Characterization of the Rhodocyclus Tenuis Photosynthetic Reaction Center

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Characterization of the Rhodocyclus tenuis photosynthetic reaction center

Ileana Agalidis a,), Anabella Ivancich b,1, Tony A. Mattioli b, FrancËoise Reiss-Husson a a Centre de GenetiqueÂÂ Moleculaire, Â Bat. Ã 24, CNRS, 91198 Gif-sur-YÕette Cedex, France bSection de Biophysique des ProteinesÂÂ et des Membranes, Departement de Biologie Cellulaire et Moleculaire, Â CEA and URA CNRS 2096, CEArSaclay, 91191 Gif-sur-YÕette Cedex, France Received 6 February 1997; revised 24 April 1997; accepted 29 April 1997

Abstract

The photosynthetic reaction centerŽ. RC from the purple non sulfur bacterium Rhodocyclus tenuis was isolated from membrane fragments by dodecyl dimethylamine oxideŽ. LDAO and purified by DEAE-chromatography in presence of Ž. dodecyl-maltoside DM . The redox midpoint potential of the primary electron donor P was determined to be Em s515 mV"20 mV. The Pq absorption band peaks at 1260 nm. Fourier transform resonance Raman spectra are consistent with a

dimericŽ. Bchl2 structure of P where both acetyl carbonyl groups of P are hydrogen bonded, one with histidine, the other with a tyrosine residue. The primary quinoneŽ. QA was identified to be menaquinone 8 and the secondary one Ž. QB q y ubiquinone 8. The secondary activity depends on the nature of the detergent. At pH 8 in DM solution, the rate of the P Q B y1 y1 back reaction is 0.1 s and QB does not function as a two-electron gate. With LDAO addition, the rate became 0.3 s and y multiple binary oscillations of QB were observed. 10 mM orthophenanthroline blocks 50% of the electron transfer to QB Ž. I50 s10 mM . This high sensitivity was correlated with the nature of the residue at the position L226. q 1997 Elsevier Science B.V.

Keywords: Bacterial reaction center; Primary donor; Quinone; Resonance Raman spectra; Ž.Rhodocyclus tenuis

1. Introduction

RhodocyclusŽ. Rc. tenuis is a facultative purple Abbreviations: Bchl, bacteriochlorophyll; Bphe, bacteriopheo- non sulfur bacterium which belongs, together with phytin; Cyt, cytochrome; DAD, diaminodurene; DM, dodecyl Rc. purpureus and RubriÕiÕaxŽ. RÕ. gelatinosus,to maltoside; FT, Fourier transform; LDAO, lauryldimethylamine oxide; OD, optical density; P, primary donor; RC, reaction a separate group which is different from the other center; C., Chromatium; Cf., Chloroflexus; Rsp., Rhodospiril- purple non sulfur bacteria. More precisely, it is clas- lum; Rb., Rhodobacter; Rc., Rhodocyclus; RÕ., RubriÕiÕax; sified in the b subclass of the Proteobacteria, accord- Rps., Rhodopseudomonas ) ing to the phylogenetic analysis based on the 16S Corresponding author. Fax: q33 01 69823802; ribosomal RNAwx 1,2 . One of the distinctive proper- E-mail: [email protected] 1 ties of Rc. tenuis concerns the morphology of its cell Present address: Spectroscopie des Complexes Polymetal-Â liques et des Metalloproteines,ÂÂ SCIBrDRFMC, CEArGrenoble, membrane, which is devoid of any invaginations; 17 rue des Martyrs, 38054 Grenoble Cedex, France. when cells adapt to photosynthetic growth conditions

Žwhich requires the insertion of the photosynthetic tion on the quinones and the primary electron donor, apparatus in the cytoplasmic membrane. they simply whose oxidation midpoint potential should be redox- increase their size, and they do not develop chro- compatible with the other proteins involved, such as matophores as in other purple bacteriawx 3 . In compar- the HiPIP and the RC-associated tetraheme. ison with a large body of knowledge accumulated Despite the phylogenetic distance between purple about the photosynthetic apparatus of purple non bacteria of the b and a-subgroupsŽ the later includ- sulfur bacteria, few data are available for Rc. tenuis, ing Rhodobacter sphaeroides, Rb. capsulatus, Rho- but they indicate substantial differences. Original fea- dospirillum rubrum., a high homology exists at the tures of the light harvesting complexesŽ LHI and level of their photosynthetic RCs, as seen by the LHII. of Rc. tenuis have been described recentlywx 4 ; sequence comparisons of the so-called L and M according to the sequences of its polypeptides, the polypeptides of several purple bacteria, including Rc. peripheral antenna, LHII, appeared to be of a mixed tenuis wx13 . It was thus assumed that a horizontal character, being related both to LHI and LHII com- gene transfer occurred from an ancestor species of plexes of other purple bacteria. Like Rps. Õiridis, this the a-subclasswx 14 . It is not known, however, if this bacterium contains a RC associated tetraheme Cyt c homology extends to the third RC polypeptide, the but having different values of its midpoint redox so-called H subunit, which has not yet been se- Ž potentials Em1s385 mV, E m2s320 mV, E m3 s quenced for any species in the Rhodocyclus genera, . 115 mV, Em4 s70 mVwx 5 and probably different and which is less strictly conserved than L and M in heme orientations. On the other hand the presence of bacteria of the a-group. a variety of soluble electron transport proteins but the In an effort to obtain a more detailed knowledge

Cyt c2 have been signaled in this bacterium, among on the structure of RCs from the b-subclass of purple which the high potential iron proteinŽ. HiPIPwx 6 , the bacteria, several properties of the RC from RÕ. three-dimensional structure of which is knownwx 7 ; gelatinosus have been recently characterizedwx 15 , recently, the direct involvement of HiPIP in the and differences have been observed in the energetics electron transport between the cytochrome bc1 com- of the quinone acceptor complex, at the level of QA plex and the RC-associated tetraheme cytochrome and QB , as compared to Rb. sphaeroides and Rb. has been demonstrated in RÕ. gelatinosus wx8 and capsulatus. In this work we extend our studies to Rc. also in Rc. tenuis ŽVermeglio,Â personal communica- tenuis and report the isolation and characterization of tion. . the RC from this bacterium. We have shown that its Currently, there is a great research interest in the structural as well as functional properties share com- membrane-diffusible quinones and soluble redox pro- mon features with bacterial RCs belonging to differ- teins mediating electron transport between cyt bc1 ent branches of their evolutionary tree. and the reaction centerŽ for a review, see Meyer and Donohuewx 9. . This has been recently stimulated with the finding that HiPIPs are directly involved in elec- 2. Materials and methods tron donation to RCs in such organisms as RÕ. gelatinosus wx8, Rhodoferax fermentans w10 x , Rc. 2.1. Reaction center isolation tenuis Ž.Vermeglio,Â A., personal communication , as well as in Chromatium inosum wx11 . Since HiPIPs are Rc. tenuis Ž.strain ATTC 25093 was grown anaer- less efficient as electron donors than are those related obically in the light. The membrane fraction was to Cyt c2 Ž.common in Rhodospirillaceae , photosyn- isolated as described inwx 16 and was suspended at a thetic electron transport utilizing HiPIPs may be con- concentration yielding an optical density in a 1-cm Ž Ž. sidered more primitive than those using Cyt c2 see cuvette of 50 at 860 nm OD860 s50 in 0.1 M Na Meyer et al.wx 12. . It is clear that to understand these phosphate buffer pH 7.5 containing 9% glycerol and electron transport processes, the redox proteins in- 5 mM Na ascorbate. This suspension was incubated volved should be fully characterized. Towards this in 3±4 mgrml dodecyldimethylamine oxideŽ. LDAO end, we have undertaken a comparative characteriza- Ž.Fluka, Biochemica for 1 h at 268C. After centrifugation of the Rc. tenuis reaction centers placing atten- tion at 430 000=g for 90 min the supernatant was I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 33 recovered; it contained crude RC and residual light staining, and identified as ubi- or menaquinones by harvesting antenna. Eventually, the pellets were re- comparison with UQ88 and MKŽ. Hofmann La Roche suspended and incubated again as above except that chromatographed in parallel. glycerol was omitted and the LDAO concentration We observed that quinone analysis in reaction was lowered to 0.9±1 mgrml. Centrifugation was center samples required removal of DM before ex- carried out at 200 000=g for 90 min and a second traction. Thus the samples were freed from detergent supernatant containing RC was recovered. Both su- by overnight treatment at 58C with Biobeads SM-2 pernatants were combined and precipitated by ammo- Ž.Biorad ; the beads were eliminated by filtration and nium sulfateŽ. 40% saturation at 48C for 20 min. The the turbid reaction center samples were analyzed as precipitate was recovered after 15 min centrifugation above. at 2000=g and suspended in a few mls of 0.1 M Tris-HCl pH 8 buffer, containing 0.1 M NaCl, 9% 2.4. FPLC gel filtration glycerol, 5 mM Na ascorbate and 0.4 mgrml dodecyl maltosideŽ. DM, Sigma . The solution was kept The apparent molecular weight of the detergent-RC overnight at 48C, and then desalted onto a PD10 complex was determined on a Superose 12 HR 10r30 Ž.G-25 Sephadex, Pharmacia column equilibrated with columnŽ. Pharmacia , equilibrated with a 50 mM Tris-

10 mM Tris-HCl pH 8, 0.2 mgrml DM. The sample SO42 H buffer containing 50 mM SO 4 Na 2 and 0.2 was then adsorbed on a DEAE Sepharose column mgrml DM. A 100 ml RC sample was injected and equilibrated with the same buffer, and eluted with a eluted with the same buffer at 0.5 mlrmin. Ab- 0±0.27 M NaCl gradient. The RC was eluted at about sorbance at 410 nm was followed with a Beckman

100 mM NaCl; fractions with absorbance ratios A 280 160 absorbance monitor. In some runs, 100 ml frac- nmrA 800 nm between 1.2 and 1.4 were pooled and tions were collected and their absorption spectra were concentrated by ultrafiltrationŽ first on a Amicon recorded with a Cary 2300 spectrophotometer. Cali- XM50 membrane then with a Centricon 100 concen- bration of the column was done with water-soluble trating system. . proteinsŽ thyroglobulin, catalase, transferrin, ovalbu- min, and myoglobin. .

2.2. Preparation of QB -depleted RC 2.5. Electrophoresis Ž. A RC solution OD800 s2 was incubated in 10 mM Tris-HCl buffer, pH 8, in the presence of 8 SDS-polyacrylamide gel electrophoresis was car- mgrml LDAO for 30 min at 308C and then adsorbed ried outwx 18 on minigels with 12.5% polyacrylamide on a DEAE-Sepharose column equilibrated with 10 in the resolving gel and 4% in the stacking gel. Heme mM Tris-HCl, 1 mgrml LDAO, pH 8. In order to staining was performedwx 19 , then the gel was eliminate solubilized QB and degraded pigments, the destained and stained further with Coomassie Blue column was successively washed with 3 vols. of R250 or silver. equilibration buffer followed by 4 volumes of the same buffer but containing 0.2 mgrml DM. Finally, 2.6. Spectral measurements the RC fraction was eluted with 0.3 M NaCl. Absolute and differential absorption spectra were 2.3. Extraction and identification of quinones recorded on a Cary 2300 spectrophotometer equipped for cross-illumination; concentration of the RC was

Quinones were extracted from Rc. tenuis mem- determined from the absorbance in the Qy band using brane fragments and lipids were removed using a the extinction coefficient measured for Rb. modified Bligh and Dyer method as described inwx 17 . sphaeroides RCwx 20 . Tetrahemic Cyt c content was Extracts were chromatographed on activatedŽ 30 min, estimated by assuming a value of 96 My1 Pcmy1 for 1208C.Ž. HPTLC silica gel plates Merck developped the global differential absorption coefficient in the Ž. ŽŽ . with petroleum etherrdiethyl-ether 85r15 vrv. Cyt a-band Dered-ox 553 nmy538 nm , corre- Spots were revealed by UV fluorescence and I 2 sponding to four hemes per cytochromewx 21 . Redox 34 I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 titrations of membrane fragments were carried out by using a flow cell with an external reaction vessel. The light-induced absorbance changes due to the donor PrPq were measured between 850 and 900 nm at a series of ambient redox potentials, monitored by a combined AgrAgCl-Pt electrodeŽ. Ingold . The electrode was calibrated using saturated quinhydrone solutions prepared at pH 5 and 7.4 as described else- wherewx 22 . Low-temperatureŽ. 77 K absorption spectra were recorded on a Aminco-Chance DW2 spectrophotometer. Flash experiments were done on a home-made single beam spectrophotometerwx 23 . Near-infrared Fourier transformŽ. FT resonance Raman spectra of isolated RCs were recorded using a Bruker IFS 66 interferometer coupled to a Bruker Fig. 1. Optical absorption spectrum of Rc. tenuis RCs in 10 mM FRA 106 Raman module equipped with a continuous, Tris, pH 8, 0.2 mgrml DM; in this sample 0.4 tetraheme Cyt c was still associated per RC. Inset: another sample where only 0.1 diode-pumped Nd:YAG laserwx 24 . Reaction center Cyt c was bound per RC. samples were measured at both room and low tem- peratureŽ. 10 K and were excited with 180 mW and 200 mW of 1064 nm laser radiation, respectively. For accessory Bchls, and Bphes, respectively as well as a the low temperature experiments, the samples were band at 597 nm corresponding to the Qx transition of held in a gas flow cryostatŽ. SMC-TBT, France Bchls. Other bands are observed at 532 nm regulated by the circulation of cold helium gas. The Ž.Ž.carotenoid and Bphe , 496 nm carotenoid and at spectral resolution was 4 cmy1. The minor fluores- 404, 386 and 366 nm. These latter three bands are cent backgrounds of the spectra were corrected by a attributed to the Soret transitions of the hemes, Bphes, polynomial fit and the maxima of the Raman bands and Bchls respectively. The above spectral features were determined both by deconvolution and second are very similar to those of other purified RCs which derivative analyses. contain a-type bacteriochlorins and carotenoid, such For the FT Raman experiments, RC samples were as Rb. sphaeroides wx25 . The strong similarity of the concentrated to ca. 100 OD at 867 nm using a optical absorption spectrum and of the relative inten-

Centricon microconcentrating systemŽ. Amicon and sities of the Qy absorption bands of P, Bchls and were poised to their reducedŽ. P or oxidized Ž Pq. Bphes with those of Rb. sphaeroides RC, strongly states with sodium ascorbate or potassium ferri- suggest that the Bchl arBphe a ratio in the Rc. cyanide, respectively. tenuis RC is 2:1 and that the general arrangement of these pigments is similar for these two RCs. Identifi- cation of the carotenoid moleculeŽ. s bound to the RC 3. Results is currently under study. Besides the two peaksŽ. 366 and 386 nm belonging 3.1. Isolation and composition of Rc. tenuis RC to Bchls and Bphes, the Soret spectral region shows an extra peak at about 410 nm likely due to a Rc. tenuis RC was solubilized from cytoplasmic tetraheme cytochrome c associated to the RC. The membrane fragments with LDAO, precipitated by presence of a tetraheme Cyt c has already been ammonium sulfate and purified in presence of DM by reported in this bacteriumwx 5 ; association of such a anion exchange chromatography. A typical optical cyt with the RC has been observed in other purple absorption spectrum of purified RCs is shown in Fig. bacteriawx 26 . A gradual loss of Cyt c during the 1 and displays characteristic near-infrared absorption isolation procedure was observed and was likely due bands at 868, 801, and 753 nm corresponding to the to the detergent treatmentsŽ. by LDAO and DM as

Qy transitions of the primary donorŽ. see below , the well as to ammonium sulfate precipitation. In purified I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 35

RC preparations the content of Cyt c per RC was variable in the range 0.1±0.4; in Fig. 1, 0.4 tetraheme cytochrome c was present per RC whereas in another preparationŽ. shown in the inset of Fig. 1 only 0.1 Cyt crRC was left. During further analysis of a RC sample by HPLC gel filtration, the RC was eluted in a single peak but a variable cytrRC ratio was observed across the elution peak itself; thus the unbinding of cyt was still occurring during chromatography. Despite this somewhat loose association, in a RC sample prereduced by DADrascorbate, the residual Cyt c was still photooxidized by the RC, as demonstrated by multiflash experimentsŽ. data not shown . When Rc. tenuis RCs were isolated and purified in the presence of LDAO they were less stable than Fig. 3. Near-IR absorption spectra of purified RC recorded in the in DM, as observed by a fast, irreversible decrease of dark and continuous light, respectively. The photooxidation of the donor is indicated by a simultaneous bleaching at 870 nm and the donor Qy absorption band and by the pheophytin- the rise of a weak absorption band at 1260 nm. The unbleached isation of the BchlŽ. data not shown . Therefore LDAO fraction is due to QB -depleted RCs, as we observed by a total was used only for extracting the RC from the mem- bleaching after UQ6 and LDAO additionŽ. not shown . brane fragments, and DM was used for purification on column. However, even in the presence of DM the RC underwent a partial degradation after 2±3 weeks 2. . Another weak component of 43 kDa was not at 48C, detected by a decrease in amplitude of the stained by Coomassie blue and revealed only by donor Qy absorption bandŽ. data not shown . heme staining and by silver staining. It was attributed By HPLC gel filtration in presence of DM the to the residual tetraheme Cyt c polypeptide. apparent M.W. of the native RC-detergent complex was estimated to be 150 kDa. After SDS denatura- 3.2. Spectral and redox properties of the primary tion, the electrophoretic pattern of purified RC indi- donor cated the presence of three polypeptides with apparent M.W. of 25, 29 and 36 kDa which likely corre- Photochemical activity was verified in the isolated sponded to L, M, and H subunits respectivelyŽ Fig. RC by the light-induced bleaching of the primary donor band at 867 nm and the simultaneous appearance of an absorption band at 1260 nm, attributed to the cation radical Pq Ž. see Fig. 3 . This latter band was shifted 10 nm to longer wavelengthŽ see inset of Fig. 3. relative to the 1250 nm Pq band determined in Rb. sphaeroides RCwx 27 . The ratio of the absorbance maxima at 868 nmŽ. in state P and 1260 nm Žin state Pq. was about 10, similar to what has been observed for Rb. sphaeroides and Rsp. rubrum RCs

wx28 . At 77 K the Qy band maximum was observed at 894 nmŽ. data not shown , which is slightly red-shifted with respect to its position in Rb. sphaeroides RC, i.e., 890 nm. In the room temperature light-minus-dark Fig. 2. SDS gel electrophoresis of purified Rc. tenuis RCŽ two spectrum of Rc. tenuis membrane fragments, the left lanes, differing by loads.Ž. and marker proteins right lane . The gel was stained with Coomassie blue; the arrow shows the maximum bleaching of the dimer Qy band was lo- position of the cytc polypeptide, previously revealed by heme cated at 890 nm, which corresponded to a 23 nm staining. red-shift relative to its position in isolated RC. This 36 I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 . M Residue L226

m 2 o-phen concentration required to inhibit the f 77 88 88 99 10 10 numbering which is related to the degree of inhibition by 10 10 H-bond donor to the C acetyl carbonyl group of the Bchl of P c sphaeroides . Rb Ž. chemical nature of the secondary quinone; e Ž. Ž.Ž g crystallographic structures, and inferred from primary sequences and FT Raman data for the other bacteria . RC with other known bacterial RCS tenuis w x w x wx wx w x abcdefg sphaeroides . wxwxwx wx w x wx wx w x wx wx wx wx wx w x . Rc Rb Ž. chemical nature of the Primary quinone; Ž. Ž. Ž. d ) P mV H-bond donor to P H-bond donor to P Q Q I and r q ))))))))) wxwx wx wx wx w x wx wx wx AB y P 2 mLMAb50 iridis Õ E 420 79 ; 386 80 none Phe L207 82 Tyr M183 82 MK MK 83 n.d. n.d. 515400 76502 34 ;500 526 77 ; 34450 520 31 78 ; 495 67,29 His L137 34 His His His L168 L168 His 168 6,63,65 57,66 32,81 none TyrM195 Tyr M196 57,66 34 none Tyr MK MK UQ UQ 52 UQ MK 47 UQ 30 50 25 UQ n.d. 18 MK 200 Ala 52 57,66 3 UQ 51 n.d. Thr 63,24 Ser 81 10 Ala . Rps Nagashima personal communication. a b ))

g a Ž. Ž .Ž. b Ž. iridis q Õ P redox midpoint potential of the primary donor between pH 7 and 8 in membrane fragments and in isolated RCs ; in b and c subscripts the amino acid residues gelatinosus sphaeroides aurantiacus . tepidium r . . . This work; H-bond donor to the C acetyl carbonyl group of the Bchl of P associated with the L subunit; P . Õ Rb Rc tenuis R Rps electron transfer between Q and Q at 50 percent; amino acid residue at position L226 Table 1 Comparison of several properties of n.d., not determined. a b associated with the M subunit; ) Bacterium class o-phen see text . were identified in C Cf I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 37

3.3. Near-infrared FT resonance Raman spectra of the reduced() P and oxidized ( Pq) Primary Donor of Rc. tenuis

As mentioned before, the electronic absorption spectrum of Rc. tenuis RCŽ. Fig. 1 is similar to that of Rb. sphaeroides, with an absorption maximum at ca. 870 nm arising from P and a Pq absorption band at 1260 nm. Thus, under our experimental conditions, we can expect a selectiveŽ. pre resonance Raman enhancement of the primary donor, PŽ via its 870 nm absorption transition. in the FT Raman spectrum of reduced Rc. tenuis RCs excited with 1064 nm, simi- Fig. 4. Chemical redox titration of light-induced absorbance lar to that observed for the dimeric primary donors in changes of P in Rc. tenuis cytoplasmic membranes suspended in RCs of Rb. sphaeroides Žwx24 and other Bchl a-con- 50 mM Tris-HCl buffer pH 8. Potassium ferricyanide and sodium taining RCs 30,32±34 . Bands in the FT Raman dithionite were added in order to increase and decrease respec- wx tively the redox potential of the cytoplasmic membranes suspen- spectra of RCs poised in their neutral P state which sion prior to illumination. Closed rectangles: oxidative titration; bleach upon Pq formation are attributable to P while open rectangles and triangles: reductive titration. The line repre- those new bands appearing in the Pq FT Raman sents the fit of the data to the Nernst equation Ž.ns1 . The spectraŽ due to a genuine resonance enhancement via Ž. calculated midpoint potential is 515"15 mV. the 1260 nm transition. are assignable to Pq wx24,30,32±36 . Fig. 5 shows the room temperature FTŽ. pre resonance Raman spectrum of Rc. tenuis RCs, in the large difference could reflect specific interactions of 1550±1800 cmy1 spectral region, excited with 1064 the RC with its lipoproteic environment within the nm light and in both the reduced PŽ. Fig. 5A and membrane, which drastically changed once the RC oxidized Pq Ž. Fig. 5B states. In Fig. 5A the bands at was extracted and solubilized by the detergentwx 29 , andror the RC-associated tetraheme cytochromewx 30 . In order to determine the electrochemical properties of the donor, we measured the redox midpoint potential of P in Rc. tenuis cytoplasmic membranes by measuring light induced absorbance changes due to Pq formation at different redox statesŽ. Fig. 4 as well as that in Rb. sphaeroides chromatophores for comparison. A redox titration could not be performed on isolated Rc. tenuis RC because P was gradually damaged during the oxidative treatment. In these measurements the tetraheme cytochrome remains fully oxidized and thus cannot interfere by reducing Pq.A value of Em s515"20 mV at pH 8 was obtained for the PrPq couple in Rc. tenuis membranesŽ see Fig. 4. , as compared to 450 mV in Rb. sphaeroides strain Y chromatophoresŽ. not shown , this latter value being in good agreement with that already published Fig. 5. Room temperature FT Raman spectra of Rc. tenuis RCs excited with 1064 nm laser radiation, and poised in their PŽ. A wx31 . Thus the midpoint redox potential of the donor and Pq Ž. B states. The 1550±1800 cmy1 spectral region shown is about 70 mV higher in Rc. tenuis than in Rb. includes the vibrational stretching modes of the Cam C methine sphaeroides chromatophoresŽ. Table 1 . bridges and the conjugated carbonyl groups of the primary donor. 38 I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46

1607, 1618, 1635, 1694Ž. broad , 1730, and 1742 due to the difference in H-bonding states of the y1 q cm , all disappear upon P formation, and thus can homologous PM acetyl groups of these two species. be assigned to P. Similarly, in Fig. 5B the bands at Comparable frequency shifts were observed when 1 1600, 1639, and 1718 cmy , clearly appear upon Pq H-bonds were genetically introduced or broken on q formation, and thus are assigned to P . Conversely, the C2 acetyl carbonyl groups of the primary donor the bands at 1657 cmy1 and 1678 cmy1 that are of Rb. sphaeroides wx35,36,39 . The broad band at ca. present in the P spectrum and persist in that of Pq 1694 cmy1 was found to be composed of 1690 and should not then arise from P. They most probably 1696 cmy1 componentsŽ by second derivative and arise from protein, the monomeric Bchl molecules, or deconvolution analyses. , and resembles the 1696 and Bphe molecules contributions, respectively 1697 cmy1 bands in the P FT Raman spectra of Cf. wx29,30,34,35 . The weak band at 1699 cmy1 in the aurantiacus wx34 and C. tepidum RCs wx 30 , respec- Pq spectrumŽ. Fig. 5B might have been masked tively, which were also both composed of two nearly y1 underneath the broad 1694 cm band in P spectrum, degenerate bands assigned to the two C9 keto car- and thus cannot be unambiguously attributed to P. bonyls of their respective primary donors. The situa- y1 In Fig. 5A, the 1607 cm band is consistent with tion is different for Rb. sphaeroides where the PM the vibrational frequency of the CaCm methine bridge and PL9 free C keto carbonyls vibrate at distinctly stretching modes of pentacoordinated Bchl molecules different frequencies, 1679 and 1691 cmy1, respec- wx37 . A similar band was observed in the P FT Raman tivelywx 24,35 . Finally, the weaker bands at 1730 spectrum of Rb. sphaeroides RCswx 24 and assigned cmy1 and 1745 cmy1 resemble those observed in the to the CaCm methine bridge stretching modes of both FT Raman spectrum of Rb. sphaeroides P, most

Bchl molecules constituting P, each with a single likely arising from the C10 carbomethoxy ester car- histidine axial ligand. The axial ligands of the Bchl bonyl groupswx 24 . molecules of the Rc. tenuis primary donor are, pre- In summary, the FT Raman data reveal that the sumably, also histidine residues. primary donor of Rc. tenuis is constituted of Bchl

The Raman vibrational frequencies of the p conju- molecules which possess two free C9 keto carbonyl gated carbonyl groups of P are expected to be ob- groups and two H-bonded C2 acetyl carbonyl groups. served in the spectral region of ca. 1620 cmy1 Ž H- This H-bond pattern is different from that of Rb. y1 bonded. to 1660 cmŽ. free if arising from C2 sphaeroides where only the PL2 C acetyl carbonyl of acetyl carbonyls, and at ca. 1660 cmy1 Ž. H-bonded to P is engaged in a H-bondŽ. Table 1 . The fact that y1 1700 cmŽ. free if arising from C9 keto carbonyl bands attributable to four conjugated carbonyl groups groupswx 38 ; the magnitude of the shift in frequency are observed in the P FT Raman spectrum of Rc. of these carbonyl group vibrators is indicative of the tenuis RCs indicates that the primary donor is consti- strength of the H-bond interaction. The bands at 1618 tuted of more than one and most likely of two cmy1 and 1635 cmy1 in the P spectrum of Rc. tenuis excitonically coupled Bchl molecules as it is in Rb. y1 in Fig. 5A are only consistent with two C2 acetyl sphaeroides RCwx 24 . The single 1607 cm band carbonyl groups, both engaged in H-bonds of differ- seen in Fig. 5A indicates that both Bchl molecules ent strengths, and the broad band at 1694 cmy1 is constituting the Rc. tenuis primary donor are coordi- consistent with a free C9 keto carbonyl group. The nated by one axial ligand each, most likely histidine, 1618 cmy1 band is reminiscent of the 1620 cmy1 as is the case for all known RCs to date. band seen in the P FT Raman spectrum of Rb. sphaeroides RCs and which was assigned to the PL 3.4. Quinone acceptor complex C2 acetyl carbonyl which is H-bonded to His L168 wx24,35 . The second, H-bonded, C2 acetyl group in For Rc. tenuis, the presence of ubiquinoneŽ. UQ Rc. tenuis is observed at 1635 cmy1, which is 18 and menaquinoneŽ. MK , both with a chain of eight cmy1 lower in frequency than the 1653 cmy1 band isoprenoid units, has already been demonstratedwx 40 . in the P FT Raman spectrum of Rb. sphaeroides Extraction and analysis of quinones from intact and assigned to the non-H-bonded C2 acetyl carbonyl of from QBA8 -less RCs allowed us to identify Q as MK PM wx 24,35 . This difference in vibrational frequency is and QB8 as UQ . I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 39

Fig. 7. Flash-induced difference spectrum of semi-ubiquinone Fig. 6. Flash-induced absorption changes and decay at 430 nm of QBy in presence of DM; same conditions as in Fig. 6 except that 2.7 mM RC solution in Tris buffer pH 8, containing 0.2 mgrml 1 mM DAD was added. DM Ž.t s10 s . Lower trace: after addition of 1 mgrml LDAO Ž.ts3.3 s . photoinduced in other bacterial RCs and playing the role of secondary electron acceptorwx 41,42 When an A single flash illumination of a solution of intact excess of UQ6 was added, a train of saturating flashes y RCs containing 0.2 mgrml DM elicited the forma- induced a single oscillation of QB as if hydroquinol q y formed after the second flash could not exchange tion of P QB radical pair, which recombined with a mean time, t , of about 10 sŽ. Fig. 6, upper trace . In with the exogenous quinone poolŽ. not shown . How- ever, if LDAOŽ. 0.5±1 mg ml was added to such a typical RCs preparations the QB site occupancy was r y 80±90%. In order to determine if the nature of the sample, multiple QB binary oscillations were ob- detergent modified the kinetics of Pq rereduction, we served, similar to those shown in Fig. 8. These tested RC solutions containing either Na cholate, observations suggested that:Ž. 1 in the absence of LDAO, UQ molecules were trapped inside the bulky Deriphat 160, C12 E 8 , or LDAOŽ. at pH 8 . Among 6 these detergents, only LDAOŽ. 0.5±1 mgrml in- dodecyl maltoside micelles, preventing them to ex- Ž. duced a change in the back reaction time relative to change with ubiquinol formed at the QB site; 2 the DM, accelerating Pq decay to about ts3±4 sŽ see Fig. 6, lower trace. . In membranes, Pq rereduction is faster, occuring with a mean time of 1±2 s onlyŽ not shown. , a value close to that found in Rb. sphaeroides RCs. In a RC sample containing DM and diaminodurol qqy Ža rapid reductant of P which prevents the P QB back reaction to occur. a single flash excitation led to y the formation of PQAB Q state with a rather long lifetimeŽ. 20±30 min . Fig. 7 shows the flash-induced y absorption profile of QB measured at least 1±2 s after the excitation flash; under these conditions there is no contribution of the residual tetraheme cytochrome to the Qy absorption band, as the photo- B y Fig. 8. Binary oscillations of semi-ubiquinoneŽ QB . observed at oxidized high potential heme is rapidly rereduced by 450 nm in RCs after a series of saturating flashes. RCs were Ž. DAD within 30±70 ms after the flash. This absorp- suspended in 10 mM Tris buffer pH 8 containing 0.5 mgrml tion profile is typical of a semi-ubiquinone radical DM, 1 mgrml LDAO, 40 mM UQ6 and 1 mM DAD. 40 I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46

conditions as above, i.e., by flash excitation in the presence of diaminodurol. The flash-induced absorption spectrumŽ. Fig. 9 has a striking resemblance with that reported for the semi-menaquinone identified as y QA in Cf. auriantacus and in C. Õinosum RCs wx43,44 . Its maximum is located at 395 nm, thus close to the maximum of semi-menaquinone in vitrowx 45 . However it displays slightly different spectral features relative to the semi-menaquinones bound to Rps. Õiridis and RÕ. gelatinosus RCs, where the maxima are red-shifted to 412 nmwx 18,46 .

Fig. 9. Flash-induced difference spectrumŽ. closed points of 3.5. Herbicide sensitiÕity y semi-menaquinone QA obtained in presence of 500 mM DAD in QB -less RCs; same conditions as in Fig. 7. For comparison the Terbutryn and orthophenanthrolineŽ. o-phen , which flash-induced difference spectrum published for semime- y are known to block the electron transfer between the naquinone QA in Chl. aurantiacus RCŽ. continuous linewx 43 has been superposed but with a 5 nm red shift as to get the same two quinones, were tested on Rc. tenuis RC. In the q y position of the maximum. presence of these herbicides, the flash induced P QA radical pair relaxed with a mean time ts35 msŽ not shown. . This fast recombination time is close to the addition of LDAO led to the formation of smaller values measured in isolated bacterial RCs which have

LDAO andror DMrLDAO mixed micelles, facilitat- menaquinone as QAB and ubiquinone as Qwx 18,47,48 . ing the diffusion of UQ6 into the protein and the The sensitivity to o-phen was measured in mem- regeneration of oxidized QB . brane fragments as well as in isolated RCsŽ see Fig. In Rc. tenuis RCs, incubation with LDAOŽ 8 10. ; the inhibitor concentration required to reduce QB . mgrml as described in Section 2 resulted in an easy activity to 50%, I50 s10 mM, is within the range of extraction of 85% of the secondary quinone. The values determined for Rps. Õiridis wx49,50 and RÕ. absence of QB was checked by the back reaction rate gelatinosus RCswx 51Ž. Table 1 . The inhibition power which was the same as in the presence of herbicide of this herbicide was about 10 times smaller in Rb. Ž y1.Ž . ka s28 s see below . A gradual recovery of sphaeroides and Rb. capsulatus RCswx 52±54 . The secondary activityŽ. up to 80% took place after incu- sensitivity to terbutryn was determined in isolated bation of QB6 -depleted RCs with an excess of UQ , Rc. tenuis RC; the value of I50,5mM, is close to provided that LDAOŽ. 0.5±1 mgrml was added to the RC solution already containing DM. This was demonstrated by a decrease in the Pq rereduction rate Ž y1. after a flash k b s3.3 s and the concomitant appearance of multiple flash-induced binary oscilla- y tions of QB observed at 450 nm in the presence of DADŽ. see Fig. 8 . It is of note that secondary activity could not be reconstituted in a DM solution in the absence of LDAO; this fact confirms our hypothesis that quinones cannot be exchanged between DM micelles and the protein.

Identification of QA required the measurement of y QA absorption spectrum without the eventual interference of the secondary quinone QB . Thus we used Fig. 10. Inhibition of electron transfer from Qy to Q by o-phen y AB QB depleted RCs for these measurements. In these in cytoplasmic membranesŽ. closed points and in isolated RCs y Ž. samples, the state PQA was formed with the same open points ; I50 s10 mM. I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 41

Table 2 q y Detergent effects upon various kinetic parameters in isolated Rc. tenuis RCs: k bAB, the P QQ recombination rate; K 2, the apparent y equilibrium constant between QAB Q and Q AB Q states, and DG, the free energy difference between these states Ž.y1 Ž. Detergent k b2s K yDG mV Ž. C12 M 0.2 mgrml 0.1 300 134 Ž. Ž. C12 M 0.2 mgrml qC 12 DAO 0.5 mgrml 0.2 142 117 Ž. Ž. C12 M 0.2 mgrml qC 12 DAO 1 mgrml 0.33 86 105 Membrane fragments 0.66±0.5 42±50 92

Values of these parameters measured in membrane fragments are shown for comparison. those already determined for Rps. Õiridis wx46,50 and observed during the isolation of RCs belonging to Rb. sphaeroides wx52 . other bacterial species like RÕ. gelatinosus wx18 , C. Õinosum, C. tepidum and Cf. aurantiacus wx26 . By 3.6. Energetics of quinone acceptor complex in Rc. contrast, the tetraheme Cyt c remains firmly associ- tenuis; dependence on detergents ated in Rps. Õiridis RC in a 1:1 ratio even though high LDAO concentrations is required for the RC q y The rate of P QA recombination has the same purificationwx 56 . Therefore, some structural differ- Ž value in isolated RC as in membrane fragments ka s ences seem to exist between the Cyt c binding site of 28 sy1. irrespective of the kind of detergent present the RCs belonging to the species cited above and that qy in the RC solutionŽ. not shown . In contrast P QB of Rps. Õiridis. Salt bridges observed in the Rps. y1 back reaction rate k b varies between 0.66±0.1 s Õiridis RC structure between residues of the Cyt c depending on the environment of the RCŽ see Table and those of L and M periplasmic surfaceswx 57 could 2. . Therefore, in this case the apparent equilibrium be absent in these RCs altogether andror the hy- y y constant K 2ABABof the electron transfer Q Q lQQ drophobic contacts could be weaker. defined as K 2bask rk wx55 , depends essentially on At variance with RC preparations from photosyn- kb2b. Table 2 shows the values of K , k and DG, the thetic bacteria belonging to the a-group, such as Rb. y y free energy gap between QABQ and Q AB Q states of sphaeroides, Rb. capsulatus, Rsp. rubrum, those Ž yDGrkT . RCs defined by K 2 se , in different deter- from Rc. tenuis and RÕ. gelatinosus Ð although not gent solutions and in membrane fragments, as well. to the same extent Ð were slowly inactivated when

We can see that for RCs in DM, k b is very slow and purified in presence of LDAO as seen by their photo- thus K 2 and DG reach their highest values. When chemistry and changes in optical spectra. In agree- LDAO is added, Pq relaxation becomes faster, ap- ment with previous workwx 58,59 , we have experi- proaching the rate measured in the native state; in the mental evidence that LDAO dissociates the H subunit same way, the other parameters depending on k b from RÕ. gelatinosus RCs extracted from the wild decreaseŽ. see Table 2 . type as well as from a mutant without the tetraheme Cyt c ŽI. Agalidis and F. Reiss-Husson, unpublished results. . This reduced stability and higher sensitivity 4. Discussion towards this detergent might reflect differences in the quaternary structure of these RCs. A comparative In this paper, the isolation and several important analysis of the amino acid sequences of the H sub- functional and structural properties of the Rc. tenuis units in these various RCs will be useful in this RC are reported. In vivo, this RC is functionally respect. We also observed that less LDAO and no associated with a tetraheme Cyt c which is involved herbicide was needed to remove UQ from the QB site in the fast rereduction of the photooxidized Bchl in Rc. tenuis as compared to Rb. sphaeroides, where dimerwx 26 . However the tetraheme Cyt c is gradually a more drastic treatment is neededŽ i.e., incubation detached from the RC all along the extraction and with 1±2% LDAO and 1mM o-phenwx 60. . The facile purification steps. A slow unbinding has already been liberation of QB by LDAO in Rc. tenuis is consistent 42 I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 with the susceptibility of the RC towards this deter- at ca. 1653 cmy1. In contrast, Rps. Õiridis, Ro. gent. denitrificans, Cf. aurantiacus and C. tepidum possess a Tyr residue at position M197 of Rb. 4.1. The primary donor of Rc. tenuis sphaeroides wx30,64 . In the 3-dimensional crystal structure of the Rps. Õiridis RC, this Tyr residue q The optical absorption spectra of P and PŽ. Fig. 3 forms a H-bond to the C2M acetyl carbonyl of P and their corresponding FT Raman spectraŽ. Fig. 5 wx30,57,64,66 The FT Raman spectra of Cf. aurantia- suggest that, similar to other purple bacterial RCs, the cus wx34 and C. tepidum wx30 RCs showed that this primary donor of Rc. tenuis is constituted of two carbonyl is also H-bonded since the observed vibra- excitonically coupled Bchl a molecules, consistent tional frequency was ca. 1632±1635 cmy1, in agree- with what is known for other bacterial primary donors. ment with the shifts observed in the FYŽ. M197 RC The FT Raman data presented in this work indicate mutant from Rb. sphaeroides wx39 . The sequence that both Bchl molecules constituting the Rc. tenuis alignment of the M subunit of Rc. tenuis ŽNagashima, primary donor possess one axial ligand eachŽ most personal communication. with that of Rb. likely histidine residues. . The determined H-bond sphaeroides RCs shows that the residue at the equiv- pattern for the primary donor is the following:Ž. i two alent position of PheŽ. M197 of Rb. sphaeroides is

H-bonded C2 acetyl carbonyl groups with observed indeed a tyrosine. Thus, we propose that this tyrosine y1 y1 vibrational frequencies at 1618 cm and 1635 cm residue is the H-bond donor to the PM2 C acetyl andŽ. ii two C9 keto carbonyl groups which are free carbonyl of P in Rc. tenuis Ž.Table 1 . from such protein interactions, vibrating at 1690 cmy1 It is interesting that some similarities are found and 1696 cmy1 Ž. Fig. 5A . between P electronic absorption spectra of Rc. tenuis Sequence alignment of the L subunits of the RCs and FYŽ. M197 Rb. sphaeroides mutant RCs in vivo: from several purple bacteriaŽ see Nagashima et al. the Qy band is broad and redshifted at 890 and 880 wx61. shows that the His L168, which forms a H-bond nm in Rc. tenuis and the Rb. sphaeroides FYŽ. M197 to the PL2 C acetyl carbonyl in Rb. sphaeroides mutantwx 39 respectively, when compared to the iso- wx62,63 is strongly conserved. The observed vibra- lated RCs. As well, the Pq absorption band is red- tional frequency for this acetyl carbonyl of the Rb. shifted to 1260 nm in both isolated RCs. These sphaeroides primary donor is 1620 cmy1 wx 24 ; for spectral modifications altogether might be the result RÕ.gelatinosus wx32 , the corresponding band is 4 of the extra H bonding of the dimer to Tyr M197. y1 cm downshifted in frequency as it is for C. tepidum Nevertheless, the extra H-bonding of PM2 C acetyl to wx30 indicating a stronger H-bond for these latter TyrM197 does not necessary correlate with a red species. Thus, a similar H-bond as that found in Rb. shift of the near infrared absorption band of Pq. For sphaeroides P is expected on the PL2 C acetyl car- example, in C. tepidum where this extra H bond bonyl group in Rc. tenuis, consistent with the ob- exists, the Pq absorption band is blue shifted to 1240 served 1618 cmy1 band. Sequence alignment data nmwx 30 . indicate that His L168 Ž.Rb. sphaeroides numbering The deduced structure of the microenvironment of is conserved in Rc. tenuis ŽNagashima, personal the primary donor of Rc. tenuis can be correlated communication. and is most likely the H-bond donor with the PrPq midpoint redox potential of RCs to the analogous C2L acetyl carbonyl of PŽ. Table 1 Ž.Table 1 . Specifically, the His-donated H-bond on y1 which gives rise to the 1618 cm bandŽ. Fig. 5A . the acetyl carbonyl of PL should maintain the redox On the other hand, the sequence alignment of the potential near q500 mV as in the case of Rb. M subunits from several purple bacteriawx 64 shows sphaeroides RCwx 29,67 . Moreover, the extra H-bond that Rb. sphaeroides, like Rb. capsulatus, Rsp. on the PM acetyl carbonyl group of Rc. tenuis RC, as rubrum, and RÕ. gelatinosus possess a Phe at posi- for C. tepidum wx30 and for FYŽ. M197 mutant RC tion M197. In the crystal structure of Rb. sphaeroides from Rb. sphaeroides wx39 , should modestly raise the RCwx 62,63 as well as the FT Raman data on all the redox midpoint potential by at least 25 mV. Thus, above mentioned specieswx 32,65 , no H-bond is ob- assuming structural analogy and supposing that the served on the PM2 C acetyl carbonyl group, vibrating additive effect of H-bonding on the primary donor I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 43 redox midpoint potential observed for Rb. the in vitro spectrum. as compared to those of Rps. sphaeroides wx36 is also applicable to Rc. tenuis, the Õiridis wx46 and RÕ. gelatinosus wx18 as if, in the redox potential of Rc. tenuis P should be somewhat latter cases, the electrochromic shifts due to interac- higher than that of Rb. sphaeroides Ž.see Table 1 . tions with chlorin pigments were stronger.

This prediction agrees well with our experimental We have shown that QB activity is strongly depen- value of 515"20 mV for the membrane fragments. dent on the kind of detergent contained in the RC Similar values were determined for the special pair in solution. After purification of the RC in the presence

C. tepidum which also has two H-bonded C2 acetyl of DM, secondary activity assays in the presence of carbonyl groups donated by a His and a Tyr residues. this surfactant have shown the absence of multiple The observed redox potential in this case was 526"5 semiquinone oscillations in intact RCs, as well as the mV for isolated RCs and 502"10 mV for membrane impediment to reconstitute QB activity in those de- fragmentswx 30 . void of the secondary quinone. These results suggest From the FT Raman results it is also possible to that the exogeneous Q6 pool was exclusively parti- estimate the degree of localization of the resulting tioned in DM micelles, and UQ6 molecules could positive charge on the primary donor in its cation neither move to QB site to eventually replace the q radical state P ;in Rb. sphaeroides RC, the positive quinolŽ. QH2BB nor reconstitute Q activity in Q -less charge distribution was reported to be ca. 80% local- RCs. Nevertheless, LDAO addition allowed the re- ized on PL wx 24 . Under similar considerations, 69% covery of normal activity at the level of the sec- localization on one Bchl moleculeŽ most probably ondary quinone. The best illustration is the full recon-

PL . of the primary donor of Rc. tenuis is estimated stitution of functional QB in RC devoid of secondary which indicates that the q charge is more delocal- activity in presence of a UQ6 exogeneous pool, ized over P than it is in Rb. sphaeroides. Very demonstrated by the appearance of flash-induced bi- similar delocalization was estimated for the primary nary oscillations of the semi-ubiquinoneŽ. see Fig. 8 . donor of C. tepidum that indeed exhibits a more In order to understand the different effects of these symmetric protein interaction, i.e., two H-bonded detergents we should take into account some of their acetyl carbonyl groupswx 30 . properties, and also what is known about their interactions with photochemical RCs. 4.2. Quinone acceptor properties Studies of Rps. Õiridis and Rb. sphaeroides RCs in crystalswx 68,69 as well as in micellar solutions of The Rc. tenuis RC possesses, like Rps. Õiridis the latterwx 70 have shown that detergents like LDAO wx46 , C. Õinosum wx44 and RÕ. gelatinosus wx18 a and OG bind essentially to the hydrophobic trans- mixed quinone-acceptor complex with MK as QA and membrane regions of the protein very likely in a UQ as QB Ž. Table 1 . In a previous reportwx 16 we monolayer type of arrangement. A similar organiza- tried to determine the flash-induced absorption pro- tion can be expected for DM. LDAO and DM are file of the primary quinone anion in isolated RCs in both nonionic and their alkyl chains are identical. presence of terbutryn and DAD. However, in these However, the dissacharide head group of DM is experimental conditions the herbicide was unable to bulkier than the amine oxide group of LDAO; ac- y effectively displace QBB andror Q and we obtained cordingly, the DM micelles are non-spherical and an absorption spectrum which was an overlap of both bigger Ž.ns130 monomers, Ms66 kDa than those yy Ž Ž. QAB and Q flash-induced absorption bands see Fig. of LDAO ns69 monomers, Ms15 kDawx 71 . The 3 inwx 16. . Therefore in order to avoid the interference cmc molar values differ also notablywx 72 , being 10 of the secondary quinone, we measured the absorp- times lower for DM. In solutions containing RCs, y tion band of QABin RCs depleted of Q . The absorp- UQ6 molecules should partition between the micelles tion spectrum obtained now for this bound semi- and the detergent belt of the protein. They might be menaquinone 8 speciesŽ. Fig. 9 is quite similar to fully embedded in the DM micelles, but poorly solu- those reported for C. Õinosum wx44 and Cf. aurantia- bilized by the small LDAO ones; in the later case, the cus wx43 . One may observe that in these three bacte- remaining UQ pool becomes available to the protein rial species the spectra are blue-shiftedŽ thus closer to detergent belt, where the regeneration of QB could 44 I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 occur. In a similar fashion LDAOrDM mixed mi- transfer was inhibited at a very low concentration of celles may have a reduced size and a different shape o-phen with I50 s10 mM. Similar values were deter- as compared to pure DM, thus being able to share the mined for Rps. Õiridis wx49,50 and RÕ. gelatinosus quinones with the RC. wx51 RCs. This sensitivity distinguishes these species Another differential effect of the interaction of altogether with respect to Rb. sphaeroides and Rb. these detergents with Rc. tenuis RC is observed on capsulatus which have I50 values in the range 100± qy P QB recombination rateŽ. see Fig. 6 . In the pres- 200 mMwx 52±54 . The degree of inhibition by o-phen ence of DM this light-induced state relaxed very was correlated with the residue present in the position Ž y1.Ž . slowly ka s0.1 s see Table 2 , indicating that L226wx 54 ; bacteria which have a Thr residue in the the electron would take a direct pathway through the position L226 are weakly inhibited, whereas those protein to return to Pq wx 73 . Subsequent addition of which have an Ala residue at this position such as LDAO induced the decrease of the Pq relaxation Rps. Õiridis wx50 and the ThrL226™Ala Rb. capsu- time to about 0.3 sy1, closer to the value determined latus mutantwx 54 , or even a Ser residue, like in RÕ. in the native stateŽ. see Table 2 . We assume that this gelatinosus wx51 , were shown to be highly sensitive. It change in kinetics reflects a different interaction of was supposed that a smaller residue in position L226 DM with the protein, relative to LDAO, which in may allow an easier diffusion of o-phen to its binding y turn should change the energy level of QBBrQ , i.e., sitewx 54 . These results are now extended to Rc. Žqy qy.Ž Ž the free energy difference DG PQAByP Q see tenuis, where L226 is also an Ala residue Nagashima, Table 2. . The fact that Pq rereduction rate could be personal comunication. and which at the same time is modulated by the detergents might be an indication strongly inhibited by o-phenŽ. Table 1 . Structural of some specific local conformational changes in the similarities of the QB site between Rc. tenuis and microenvironment of pigments induced by the inter- Rps. Õiridis are reinforced by these observations. action with the bound surfactant molecules. From the whole kinetic behavior of the secondary acceptor, it turns out that no exchange of UQ or very Acknowledgements little occurred at the QB site in DM solutions, as if UQ diffusion between DM micelles and RC-deter- We are grateful to Dr. K. Nagashima who kindly gent complex was almost absent; in contrast, a faster provided us with the unpublished primary sequences quinone exchange with a weak affinity for Q binding of the L and M subunits of Rc. tenuis reaction seemed to proceed in presence of LDAO, as in one of center. We thank Dr. P. Sebban and Dr. C. Vernotte the reaction models proposed by Shinkarev and for their useful discussion and the critical reading of Wraightwx 74 . the manuscript. We are indebted to Mrs. M.C. Gonet In the Rc. tenuis RC, as already shown for other for her assistance in growing Rc. tenuis cells and bacterial RCswx 75 , the Hq concentration is capable preparing the membranes. of modifying Pq rereduction rate after a flash. Pre- q y liminary P QQABrecombination reaction rate measurements in DM solution, at several pH values References between 6 and 9.5, have shown an increase in rate from 0.043 to 0.27 sy1 Ž I. Agalidis, unpublished wx1 C.R. Woese, Microbiol. Rev. 51Ž. 1987 221±271. results. . From these rate constants, it turns out that wx2 A. Willems, M. Gillis, J. De Ley, Intern. J. Syst. Bacteriol. 41Ž. 1991 65±73. q y q y the free energy gap between P QABand P Q states wx3 B. Wakim, J.R. Golecki, J. Oelze, FEMS Microbiol. Lett. 4 below pH 8 is larger than 100 mV. Then it is Ž.1978 199±201. q y reasonable to assume that the pathway of P QB wx4 Q.H. Hu, R.A. Brunisholz, G. Frank, H. Zuber, Eur. J. recombination essentially occurs by a tunnelling Biochem. 238Ž. 1996 381±390. mechanism to the ground state 73 ; conversely, at pH wx5 L. Menin, P. Parot, B. Scoepp, P. Richaud, A. Vermeglio,Â wx in: P.E. MathisŽ. Ed. , Photosynthesis: from Light to Bio- values higher than 8 the indirect pathway through the sphere, vol. II, Kluwer Academic Publishers, Dordrecht, q y higher lying P QA state seems to dominate. The Netherlands, 1995. y In the Rc. tenuis RC the QABto Q electron wx6 R.G. Bartsch, Biochim. Biophys. Acta 1058Ž. 1991 28±30. I. Agalidis et al.rBiochimica et Biophysica Acta 1321() 1997 31±46 45

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