appreciablydecreased in mutants in which the glutamate was replaced byother residues (16, 48). Therefore, it has been inferred thatthe H bonding of quinol to glutamate stabilizes the bindingof quinol to the site. On the other hand, it has been proposedthat the carboxyl group of Glu303 is the acceptor of the secondH⁺ released by QH₂ oxidation (16). Although the Q₀ pocket inthe R. gelatinosus cyt bc₁ complex contains the PVWY motif,the ability of the enzyme to turn over demonstrates that Gluis not indispensable for binding QH₂. It is likely that theH bonding to ISP in addition to other interactions with thenearby residues is sufficient to stabilize the quinol in itsactive site.

The resistance to antimycin usually involves displacement ofthe quinone from the Q_i site. Several authors have shown thatsome substitutions of residues by site-directed mutagenesisin Rhodobacter capsulatus PetB conferred resistance to antimycinand HQNO (for reviews see references 7 and 14). Substitutionsof residues involved in the resistance to antimycin at the Q_isites of different organisms have been mapped (7, 14). Amongthe residues contributing to the folding of the R. gelatinosusQ_i pocket, relevant substitutions (I49F, A55M and I254T) thatmay affect antimycin access or binding to the pocket were found.However, due to the low conservation of the Q_i site sequences,it is difficult to ascertain the residues responsible for resistanceto antimycin in the R. gelatinosus cyt bc₁ complex.

A. vinosum cyt bc₁ has properties that appear similar to thoseof R. gelatinosus in that it is resistant to inhibitors of bothQ₀ and Q_i sites. The resistance of both species to stigmatellincould be correlated to the E303V substitution in the cyt b subunit.Nevertheless, part of these results are different from previouslypublished results for A. vinosum chromatophores that showedinhibition of the cyt bc₁ complex by both antimycin and myxothiazol(6, 11, 44). However, in this paper, we have measured resistanceto inhibitors and identified the sequence flanking the PVWYloop on the same strain to get reliable results. The resistanceto myxothiazol and to antimycin, which is controversial andwhich disagrees with previous works, was found for A. vinosumstrains D and DSM180T.

In the R. gelatinosus and A. vinosum PetA sequences (not shown),a substitution (Y165F) adjacent to the cluster ligand domainof the Rieske protein was identified. Site-directed mutagenesisexperiments with yeast (20) and Rhodobacter sphaeroides (27)have shown that replacing the amino acids forming OH-S bondsto the cluster (S163 and Y165) by residues unable to provideH bonds decreases the midpoint redox potential of the proteinas well as the catalytic activity of the cyt bc₁ complex. Thereforesubstitution Y165F in the cluster binding domain of the Rieskeprotein in R. gelatinosus could result in a midpoint potentiallower than that for yeast or Rhodobacter sphaeroides. Mutationsin the Rieske protein that confer resistance to stigmatellinin Rhodobacter capsulatus, in particular mutations that perturbedQ₀ site occupancy, have been reported (8). In the absence ofexperimental data, sequence alignment does not give any informationon the possible contribution of the R. gelatinosus ISP subunitto the resistance to Q₀ inhibitors.

In addition to the PVWY motif and the Y165F substitution inthe ISP subunits, the extra residue found between the conservedheme-binding histidines in helix IV could generate differentredox potential properties of the R. gelatinosus cyt bc₁ complex.Indeed, it was shown by Kuras et al. (31) that addition of anextra residue in this spacing region by site-directed mutagenesisof petB from Rhodobacter sphaeroides increases the potentialof the b_L heme, shifting the potential difference between thetwo b hemes from 140 mV in the wild-type to 55 mV in the mutantstrain. Therefore, this arrangement in helix IV, as well asthe substituted residues in the cyt b and the ISP subunits ofthe R. gelatinosus cyt bc₁ complex, could account for a modifiedstructure of this enzyme at the level of the Q₀ and Q_i pockets,impeding the binding of the inhibitors.

Given the high level of resistance to quinone analogs in thecyt bc₁ complex from R. gelatinosus, it seems likely that thenature and the structure of the quinone binding sites in cytbc₁ are significantly different from those of the correspondingsites in cyt bc₁ of Rhodobacter species. This study of a bacterialstrain belonging to the ß subclass of purple bacteriapoints to interesting features that help us to focus on importantresidues implicated in the functioning of the cyt bc₁ complex.These assumptions should be verified by site-directed mutagenesis,and purification of the complex is in progress. This will allowa deeper understanding of the functioning of this crucial enzyme.

ACKNOWLEDGMENTS

We thank E. Nabedrik and S. Ardrianambinitsoa for the gift ofA. vinosum strain D. We are grateful to M. C. Gonnet for technicalassistance and to ESGS for DNA sequencing. We thank F. Reiss-Hussonand L. Sperling for critical reading of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Centre de Génétique Moléculaire CNRS (UPR-2167) Associé à l'Université Pierre et Marie Curie, BÂt. 24, Ave. de la Terrasse, 91198 Gif sur Yvette Cedex, France. Phone: 33-1-69823137. Fax: 33-1-69823230. E-mail: astier{at}cgm.cnrs-gif.fr.

REFERENCES

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