The Reaction Center of Green Sulfur Bacteria1

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Review The reaction center of green sulfur bacteria1

G. Hauska a;*, T. Schoedl a, Herve¨ Remigy b, G. Tsiotis c

a Lehrstuhl fu«r Zellbiologie und P£anzenphysiologie, Fakulta«tfu«r Biologie und Vorklinische Medizin, Universita«t Regensburg, 93040 Regenburg, Germany b Biozentrum, M.E. Mu«ller Institute of Microscopic Structural Biology, University of Basel, CH-4056 Basel, Switzerland c Division of Biochemistry, Department of Chemistry, University of Crete, 71409 Heraklion, Greece

Received 9 April 2001; received in revised form 13 June 2001; accepted 5 July 2001

Abstract

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA^D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary

electron acceptor A0, which is a Chl a-derivative and FeS-center FX. An equivalent to the electron acceptor A1 in Photosystem I, which is tightly bound phylloquinone acting between A0 and FX, is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A0 and FX in the two systems. The subunit PscB contains the two FeS-centers FA and FB. STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this

protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)3(PscA)2PscB(PscC)2PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840. Also the way of NAD-reduction is unknown, since a gene equivalent to ferredoxin- NADP reductase is not present in the genome. ß 2001 Elsevier Science B.V. All rights reserved.

Abbreviations: (B)Chl, (bacterio)chorophyll; C., Chlorobium; cyt, cytochrome; FMO, Fenna^Mathews^Olson BChl a-protein; FA,FB and FX, FeS-clusters A, B and X, respectively; FNR, ferredoxin-NADP reductase; GSB, green sulfur bacteria; MQ, menaquinone; PSI and PSII, Photosystems I and II; PscA^D, protein subunits of the RC from GSB following the nomenclature of D.A. Bryant [44]; RC, reaction center; RT, room temperature; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis; STEM, scanning transmission electron microscopy * Corresponding author. Fax: +49-941-943-3352. E-mail address: [email protected] (G. Hauska). 1 Dedicated to the memory of Jan Amesz.

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Keywords: Green sulfur bacteria; Homodimeric P840 reaction center; FeS type reaction center; Photosynthetic electron transport; Energy transfer; Bacteriochlorophyll protein; Menaquinone; Cytochrome; Scanning transmission electron microscopy particle analysis

1. Introduction has recently been obtained [8,9], as detailed elsewhere [10]. The photosynthetic reaction centers (RCs) of aero- In this review we will update the essentials of the tolerant organisms contain a heterodimeric core, homodimeric reaction center from Chlorobiaceae, built by two strongly homologous polypeptides. which have been summarized before [11,12]. After a Each of them contributes ¢ve transmembrane pep- brief description of the outer antenna system we will tide helices to hold a pseudosymmetric double set discuss the progress made on isolation procedures, of redox components, like in two hands. This holds on analysis of pigments, genes and proteins, as well for Q- as well as for FeS-type RCs [1], as is amply as on the spectroscopy of energy and electron trans- documented by the crystal structure, which is avail- fer within the RC. The particle structure will also be able for purple bacteria since 1985 [2], more recently presented here for comparison to the structure of the for PSI [3,4], and just has been published for PSII [5]. PSI-RC in the accompanying article by Fromme et Interestingly, only one branch of the double set al. [4]. For several further aspects of the GSB RC the seems to be used in physiological electron transfer. reader is referred to other contributions for this issue Why is that so? (FeS-centers/Vassiliev et al., transient EPR spectros- Clues to this unsolved question may come from copy/van der Est, evolution/Nitschke et al.). The ho- homodimeric RCs, of the green sulfur bacteria modimeric RC of Heliobacteria will also be adressed, (GSB, i.e., Chlorobiaceae) and the Heliobacteria, for details see the accompanying article by Neerken both living strictly anaerobic. They resemble the and Amesz. PSI-RC, with FeS-clusters as terminal electron acceptors, but the two branches of transmembrane electron transfer are held by two identical proteins 2. Energy transfer from the outer antenna [6,7]. Unfortunately high structural resolution has not been achieved yet for the homodimeric RCs, In the photosynthetic units of the di¡erent photo- only a gross structure (2 nm resolution) of the RC systems the excited states of the pigments migrate from Chlorobium tepidum by STEM particle analysis from the outer to the inner antennae and are ¢nally

Fig. 1. The antenna system of green sulfur bacteria. Numbers following the designations of the pigments indicate absorption maxima.

BBABIO 45074 15-10-01 262 G. Hauska et al. / Biochimica et Biophysica Acta 1507 (2001) 260^277 trapped in the RC. For a general and comprehensive e¤cient energy transfer already is damaged by isolat- treatment of these energy transfer processes the read- ing the membranes (see below). Indeed, FMO is er is referred to van Grondelle et al. 1994 ([13]; for rather loosely bound to the RC and is easily lost PSI see Gobets and van Grondelle in this issue). during isolation. Excitation transfer measurements In the photosynthetic apparatus of GSB light en- in intact cells may clarify the situation. ergy is funneled to the RC in the cytoplasmic membrane by a unique peripheral antenna system, the so- called chlorosome [14,15], as depicted in Fig. 1. It 3. Composition captures light very e¤ciently with some 200 000 bacteriochlorophyll (BChl) c, d or e-molecules per chlo- 3.1. Protein subunits in isolated reaction centers rosome (M. Miller, personal communication) and constitutes the largest outer antenna known, with a The isolation of the RC from GSB started with the photosynthetic unit of several thousand chlorophyll mechanical separation of the chlorosomes in the molecules per RC (J. Olson, K. Matsuura, personal early seventies [23,24]. Subsequently the dissolution communications). With 5000 chlorophylls per RC of the membrane by Triton X-100 and fractionation there would be some 40 RCs per chlorosome. BChl was systematically studied, ¢rst by Jan Amesz and c, d and e in the chlorosome are arranged in non- his collaborators working on Prosthecochloris aes- proteinaceous, tubular stacks with an absorption tuarii [25,26]. Meanwhile protocols using either Tri- maximum at 720^750 nm. Energy transfer proceeds ton X-100 and/or alkyl glycosides are available for from these rods, via a so-called baseplate of BChl several species of GSB, which include Chlorobium a-795 (see [16]) to the Fenna^Mathews^Olson BChl limicola f.sp. thiosulfatophilum [27^29], C. tepidum a-protein (FMO), with an absorption peak at 808 nm. [28,30] and C. vibrioforme [31]. These procedures FMO likely transfers the excitation to the RC, with have been reviewed before [11,12] and do not need BChl a-840 as the primary electron donor P840 and to be described in detail here. Our present knowledge Chl a-670 as the primary electron acceptor A0 (see is summarized in Table 1 together with the following below). Heliobacteria do not have chlorosomes and statements: lack an extended antenna. The only other photosynthetic organisms with chlorosomes are the green non- 1. Functionally intact isolates of the RC from GSB sulfur bacteria (Chloro£exaceae), which lack the contain three FeS-centers, show stable charge sep- FMO-protein. These are aerotolerant organisms aration with electron transfer to the terminal FeS- and thus contain a heterodimeric RC, which is of center and accordingly lack fast recombination the Q-type [14,15]. rates from preceding electron acceptors (see be- The energy transfer in chlorosomes is e¤ciently low). They should be capable to reduce ferredoxin quenched under oxidizing conditions [14,15]. Chloro- [32,33] and to catalyze transmembrane charge sep- bium quinone which is enriched in chlorosomes has aration after reconstitution into lipid vesicles [34]. been envisaged as the responsible redox regulator 2. Such RC preparations from Chlorobium limicola [17], more recently the involvement of FeS-proteins f.sp. thiosulfatophilum [29], C. tepidum [28,30] in the chlorosome envelope is discussed [18]. Also and C. vibrioforme [31] contain the ¢ve polypep- within the BChl a-molecules of the FMO-protein en- tides, PscA^D plus FMO. ergy transfer is attenuated under oxidizing conditions 3. The core of the RC is built by two copies of the [14], involving Tyr-radicals [19]. Both quenching large integral membrane protein PscA and one mechanisms contribute to save the photosynthetic copy of the peripheral protein PscB. PscA binds apparatus from damage by oxygen. In this context the primary electron donor P840, the primary the surprisingly low e¤ciency estimated for energy electron acceptor A0 and 4Fe4S-cluster FX, PscB transfer ( 6 30%) from FMO to the RC may be rel- binds the two terminal 4Fe4S-clusters FA and evant, which was found not only for isolated RCs FB, also called center 1 and 2 (see Vassiliev et but also for membranes [14,15,20^22]. Possibly the al., this issue). It is related to bacterial ferredoxin interaction between FMO and the RC required for [35,36].

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4. An intact core of the RC, only containing PscA coded by the transcription unit pscAB which has and -B, but ful¢lling the above criteria has been been sequenced for C. limicola f.sp. thiosulfatophilum isolated only from P. aestuarii [37,38]. It seems [6] and C. tepidum [45,46], while PscC, a peculiar that the interaction of PscA and PscB is more cytochrome c [47], PscD [28] and FMO [48] are en- stable in this organism, which is relatively distant coded by separate loci. Meanwhile genome sequenc- to the other GSB studied [39]. Unfortunately the ing has been completed for C. tepidum (see corre- psc-genes have not been sequenced for P. aestu- sponding website of NCBI), which con¢rms the arii. sequences of the pscAB-transcription unit and of 5. Solubilization by detergent leads to partial remov- the other genes. al of the FMO-protein which destabilizes the re- sidual complex, with loss of FeS-centers and of 3.2.1. The gene pscA the subunits PscB and -D. The interaction of the In con¢rmation of earlier evidences for GSB [6] three proteins is indicated by the observation that and Heliobacteria [7] pscA is the only gene in the PscD copuri¢es with FMO and PscB from RCs genome of C. tepidum with the required signatures and by cross-linking experiments (H. Rogl, un- for the large transmembrane core protein of a FeS- published). The loss of FeS-centers is re£ected type RC, in contrast to the two genes psaA and psaB by fast recombination of P840 with earlier elec- coding for the PSI-RC subunits (see [4]). Undoubt- tron acceptors [40,41]. edly, therefore, the core of the RC in GSB is a homodimer formed by two identical proteins. The gene 3.2. Genes pscA from C. tepidum codes for a 82 kDa-protein of 731 amino acids [45,46].The primary structure is 95% The two RC-core proteins PscA and PscB are en- identical to the one from C. limicola [6]. However,

Table 1 Proteins, pigments and redox components in isolated FeS-type reaction centers

Proteins Tetrapyrrols Carotenoids FeS-centers A1-Quinones Denotation/ Size Forms/ Function Forms/ Denotation/ Type/tightly number (kDa) number number type/number bound Green sulfur bacteria RC-core PscA/2 82 BChl a/16 P840+antenna 2 X/4Fe4S/1 MQ7/none

Chl a/4 A0+antenna PscB/1 24 ^ ^ A,B/4Fe4S/2 ^ Additional proteins PscC/2 23 Heme-c/2 e-Donor ^ ^ ^ PscD/1 15 ^ ? ^ ^ ^ FMO/6 40 BChl a/42 Antenna ^ ^ ^

Heliobacteria RC-core PshA 68 BChl g/35 P798+antenna 1^2 X/4Fe4S/1 MQ5-10/none

OH-Chl a/2 A0+antenna PshB ? ^ A,B/4Fe4S/2 ^ Additional proteins ?

Photosystem I RC-core PsaA/B 83/82 Chl a/85 P700, 20 (5 cis) X/4Fe4S/1 Phyllo-Q/2

A0+antenna PsaC 8 ^ A,B/4Fe4S/2 ^ Additional PsaD^F,I^M,X Chl a/10 Antenna 2 proteins: 10 The compositions for the RC from GSB with respect to redox centers and polypeptides [11,12], and pigments [38,42] are shown in comparison to the Heliobacterium Heliobacillus mobilis ([7,43], see Neerken and Amesz, this issue) and PSI from the cyanobacterium Synechococcus elongatus [4]. PshA and PshB denote the RC subunits in H. mobilis corresponding to PscA and PscB of GSB (see [44]).

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Fig. 2. Alignment of the C-terminal region in the core subunits of FeS-type reaction centers binding the redox centers P, A0,A1 and FX. Identical residues to PscA of Chlorobium are in bold, conserved residues are underscored by asterisks. Italics framed by diagonal strokes indicate the two transmembrane helices IX and X (tmhIX and X). Cytoplasmic and periplasmic ends are shown by the letters c and p on top of the alignment. Residues involved in pigment and FeS-cluster binding are highlighted by arrows; P, Cp2 and 3 stand for the ¢rst (`special'), second and third pair of chlorophylls in the RC-core, Cp2 and 3 forming the electron acceptor A0 in PSI (see [4]; Fromme et al., this issue); A1 stands for the secondary electron acceptor which is phylloquinone in PSI, and FeS-X is the ¢rst of three 4Fe4S-clusters. For the Ps-nomenclature of the RC-subunits see Bryant [44]. C. lim, C. tep and H. mob stand for Chlorobium limicola, Chlorobium tepidum and Heliobacillus mobilis, respectively. one striking di¡erence was found: Residues 285 to ment of the region binding the redox components 296 in C. tepidum read AIGYINIALGCI which are P840, A0,A1, and FX is shown in Fig. 2. It starts HLRHQHRAW-VI in C. limicola. Only 19 histidines with a peptide exposed to the cytoplasmic surface per PscA are present in C. tepidum compared to 21 in which contributes two cysteines to bind the 4Fe4S- C. limicola (at position 589 C. tepidum carries a H cluster FX between the heterodimer of PsaA/B in PSI instead of a Q). The two core proteins of PSI contain or the homodimers of PscA and PshA. Nine residues about the double number of histidines, 42 in PsaA are identical in a stretch of 12, which is the most and 39 in PsaB, in accordance with a denser popu- highly conserved part of the whole alignment. The lation by chlorophylls. The corresponding PshA-pro- crystal structures clearly show that the primary tein from Heliobacteria is only 609 residues long but charge separation in Q-type and FeS-type RCs in- contains 25 histidines [7], and binds chlorophylls volves a consortium of three pairs of chlorine-tetra- with an intermediate density (Table 1). pyrrols. These are three pairs of Chl a in PSI [4], the Sequence alignment of these large subunits in FeS- special pair of the primary donor P700 and two more type RCs [6,7,45] suggest that the 11 transmembrane denoted Cp2 and Cp3, functioning as the primary helices in PsaA/B of PSI are conserved in Chlorobium acceptor A0. The special pair of P700 in PSI, P840 PscA, as well as in PshA of Heliobacillus. Overall in GSB and P798 in Heliobacteria is bound to a identities are low between GSB and PSI as well as conserved histidine in the middle of the transmem- between GSB and Heliobacillus (only about 17% in brane helix X (Fig. 2), while the binding residues in each pairwise comparison), but are particularly sig- PsaA/B for Cp2 (an asparagine in transmembrane ni¢cant in the C-terminal portion, which holds the helix IX which holds the chlorophyll via a water redox components between the ¢ve putative trans- molecule) as well as for Cp3 (a methionine close to membrane helices VII to XI. This fold is common the cytoplasmic end of transmembrane helix X) are to FeS-type as well as Q-type RCs, what has been neither conserved in PscA nor in PshA. elaborated in detail by Schubert et al. [3] and further The secondary electron acceptor A1 in the RC of substantiated by the recently obtained, re¢ned struc- PSI is phylloquinone, and is bound in van der Waals tures for the RCs of PSI [4] and PSII [5]. An align- distance to the tryptophan of the conserved peptide

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RGYWQE of PsaA/B [3,4]. This tryptophan is not conserved, neither in GSB nor in Heliobacteria, and this may be the reason for less tight binding of MQ to the RC in these organisms compared to phylloquinone in PSI.

3.2.2. The gene pscB The second gene of the transcription unit has been sequenced for C. limicola [6], C. tepidum [45,46] and C. vibrioforme [49]. Theses genes, known as pscB, code for 23 kDa-proteins of 230 or 231 residues. They are about 90% identical. The di¡erences are largely con¢ned to the N-terminal region with a re- petitive sequence, enriched in proline, alanine and lysine which is also found in other proteins from GSB, like PscA and cytochrome b [6,50]. This positively charged extension probably is responsible for the slow migration in SDS^PAGE, with an apparent Fig. 3. Absorption spectra of reaction centers from green sulfur Mr of 32 kDa [51]. The C-terminal, more conserved part of the PscB-proteins resembles PsaC of PSI and bacteria. The ¢gure shows the absorption spectra at RT and 6 K for P. aestuarii (a,b) and C. tepidum (c,d). Spectra b^d are harbors the FeS-binding peptides with four cysteine shifted upwards for clarity (the ¢gure represents Fig. 1A from in each one. The folding of this part corresponds to [32]; courtesy H.P. Permentier). bacterial ferredoxin with two 4Fe4S-clusters [35,36], the ¢rst three and the last of the eight cysteine binding FB, the rest FA [52]. The exchange of the two heme c-binding peptide close to the C-terminus positively charged residues KR between the sixth [47]. The gene pscD codes for a 15 kDa-protein and the seventh cysteine in PsaC for the neutral res- with positive net charge of no obvious relation, idues SA in PscB has been advocated to explain the which may be involved in stabilization of PscB drop in redox potential of FA (see Fig. 7) in GSB and/or in the interaction with ferredoxin [28,33]. compared to PSI [6]. This was subsequently substan- The fmo-gene coding for the intermediary BChl tiated by targeted mutation of PsaC in PSI from a-antenna, the 40 kDa FMO-protein has been se- Chlamydomonas reinhardtii [53]. quenced for C. tepidum [48] after the amino acid se- The pscB-genes from C. tepidum and from C. vi- quence had been elucidated for P. aestuarii [55]. The brioforme have been expressed in Escherichia coli and sequences are almost identical. At present sequence their FeS-clusters have been reconstituted [45,46,49]. information from fmo is used to establish the phylo- Unfortunately, sequences for the psc-genes from P. genetic relations within the GSB [56]. aestuarii are not known yet. They may provide the clues for the more stable isolate of a PscA/B-RC- 3.3. Pigments core. Di¡erences in the PscB-protein from P. aestuarii and from other GSB are indicated by a lack of The RC-core of GSB contains 16 BChl a and four immunological cross reaction [54] and by di¡erent Chl a (Table 1), eight and two for each PscA-protein migration in SDS^PAGE [38]. [38,42]. Twenty chlorines per a mass of 164 kDa is only about 1/4 of the pigmentation in PsaA/B of PSI 3.2.3. Genes for other subunits with 85 Chl a per 165 kDa. Interestingly, PshA of The gene pscC codes for a cytochrome c with an Heliobacteria with 37 chlorines [43] per 136 kDa [7] is K-band absorbing at 551 nm in reduced form [11,12]. signi¢cantly more densely pigmented than the RC of Its unusual primary structure suggests three trans- GSB (Table 1). membrane helices at the N-terminus and has the Fig. 3 shows the spectra at RT and 6 K for the

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RC-cores of C. tepidum and P. aestuarii (Fig. 1 in di¡erential extraction of BChl a bound to FMO and [38], courtesy H.P. Permentier). In the 2nd derivative to RC with aqueous organic solvent [42]. of the spectra at 6 K eight Qy-transitions can be The four Chl a-molecules in the RC of GSB are discerned. They peak at 778/777, 784/784, 796/797, esteri¢ed to 2,6-phytadienol [58]. The RT-absorption 806/800, 818/809, 825/820, 832/831 and 837/837 nm peak at 670 nm splits into four components at 6 K ^ for C. tepidum/ P. aestuarii, respectively. For C. limi- two closely spaced maxima at 668 and 670 nm and cola the low T-spectrum has been ¢tted with seven two shoulders, at 662 and 675 nm, for C. tepidum as major spectral components absorbing at 797, 804, well as for P. aestuarii [32]. This splitting is probably 810, 816, 824, 832 nad 837 nm [57]. The components caused by electronic interaction of the four closely re£ect the di¡erent environment of the eight BChl spaced chlorophylls, and thus may resemble Cp2 and a-molecules and/or electronic interaction. Cp3 (see Fig. 2), the second and third pair of Chl a 2 Two of the 16 BChl a-molecules are 13 -epimers which constitute A0 in the RC-core of PSI [4]. A0 in (BChl aP), and are considered to form the special pair Heliobacteria may be simpler with only two mole- of the primary donor P840 [58], in accordance with cules of 81-OH-Chl a [43,63]. It should be noted, the re¢ned crystal structure for the PSI-RC which however, that again a Chl a-derivative constitutes reveals that P700 is formed by a heterodimer of the primary acceptor A0, absorbing to the blue one Chl a and one 132-epimer Chl aP [4]. Since also from P789 at 668 nm. in P798 of Heliobacteria two of the BChl g are BChl The RC of GSB contains two carotenoids on a gP [59], 132-epimers seem to be a general feature of P molar basis, one per 10 chlorophylls (Table 1). In in FeS-type RCs. P. aestuarii equal amounts of rhodopsin and chloro- In GSB the RC is associated with the 40 kDa bactene are present, while in C. tepidum four deriv- FMO-protein. Since its crystal structure was the ¢rst atives of chlorobactene and/or Q-carotene occur to be elucidated for a chlorophyll protein [60,61] it which have been separated by HPLC [38]. The core may be the spectroscopically best characterized chlo- of the aerotolerant PSI-RC contains substantially rophyll consortium by now (see [14,15]). It carries more carotenoids, almost one per four chlorophylls. seven BChl a-molecules with three major low-T Qy- A total of 22 are organized in six clusters with two, absorptions at 805, 816 and 825 nm [20] and forms three and six molecules [4]. Five of the 22 are cis- stable trimers. Two of them bind to the RC as de- isomers. They have been detected before to occur in picted in Figs. 1 and 8 ([9,38], see Fig. 4b). Together PSI and other RCs including C. tepidum and are they make up for 58 BChl a-molecules, 42 from two considered especially for photoprotection of RCs FMO-trimers plus 16 from the RC-core (Table 1). [64]. The RC of the anaerobic Heliobacteria contains This accounts for all the BChl a present in mem- even less carotenoid than GSB, only 1^2 molecules of branes of GSB, outside the chlorosomes [38,42]2. neurosporene are present per 37 chlorophylls [63]. The amount is lower than previous estimations because the average extinction coe¤cient of BChl a bound to FMO was found to be signi¢cantly higher 4. Particle structure than of BChl a bound to the RC. For the Qy-absorption peaks at RT the ratio is 1.7, as determined by A high-resolution structure is required to ¢nd out whether the two electron transfer branches are completely symmetrical in the homodimeric core structure (PscA)2PscB of GSB. Unfortunately, neither 2 Griesbeck et al. [42] arrived at 5 FMO-proteins/RC in the 2D- nor 3D-crystals have been obtained to date. Un- membrane, which is less than 2 trimers. They used an extinction til now only low-resolution images of RC-particles coe¤cient of 76 mM31cm31 for BChl a in a mixture of 20%meth- by STEM were obtained [8^10]. Electron micro- anol and 80% acetone [62]. According to the recently determined graphs of the particles for two forms of the RC values of 55 for pure methanol and 69 mM31cm31 for pure acetone by Permentier et al. [38] this extinction coe¤cient more from C. tepidum which band at di¡erent densities likely is about 63 mM31cm31, yielding 6 FMO-proteins, i.e., in sucrose gradients [28] are shown in Fig. 4 again. 2 trimers per RC in the membrane. In the upper band a subcomplex of PscA and cyto-

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chrome c-551 (PscC) is concentrated (Fig. 4a) while in the lower band the functionally intact complex containing the subunits PscA^D plus FMO is col- lected (Fig. 4b). The dominant particle in Fig. 4a has a mass of 248 kDa which accommodates two copies of PscA and 1^2 PscC-proteins [8]. Image analysis using eight projections of the elongated particle yield average dimensions of 13.5U7.7 nm for the top view (probably perpendicular to the membrane plane), and 13.9U. 5.8 nm for side view (probably in the membrane plane). The structure re£ects a dimer with two centers of mass on each side of a cavity, as is expected for a homodimer of PscA. Its dimensions are similar to the core of the PSI-RC with the PsaA/B heterodimer [4]. An asymmetry in the top view probably corresponds to the cytochrome PscC which thus is attached to (PscA)2 from the side in the membrane. Only one PscC is bound to (PscA)2 in the dominant particle of Fig. 4a, but spectroscopic evidence exists for two cytochromes c-551 functioning in the intact RC (see below). The dominant particle in the electron micrograph for the intact complex (Fig. 4b) corresponds to a mass of 454 kDa and shows the elongated structure for the RC again, with dimensions of about 15U8U6 nm, plus one trimer of FMO attached to it. It corresponds to a subunit composition of (FMO)3(PscA)2PscBCD (3U40+2U82+24+23+15 plus 41 for chlorophylls = 387 kDa, leaving 67 kDa for bound detergent, lipid and cofactors). A few particles with two bound FMO-trimers are observed (arrow) which we consider to represent the intact RC-complex in the membrane. In comparison to the RC-core particles (Fig. 4a), a protrusion from the surface which binds the FMO is observed which probably represents the subunit PscB with FeS-centers FA and FB, very much like PsaC in PSI [4]. PscD may contribute to this extra mass. Fig. 4. STEM electron micrographs of RC-particles from Chlo- Free FMO-trimers are also present in both frac- robium tepidum. Panel a shows the particles in the upper band tions of the RC (arrowheads in Fig. 4a,b). They have of the sucrose density gradient, which represents an RC-sub- a mass of 183 kDa [9] which is made up by 3U40 complex with the subunits PscA and PscC [8]; panel b shows kDa for the protein and 3U7 kDa for the chloro- the particles in the lower band containing functionally intact phylls. The high-resolution crystal structure of FMO RC consisting of PscA^D plus FMO-trimers [9]. The bars correspond to 5 nm. Arrowheads point to detached FMO-trimers; is known for P. aestuarii [55,60] and for C. tepidum the arrow in b points to an RC with two bound FMO-trimers. [61]. Fig. 5 compiles the STEM images for a side view of the 454 kDa FMO-RC particle (Fig. 5a) and of

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Fig. 5. Images of the FMO^RC-complex and of the FMO-trimer from Chlorobium tepidum. The image in a represents the side view of the 454 kDa RC particle consisting of PscA^D plus one FMO-trimer (Fig. 5a of [9]), the one in b shows a top view of the FMO- trimer particle according to [8]; the projections in c and d correspond to [61] (courtesy J.P. Allen). The bars represent 5 nm. the top view for the FMO-trimer (Fig. 5b). For com- with the RC and had been crystallized out from a parison of the dimensions the high-resolution struc- RC-preparation (A. Ben-Shem, N. Nelson, unpub- ture in side view (Fig. 5c; space ¢lling) and in top lished results). The results resemble the published view (Fig. 5d; back bone) of the FMO-trimer are structures for the FMO trimer [55,61], but an addi- included in Fig. 5 ([61], courtesy of J.P. Allen). The tional mass which ¢ts an extra chlorophyll is found particle image in Fig. 5a suggests that the FMO- in van der Waals-distance to the loop connecting L- trimer is bound in its side view with the mass center, sheets 7 and 8 in FMO. Such an extra chlorophyll probably re£ecting the superposition of two FMO- could serve the energy transfer from FMO to the proteins, distant to the protrusion from the RC. It RC. further demonstrates that the FMO-trimers are completely peripheral structures, and are not partially embedded in the membrane [61]. From this position 5. Energy transfer within the reaction center and and the placement of the BChls in FMO in Fig. 5c it primary charge separation is obvious that the distance to the chlorophylls in the RC is rather large. New observations on the FMO- Energy transfer in P840-RC is low (23%) from structure may be important in this context. The carotenoids [20] but very e¤cient among the chloro- structure for C. tepidum has been solved once phylls. The distinct peaks in the Qy-region (Fig. 3) more, this time for FMO which had been copuri¢ed allow well for photoselective laser spectroscopy. Re-

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excitation energy from BChl a-837 to P840 (`di¡u- sion limitation', bottom scheme). In the ¢rst case the higher e¤ciency of charge separation by excitation of A0 is achieved by an alternative pathway in which excited A0 attracts an electron from P in the ground state: PA*CPA3. Limitation by energy transfer from BChl a closest to P840 is a good alternative possibility, however, in view of the relative large distance of the corresponding Chl a to P700 in PSI [4], and it is likely that energy transfer from close by A0 to P840 is more e¤cient. Whatever the accelerating e¡ect exerted by A0*, electron transfer from P840, or excitation transfer to P840, primary charge separation in polychromatic light should not be monopha- sic, and should have a faster component following A0* compared to BChl a*. The redox potential of P840 is 240 mV [23,67] and the rates of primary charge separation (10^30 ps), of 3 recombination of the primary radical pair P840 A0 to the triplet state of P840 (20^35 ns), of the triplet decay (90 Ws), and of the forward electron transfer 3 Fig. 6. Two models for energy transfer and charge separation from A0 (600 ps) have been determined early by in the P840-reaction center. The scheme on the top represents laser £ash spectroscopy [68,69], as summarized be- the trap-limited model, the bottom scheme the di¡usion limited fore [11,12] and presented in Fig. 7 again. The cor- model (see [13], Gobets and van Grondelle (this issue), and text responding rates for these early steps of forward elec- for explanation). tron transport are faster in the PSI-RC, 1^3 ps for the primary charge separation and 20^50 ps for re- 3 cent results on FMO-free RC-core preparations, oxidation of A0 by A1 have been put forward (see both at RT [20] and cryogenic T [65], show that [70,71]). However the actual rate of the primary energy transfer to BChl a-837 in the electronically charge separation is blurred by the ¢nal energy trans- coupled system is completed within 2 ps, which is fer from BChl a-837* to P840 (Fig. 6). The measure- followed by transfer to P840 and primary charge ment of P840 is especially complicated in the 840 separation in some 25 ps. The excitation energy is nm region, because of overlapping absorption also distributed within 2 ps if it comes from Chl changes from excited bacteriochlorophyll singlet a-670, which constitutes the primary electron accep- and triplet states [20,41,57,65]. The slowly decaying tor A0 (see above). However, in this case charge sep- absorption decrease in RC-core complexes at 840 nm aration surprisingly is even more e¤cient. The very is attributed to P840, and amounts to somewhat same observation has been made with RC complexes less than 10% of the total absorption at this wave- from Heliobacteria upon selective excitation of length [41]. P840 can be more conveniently mea- the electron acceptor A0, which is OH-Chl a-670 sured at 1150 nm [72,73] or also at 605 nm [74]. [43,63]. Both cases represent well selectable examples However, ultrafast laser spectroscopy at these wave- of a more general phenomenon in energy transfer of lengths has not been carried out yet. Photovoltage RCs, which has also been observed for purple bac- studies arrived at a rate of 50 ps for the primary teria and PSI ([66], see Gobets and van Grondelle, charge separation, and at 600 ps for the subsequent 3 this issue). In Fig. 6 the two explanations for this electron transfer step [75,76], presumably from A0 to observation are depicted. The system is either limited Fx (Fig. 7). by the rate of charge separation (`trap limitation', Photoreduction of A0 measured at 670 nm [68,77] top scheme in Fig. 6), or by the ¢nal transfer of leads to a complex spectral change which may in-

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one red-shifted C9-carbonyl stretching mode in the resonance Raman spectrum upon oxidation of P840 to about half the extent measured for the purple bacterial RC can be interpreted as an even distribution of the unpaired electron [81]. This is in accordance with a symmetric core with two equivalent electron transfer branches, suggested by the homodimeric structure (see above). In FTIR spectra two C9-carbonyl stretching modes are visible, however [78].

6. Secondary electron transfer

Fig. 7 depicts the electron transfer steps in the P840-RC with rates and redox potentials. It is based on previous schemes [11,12], but incorporates new data. In particular the quinoid acceptor A1 is not placed between A0 and FX but into a side path. The corresponding electron transfer steps in the RC of PSI can be found in [70,71], of Heliobacteria in the accompanying article of Neerken and Amesz.

Fig. 7. Redox potentials and rates of electron transfer in the 6.1. The secondary electron acceptor A1 P840-reaction center. The rates are given for isolated RCs at RT, references are given in the accompanying text; Fd stands The electron acceptor A1 in PSI has been identi¢ed for ferredoxin. Corresponding schemes for PSI can be found in as phylloquinone in the mid eighties [82,83] and its the accompanying articles in this issue by Brettel and Leibl or function between A and F has been extensively Itoh et al., for Heliobacteria in the article by Neerken and 0 X Amesz. documented [70,71]. Meanwhile two symmetrically bound phylloquinones are clearly visible in the high-resolution X-ray structure of the PSI-RC [4]. volve more than one Chl a-molecule [20,65], in anal- Originally an equivalent role of MQ-7 as electron ogy to PSI where the core structure contains three acceptor A1 in the RC of GSB was considered [84] pairs of Chls a, one for P700 and two for A0 [4]. In on the basis of a photoaccumulated semiquinone Heliobacteria the spectral change at 670 nm is sim- radical in membranes [85,86], and even in isolated pler and may involve a single pair of OH-Chl a mol- RCs [87]. However, doubts on the obligatory role 3 ecules [43]. The recombination rate of P840 A0 to of MQ as an intermediate have arisen, because prep- the triplet of P840 is 20^35 ns in RC-core prepara- arations completely devoid of MQ [34,38] but capa- tions [68], and 19 ns (Fig. 7) in over-reduced prepa- ble of electron transfer to the terminal FeS-clusters rations containing the subunits PscA^D and the [88] have been obtained. It is critical for this removal FMO-protein [73]. to separate quinone containing detergent micelles The unpaired electron of P840 is rather evenly from the RC on sucrose density gradients or by gel distributed over the two chlorophylls in the special ¢ltration [34,38]. Otherwise MQ-7 and may still be pair compared to other RCs. This is indicated by reduced in photoaccumulation experiments resulting several magnetospectroscopic parameters (see in an A1-like semiquinone radical [87]. However, [11,78,79]), including the narrow line width of the Kusumoto et al. found no evidence in such a prepa- EPR spectrum of P840, the zero ¢eld splitting pa- ration that MQ acts as an intermediate electron ac- rameters of the P840 triplet, ENDOR and special ceptor between A0 and FX, although about 1 MQ/ TRIPLE spectra [80]. Also the observation of only RC was left [73]. No appropriate transient for a

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Fig. 8. Membrane topography of the P840-reaction center and other electron transport components in green sulfur bacteria. SQR and

FCC stand for sul¢de^quinone reductase; £avocytochrome c,bL and bH for low-potential and high-potential heme b; and Qi and Qo for inner and outer quinone binding site, respectively.

semiquinone in the UV was observed, and in £ash tron acceptor between A0 and FX is the observation titration experiments charge recombination with that the recombination rate from FX to P840 is sig- P840 was slow after each of the ¢rst three £ashes, ni¢cantly slower in the P840-RC (17 ms in Fig. 7; see 3 but occurred from A0 in 19 ns after the fourth £ash [89]) in comparison to the PSI-RC (around 1 ms; see (Fig. 7), leaving no room for an additional electron [71]). However, the situation is too complicated to acceptor in the path to the three terminal FeS-centers simply rule out the participation of MQ in forward at RT. Less tight binding of MQ in the A1-site of the electron transport. As discussed by Kusumoto et al. 3 P840-RC indeed is indicated by sequence comparison [73], the reaction time of 600 ps for oxidation of A0 (see Section 3.2.1). However, MQ may still accept in the P840-RC ([69,75,76]; Fig. 7) compared to 20^ electrons, but in a side path (Fig. 7). This would 50 ps for the PSI-RC (see [70,71]) is still too fast for explain why the EPR signal of a semiquinone radical electron transport from A0 to FX, unless the distance can be observed in photoaccumulation experiments between these two components is correspondingly [85^87]. Under physiological conditions the fully re- smaller (20 Aî center to center in the PSI-RC, see duced state may be formed, either by double reduc- [4,71]). Alternatively the electron may leave A1 faster tion or by dismutation of the two semiquinones in to FX than it reaches it from A0 [73]. Moreover, in the A1-sites. MQH2 may exchange with the quinol time resolved EPR an intermediate electron acceptor pool serving ATP formation via cyclic electron trans- is indicated from the change in the polarization pat- port (Fig. 8). In accordance with a lack of an elec- tern of the radical pair after charge separation in the

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P840-RC, also at RT [79]. Furthermore, in whole cently in whole cells [90], as well as by optical spec- cells of GSB at cryogenic T electrons photooxidation troscopy in the blue on isolated RCs [12,89]. Discov- of P840 is stable in the dark without concomitant ery and assignment has been reviewed before [11,12], reduction of the FeS-centers ([90], see below), so thus we will focus on more recent results. They are the electrons must reach an alternative electron ac- also dealt with in the contribution of Vassiliev et al. ceptor beyond A0. to this issue [102]. The situation in Heliobacteria is similar where MQ The EPR spectra of all three clusters are known by is also present in RC preparations [91] and a semi- now, the complete g-tensor of FX has been deter- quinone can be photoaccumulated [86], but no cor- mined only recently [90]. The line widths are some- responding spectral change in the UV is observed what broader in GSB than in PSI [90], but may be [92] and MQ can be extracted without e¡ect of additionally broadened after solubilization of the RC charge separation to the terminal acceptors [93]. from the membranes by detergents [88,104]. The Thus the question as to the existence of an electron spectrum of reconstituted PscB is even broader, too acceptor between A0 and FX in GSB and Heliobac- broad for the distinction of centers FA and FB teria is not settled. [45,46,49]. Rebinding to the RC has been achieved, A few complicating observations on A1 in PSI are but the expected narrowing of the EPR spectrum has also worth mentioning here. The contradiction that not been documented yet. Such sharpening of the irradiation destroys phylloquinone but does not in- EPR spectrum is observed for PsaC upon rebinding hibit the EPR signal of the A1-radical still stands, to PSI (see [102,105]) and the immobilization is ob- questioning the assignment to semiphylloquinone vious in the crystal structure, showing how an arm ([94], see [70]). Should the story of the electron do- from PsaD clamps PsaC (see [4]). According to nating tyrosine Z in PSII, which originally was Nitschke et al. [103] FA and FB in membranes are thought to be a special plastoquinone molecule [95] not reducible by dithionite, and thus are more neg- be repeated for PSI, and the A1-radical turn out to ative than in PSI. They become more reducible in be a reduced amino acid residue? isolated RCs depending on the preparations Also A1 in PSI can be fully reduced to phylloqui- [88,89,104], and not on the strain in use [88]. At nol under certain conditions [96], and is not totally cryogenic T only center FB is partially reduced in immobile. Its exchange with free phylloquinone is membranes and isolated RCs of GSB [88,103], while stimulated in the light, as studied by the e¡ect of in PSI it is center FA (see [102,105]). Thus the relative the deuterated form on the spin polarized EPR spec- values for the redox potentials of FA and FB may be trum [97,98]. Thus also for PSI it is conceivable that turned around, FA being more reducing than FB in under certain conditions phylloquinol is formed and GSB compared to PSI (Fig. 7). As discussed in Sec- may contribute to ATP formation in cyclic electron tion 3.2.2, this is also suggested by the lack of two transport. Interestingly in this context, in Synocho- positive charges next to the cluster binding cysteines, cystis phylloquinone is readily replaced by plastoqui- which in PsaC of PSI stabilize the reduced form of none, if synthesis of phylloquinone is blocked by FA. In apparent contradiction to this is the observa- mutations [99^101]. tion that in a £ash series the escape rate of electrons from isolated P840-RCs to external oxidants is high- 6.2. The FeS-centers est after the second £ash [89]. This suggests that like in PSI, the exposed site of ferredoxin reduction, 3 As discussed above, the electron from A0 arrives which is FB (see [102]), should be more negative. at FX in 600 ps ([69,75,76]; Fig. 7), which is faster The situation is more complicated than in a static than the transfer from A1 in PSI-RCs (15^200 ns; view, though. In whole cells of GSB photoreduction [70,71]). of FeS-centers is totally blocked at cryogenic temper- The three FeS-centers in the P840-RC of GSB ature [90], while in membranes and isolated RCs part (Fig. 7), which resemble FX,FA and FB from PSI of FB is reduced upon illumination [88,103]. This [70,102], have been studied extensively by EPR in may be explained either by a change in spin states membranes [103], in isolated RCs [88,90] and re- at lower temperature, or by a loss of conformational

BBABIO 45074 15-10-01 G. Hauska et al. / Biochimica et Biophysica Acta 1507 (2001) 260^277 273 dynamics below the glass temperature at about 200 is identical to the 32 kDa-tetraheme cytochrome c K [90]. Conformational dynamics may be required isolated and characterized from C. limicola f. thiosul- for e¤cient electron transfer to the terminal accep- fatophilum ([108], see [11,109]). In C. tepidum, how- tors, like as discussed for PSI [70]. A transition to a ever, the photo-oxidizable cytochrome c-553 has faster recombination rate of electrons from earlier been considered to be a smaller, water soluble spe- acceptors to P840, observed at 200 K in optical cies, with a mass of 10 kDa [106]. This discrepancy spectroscopy advocates the second explanation [41]. may re£ect di¡erences among the species of GSB. In In a recent study on serial £ash spectroscopy of spite of the long standing question [11,109], the na- the functionally competent RC from C. tepidum, ture of the physiological electron donor to P840 still which contains the ¢ve subunits PscA^D and FMO remains unclear. (Table 1), the spectral transients of the three FeS- centers in the blue, from 410 to 480 nm, and of 6.4. Ferredoxin and NAD+ reduction P840 in the IR at 1150 nm were studied simultane- ously [89]. The three centers were sequentially re- As stated above, functionally intact P840-RCs duced yielding transients of equal spectra and ampli- from GSB containing the three FeS-clusters FX,FA tudes. The forward rates were faster than the Ws- and FB [30,31] are able to reduce ferredoxin at high resolution of the £ash set up, but the ms-recombina- rate. This has been demonstrated for C. vibrioforme tion rates given in Fig. 7 were determined by a com- with 2Fe2S-ferredoxin from plant chloroplasts or prehensive computational analysis, taking into ac- with 2(4Fe4S)-ferredoxin from Clostridium pasteuria- count all possible electron transfer reactions to num [32], as well as recently for C. tepidum, with each P840. of the four di¡erent 2(4Fe4S)-ferredoxins isolated from that organism [33]. In both cases ferredoxin 6.3. Electron donation by cytochromes c reduction was measured as reduction of NADP using ferredoxin-NADP reductase (FNR) from spi- The rate of electron donation from cytochrome c nach. Interestingly, a gene for FNR is missing from to P840 is 7 Ws in whole cells, and 100 Ws in isolated the genome of C. tepidum (D. Bryant, personal com- RCs (Fig. 7). The subunit PscC of the P840-RC (Ta- munication). Thus, ferredoxin which is reduced by ble 1), a hydrophobic cytochrome c-551 with three the RC cannot be the reductant for NAD in GSB putative membrane spanning helices, speci¢cally oc- as considered so far (see [46]). On the other hand, curs in GSB [47]. It copuri¢es with the P840-RC [27^ FNR from spinach, with a 2Fe2S-ferredoxin as phys- 31] and donates electrons to P840 with a rate of iological reaction partner, and FNR from GSB about 100 Ws in isolated RCs [29,73]. Two copies which interacts with 2(4Fe4S)-ferredoxin may be to- of the cytochrome are bound to intact isolated RCs tally unrelated enzymes (H. Sakurai, personal com- ([31,57], see Fig. 8), which are equivalent and in rap- munication). Surprisingly, the genes for a NADH id redox equilibrium with P840 [29,73]. The midpoint dehydrogenase complex plus a quinol oxidase are potential is 53 mV more negative [73] than the one of present, much like for the respiratory chain in E. P840 (+240 mV in [23,67], +230 mV in [106]). The coli, although GSB are obligate phototrophs. This rate of electron transfer depends on the viscosity of unexpected dehydrogenase may serve three functions. the suspending medium [104], thus is controlled by It may be driven uphill, in reverse by the electro- di¡usion on the aqueous surface. In whole cells pho- chemical proton potential, to reduce NAD with tooxidation of cytochrome c is considerably faster (7 sul¢de, and/or together with quinol oxidase may Ws; U. Feiler, W. Nitschke, unpublished observation, serve as yet another mechanism for protection from see [11]), and in membranes photooxidation is bipha- oxygen. The gene for the FMN-binding subunit of sic, with rates of 7 and 70 Ws [67]. Furthermore, the NADH dehydrogenase is missing, however, and the absorption peak in membranes and cells is found at complex may rather function as proton translocating 553 rather than at 551 nm [23,67,106]. Thus a cyto- ferredoxin-menaquinone oxidoreductase in cyclic chrome di¡erent from PscC has been considered as electron transport around the P840-RC(Oh-Oka, the immediate electron donor in vivo, which possibly personal communication). Together with the mena-

BBABIO 45074 15-10-01 274 G. Hauska et al. / Biochimica et Biophysica Acta 1507 (2001) 260^277 quinol-cyt c oxidoreductase activity of the cyt bc- tween the Rieske FeS-protein and PscC, the cyt c- complex (Fig. 8) it may contribute to the formation 551, has been put forward [112]. of the electrochemical proton potential for ATP syn- A1, which represents RC-bound MQ, is shown in thesis. Fig. 8 to accept electrons in a side path. The MQH2 formed is thought to exchange with the MQ-pool, 6.5. Membrane topography and reconstitution into like plastoquinol does from the QB-site in the RC lipid vesicles of PSII. MQH2 is oxidized by the cyt bc-complex which translocates protons via the so-called Q-cycle Fig. 8 summarizes our view of the topographical mechanism. This mechanism which is a feature of all organization of the P840-RC and of other electron the cyt bc-complexes, in photosynthesis as well as in transport components in the membrane of GSB, as it respiration [113], is indicated by oxidant-induced re- is suggested by current evidence. It remains to be duction of cyt b, which has also been measured in proven by a high-resolution structure, but on the membranes of GSB [110]. basis of magneto-spectroscopic evidence (see above) As depicted in Fig. 8, electrons from sul¢de enter we assume that the redox components P840, A0,A1 the redox system of GSB in two ways (see [114]), and FX are arranged in asymmetric double set within either via £avocytochrome c (FCC; [115]), or via the core of the homodimeric P840-RC. The two cop- sul¢de-quinone reductase (SQR; [116]). ies of the large 82 kDa subunit are held together by Isolated, functionally intact P840-RC complexes of P840, via the conserved histidine in the tenth trans- the subunit composition (FMO)3(PscA)2PscB-D membrane helix and by the 4Fe4S-cluster of FX (see have been shown to translocate protons when incor- Section 3.2.1). In accordance with the STEM particle porated into lipid vesicles [34]. In this case phenazi- analysis (see Section 4) we suggest that the subunit nium methosulfate replaced the proton translocating PscB with the two FeS-centers FA and FB protrudes system of MQH2 plus cyt bc-complex. It probably is from the cytoplasmic side of the core to reduce fer- reduced and takes up a proton at FeS-cluster FB [86], redoxin. It is pictured in an asymmetric way in Fig. and is reoxidized with simultaneous proton liberation 8, and indeed, binding of a ligand to a homodimeric by P840 and/or cyt c-551. protein complex may cause asymmetry [111]. Two trimers of FMO together with two copies of PscD £ank this protrusion, leaving little space for the re- 7. Conclusion and open questions duction of ferredoxins. These are rather small proteins, however [33]. In addition, two copies of the cyt The homodimeric RCs of GSB, together with the c-551-subunit PscC are bound in a symmetric way, one from Heliobacteria ([43], see Neerken and with their hemes exposed to the aqueous phase of the Amesz, this issue) undoubtedly are of complementa- periplasmic membrane surface [107]. ry value to the PSI-RC [4], contributing basic knowl- Although the nature of the physiological electron edge as well as technical opportunities to answer donor is still unclear, for simplicity only PscC with open questions in photosynthesis. heme c-551 is shown in Fig. 8 to donate electrons to Such questions are: P840. For the same reason it is shown to be reduced by the Rieske FeS-protein of the cytochrome bc-com- 1. Why are RCs of oxygen tolerant organisms heter- plex [50,110] during electron transport from mena- odimers? Why do they contain two branches of quinol (MQ). This view is based on the observations electron transfer components in pseudosymmetric that PscC copuri¢es not only with the P840-RC, but arrangement through the membrane, and why also with cyt b [27], and that in a modi¢ed prepara- is one branch preferred? Are the homodimeric tion procedure using dodecyl maltoside the Rieske RCs of GSB and Heliobacteria totally symmetric, FeS-protein remains bound to the P840-RC (A. with two equivalent sets of electron transfer Ben-Shem, N. Nelson, unpublished). However, spec- components? How did the di¡erent types troscopic evidence for an additional, membrane of RCs evolve ([117], see Nitschke et al., this bound cyt c-556 of 17 or 21 kDa, functioning be- issue)?

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