The reaction center of green sulfur bacteria 1

Biochimica et Biophysica Acta 1507 (2001) 260^277
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Review

The reaction center of green sulfur bacteria1
G. Hauska
a

aY

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

c

Lehrstuhl fu«r Zellbiologie und P£anzenphysiologie, Fakulta«t fu«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)2 PscB(PscC)2 PscD. 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 ferredoxinNADP‡ 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: (G. Hauska).
1
Dedicated to the memory of Jan Amesz.
0005-2728 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 5 - 2 7 2 8 ( 0 1 ) 0 0 2 0 0 - 6

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261

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
The photosynthetic reaction centers (RCs) of aerotolerant organisms contain a heterodimeric core,
built by two strongly homologous polypeptides.
Each of them contributes ¢ve transmembrane peptide helices to hold a pseudosymmetric double set
of redox components, like in two hands. This holds
for Q- as well as for FeS-type RCs [1], as is amply
documented by the crystal structure, which is available for purple bacteria since 1985 [2], more recently
for PSI [3,4], and just has been published for PSII [5].
Interestingly, only one branch of the double set
seems to be used in physiological electron transfer.
Why is that so?
Clues to this unsolved question may come from
homodimeric RCs, of the green sulfur bacteria
(GSB, i.e., Chlorobiaceae) and the Heliobacteria,
both living strictly anaerobic. They resemble the
PSI-RC, with FeS-clusters as terminal electron acceptors, but the two branches of transmembrane
electron transfer are held by two identical proteins
[6,7]. Unfortunately high structural resolution has
not been achieved yet for the homodimeric RCs,
only a gross structure (2 nm resolution) of the RC
from Chlorobium tepidum by STEM particle analysis

has recently been obtained [8,9], as detailed elsewhere
[10].
In this review we will update the essentials of the
homodimeric reaction center from Chlorobiaceae,

which have been summarized before [11,12]. After a
brief description of the outer antenna system we will
discuss the progress made on isolation procedures,
on analysis of pigments, genes and proteins, as well
as on the spectroscopy of energy and electron transfer within the RC. The particle structure will also be
presented here for comparison to the structure of the
PSI-RC in the accompanying article by Fromme et
al. [4]. For several further aspects of the GSB RC the
reader is referred to other contributions for this issue
(FeS-centers/Vassiliev et al., transient EPR spectroscopy/van der Est, evolution/Nitschke et al.). The homodimeric RC of Heliobacteria will also be adressed,
for details see the accompanying article by Neerken
and Amesz.
2. Energy transfer from the outer antenna
In the photosynthetic units of the di¡erent photosystems the excited states of the pigments migrate
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.

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trapped in the RC. For a general and comprehensive
treatment of these energy transfer processes the reader is referred to van Grondelle et al. 1994 ([13]; for
PSI see Gobets and van Grondelle in this issue).
In the photosynthetic apparatus of GSB light energy is funneled to the RC in the cytoplasmic membrane by a unique peripheral antenna system, the socalled chlorosome [14,15], as depicted in Fig. 1. It
captures light very e¤ciently with some 200 000 bacteriochlorophyll (BChl) c, d or e-molecules per chlorosome (M. Miller, personal communication) and

constitutes the largest outer antenna known, with a
photosynthetic unit of several thousand chlorophyll
molecules per RC (J. Olson, K. Matsuura, personal
communications). With 5000 chlorophylls per RC
there would be some 40 RCs per chlorosome. BChl
c, d and e in the chlorosome are arranged in nonproteinaceous, tubular stacks with an absorption
maximum at 720^750 nm. Energy transfer proceeds
from these rods, via a so-called baseplate of BChl
a-795 (see [16]) to the Fenna^Mathews^Olson BChl
a-protein (FMO), with an absorption peak at 808 nm.
FMO likely transfers the excitation to the RC, with
BChl a-840 as the primary electron donor P840 and
Chl a-670 as the primary electron acceptor A0 (see
below). Heliobacteria do not have chlorosomes and
lack an extended antenna. The only other photosynthetic organisms with chlorosomes are the green nonsulfur bacteria (Chloro£exaceae), which lack the
FMO-protein. These are aerotolerant organisms
and thus contain a heterodimeric RC, which is of
the Q-type [14,15].
The energy transfer in chlorosomes is e¤ciently
quenched under oxidizing conditions [14,15]. Chlorobium quinone which is enriched in chlorosomes has
been envisaged as the responsible redox regulator
[17], more recently the involvement of FeS-proteins
in the chlorosome envelope is discussed [18]. Also
within the BChl a-molecules of the FMO-protein energy transfer is attenuated under oxidizing conditions
[14], involving Tyr-radicals [19]. Both quenching
mechanisms contribute to save the photosynthetic
apparatus from damage by oxygen. In this context
the surprisingly low e¤ciency estimated for energy
transfer ( 6 30%) from FMO to the RC may be relevant, which was found not only for isolated RCs
but also for membranes [14,15,20^22]. Possibly the

interaction between FMO and the RC required for

e¤cient energy transfer already is damaged by isolating the membranes (see below). Indeed, FMO is
rather loosely bound to the RC and is easily lost
during isolation. Excitation transfer measurements
in intact cells may clarify the situation.
3. Composition
3.1. Protein subunits in isolated reaction centers
The isolation of the RC from GSB started with the
mechanical separation of the chlorosomes in the
early seventies [23,24]. Subsequently the dissolution
of the membrane by Triton X-100 and fractionation
was systematically studied, ¢rst by Jan Amesz and
his collaborators working on Prosthecochloris aestuarii [25,26]. Meanwhile protocols using either Triton X-100 and/or alkyl glycosides are available for
several species of GSB, which include Chlorobium
limicola f.sp. thiosulfatophilum [27^29], C. tepidum
[28,30] and C. vibrioforme [31]. These procedures
have been reviewed before [11,12] and do not need
to be described in detail here. Our present knowledge
is summarized in Table 1 together with the following
statements:
1. Functionally intact isolates of the RC from GSB
contain three FeS-centers, show stable charge separation with electron transfer to the terminal FeScenter and accordingly lack fast recombination
rates from preceding electron acceptors (see below). They should be capable to reduce ferredoxin
[32,33] and to catalyze transmembrane charge separation after reconstitution into lipid vesicles [34].
2. Such RC preparations from Chlorobium limicola
f.sp. thiosulfatophilum [29], C. tepidum [28,30]
and C. vibrioforme [31] contain the ¢ve polypeptides, PscA^D plus FMO.
3. The core of the RC is built by two copies of the
large integral membrane protein PscA and one

copy of the peripheral protein PscB. PscA binds
the primary electron donor P840, the primary
electron acceptor A0 and 4Fe4S-cluster FX , PscB
binds the two terminal 4Fe4S-clusters FA and
FB , also called center 1 and 2 (see Vassiliev et
al., this issue). It is related to bacterial ferredoxin
[35,36].

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4. An intact core of the RC, only containing PscA
and -B, but ful¢lling the above criteria has been
isolated only from P. aestuarii [37,38]. It seems
that the interaction of PscA and PscB is more
stable in this organism, which is relatively distant
to the other GSB studied [39]. Unfortunately the
psc-genes have not been sequenced for P. aestuarii.
5. Solubilization by detergent leads to partial removal of the FMO-protein which destabilizes the residual complex, with loss of FeS-centers and of
the subunits PscB and -D. The interaction of the
three proteins is indicated by the observation that
PscD copuri¢es with FMO and PscB from RCs
and by cross-linking experiments (H. Rogl, unpublished). The loss of FeS-centers is re£ected
by fast recombination of P840‡ with earlier electron acceptors [40,41].

3.2. Genes
The two RC-core proteins PscA and PscB are en-

263

coded by the transcription unit pscAB which has
been sequenced for C. limicola f.sp. thiosulfatophilum
[6] and C. tepidum [45,46], while PscC, a peculiar
cytochrome c [47], PscD [28] and FMO [48] are encoded by separate loci. Meanwhile genome sequencing has been completed for C. tepidum (see corresponding website of NCBI), which con¢rms the
sequences of the pscAB-transcription unit and of
the other genes.
3.2.1. The gene pscA
In con¢rmation of earlier evidences for GSB [6]
and Heliobacteria [7] pscA is the only gene in the
genome of C. tepidum with the required signatures
for the large transmembrane core protein of a FeStype RC, in contrast to the two genes psaA and psaB
coding for the PSI-RC subunits (see [4]). Undoubtedly, therefore, the core of the RC in GSB is a homodimer formed by two identical proteins. The gene
pscA from C. tepidum codes for a 82 kDa-protein of
731 amino acids [45,46].The primary structure is 95%
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/
number

Size
(kDa)

Forms/
number

Function

Forms/
number

Denotation/
type/number

Type/tightly
bound

PscA/2

82

X/4Fe4S/1

MQ7/none

24
23

15
40

P840+antenna
A0 +antenna

2

PscB/1
PscC/2
PscD/1
FMO/6

BChl a/16
Chl a/4
^
Heme-c/2
^
BChl a/42

^
^
^
^

A,B/4Fe4S/2
^
^
^

^
^
^
^

Heliobacteria
RC-core

PshA

68

BChl g/35
OH-Chl a/2

1^2

X/4Fe4S/1

MQ5-10/none

Additional proteins

PshB
?

?

P798+antenna
A0 +antenna

^

A,B/4Fe4S/2

^

Photosystem I
RC-core

PsaA/B

83/82

Chl a/85

20 (5 cis)

X/4Fe4S/1

Phyllo-Q/2

PsaC
PsaD^F,I^M,X

8

P700,
A0 +antenna

Chl a/10

Antenna

^
2

A,B/4Fe4S/2

^

Green sulfur bacteria
RC-core

Additional proteins

Additional
proteins: 10

e-Donor
?
Antenna

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
296 in C. tepidum read AIGYINIALGCI which are
HLRHQHRAW-VI in C. limicola. Only 19 histidines
per PscA are present in C. tepidum compared to 21 in
C. limicola (at position 589 C. tepidum carries a H
instead of a Q). The two core proteins of PSI contain
about the double number of histidines, 42 in PsaA
and 39 in PsaB, in accordance with a denser population by chlorophylls. The corresponding PshA-protein from Heliobacteria is only 609 residues long but
contains 25 histidines [7], and binds chlorophylls
with an intermediate density (Table 1).
Sequence alignment of these large subunits in FeStype RCs [6,7,45] suggest that the 11 transmembrane
helices in PsaA/B of PSI are conserved in Chlorobium
PscA, as well as in PshA of Heliobacillus. Overall
identities are low between GSB and PSI as well as
between GSB and Heliobacillus (only about 17% in
each pairwise comparison), but are particularly signi¢cant in the C-terminal portion, which holds the
redox components between the ¢ve putative transmembrane helices VII to XI. This fold is common

to FeS-type as well as Q-type RCs, what has been
elaborated in detail by Schubert et al. [3] and further
substantiated by the recently obtained, re¢ned structures for the RCs of PSI [4] and PSII [5]. An align-

ment of the region binding the redox components
P840, A0 , A1 , and FX is shown in Fig. 2. It starts
with a peptide exposed to the cytoplasmic surface
which contributes two cysteines to bind the 4Fe4Scluster FX between the heterodimer of PsaA/B in PSI
or the homodimers of PscA and PshA. Nine residues
are identical in a stretch of 12, which is the most
highly conserved part of the whole alignment. The
crystal structures clearly show that the primary
charge separation in Q-type and FeS-type RCs involves a consortium of three pairs of chlorine-tetrapyrrols. These are three pairs of Chl a in PSI [4], the
special pair of the primary donor P700 and two more
denoted Cp2 and Cp3, functioning as the primary
acceptor A0 . The special pair of P700 in PSI, P840
in GSB and P798 in Heliobacteria is bound to a
conserved histidine in the middle of the transmembrane helix X (Fig. 2), while the binding residues in
PsaA/B for Cp2 (an asparagine in transmembrane
helix IX which holds the chlorophyll via a water
molecule) as well as for Cp3 (a methionine close to
the cytoplasmic end of transmembrane helix X) are
neither conserved in PscA nor in PshA.
The secondary electron acceptor A1 in the RC of
PSI is phylloquinone, and is bound in van der Waals
distance to the tryptophan of the conserved peptide

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265

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 repetitive 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
Mr of 32 kDa [51]. The C-terminal, more conserved
part of the PscB-proteins resembles PsaC of PSI and
harbors the FeS-binding peptides with four cysteine
in each one. The folding of this part corresponds to
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
positively charged residues KR between the sixth
and the seventh cysteine in PsaC for the neutral residues SA in PscB has been advocated to explain the
drop in redox potential of FA (see Fig. 7) in GSB
compared to PSI [6]. This was subsequently substantiated by targeted mutation of PsaC in PSI from
Chlamydomonas reinhardtii [53].
The pscB-genes from C. tepidum and from C. vibrioforme have been expressed in Escherichia coli and

their FeS-clusters have been reconstituted [45,46,49].
Unfortunately, sequences for the psc-genes from P.
aestuarii are not known yet. They may provide the
clues for the more stable isolate of a PscA/B-RCcore. Di¡erences in the PscB-protein from P. aestuarii and from other GSB are indicated by a lack of
immunological cross reaction [54] and by di¡erent
migration in SDS^PAGE [38].
3.2.3. Genes for other subunits
The gene pscC codes for a cytochrome c with an
K-band absorbing at 551 nm in reduced form [11,12].
Its unusual primary structure suggests three transmembrane helices at the N-terminus and has the

Fig. 3. Absorption spectra of reaction centers from green sulfur
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
shifted upwards for clarity (the ¢gure represents Fig. 1A from
[32]; courtesy H.P. Permentier).

heme c-binding peptide close to the C-terminus
[47]. The gene pscD codes for a 15 kDa-protein
with positive net charge of no obvious relation,
which may be involved in stabilization of PscB
and/or in the interaction with ferredoxin [28,33].
The fmo-gene coding for the intermediary BChl
a-antenna, the 40 kDa FMO-protein has been sequenced for C. tepidum [48] after the amino acid sequence had been elucidated for P. aestuarii [55]. The
sequences are almost identical. At present sequence
information from fmo is used to establish the phylogenetic relations within the GSB [56].
3.3. Pigments
The RC-core of GSB contains 16 BChl a and four
Chl a (Table 1), eight and two for each PscA-protein
[38,42]. Twenty chlorines per a mass of 164 kDa is

only about 1/4 of the pigmentation in PsaA/B of PSI
with 85 Chl a per 165 kDa. Interestingly, PshA of
Heliobacteria with 37 chlorines [43] per 136 kDa [7] is
signi¢cantly more densely pigmented than the RC of
GSB (Table 1).
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
[38], courtesy H.P. Permentier). In the 2nd derivative
of the spectra at 6 K eight Qy -transitions can be
discerned. They peak at 778/777, 784/784, 796/797,
806/800, 818/809, 825/820, 832/831 and 837/837 nm
for C. tepidum/ P. aestuarii, respectively. For C. limicola the low T-spectrum has been ¢tted with seven
major spectral components absorbing at 797, 804,
810, 816, 824, 832 nad 837 nm [57]. The components
re£ect the di¡erent environment of the eight BChl
a-molecules and/or electronic interaction.
Two of the 16 BChl a-molecules are 132 -epimers
(BChl aP), and are considered to form the special pair
of the primary donor P840 [58], in accordance with
the re¢ned crystal structure for the PSI-RC which
reveals that P700 is formed by a heterodimer of
one Chl a and one 132 -epimer Chl aP [4]. Since also

in P798 of Heliobacteria two of the BChl g are BChl
gP [59], 132 -epimers seem to be a general feature of P
in FeS-type RCs.
In GSB the RC is associated with the 40 kDa
FMO-protein. Since its crystal structure was the ¢rst
to be elucidated for a chlorophyll protein [60,61] it
may be the spectroscopically best characterized chlorophyll consortium by now (see [14,15]). It carries
seven BChl a-molecules with three major low-T Qy absorptions at 805, 816 and 825 nm [20] and forms
stable trimers. Two of them bind to the RC as depicted in Figs. 1 and 8 ([9,38], see Fig. 4b). Together
they make up for 58 BChl a-molecules, 42 from two
FMO-trimers plus 16 from the RC-core (Table 1).
This accounts for all the BChl a present in membranes of GSB, outside the chlorosomes [38,42]2 .
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
than of BChl a bound to the RC. For the Qy -absorption peaks at RT the ratio is 1.7, as determined by

2
Griesbeck et al. [42] arrived at 5 FMO-proteins/RC in the
membrane, which is less than 2 trimers. They used an extinction
coe¤cient of 76 mM31 cm31 for BChl a in a mixture of 20%methanol and 80% acetone [62]. According to the recently determined
values of 55 for pure methanol and 69 mM31 cm31 for pure
acetone by Permentier et al. [38] this extinction coe¤cient more
likely is about 63 mM31 cm31 , yielding 6 FMO-proteins, i.e.,
2 trimers per RC in the membrane.

di¡erential extraction of BChl a bound to FMO and
to RC with aqueous organic solvent [42].
The four Chl a-molecules in the RC of GSB are
esteri¢ed to 2,6-phytadienol [58]. The RT-absorption
peak at 670 nm splits into four components at 6 K ^

two closely spaced maxima at 668 and 670 nm and
two shoulders, at 662 and 675 nm, for C. tepidum as
well as for P. aestuarii [32]. This splitting is probably
caused by electronic interaction of the four closely
spaced chlorophylls, and thus may resemble Cp2 and
Cp3 (see Fig. 2), the second and third pair of Chl a
which constitute A0 in the RC-core of PSI [4]. A0 in
Heliobacteria may be simpler with only two molecules of 81 -OH-Chl a [43,63]. It should be noted,
however, that again a Chl a-derivative constitutes
the primary acceptor A0 , absorbing to the blue
from P789 at 668 nm.
The RC of GSB contains two carotenoids on a
molar basis, one per 10 chlorophylls (Table 1). In
P. aestuarii equal amounts of rhodopsin and chlorobactene are present, while in C. tepidum four derivatives of chlorobactene and/or Q-carotene occur
which have been separated by HPLC [38]. The core
of the aerotolerant PSI-RC contains substantially
more carotenoids, almost one per four chlorophylls.
A total of 22 are organized in six clusters with two,
three and six molecules [4]. Five of the 22 are cisisomers. They have been detected before to occur in
PSI and other RCs including C. tepidum and are
considered especially for photoprotection of RCs
[64]. The RC of the anaerobic Heliobacteria contains
even less carotenoid than GSB, only 1^2 molecules of
neurosporene are present per 37 chlorophylls [63].
4. Particle structure
A high-resolution structure is required to ¢nd out
whether the two electron transfer branches are completely symmetrical in the homodimeric core structure (PscA)2 PscB of GSB. Unfortunately, neither
2D- nor 3D-crystals have been obtained to date. Until now only low-resolution images of RC-particles
by STEM were obtained [8^10]. Electron micrographs of the particles for two forms of the RC
from C. tepidum which band at di¡erent densities

in sucrose gradients [28] are shown in Fig. 4 again.
In the upper band a subcomplex of PscA and cyto-

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Fig. 4. STEM electron micrographs of RC-particles from Chlorobium tepidum. Panel a shows the particles in the upper band
of the sucrose density gradient, which represents an RC-subcomplex with the subunits PscA and PscC [8]; panel b shows
the particles in the lower band containing functionally intact
RC consisting of PscA^D plus FMO-trimers [9]. The bars correspond to 5 nm. Arrowheads point to detached FMO-trimers ;
the arrow in b points to an RC with two bound FMO-trimers.

267

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 collected (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)2 PscBCD
(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.
Free FMO-trimers are also present in both fractions of the RC (arrowheads in Fig. 4a,b). They have
a mass of 183 kDa [9] which is made up by 3U40
kDa for the protein and 3U7 kDa for the chlorophylls. The high-resolution crystal structure of FMO
is known for P. aestuarii [55,60] and for C. tepidum
[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 FMOtrimer 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 comparison of the dimensions the high-resolution structure in side view (Fig. 5c; space ¢lling) and in top
view (Fig. 5d; back bone) of the FMO-trimer are
included in Fig. 5 ([61], courtesy of J.P. Allen). The
particle image in Fig. 5a suggests that the FMOtrimer is bound in its side view with the mass center,
probably re£ecting the superposition of two FMOproteins, distant to the protrusion from the RC. It
further demonstrates that the FMO-trimers are completely peripheral structures, and are not partially
embedded in the membrane [61]. From this position
and the placement of the BChls in FMO in Fig. 5c it
is obvious that the distance to the chlorophylls in the
RC is rather large. New observations on the FMOstructure may be important in this context. The
structure for C. tepidum has been solved once
more, this time for FMO which had been copuri¢ed

with the RC and had been crystallized out from a
RC-preparation (A. Ben-Shem, N. Nelson, unpublished results). The results resemble the published
structures for the FMO trimer [55,61], but an additional mass which ¢ts an extra chlorophyll is found
in van der Waals-distance to the loop connecting Lsheets 7 and 8 in FMO. Such an extra chlorophyll
could serve the energy transfer from FMO to the
RC.
5. Energy transfer within the reaction center and
primary charge separation
Energy transfer in P840-RC is low (23%) from
carotenoids [20] but very e¤cient among the chlorophylls. The distinct peaks in the Qy -region (Fig. 3)
allow well for photoselective laser spectroscopy. Re-

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Fig. 6. Two models for energy transfer and charge separation
in the P840-reaction center. The scheme on the top represents
the trap-limited model, the bottom scheme the di¡usion limited
model (see [13], Gobets and van Grondelle (this issue), and text
for explanation).

cent results on FMO-free RC-core preparations,
both at RT [20] and cryogenic T [65], show that
energy transfer to BChl a-837 in the electronically
coupled system is completed within 2 ps, which is
followed by transfer to P840 and primary charge
separation in some 25 ps. The excitation energy is
also distributed within 2 ps if it comes from Chl
a-670, which constitutes the primary electron acceptor A0 (see above). However, in this case charge separation surprisingly is even more e¤cient. The very
same observation has been made with RC complexes
from Heliobacteria upon selective excitation of
the electron acceptor A0 , which is OH-Chl a-670
[43,63]. Both cases represent well selectable examples
of a more general phenomenon in energy transfer of
RCs, which has also been observed for purple bacteria and PSI ([66], see Gobets and van Grondelle,
this issue). In Fig. 6 the two explanations for this
observation are depicted. The system is either limited
by the rate of charge separation (`trap limitation',
top scheme in Fig. 6), or by the ¢nal transfer of

269

excitation energy from BChl a-837 to P840 (`di¡usion 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*CP‡ A3 . 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 monophasic, 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
recombination of the primary radical pair P840‡ A3
0
to the triplet state of P840 (20^35 ns), of the triplet
decay (90 Ws), and of the forward electron transfer
from A3
0 (600 ps) have been determined early by
laser £ash spectroscopy [68,69], as summarized before [11,12] and presented in Fig. 7 again. The corresponding rates for these early steps of forward electron transport are faster in the PSI-RC, 1^3 ps for
the primary charge separation and 20^50 ps for reoxidation of A3
0 by A1 have been put forward (see
[70,71]). However the actual rate of the primary
charge separation is blurred by the ¢nal energy transfer from BChl a-837* to P840 (Fig. 6). The measurement of P840‡ is especially complicated in the 840
nm region, because of overlapping absorption
changes from excited bacteriochlorophyll singlet
and triplet states [20,41,57,65]. The slowly decaying

absorption decrease in RC-core complexes at 840 nm
is attributed to P840‡ , and amounts to somewhat
less than 10% of the total absorption at this wavelength [41]. P840‡ can be more conveniently measured at 1150 nm [72,73] or also at 605 nm [74].
However, ultrafast laser spectroscopy at these wavelengths has not been carried out yet. Photovoltage
studies arrived at a rate of 50 ps for the primary
charge separation, and at 600 ps for the subsequent
electron transfer step [75,76], presumably from A3
0 to
Fx (Fig. 7).
Photoreduction of A0 measured at 670 nm [68,77]
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
P840-reaction center. The rates are given for isolated RCs at
RT, references are given in the accompanying text; Fd stands
for ferredoxin. Corresponding schemes for PSI can be found in
the accompanying articles in this issue by Brettel and Leibl or
Itoh et al., for Heliobacteria in the article by Neerken and
Amesz.

volve more than one Chl a-molecule [20,65], in analogy to PSI where the core structure contains three
pairs of Chls a, one for P700 and two for A0 [4]. In
Heliobacteria the spectral change at 670 nm is simpler and may involve a single pair of OH-Chl a molecules [43]. The recombination rate of P840‡ A3
0 to
the triplet of P840 is 20^35 ns in RC-core preparations [68], and 19 ns (Fig. 7) in over-reduced preparations containing the subunits PscA^D and the
FMO-protein [73].
The unpaired electron of P840‡ is rather evenly
distributed over the two chlorophylls in the special
pair compared to other RCs. This is indicated by
several magnetospectroscopic parameters (see
[11,78,79]), including the narrow line width of the
EPR spectrum of P840‡ , the zero ¢eld splitting parameters of the P840 triplet, ENDOR and special
TRIPLE spectra [80]. Also the observation of only

6.1. The secondary electron acceptor A1
The electron acceptor A1 in PSI has been identi¢ed
as phylloquinone in the mid eighties [82,83] and its

function between A0 and FX has been extensively
documented [70,71]. Meanwhile two symmetrically
bound phylloquinones are clearly visible in the
high-resolution X-ray structure of the PSI-RC [4].
Originally an equivalent role of MQ-7 as electron
acceptor A1 in the RC of GSB was considered [84]
on the basis of a photoaccumulated semiquinone
radical in membranes [85,86], and even in isolated
RCs [87]. However, doubts on the obligatory role
of MQ as an intermediate have arisen, because preparations completely devoid of MQ [34,38] but capable of electron transfer to the terminal FeS-clusters
[88] have been obtained. It is critical for this removal
to separate quinone containing detergent micelles
from the RC on sucrose density gradients or by gel
¢ltration [34,38]. Otherwise MQ-7 and may still be
reduced in photoaccumulation experiments resulting
in an A1-like semiquinone radical [87]. However,
Kusumoto et al. found no evidence in such a preparation that MQ acts as an intermediate electron acceptor between A0 and FX , although about 1 MQ/
RC was left [73]. No appropriate transient for a

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271

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
titration experiments charge recombination with
P840‡ was slow after each of the ¢rst three £ashes,
but occurred from A3
0 in 19 ns after the fourth £ash
(Fig. 7), leaving no room for an additional electron
acceptor in the path to the three terminal FeS-centers
at RT. Less tight binding of MQ in the A1 -site of the
P840-RC indeed is indicated by sequence comparison
(see Section 3.2.1). However, MQ may still accept
electrons, but in a side path (Fig. 7). This would
explain why the EPR signal of a semiquinone radical
can be observed in photoaccumulation experiments
[85^87]. Under physiological conditions the fully reduced state may be formed, either by double reduction or by dismutation of the two semiquinones in
the A1 -sites. MQH2 may exchange with the quinol
pool serving ATP formation via cyclic electron transport (Fig. 8). In accordance with a lack of an elec-

tron acceptor between A0 and FX is the observation
that the recombination rate from FX to P840 is signi¢cantly slower in the P840-RC (17 ms in Fig. 7; see
[89]) in comparison to the PSI-RC (around 1 ms; see
[71]). However, the situation is too complicated to
simply rule out the participation of MQ in forward
electron transport. As discussed by Kusumoto et al.
[73], the reaction time of 600 ps for oxidation of A3
0
in the P840-RC ([69,75,76]; Fig. 7) compared to 20^
50 ps for the PSI-RC (see [70,71]) is still too fast for
electron transport from A0 to FX , unless the distance
between these two components is correspondingly
î center to center in the PSI-RC, see

smaller (20 A
[4,71]). Alternatively the electron may leave A1 faster
to FX than it reaches it from A0 [73]. Moreover, in
time resolved EPR an intermediate electron acceptor
is indicated from the change in the polarization pattern of the radical pair after charge separation in the

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P840-RC, also at RT [79]. Furthermore, in whole
cells of GSB at cryogenic T electrons photooxidation
of P840 is stable in the dark without concomitant
reduction of the FeS-centers ([90], see below), so
the electrons must reach an alternative electron acceptor beyond A0 .
The situation in Heliobacteria is similar where MQ
is also present in RC preparations [91] and a semiquinone can be photoaccumulated [86], but no corresponding spectral change in the UV is observed
[92] and MQ can be extracted without e¡ect of
charge separation to the terminal acceptors [93].
Thus the question as to the existence of an electron
acceptor between A0 and FX in GSB and Heliobacteria is not settled.
A few complicating observations on A1 in PSI are
also worth mentioning here. The contradiction that
irradiation destroys phylloquinone but does not inhibit the EPR signal of the A1 -radical still stands,
questioning the assignment to semiphylloquinone
([94], see [70]). Should the story of the electron donating tyrosine Z in PSII, which originally was
thought to be a special plastoquinone molecule [95]

be repeated for PSI, and the A1 -radical turn out to
be a reduced amino acid residue?
Also A1 in PSI can be fully reduced to phylloquinol under certain conditions [96], and is not totally
immobile. Its exchange with free phylloquinone is
stimulated in the light, as studied by the e¡ect of
the deuterated form on the spin polarized EPR spectrum [97,98]. Thus also for PSI it is conceivable that
under certain conditions phylloquinol is formed and
may contribute to ATP formation in cyclic electron
transport. Interestingly in this context, in Synochocystis phylloquinone is readily replaced by plastoquinone, if synthesis of phylloquinone is blocked by
mutations [99^101].
6.2. The FeS-centers
As discussed above, the electron from A3
0 arrives
at FX in 600 ps ([69,75,76]; Fig. 7), which is faster
than the transfer from A1 in PSI-RCs (15^200 ns;
[70,71]).
The three FeS-centers in the P840-RC of GSB
(Fig. 7), which resemble FX , FA and FB from PSI
[70,102], have been studied extensively by EPR in
membranes [103], in isolated RCs [88,90] and re-

cently in whole cells [90], as well as by optical spectroscopy in the blue on isolated RCs [12,89]. Discovery and assignment has been reviewed before [11,12],
thus we will focus on more recent results. They are
also dealt with in the contribution of Vassiliev et al.
to this issue [102].
The EPR spectra of all three clusters are known by
now, the complete g-tensor of FX has been determined only recently [90]. The line widths are somewhat broader in GSB than in PSI [90], but may be
additionally broadened after solubilization of the RC
from the membranes by detergents [88,104]. The
spectrum of reconstituted PscB is even broader, too

broad for the distinction of centers FA and FB
[45,46,49]. Rebinding to the RC has been achieved,
but the expected narrowing of the EPR spectrum has
not been documented yet. Such sharpening of the
EPR spectrum is observed for PsaC upon rebinding
to PSI (see [102,105]) and the immobilization is obvious in the crystal structure, showing how an arm
from PsaD clamps PsaC (see [4]). According to
Nitschke et al. [103] FA and FB in membranes are
not reducible by dithionite, and thus are more negative than in PSI. They become more reducible in
isolated RCs depending on the preparations
[88,89,104], and not on the strain in use [88]. At
cryogenic T only center FB is partially reduced in
membranes and isolated RCs of GSB [88,103], while
in PSI it is center FA (see [102,105]). Thus the relative
values for the redox potentials of FA and FB may be
turned around, FA being more reducing than FB in
GSB compared to PSI (Fig. 7). As discussed in Section 3.2.2, this is also suggested by the lack of two
positive charges next to the cluster binding cysteines,
which in PsaC of PSI stabilize the reduced form of
FA . In apparent contradiction to this is the observation that in a £ash series the escape rate of electrons
from isolated P840-RCs to external oxidants is highest after the second £ash [89]. This suggests that like
in PSI, the exposed site of ferredoxin reduction,
which is FB (see [102]), should be more negative.
The situation is more complicated than in a static
view, though. In whole cells of GSB photoreduction
of FeS-centers is totally blocked at cryogenic temperature [90], while in membranes and isolated RCs part
of FB is reduced upon illumination [88,103]. This
may be explained either by a change in spin states
at lower temperature, or by a loss of conformational

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dynamics below the glass temperature at about 200
K [90]. Conformational dynamics may be required
for e¤cient electron transfer to the terminal acceptors, like as discussed for PSI [70]. A transition to a
faster recombination rate of electrons from earlier
acceptors to P840‡ , observed at 200 K in optical
spectroscopy advocates the second explanation [41].
In a recent study on serial £ash spectroscopy of
the functionally competent RC from C. tepidum,
which contains the ¢ve subunits PscA^D and FMO
(Table 1), the spectral transients of the three FeScenters in the blue, from 410 to 480 nm, and of
P840 in the IR at 1150 nm were studied simultaneously [89]. The three centers were sequentially reduced yielding transients of equal spectra and amplitudes. The forward rates were faster than the Wsresolution of the £ash set up, but the ms-recombination rates given in Fig. 7 were determined by a comprehensive computational analysis, taking into account all possible electron transfer reactions to
P840‡ .
6.3. Electron donation by cytochromes c
The rate of electron donation from cytochrome c
to P840‡ is 7 Ws in whole cells, and 100 Ws in isolated
RCs (Fig. 7). The subunit PscC of the P840-RC (Table 1), a hydrophobic cytochrome c-551 with three
putative membrane spanning helices, speci¢cally occurs in GSB [47]. It copuri¢es with the P840-RC [27^
31] and donates electrons to P840‡ with a rate of
about 100 Ws in isolated RCs [29,73]. Two copies
of the cytochrome are bound to intact isolated RCs
([31,57], see Fig. 8), which are equivalent and in rapid redox equilibrium with P840 [29,73]. The midpoint
potential is 53 mV more negative [73] than the one of
P840 (+240 mV in [23,67], +230 mV in [106]). The
rate of electron transfer depends on the viscosity of
the suspending medium [104], thus is controlled by

di¡usion on the aqueous surface. In whole cells photooxidation of cytochrome c is considerably faster (7
Ws; U. Feiler, W. Nitschke, unpublished observation,
see [11]), and in membranes photooxidation is biphasic, with rates of 7 and 70 Ws [67]. Furthermore, the
absorption peak in membranes and cells is found at
553 rather than at 551 nm [23,67,106]. Thus a cytochrome di¡erent from PscC has been considered as
the immediate electron donor in vivo, which possibly

273

is identical to the 32 kDa-tetraheme cytochrome c
isolated and characterized from C. limicola f. thiosulfatophilum ([108], see [11,109]). In C. tepidum, however, the photo-oxidizable cytochrome c-553 has
been considered to be a smaller, water soluble species, with a mass of 10 kDa [106]. This discrepancy
may re£ect di¡erences among the species of GSB. In
spite of the long standing question [11,109], the nature of the physiological electron donor to P840‡ still
remains unclear.
6.4. Ferredoxin and NAD+ reduction
As stated above, functionally intact P840-RCs
from GSB containing the three FeS-clusters FX , FA
and FB [30,31] are able to reduce ferredoxin at high
rate. This has been demonstrated for C. vibrioforme
with 2Fe2S-ferredoxin from plant chloroplasts or
with 2(4Fe4S)-ferredoxin from Clostridium pasteurianum [32], as well as recently for C. tepidum, with each
of the four di¡erent 2(4Fe4S)-ferredoxins isolated
from that organism [33]. In both cases ferredoxin
reduction was measured as reduction of NADP‡ using ferredoxin-NADP‡ reductase (FNR) from spinach. Interestingly, a gene for FNR is missing from
the genome of C. tepidum (D. Bryant, personal communication). Thus, ferredoxin which is reduced by
the RC cannot be the reductant for NAD‡ in GSB
as considered so far (see [46]). On the other hand,
FNR from spinach, with a 2Fe2S-ferredoxin as physiological reaction partner, and FNR from GSB
which interacts with 2(4Fe4S)-ferredoxin may be totally unrelated enzymes (H. Sakurai, personal communication). Surprisingly, the genes for a NADH

dehydrogenase complex plus a quinol oxidase are
present, much like for the respiratory chain in E.
coli, although GSB are obligate phototrophs. This
unexpected dehydrogenase may serve three functions.
It may be driven uphill, in reverse by the electrochemical proton potential, to reduce NAD‡ with
sul¢de, and/or together with quinol oxidase may
serve as yet another mechanism for protection from
oxygen. The gene for the FMN-binding subunit of
NADH dehydrogenase is missing, however, and the
complex may rather function as proton translocating
ferredoxin-menaquinone oxidoreductase in cyclic
electron transport around the P840-RC(Oh-Oka,
personal communication). Together with the mena-

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quinol-cyt c oxidoreductase activity of the cyt bccomplex (Fig. 8) it may contribute to the formation
of the electrochemical proton potential for ATP synthesis.
6.5. Membrane topography and reconstitution into
lipid vesicles
Fig. 8 summarizes our view of the topographical
organization of the P840-RC and of other electron
transport components in the membrane of GSB, as it
is suggested by current evidence. It remains to be
proven by a high-resolution structure, but on the

basis of magneto-spectroscopic evidence (see above)
we assume that the redox components P840, A0 , A1
and FX are arranged in asymmetric double set within
the core of the homodimeric P840-RC. The two copies of the large 82 kDa subunit are held together by
P840, via the conserved histidine in the tenth transmembrane helix and by the 4Fe4S-cluster of FX (see
Section 3.2.1). In accordance with the STEM particle
analysis (see Section 4) we suggest that the subunit
PscB with the two FeS-centers FA and FB protrudes
from the cytoplasmic side of the core to reduce ferredoxin. It is pictured in an asymmetric way in Fig.
8, and indeed, binding of a ligand to a homodimeric
protein complex may cause asymmetry [111]. Two
trimers of FMO together with two copies of PscD
£ank this protrusion, leaving little space for the reduction of ferredoxins. These are rather small proteins, however [33]. In addition, two copies of the cyt
c-551-subunit PscC are bound in a symmetric way,
with their hemes exposed to the aqueous phase of the
periplasmic membrane surface [107].
Although the nature of the physiological electron
donor is still unclear, for simplicity only PscC with
heme c-551 is shown in Fig. 8 to donate electrons to
P840‡ . For the same reason it is shown to be reduced
by the Rieske FeS-protein of the cytochrome bc-complex [50,110] during electron transport from menaquinol (MQ). This view is based on the observations
that PscC copuri¢es not only with the P840-RC, but
also with cyt b [27], and that in a modi¢ed preparation procedure using dodecyl maltoside the Rieske
FeS-protein remains bound to the P840-RC (A.
Ben-Shem, N. Nelson, unpublished). However, spectroscopic evidence for an additional, membrane
bound cyt c-556 of 17 or 21 kDa, functioning be-

tween the Rieske FeS-protein and PscC, the cyt c551, has been put forward [112].
A1 , which represents RC-bound MQ, is shown in
Fig. 8 to accept electrons in a side path. The MQH2

formed is thought to exchange with the MQ-pool,
like plastoquinol does from the QB -site in the RC
of PSII. MQH2 is oxidized by the cyt bc-complex
which translocates protons via the so-called Q-cycle
mechanism. This mechanism which is a feature of all
the cyt bc-complexes, in photosynthesis as well as in
respiration [113], is indicated by oxidant-induced reduction of cyt b, which has also been measured in
membranes of GSB [110].
As depicted in Fig. 8, electrons from sul¢de enter
the redox system of GSB in two ways (see [114]),
either via £avocytochrome c (FCC; [115]), or via
sul¢de-quinone reductase (SQR; [116]).
Isolated, functionally intact P840-RC complexes of
the subunit composition (FMO)3 (PscA)2 PscB-D
have been shown to translocate protons when incorporated into lipid vesicles [34]. In this case phenazinium methosulfate replaced the proton translocating
system of MQH2 plus cyt bc-complex. It probably is
reduced and takes up a proton at FeS-cluster FB [86],
and is reoxidized with simultaneous proton liberation
by P840‡ and/or cyt c-551.
7. Conclusion and open questions
The homodimeric RCs of GSB, together with the
one from Heliobacteria ([43], see Neerken and
Amesz, this issue) undoubtedly are of complementary value to the PSI-RC [4], contributing basic knowledge as well as technical opportunities to answer
open questions in photosynthesis.
Such questions are:
1. Why are RCs of oxygen tolerant organisms heterodimers? Why do they contain two branches of
electron transfer components in pseudosymmetric
arrangement through the membrane, and why
is one branch preferred? Are the homodimeric
RCs of GSB and Heliobacteria totally symmetric,

with two equivalent sets of electron transfer
components? How did the di¡erent types
of RCs evolve ([117], see Nitschke et al., this
issue)?

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2. Why is the special pair P in FeS-type RC made of
132 -epimers of BChl a or g, or Chl a [4,58,59]?
3. Why are the acceptors A0 in GSB and Heliobacteria Chl a-derivatives, absorbing at shorter wavelength than the main pigments? This fact o¡ers
valuable possibilities for spectroscopic photoselection, but what is its phylogenetic message?
4. What is the nature of the quinoid electron acceptor A1 in GSB, and in what way is MQ involved?
Is the function of phylloquinone as acceptor A1 in
PSI settled already?
5. How is the comparatively high content of PSI in
carotenoids related to oxygen tolerance? What is
the role of the carotenoids in the RCs of the anaerobic organisms?
6. What is the nature of the cyt c electron donor for
the RCs of GSB and Heliobacteria? What is their
phylogenetic relation ([109], see Nitschke et al.,
this issue)?
7. What are the consequences from the fact that a
gene for FNR is missing, but genes for a NADHdehydrogenase complex and for a quinol oxidase
are present in the genome of C. tepidum (D. Bryant, personal communication)?

[12]
[13]

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