A New Cytochrome Subunit Bound to the Photosynthetic Reaction Center in the Purple Bacterium, Rhodovulum sulfidophilum *

The nucleotide sequence of the pufoperon, which contains the genes encoding the B870 light-harvesting protein and the reaction center complex of the purple photosynthetic bacterium, Rhodovulum sulfidophilum, was determined. The operon, which consisted of six genes, pufQ,pufB, pufA, pufL, pufM, and pufC, is a new variety in photosynthetic bacteria in the sense thatpufQ and pufC coexist. The amino acid sequence of the cytochrome subunit of the reaction center deduced from thepufC sequence revealed that this cytochrome contains only three possible heme-binding motifs; the heme-1-binding motif of the corresponding tetraheme cytochrome subunits was not present. This is the first exception of the “tetraheme” cytochrome family in purple bacteria and green filamentous bacteria. The pufC sequence also revealed that the sixth axial ligands to heme-1 and heme-2 irons were not present in the cytochrome either. This cytochrome was actually detected in membrane preparation as a 43-kDa protein and shown to associate functionally with the photosynthetic reaction center as the immediate electron donor to the photo-oxidized special pair of bacteriochlorophyll. This new cytochrome should be useful for studies on the role of each heme in the cytochrome subunit of the bacterial reaction center and the evolution of proteins in photosynthetic electron transfer systems.

The photosynthetic pigment-protein system of purple bacteria consists of a reaction center (RC) 1 complex and two lightharvesting 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 c 2 (1)(2)(3)(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 distinguish-able 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)(18)(19)(20). In R. gelati-nosus, 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)(18)(19). The pufQ and pufX genes have been reported only in two Rhodobacter species (17)(18)(19)(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.

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 g/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, 5Ј-AGAGGGAGCTCGCATGA-3Ј and 5Ј-CCGGGTTTGTAGT-GGAA-3Ј, were well conserved in most purple bacteria at the 5Ј end of the bchZ gene encoding an enzyme for bacteriochlorophyll biosynthesis and the 3Ј 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 ϫ g for 20 min and 280,000 ϫ 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 ϫ 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).

RESULTS
Structure of the puf Operon of R. sulfidophilum-A 10-kb DNA fragment showing a positive hybridizing signal to a po-lymerase 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).
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)(36)(37)(38). An incompletely sequenced ORF found downstream of pufC in R. sulfidophilum showed a high identity to the 5Ј 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 GenBank TM 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 Ϫ28.1 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 5Ј-3Ј 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.
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.
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).
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 c 1 in the cytochrome bc 1 complex.
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  R. sulfidophilum (Rdv. sul), R. capsulatus (Rba. cap), and R. sphaeroides  (Rba. sph). Identical amino acids are indicated by asterisks.
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). 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 hemestaining 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 photooxidized 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 bc 1 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 biosynthe- sis (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 Ϫ380, Ϫ20, and ϩ360 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.