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

1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês

10.1074/jbc.274.16.10795

1083-351X

Shinji Masuda, Makoto Yoshida, Kenji V. P. Nagashima, Keizo Shimada, Katsumi Matsuura,

Genomics and Phylogenetic Studies

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 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) 1The abbreviations used are: RC, reaction center; ORF, open reading frame; kb, kilobase(s); MOPS, 4-morpholinopropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. 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 c 2 (1Dutton P.L. Prince R.C. Clayton R.K. Sistron W.R. The Photosynthetic Bacteria: Reaction-center-driven Cytochrome Interactions in Electron and Proton Translocation and Energy Coupling. Plenum Press, New York1978: 525-570Google Scholar, 2Matsuura K. Shimada K. Current Research in Photosynthesis. Kluwer Academic Publishing, Dordrecht, The Netherlands1990: 193-196Crossref Google Scholar, 3Matsuura K. J. Plant Res. 1994; 107: 191-200Crossref Scopus (16) Google Scholar, 4Nitschke W. Dracheva S.M. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Reaction Center Associated Cytochromes. Kluwer Academic Publishing, Dordrecht, The Netherlands1995: 775-805Google Scholar). A three-dimensional structure of the RC of Blastochloris (formerly calledRhodopseudomonas) viridis showed that the cytochrome subunit has four c-type hemes aligned along the long axis of this subunit (5Deisenhofer J. Epp O. Miki K. Huber R. Michel H. Nature. 1985; 318: 618-624Crossref PubMed Scopus (2575) Google Scholar). These four hemes are distinguishable in terms of the peak wavelengths of the α-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 (6Dracheva S.M. Drachev L.A. Konstantinov A.A. Semenov A.Y. Skulachev V.P. Arutjunjan A.M. Shuvalov V.A. Zaberezhnaya S.M. Eur. J. Biochem. 1988; 171: 253-264Crossref PubMed Scopus (148) Google Scholar, 7Vermeglio A. Richaud P. Breton J. FEBS Lett. 1989; 243: 259-263Crossref Scopus (47) Google Scholar, 8Alegria G. Dutton P.L. Biochim. Biophys. Acta. 1991; 1057: 239-257Crossref PubMed Scopus (96) Google Scholar). 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 (4Nitschke W. Dracheva S.M. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Reaction Center Associated Cytochromes. Kluwer Academic Publishing, Dordrecht, The Netherlands1995: 775-805Google Scholar). Amino acid sequences of the cytochrome subunits of various purple bacteria have been reported, the sequence identities among the subunits being over 40% (9Nagashima K.V.P. Sakuragi Y. Shimada K. Matsuura K. Photosynth. Res. 1998; 55: 349-355Crossref Google Scholar). 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 (10Dracheva S. Williams J.A. Van Driessche G. Van Beeumen J.J. Blankenship R.E. Biochemistry. 1991; 30: 11451-11458Crossref PubMed Scopus (24) Google Scholar). 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 (11Weyer K.A. Lottspeich F. Gruenberg H. Lang F. Oesterhelt D. Michel H. EMBO J. 1987; 6: 2197-2202Crossref PubMed Google Scholar). 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 (12Osyczka A. Nagashima K.V.P. Sogabe S. Miki K. Yoshida M. Shimada K. Matsuura K. Biochemistry. 1998; 37: 11732-11744Crossref PubMed Scopus (30) Google Scholar), which is consistent with a suggestion by Knaff et al. (13Knaff D.B. Willie A. Long J.E. Kriauciunas A. Durham B. Millett F. Biochemistry. 1991; 30: 1303-1310Crossref PubMed Scopus (55) Google Scholar). 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 (9Nagashima K.V.P. Sakuragi Y. Shimada K. Matsuura K. Photosynth. Res. 1998; 55: 349-355Crossref Google Scholar). 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, α and β subunits, which bind bacteriochlorophyll and carotenoids. In purple photosynthetic bacteria, β and α polypeptides of the LH1 and the L and M polypeptides of RC are encoded by pufB, pufA, pufL, andpufM 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 (14Youvan D.C. Bylina E.J. Alberti M. Begusch H. Hearst J.E. Cell. 1984; 37: 949-957Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 15Michel H. Weyer K.A. Gruenberg H. Dunger I. Oesterhelt D. Lottspeich F. EMBO J. 1986; 5: 1149-1158Crossref PubMed Scopus (212) Google Scholar, 16Bauer C.E. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Regulation of Photosynthesis Gene Expression. Kluwer Academic Publishing, Dordrecht, The Netherlands1995: 1221-1234Google Scholar). In species with the bound cytochrome subunit, thepufC 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 ofpufB and the pufX gene downstream ofpufM (17Bauer C.E. Young D.A. Marrs B.L. J. Biol. Chem. 1988; 263: 4820-4827Abstract Full Text PDF PubMed Google Scholar, 18Davis J. Donohue T.G. Kaplan S. J. Bacteriol. 1988; 170: 320-329Crossref PubMed Google Scholar, 19Farchaus J.W. Gruenberg H. Oesterhelt D. J. Bacteriol. 1990; 172: 977-985Crossref PubMed Google Scholar, 20Lilburn T.G. Haith C.E. Prince R.C. Beatty J.T. Biochim. Biophys. Acta. 1992; 1100: 160-170Crossref PubMed Scopus (90) Google Scholar). In R. gelatinosus, two unidentified ORFs were detected in the puf operon (21Nagashima K.V.P. Matsuura K. Ohyama S. Shimada K. J. Biol. Chem. 1994; 269: 2477-2484Abstract Full Text PDF PubMed Google Scholar). An unidentified ORF was also found in Acidiphilium rubrum puf operon (22Nagashima K.V.P. Matsuura K. Wakao N. Hiraishi A. Shimada K. Plant Cell Physiol. 1997; 38: 1249-1258Crossref PubMed Scopus (30) Google Scholar). Purple photosynthetic bacteria are classified into three subclasses, α, β, and γ, based on the nucleotide sequences of 16 S rRNA. The α subclass is further divided into four subgroups, α1 to α4 (23Woese C.R. Microbiol. Rev. 1987; 51: 221-271Crossref PubMed Google Scholar). The α3 subgroup contains three genera of photosynthetic species,Rhodobacter, Rhodovulum, andRoseobacter. The genus Rhodobacter contains freshwater species, whereas the other two genera consist of marine species (24Hiraishi A. Ueda Y. Int. J. Syst. Bacteriol. 1994; 44: 15-23Crossref Scopus (108) Google Scholar). The nucleotide sequence data for puf operon are available in the species of Rhodobacter andRoseobacter but not in the Rhodovulum species. It has been reported that the RC of Roseobacter denitrificanscontains the cytochrome subunit (2Matsuura K. Shimada K. Current Research in Photosynthesis. Kluwer Academic Publishing, Dordrecht, The Netherlands1990: 193-196Crossref Google Scholar, 3Matsuura K. J. Plant Res. 1994; 107: 191-200Crossref Scopus (16) Google Scholar, 25Kortluke C. Breese K. Gad'on N. Labahn A. Drews G. J. Bacteriol. 1997; 179: 5247-5258Crossref PubMed Google Scholar), although closely related species such as R. capsulatus and R. sphaeroidesdo not contain this subunit (1Dutton P.L. Prince R.C. Clayton R.K. Sistron W.R. The Photosynthetic Bacteria: Reaction-center-driven Cytochrome Interactions in Electron and Proton Translocation and Energy Coupling. Plenum Press, New York1978: 525-570Google Scholar, 17Bauer C.E. Young D.A. Marrs B.L. J. Biol. Chem. 1988; 263: 4820-4827Abstract Full Text PDF PubMed Google Scholar, 18Davis J. Donohue T.G. Kaplan S. J. Bacteriol. 1988; 170: 320-329Crossref PubMed Google Scholar, 19Farchaus J.W. Gruenberg H. Oesterhelt D. J. Bacteriol. 1990; 172: 977-985Crossref PubMed Google Scholar). The pufQ andpufX genes have been reported only in twoRhodobacter species (17Bauer C.E. Young D.A. Marrs B.L. J. Biol. Chem. 1988; 263: 4820-4827Abstract Full Text PDF PubMed Google Scholar, 18Davis J. Donohue T.G. Kaplan S. J. Bacteriol. 1988; 170: 320-329Crossref PubMed Google Scholar, 19Farchaus J.W. Gruenberg H. Oesterhelt D. J. Bacteriol. 1990; 172: 977-985Crossref PubMed Google Scholar, 20Lilburn T.G. Haith C.E. Prince R.C. Beatty J.T. Biochim. Biophys. Acta. 1992; 1100: 160-170Crossref PubMed Scopus (90) Google Scholar). Why species in α3 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 thepuf 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. 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. (26Nagashima K.V.P. Hiraishi A. Shimada K. Matsuura K. J. Mol. Evol. 1997; 45: 131-136Crossref PubMed Scopus (129) Google Scholar). ForR. sulfidophilum, the PYS medium was supplemented with 0.35m sodium chloride. Cells of R. denitrificanswere grown aerobically in the dark at room temperature with a medium, as described by Shioi (27Shioi Y. Plant Cell Physiol. 1986; 27: 567-572Google Scholar). 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. The R. sulfidophilum genomic cosmid library was constructed in our previous study (28.Masuda, S., Matsumoto, Y., Nagashima, K. V. P., Shimada, K., Inoue, K., Bauer, C. E., Matsuura, K., Proceedings of XIth International Congress on Photosynthesis, 1998, Kluwer Academic Publishing, Dordrecht, The Netherlands, in press.Google Scholar). The pufB and pufA and part of the pufL genes of R. sulfidophilum were amplified by polymerase chain reaction according to Hiraishi and Ueda (24Hiraishi A. Ueda Y. Int. J. Syst. Bacteriol. 1994; 44: 15-23Crossref Scopus (108) Google Scholar). 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 thebchZ gene encoding an enzyme for bacteriochlorophyll biosynthesis and the 3′ end of the L subunit of RC, respectively (26Nagashima K.V.P. Hiraishi A. Shimada K. Matsuura K. J. Mol. Evol. 1997; 45: 131-136Crossref PubMed Scopus (129) Google Scholar). 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 wholepuf 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 (29Maniatis T. Fritsch F.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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). 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 byApaI 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 (29Maniatis T. Fritsch F.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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. SDS-PAGE was carried out according to Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207516) Google Scholar). Heme staining was performed by the method of Thomas et al.(31Thomas P.E. Ryan D. Levin W. Anal. Biochem. 1976; 75: 168-176Crossref PubMed Scopus (897) Google Scholar). 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 (32Matsuura K. Fukushima A. Shimada K. Satoh T. FEBS Lett. 1988; 237: 21-25Crossref Scopus (26) Google Scholar). 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 pufgenes of other photosynthetic bacteria revealed that five of the six ORFs were pufB, pufA, pufL, pufM, and pufC, which encode the β and α 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 ofpufB showed significant sequence identities to those ofpufQ 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 thepufQ 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 (33Bauer C.E. Marrs B.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7074-7078Crossref PubMed Scopus (56) Google Scholar, 34Gong L. Lee J.K. Kaplan S. J. Bacteriol. 1994; 173: 2946-2961Crossref Google Scholar). 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 (21Nagashima K.V.P. Matsuura K. Ohyama S. Shimada K. J. Biol. Chem. 1994; 269: 2477-2484Abstract Full Text PDF PubMed Google Scholar,35Taylor D.P. Cohen S.N. Clark W.G. Marrs B.L. J. Bacteriol. 1983; 154: 580-590Crossref PubMed Google Scholar, 36Wiessner C. Dunger I. Michel H. J. Bacteriol. 1990; 172: 2877-2887Crossref PubMed Google Scholar, 37Liebetanz R. Hornberger U. Drews G. Mol. Microbiol. 1991; 5: 1459-1468Crossref PubMed Scopus (31) Google Scholar, 38Nagashima K.V.P. Matsuura K. Shimada K. Photosynth. Res. 1996; 50: 61-70Crossref PubMed Scopus (11) Google Scholar). An incompletely sequenced ORF found downstream ofpufC in R. sulfidophilum showed a high identity to the 5′ region of ORF 641 encoding the β-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 betweenpufC 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 thepuf operon of R. sulfidophilum is terminated after pufC. No other ORFs were found in R. sulfidophilum puf operon, leading to the conclusion that thepuf operon in this species is constructed with an order ofpufQ, 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 betweenpufQ and pufB and pufA andpufL (Fig. 2). The locations of these two hairpin-loop structures are the same as those in the puf operon ofR. capsulatus (Fig. 1) (17Bauer C.E. Young D.A. Marrs B.L. J. Biol. Chem. 1988; 263: 4820-4827Abstract Full Text PDF PubMed Google Scholar). The hairpin loop betweenpufA 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 thepuf operon (39Zhu Y.S. Kiley P.J. Donohue T.J. Kaplan S. J. Biol. Chem. 1986; 261: 10366-10374Abstract Full Text PDF PubMed Google Scholar, 40Klug G. Adams C.W. Belasco J. Doerge B. Cohen S.N. EMBO J. 1987; 6: 3515-3520Crossref PubMed Scopus (70) Google Scholar, 41Heck C. Rothfuchs R. Jager A. Rauhut R. Klug G. Mol. Microbiol. 1996; 20: 1165-1178Crossref PubMed Scopus (41) Google Scholar). ApufC 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) (5Deisenhofer J. Epp O. Miki K. Huber R. Michel H. Nature. 1985; 318: 618-624Crossref PubMed Scopus (2575) Google Scholar) were not conserved either. The putative α and β 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 α and β subunits, respectively) presumed to bind bacteriochlorophylls (42Zuber H. Cogdell R.J. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Structure and Organization of Purple Bacterial Antenna Complexes. Kluwer Academic Publishing, Dordrecht, The Netherlands1995: 315-348Google Scholar). 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 (4Nitschke W. Dracheva S.M. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Reaction Center Associated Cytochromes. Kluwer Academic Publishing, Dordrecht, The Netherlands1995: 775-805Google Scholar). R. sulfidophilum had the additional C-terminal sequence of the M subunit as well, although it was a little shorter than the others. Because the gene combination of thepuf 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 ofpufL (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 Rhodobacterspecies, 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 (34Gong L. Lee J.K. Kaplan S. J. Bacteriol. 1994; 173: 2946-2961Crossref Google Scholar). The 0.6-kb band in Fig. 6, therefore, was likely to contain both thepufQ 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 (39Zhu Y.S. Kiley P.J. Donohue T.J. Kaplan S. J. Biol. Chem. 1986; 261: 10366-10374Abstract Full Text PDF PubMed Google Scholar, 40Klug G. Adams C.W. Belasco J. Doerge B. Cohen S.N. EMBO J. 1987; 6: 3515-3520Crossref PubMed Scopus (70) Google Scholar). Membrane proteins from R. sulfidophilumand from phylogenetically related species, R. denitrificansand R. sphaeroides, were subjected to SDS-PAGE, and proteins containing c-type cytochromes were specifically stained (Fig. 7). The band at 43.1 kDa inR. sulfidophilum corresponds to the RC-bound cytochrome (lane 1). A similar band at 48.3 kDa was observed inR. 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 (4Nitschke W. Dracheva S.M. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Reaction Center Associated Cytochromes. Kluwer Academic Publishing, Dordrecht, The Netherlands1995: 775-805Google Scholar, 24Hiraishi A. Ueda Y. Int. J. Syst. Bacteriol. 1994; 44: 15-23Crossref Scopus (108) Google Scholar). Bands seen at 31.6, 35.7, and 34.5 kDa inlanes 1, 2, and 3, respectively, correspond to cytochrome c 1 in the cytochromebc 1 complex. The flash-induced absorbance changes in the α-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. 8 A, traces a and b). The transient spectra of cytochrome photo-oxidation are clearly seen in Fig. 8 B 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). In this study, we found new characteristics in the nucleotide sequence of the puf operon of R. sulfidophilumand 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 andpufC 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 inR. 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 thatpufC 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. sulfidophilumis 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 (12Osyczka A. Nagashima K.V.P. Sogabe S. Miki K. Yoshida M. Shimada K. Matsuura K. Biochemistry. 1998; 37: 11732-11744Crossref PubMed Scopus (30) Google Scholar, 13Knaff D.B. Willie A. Long J.E. Kriauciunas A. Durham B. Millett F. Biochemistry. 1991; 30: 1303-1310Crossref PubMed Scopus (55) Google Scholar). The study of site-directed mutagenesis inR. 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 (9Nagashima K.V.P. Sakuragi Y. Shimada K. Matsuura K. Photosynth. Res. 1998; 55: 349-355Crossref Google Scholar, 12Osyczka A. Nagashima K.V.P. Sogabe S. Miki K. Yoshida M. Shimada K. Matsuura K. Biochemistry. 1998; 37: 11732-11744Crossref PubMed Scopus (30) Google Scholar). 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 (12Osyczka A. Nagashima K.V.P. Sogabe S. Miki K. Yoshida M. Shimada K. Matsuura K. Biochemistry. 1998; 37: 11732-11744Crossref PubMed Scopus (30) Google Scholar). 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 (12Osyczka A. Nagashima K.V.P. Sogabe S. Miki K. Yoshida M. Shimada K. Matsuura K. Biochemistry. 1998; 37: 11732-11744Crossref PubMed Scopus (30) Google Scholar, 13Knaff D.B. Willie A. Long J.E. Kriauciunas A. Durham B. Millett F. Biochemistry. 1991; 30: 1303-1310Crossref PubMed Scopus (55) Google Scholar). 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 (43Nagashima K.V.P. Shimada K. Matsuura K. FEBS Lett. 1996; 385: 209-213Crossref PubMed Scopus (26) Google Scholar). 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 thepufC gene coding for the subunit, having a pufXgene 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 (19Farchaus J.W. Gruenberg H. Oesterhelt D. J. Bacteriol. 1990; 172: 977-985Crossref PubMed Google Scholar, 20Lilburn T.G. Haith C.E. Prince R.C. Beatty J.T. Biochim. Biophys. Acta. 1992; 1100: 160-170Crossref PubMed Scopus (90) Google Scholar), it may also reduce the back reaction. Thus, PufX may be a functional alternative of the cytochrome subunit in photosynthetic electron transport in theRhodobacter species. However, some purple photosynthetic bacteria, at least Rhodospirillum rubrum, have neitherpufC 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 theRhodobacter 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 (33Bauer C.E. Marrs B.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7074-7078Crossref PubMed Scopus (56) Google Scholar,34Gong L. Lee J.K. Kaplan S. J. Bacteriol. 1994; 173: 2946-2961Crossref Google Scholar). The hydropathy profile of the pufQ gene product ofR. sulfidophilum showed high similarities to those ofR. 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.