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INTRODUCTION |
The photosynthetic pigment-protein system of purple bacteria
consists of a reaction center
(RC)1 complex and two
light-harvesting complexes, LH1 and LH2. The light energy captured by
LH1 and LH2 is transferred to the RC, where the primary photochemical
reaction takes place. Two types of RC are known in purple bacteria. One
has a tightly bound subunit of a c-type cytochrome at the
periplasmic side that donates electrons to the photo-oxidized RC core
complex. The other does not have the cytochrome subunit and accepts
electrons directly from water-soluble electron carriers such as
cytochrome c2 (1-4). A three-dimensional structure of the RC of Blastochloris (formerly called
Rhodopseudomonas) viridis showed that the
cytochrome subunit has four c-type hemes aligned along the
long axis of this subunit (5). These four hemes are distinguishable in
terms of the peak wavelengths of the
-bands and the redox midpoint
potentials in B. viridis. It has been shown that the hemes
are arranged sequentially with high-low-high-low midpoint potentials
from the special pair of bacteriochlorophylls in the LM core, the core
part of the reaction center complex composed of L and M subunits and
cofactors (6-8). This alternate arrangement of hemes seems to be
conserved through the cytochrome subunits of various purple bacteria,
although its significance in the function has not been clarified
(4).
Amino acid sequences of the cytochrome subunits of various purple
bacteria have been reported, the sequence identities among the subunits
being over 40% (9). All of the sequences consistently conserve four
heme-binding motifs (Cys-Xaa-Xaa-Cys-His) and methionine and histidine
residues as the sixth axial ligands for the heme irons. The four
heme-binding motifs were also conserved in a green filamentous
bacterium, Chloroflexus aurantiacus, which is
phylogenetically distant from purple bacteria (10). Thus, the
cytochrome subunit has often been called a "tetraheme cytochrome."
When hemes are numbered according to the order in the amino acid
sequence from the N terminus, the four hemes of the cytochrome subunit
are arranged in the structure of the B. viridis RC in the
order of heme-3, heme-4, heme-2, and heme-1 from the special pair in
the membrane (11). Recently, we showed direct evidence through
mutagenesis on the cytochrome subunit of Rubrivivax
gelatinosus that electron transfer to the cytochrome subunit from
soluble cytochromes occurred via electrostatic interactions between
negatively charged amino acids surrounding heme-1 and positively
charged amino acids on the soluble cytochromes (12), which is
consistent with a suggestion by Knaff et al. (13). These
charged residues on the cytochrome subunit are well conserved among
many purple bacteria so far examined, suggesting that the most distant
heme-1 works as a direct electron acceptor from the soluble electron carriers (9). This indicates that all four hemes are involved in the
electron transfer from the soluble carrier to the special pair.
The RC complexes of purple bacteria are known to consist, at least, of
L, M, and H subunits. Light-harvesting (LH) complexes are composed of
two membrane spanning polypeptides,
and
subunits, which bind
bacteriochlorophyll and carotenoids. In purple photosynthetic bacteria,
and
polypeptides of the LH1 and the L and M polypeptides of RC
are encoded by pufB, pufA, pufL, and
pufM genes, respectively, which form an operon
called "puf operon." The H polypeptide of RC is encoded
by the puhA gene that is out of the puf operon
(14-16). In species with the bound cytochrome subunit, the
pufC gene coding for the RC-bound c-type
cytochrome is located immediately downstream of pufM in the
operon. Some species have other genes in their puf operons.
Rhodobacter sphaeroides and Rhodobacter
capsulatus have the pufQ gene upstream of
pufB and the pufX gene downstream of
pufM (17-20). In R. gelatinosus, two
unidentified ORFs were detected in the puf operon (21). An
unidentified ORF was also found in Acidiphilium rubrum
puf operon (22).
Purple photosynthetic bacteria are classified into three subclasses,
,
, and
, based on the nucleotide sequences of 16 S rRNA. The
subclass is further divided into four subgroups,
1 to
4 (23).
The
3 subgroup contains three genera of photosynthetic species,
Rhodobacter, Rhodovulum, and
Roseobacter. The genus Rhodobacter contains
freshwater species, whereas the other two genera consist of marine
species (24). The nucleotide sequence data for puf operon
are available in the species of Rhodobacter and
Roseobacter but not in the Rhodovulum species. It
has been reported that the RC of Roseobacter denitrificans
contains the cytochrome subunit (2, 3, 25), although closely related
species such as R. capsulatus and R. sphaeroides
do not contain this subunit (1, 17-19). The pufQ and
pufX genes have been reported only in two Rhodobacter species (17-20). Why species in
3 subclass
show such varied structures of RCs and puf operons has not
been determined yet.
In the present study, we determined the nucleotide sequence of the
puf operon of a purple nonsulfur bacterium, Rhodovulum sulfidophilum. Results indicate that this bacterium has a unique RC-bound cytochrome subunit that has only three heme-binding motifs, one of which, in addition, lacks the amino acid residue functioning as
the sixth ligand for the heme iron.
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EXPERIMENTAL PROCEDURES |
Media and Growth Conditions--
Cells of R. sulfidophilum and R. sphaeroides were grown
photosynthetically at 30 °C in screw-capped bottles filled with a PYS medium, as described by Nagashima et al. (26). For
R. sulfidophilum, the PYS medium was supplemented with 0.35 M sodium chloride. Cells of R. denitrificans
were grown aerobically in the dark at room temperature with a medium,
as described by Shioi (27). Escherichia coli was grown at
37 °C in a Luria-Bertani medium. When required, ampicillin (100 µg/ml; final concentration) was added to the medium.
Screening and Cloning of the puf Genes--
The R. sulfidophilum genomic cosmid library was constructed in our
previous study (28). The pufB and pufA and part
of the pufL genes of R. sulfidophilum were
amplified by polymerase chain reaction according to Hiraishi and Ueda
(24). The sequences of the two primers used for polymerase chain
reaction, 5'-AGAGGGAGCTCGCATGA-3' and 5'-CCGGGTTTGTAGTGGAA-3', were
well conserved in most purple bacteria at the 5' end of the
bchZ gene encoding an enzyme for bacteriochlorophyll
biosynthesis and the 3' end of the L subunit of RC, respectively (26).
The amplified DNA fragment was labeled with digoxigenin-dUTP as
instructed by the manufacturer (Boehringer Mannheim). This fragment was
then used as the probe for colony hybridization to screen the whole
puf operon of R. sulfidophilum (Fig. 1,
probe A). Nine positive clones were selected from the cosmid
library. Inserted DNA fragments in one of the nine cosmid vectors were
digested with EcoRI and screened by Southern blot hybridization using the same probe as described in the cosmid screening. An approximately 10-kb DNA fragment giving a positive signal
was identified and cloned into the plasmid pUC118, being named pUFS101.
This plasmid was used as the template for DNA sequencing, as described
below. DNA manipulation, colony hybridization, Southern blot
hybridization, and plasmid isolation were carried out according to a
manual of molecular cloning (29).
DNA Sequencing--
Sequencing of pUFS101 (see "Screening and
Cloning of the puf Genes") was performed using a Dye Terminator Cycle
Sequencing kit and a 310A DNA Sequencer or a 377A DNA Sequencer
(Applied Biosystems). Oligonucleotides designed to generate overlapping DNA sequences to complete the DNA sequence analysis (primer walking) were ordered from Life Technologies, Inc. The DNA sequences were analyzed using the DNASIS program (Hitachi).
Extraction of RNA and Northern Hybridization--
The total RNA
of R. sulfidophilum was extracted with a RNeasy kit
(QIAGEN). Electrophoresis of the total RNA of R. sulfidophilum was performed in 1.2% agarose gels containing
formaldehyde (40 mM MOPS, 10 mM sodium acetate,
1 mM EDTA, and 2.2 M formaldehyde, pH 7.0).
After electrophoresis, the RNA was transferred to the positively
charged nylon membranes (Boehringer Mannheim). The probe used for
hybridization was the polymerase chain reaction product used for colony
hybridization to screen the whole puf genes of R. sulfidophilum (Fig. 1, probe A) or the 1.2-kb DNA fragment corresponding to the pufC excised from pUFS101 by
ApaI endonuclease (Fig. 2, probe B). The DNA
fragment was labeled with digoxigenin-dUTP as instructed by the
manufacturer (Boehringer Mannheim). RNA Molecular Weight Marker I
(Boehringer Mannheim) was used as a molecular weight standard.
Hybridization was carried out according to a manual of molecular
cloning (29).
Preparation of Membrane Samples--
Cells of R. sphaeroides were harvested by centrifugation and washed once with
distilled water. Cells of R. sulfidophilum and R. denitrificans were harvested by centrifugation and washed once with 100 mM sodium chloride. Washed cells were then
centrifuged and suspended in a 25 mM sodium phosphate
buffer, pH 7.8, supplemented with 100 mM sodium chloride, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Cells were disrupted with sonication and treated with DNaseI.
Membrane fragments were collected by a method of differential centrifugation as a sedimented fraction between 7000 × g for 20 min and 280,000 × g for 20 min. To
obtain membrane preparations free of soluble electron carrier proteins,
the membrane preparations were suspended in a 25 mM sodium
phosphate buffer, pH 7.8, supplemented with 100 mM sodium
chloride and 0.01% Triton X-100 and centrifuged at 280,000 × g for 20 min and then resuspended in the same buffer.
Detection of Heme-containing Proteins in Membrane
Preparations--
SDS-PAGE was carried out according to Laemmli (30).
Heme staining was performed by the method of Thomas et al.
(31).
Flash-induced Absorbance Change Spectrophotometry--
The
absorbance changes due to the photo-oxidation of cytochromes induced by
xenon flash illumination in the membrane preparations free of soluble
electron carrier proteins were recorded with a single beam
spectrophotometer, as described previously (32).
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RESULTS |
Structure of the puf Operon of R. sulfidophilum--
A 10-kb DNA
fragment showing a positive hybridizing signal to a polymerase chain
reaction product containing R. sulfidophilum pufB,
pufA, and pufL genes (Fig.
1, probe A) was cloned into
pUC118 and named pUFS101. The 5.4-kb region in the inserted DNA
fragment was sequenced and analyzed, as shown in Fig.
2. This nucleotide sequence had six ORFs,
each of which had a consensus Shine-Dalgarno sequence, GGAG (one GAGG),
preceding the start codon, ATG. Comparisons with the puf
genes of other photosynthetic bacteria revealed that five of the six
ORFs were pufB, pufA, pufL, pufM, and pufC, which encode the
and
subunits of the LH1 light-harvesting complex, and the L, M, and cytochrome subunits of the RC complex, respectively. The amino acid sequence of the remaining ORF upstream of
pufB showed significant sequence identities to those of
pufQ gene products of R. capsulatus and R. sphaeroides, as shown in Fig. 3. The
ORF was identified as pufQ, because it encodes a protein
with 73 amino acids showing 37 and 38% identities to the
pufQ gene products of R. capsulatus and R. sphaeroides, respectively. The role of this gene product has not
been fully clarified yet but has been suggested to be involved in the
assembly of pigment-protein complexes and bacteriochlorophyll
biosynthesis (33, 34).

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Fig. 1.
Physical and genetic maps of the
puf operons of R. sulfidophilum, R. capsulatus, and R. denitrificans. The
genes are indicated by open boxes. Q,
pufQ; B, pufB; A,
pufA; L, pufL; M,
pufM; C, pufC; X,
pufX. The positions of putative hairpin structures are
indicated by open circles. Recognition sites by various
restriction enzymes in the DNA fragment of R. sulfidophilum
are also shown on the top. Ap, ApaI;
Bm, BamHI; Nt, NotI;
Nr, NruI; Ps, PstI;
Sm, SmaI; Sp, SphI. DNA
probes used in Southern and Northern hybridization experiments are
indicated by thick lines.
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Fig. 2.
The nucleotide sequences of puf
genes and the deduced amino acid sequences of the products of
R. sulfidophilum. The putative ribosome-binding sites are
underlined. The stop codons are indicated by
asterisks. Head-to-head arrows indicate presumed
hairpin structures giving minimum free energies in the respective
regions.
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Fig. 3.
Alignment of the PufQ amino acid sequences of
R. sulfidophilum (Rdv. sul), R. capsulatus (Rba. cap), and R. sphaeroides (Rba. sph). Identical
amino acids are indicated by asterisks.
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The upstream region of the pufQ gene of R. sulfidophilum contained a nucleotide sequence showing significant
sequence identity to bchZ, which encodes an enzyme for
bacteriochlorophyll biosynthesis found in many purple bacteria (21,
35-38). An incompletely sequenced ORF found downstream of
pufC in R. sulfidophilum showed a high identity
to the 5' region of ORF 641 encoding the
-chain of pyruvate dehydrogenase, which is located downstream of the puf operon
in R. capsulatus and R. denitrificans (DDBJ,
EMBL, and GenBankTM accession numbers Z11165 and X83392,
respectively). Two putative hairpin structures were found between
pufC and this ORF (Fig. 2). One of these structures had a
10-base pair stem part with a calculated free enthalpy of
28.1
kcal/mol followed by poly(T) residues. These findings indicate that the
puf operon of R. sulfidophilum is terminated
after pufC. No other ORFs were found in R. sulfidophilum puf operon, leading to the conclusion that the
puf operon in this species is constructed with an order of
pufQ, pufB, pufA, pufL,
pufM, and pufC, the combination of which has
not been reported previously in other purple bacteria.
Two additional putative hairpin loop structures were found between
pufQ and pufB and pufA and
pufL (Fig. 2). The locations of these two hairpin-loop
structures are the same as those in the puf operon of
R. capsulatus (Fig. 1) (17). The hairpin loop between
pufA and pufL has been suggested to work as an
mRNA decay terminator for the 5'-3' exonuclease activity, providing
the necessary mRNA stability for the proper functioning of the
puf operon (39-41).
Lack of a Heme-1-binding Motif in the Cytochrome Subunit--
A
pufC gene coding for the cytochrome subunit of RC was found
in R. sulfidophilum puf operon (Figs. 1 and 2). The deduced amino acid sequence of PufC in R. sulfidophilum consisted of
356 amino acids with the calculated molecular weight of 39,145. The protein had the highest similarity to its homologue of R. denitrificans (40% identity). An amino acid sequence alignment of
the cytochrome subunits of R. sulfidophilum and various
purple bacteria is shown in Fig. 4.
Surprisingly, one of the four conserved heme-binding motifs
(Cys-Xaa-Xaa-Cys-His), corresponding to heme-1 in the tetraheme subunit
of other species, was not detected in R. sulfidophilum, whereas three other possible heme-binding sites were conserved. Only in
this bacterium, methionine residues functioning as the axial ligands to
the first heme and the second heme irons (positions 118 and 157, respectively) (5) were not conserved either.

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Fig. 4.
Alignment of the amino acid sequences of
RC-bound cytochrome subunits (PufC) of purple bacteria. Conserved
amino acids are shaded. Possible heme-binding sites and
axial ligands to the heme irons are boxed and marked with
asterisks, respectively, with the corresponding heme
numberings.
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Gene Coding for LH1 and L and M Subunits of RC in R. sulfidophilum--
The putative
and
subunits of LH1 were
composed of 54 and 48 amino acid residues, respectively, and showed the
highest identities, exceeding 70%, with R. capsulatus. This
subunits contained almost all amino acid residues commonly conserved in
the corresponding polypeptides of other purple bacteria, including the
histidine residues (32nd and 39th of the
and
subunits,
respectively) presumed to bind bacteriochlorophylls (42). The alignment
of the C-terminal amino acid sequences of M subunits from various purple bacteria is shown in Fig. 5. The
additional 17-20 amino acids at the C terminus of the M subunit have
been reported only in bacteria having RC-bound cytochrome subunits and
are thought to contribute to the binding between the cytochrome subunit
and the LM core (4). R. sulfidophilum had the additional
C-terminal sequence of the M subunit as well, although it was a little
shorter than the others.

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Fig. 5.
Alignment of the C-terminal amino acid
sequences of M subunit of reaction center complex. The upper five
organisms have RC-bound cytochrome subunits. The lower three organisms
do not have the cytochrome.
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Analysis of Transcripts--
Because the gene combination of the
puf operon of R. sulfidophilum was revealed to be
different from those of other purple bacteria (Fig. 1) and the gene
coding for the RC-bound cytochrome was unique, as described above, we
performed Northern hybridization experiments to identify the
transcripts of this puf operon. Results are shown in Fig.
6. The total RNA was extracted from
photosynthetically grown cells. Two probes were used for Northern
hybridization (Fig. 1, probes A and B). One of
the two probes corresponding to pufQBA and the part of
pufL (probe A, Fig. 6, lane 1) was
hybridized strongly with a 0.6-kb band and weakly with an approximately
4.5-kb band. Another probe corresponding to pufC
(probe B, Fig. 6, lane 2) was only hybridized
with the approximately 4.5-kb band. In the Rhodobacter
species, the transcript corresponding to pufQ was detected
with the specific probes to the pufQ gene, and its band was
almost the same in size as the pufBA transcript (34). The
0.6-kb band in Fig. 6, therefore, was likely to contain both the
pufQ and the pufBA transcripts. The 4.5-kb
transcript probably includes the whole puf operon,
pufQ, pufB, pufA, pufL,
pufM, and pufC. The 0.6-kb transcripts were more
abundant than the 4.5-kb transcript. This difference may be due to
abundance in pufBA transcripts, a factor thought to adjust
the ratio of LH1 peptides to RC proteins (39, 40).

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Fig. 6.
Northern hybridization analysis of total RNA
from R. sulfidophilum with probes specific for the
puf DNA region. Total RNA was extracted from
R. sulfidophilum grown photosynthetically. The
digoxigenin-dUTP-labeled probe A (lane 1) and probe B
(lane 2) shown in Fig. 1 were used. The lengths of the
standard RNAs are indicated on the left.
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Detection of the RC-bound Cytochrome in Membrane
Preparations--
Membrane proteins from R. sulfidophilum
and from phylogenetically related species, R. denitrificans
and R. sphaeroides, were subjected to SDS-PAGE, and proteins
containing c-type cytochromes were specifically stained
(Fig. 7). The band at 43.1 kDa in
R. sulfidophilum corresponds to the RC-bound cytochrome
(lane 1). A similar band at 48.3 kDa was observed in
R. denitrificans (lane 2) but not in R. sphaeroides (lane 3), consistent with the presence of
the RC-bound cytochrome in the former two species and its absence in
the last species (4, 24). Bands seen at 31.6, 35.7, and 34.5 kDa in
lanes 1, 2, and 3, respectively,
correspond to cytochrome c1 in the cytochrome
bc1 complex.

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Fig. 7.
SDS-PAGE analysis of proteins containing
c-type cytochromes in the membrane. Membrane
preparations containing 20 pmol of RC were treated by acetone to remove
pigments. Proteins were solubilized by boiling for 1 min in the
presence of 2% SDS. Sediments after ultracentrifugation were
discarded. Samples were treated with 2-mercaptoethanol and subjected to
SDS-PAGE followed by heme staining. A linear gradient of 8.9-13.3% of
polyacrylamide was used for the gel. Lane 1, R. sulfidophilum; lane 2, R. denitrificans;
lane 3, R. sphaeroides.
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Photo-oxidation of the RC-bound Cytochrome--
The flash-induced
absorbance changes in the
-band region of c-type
cytochromes were observed in membrane preparations from R. sulfidophilum and the related species (Fig.
8). The absence of soluble cytochromes in
the preparation was ensured by treatment with a salt and a detergent
(see "Experimental Procedures"). Fast photo-oxidation of the
RC-bound cytochrome in R. sulfidophilum and R. denitrificans was observed as absorbance decreased at 554-540 nm,
which is a characteristic feature of the RC-bound cytochrome subunit
(Fig. 8A, traces a and b). The
transient spectra of cytochrome photo-oxidation are clearly seen in
Fig. 8B for R. sulfidophilum (circles)
and R. denitrificans (triangles). On the other
hand, no photo-oxidation of cytochromes was seen in the kinetics and spectrum of R. sphaeroides (Fig. 8, A,
trace c, and B, squares).

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Fig. 8.
Flash-induced absorbance and spectral changes
in membrane preparations. Membrane preparations free of soluble
components were suspended in 25 mM sodium phosphate buffer,
pH 7.8, supplemented with 100 mM NaCl, 0.01% Triton X-100,
10 µM 2,3,5,6-tetramethyl-p-phenylene-diamine,
100 µM ferricyanide, and 200 µM sodium
ascorbate. The data are normalized as to contain 1 µM RC.
A, the kinetic traces showing the flash-induced absorbance
changes at 554-540 nm of R. sulfidophilum (trace
a), R. denitrificans (trace b), and R. sphaeroides (trace c). Each trace is an average of four
identical measurements separated by 30-s intervals. B, the
corresponding transient spectra 250 µs after the actinic flash. Those
from R. sulfidophilum (circles), R. denitrificans (triangles), and R. sphaeroides (squares) are shown.
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DISCUSSION |
In this study, we found new characteristics in the nucleotide
sequence of the puf operon of R. sulfidophilum
and confirmed the presence and function of the product of a unique
cytochrome gene. The R. sulfidophilum puf operon contained,
from upstream, pufQ, pufB, pufA,
pufL, pufM, and pufC genes, the
combination of which has not been reported in other purple bacteria
investigated so far in the sense that both pufQ and
pufC are present in the operon. The amino acid sequence
alignment of the RC-bound cytochrome subunits of R. sulfidophilum and various purple bacteria revealed that the
heme-1-binding site (Fig. 4, position 131-135) is not conserved in
R. sulfidophilum, although three other possible heme-binding sites were observed. Methionine residues at positions 118 and 157, which are thought to be the axial ligands to heme-1 and heme-2 irons,
respectively, were not conserved either (Fig. 4). No alternatives for
the heme-1-binding site and the two ligands were found in the sequence.
Therefore, only two heme-binding sites bear similarity to those of the
tetraheme cytochrome subunits in other purple bacteria in addition to
the unusual heme-2-binding site. Northern hybridization analysis
clearly indicated that the gene coding for this unique cytochrome
subunit is transcribed as a part of puf operon as in other
purple bacteria (Fig. 6). Low stringency genomic Southern hybridization
experiments with a pufC-specific probe showed that
pufC is a single copy gene on the R. sulfidophilum chromosome (data not shown). These observations
suggest that this bacterium has lost the heme-1 from the RC-bound
cytochrome subunit.
The SDS-PAGE analysis in combination with the heme-staining method
(Fig. 7) indicates that pufC in R. sulfidophilum
is indeed translated in vivo and the product is integrated
into the membrane. Furthermore, the RC-bound cytochrome in the membrane
of R. sulfidophilum is photoactive, as shown by the
flash-induced absorbance changes (Fig. 8). The cytochrome subunit is
presumed to accept electrons from water-soluble cytochromes and to
transfer them to the photooxidized RC core complex.
It has been shown that electron transfer reactions from soluble
electron donors to the cytochrome subunit are controlled by charge
interactions (12, 13). The study of site-directed mutagenesis in
R. gelatinosus has shown that negatively charged amino acids (Glu) surrounding the heme-1 (positions 82, 113, and 129 in Fig. 4),
which are well conserved among purple bacteria, have a stimulative effect on the rate of electron transfer, suggesting that the heme-1 of
the RC-bound cytochrome subunit is a direct electron acceptor from
soluble electron donors in purple bacteria (9, 12). The absence of a
heme-1-binding domain in R. sulfidophilum suggests that the
site of interaction with soluble cytochromes is different from that in
usual purple bacteria. This idea is supported by the charge
distribution on the surface of the cytochrome subunit, because the
above-mentioned three glutamate residues that are suggested to be
important for the interaction are not conserved in R. sulfidophilum (Fig. 4). These observations suggest that the
electron transfer between the cytochrome subunit and soluble electron
donors does not occur on the surface around the heme-1 but may occur
around the other hemes of the cytochrome subunit in R. sulfidophilum. An unidentified interaction site on the cytochrome subunit will be revealed by the method of site-directed mutagenesis, as
has been done in R. gelatinosus (12).
The physiological significance of the cytochrome subunit in RC is still
unclear, because some species of purple bacteria lack this subunit.
Until now, the following properties of the subunit have been shown: 1)
the four hemes are arranged sequentially with high-low-high-low
midpoint potentials; 2) the subunit can reduce the photo-oxidized
special pair of bacteriochlorophylls faster than the soluble
cytochromes; and 3) the heme-1 of the cytochrome is a site involved in
the electron flow from soluble electron carriers, indicating that all
four hemes of the subunit are likely to be involved in electron
transfer toward the photo-oxidized special pair of bacteriochlorophylls
(12, 13). The existence of a cytochrome subunit containing only three
hemes, including one unusual heme in R. sulfidophilum,
suggests that all four hemes and the arrangement of high-low-high-low
midpoint potentials are not essential requirements for the functions of
the subunit.
We have previously reported that a R. gelatinosus mutant
lacking the cytochrome subunit is able to grow photosynthetically (43).
Possibly, the main role of the cytochrome subunit is to reduce the
photo-oxidized special pair of bacteriochlorophyll fast enough to avoid
the electron backflow ("back reaction") from the ubiquinone to the
oxidized special pair. Rhodobacter species do not have the
pufC gene coding for the subunit, having a pufX gene at that position instead (Fig. 1). Because the PufX has been suggested to be involved in efficient electron transfer from the RC to
the bc1 complex (19, 20), it may also reduce the
back reaction. Thus, PufX may be a functional alternative of the
cytochrome subunit in photosynthetic electron transport in the
Rhodobacter species. However, some purple photosynthetic
bacteria, at least Rhodospirillum rubrum, have neither
pufC nor pufX genes in the puf operon.
This bacterium may have other systems to reduce the possibilities of
back reaction.
It should be noted that the pufQ gene was found in R. sulfidophilum puf operon that had been detected only in the
Rhodobacter species (Fig. 1). This gene product was
suggested to be an integral membrane protein involved in the assembly
of pigment-protein complexes and bacteriochlorophyll biosynthesis (33,
34). The hydropathy profile of the pufQ gene product of
R. sulfidophilum showed high similarities to those of
R. capsulatus and R. sphaeroides (data not
shown). Characterization of pufQ gene in R. sulfidophilum would be useful for further understanding of its role.
Finally, the study presented here clearly demonstrated that R. sulfidophilum utilizes a unique RC-bound cytochrome subunit that
contains only three heme-binding sites. Our preliminary experiments of
the membrane redox titration showed the unique characteristic of the
subunit in that the redox potentials of these three hemes were
380,
20, and +360 mV, the middle one showing an unusual absorbance
spectrum. Further biochemical and biophysical studies of this
cytochrome subunit will help us to understand not only the
physiological significance of the RC-bound cytochrome subunit but also
the evolution of RC complexes and electron transfer systems in
photosynthetic bacteria.