Chimeric Photosynthetic Reaction Center Complex of Purple Bacteria Composed of the Core Subunits of Rubrivivax gelatinosus and the Cytochrome Subunit of Blastochloris viridis *

A gene coding for the photosynthetic reaction center-bound cytochrome subunit, pufC, ofBlastochloris viridis, which belongs to the α-purple bacteria, was introduced into Rubrivivax gelatinosus, which belongs to the β-purple bacteria. The cytochrome subunit of B. viridis was synthesized in the R. gelatinosus cells, in which the native pufC gene was knocked out, and formed a chimeric reaction center (RC) complex together with other subunits ofR. gelatinosus. The transformant was able to grow photosynthetically. Rapid photo-oxidization of the hemes in the cytochrome subunit was observed in the membrane of the transformant. The soluble electron carrier, cytochrome c 2, isolated from B. viridis was a good electron donor to the chimeric RC. The redox midpoint potentials and the redox difference spectra of four hemes in the cytochrome subunit of the chimeric RC were almost identical with those in the B. viridis RC. The cytochrome subunit of B. viridis seems to retain its structure and function in the R. gelatinosus cell. The chimeric RC and its mutagenesis system should be useful for further studies about the cytochrome subunit of B. viridis.

The L and M subunits of RC are encoded by the pufL and pufM genes, respectively, which form an operon called puf together with the pufB and pufA genes that encode the ␤ and ␣ subunits of the light-harvesting 1 complex. In species containing the RC-bound cytochrome subunit, the pufC gene coding for this subunit is located immediately downstream of the pufM gene. Amino acid sequences derived from the nucleotide sequences of the pufC genes of purple bacteria contain four CXXCH sequence motifs to bind c-type hemes (3), with an exception in the Rhodovulum species (4). The hemes have been numbered as heme-1 to heme-4 according to the order of the appearance of the respective binding motifs in the amino acid sequence (5).
The RC complex purified from a purple nonsulfur bacterium belonging to the ␣-subclass, Blastochloris viridis (formally called Rhodopseudomonas viridis) (6,7), has been crystallized (8 -10). Its crystal structure showed that the four c-type hemes are arranged in a roughly linear manner, almost perpendicularly to the membrane. The spectrophotometric and thermodynamic properties of the four hemes and their arrangement in the structure have been extensively studied (11)(12)(13)(14)(15)(16). The four hemes have high-low-high-low midpoint potentials from the special pair to the periplasmic space. They are distinguishable in terms of the peak positions in their ␣-absorption bands. A gene manipulation system for B. viridis was established previously (17), and some studies had been done using sitedirected mutagenesis manipulated into the LM core complex and into the cytochrome subunit of B. viridis (18). B. viridis cannot be inoculated aerobically and grows with a doubling time of 24 h under microaerobic respiratory conditions (17). The very low growth rate makes it difficult to obtain a sufficient number of the mutant strains of B. viridis. The expression of the B. viridis cytochrome subunit gene in Escherichia coli cells was also challenged. However, it resulted in the accumulation of the inclusion body without heme incorporation (19).
The cytochrome subunit of Rubrivivax gelatinosus, which belongs to the ␤-subclass of purple bacteria, has also been studied and shown to be similar to that of B. viridis in terms of the redox potentials of hemes (20). R. gelatinosus grows well under respiratory conditions (about 2 h of the doubling time) as well as under photosynthetic conditions (about 2.5 h of the doubling time); therefore, it is easy to introduce mutations into the photosynthetic apparatus (21). The photosynthetic growth rate of the R. gelatinosus mutant lacking the cytochrome subunit was about a half of that of the wild type, showing that the cytochrome subunit is not essential but is advantageous for photosynthesis (21). Based on the mutational replacements of charged amino acid residues distributed on the surface of the subunit, Osyczka et al. (22)(23)(24) have shown that the low potential heme-1 located at the most distant position from the special pair is a direct electron acceptor from the soluble electron * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Several water-soluble electron carrier proteins are present in the periplasmic space of R. gelatinosus. Three of these proteins (high potential iron-sulfur protein (HiPIP), high potential cytochrome c 8 , and low potential cytochrome c 8 ), have been shown to work as the electron donors to the RC-bound cytochrome (25)(26)(27). The physiological electron donor to the B. viridis RC is the cytochrome c 2 . The ionic strength dependence studies on the reaction of the cytochrome c 2 with the B. viridis RC have suggested that the heme-1 of the cytochrome subunit is the primary electron acceptor from the cytochrome c 2 (28). However, direct evidence for the precise interaction mechanisms between the soluble electron donor and heme-1 in B. viridis is not yet available.
In this study, the gene for the cytochrome subunit of R. gelatinosus was replaced by that of B. viridis using a common restriction site at a conserved DNA region in both species. The mutagenized cells of R. gelatinosus produced a chimeric RC complex containing the cytochrome subunit derived from B. viridis and the other subunits from the host. The chimeric RC was isolated and characterized.

EXPERIMENTAL PROCEDURES
Bacteria and Growth Media-R. gelatinosus strain IL144RL2, which is a spontaneous mutant of the wild-type strain IL144 and shows greatly depressed production of the light-harvesting 2 complex, was used as a host strain for gene manipulations in this study. The R. gelatinosus strain ⌬C constructed in a previous study was also used in this study, in which the pufC gene was knocked out by insertion of a kanamycin resistance gene (22). R. gelatinosus strains were grown under aerobic-dark or anaerobic-light conditions at 30°C with a PYS medium (21). B. viridis cells were grown under anaerobic-light conditions at 30°C in a malate-basal salt medium containing 0.5% sodium malate, 0.1% yeast extract (Difco), 0.1% ammonium sulfate, 1% basal salt solution (29), 0.1% vitamin solution (29), and 20 mM sodium phosphate (pH 7.0). E. coli strain JM109 was used as a host for plasmids and grown aerobically with a Luria-Bertani medium or an SOB medium at 37°C (30). When needed, ampicillin, kanamycin, or chloramphenicol was added to the E. coli and R. gelatinosus cultures at a final concentration of 50 g/ml.
Plasmid Construction and Transformation-The plasmid pGELPUF containing the 4.6-kb whole puf operon of R. gelatinosus was created by connecting two DNA fragments, pGH37 and pGI7, previously cloned from the genomic DNA library of R. gelatinosus strain IL144 (31). The 3.7-kb region flanked by NotI restriction sites within the puf operon of R. gelatinosus was replaced by an ampicillin resistance gene. This plasmid was named p⌬PUF2 (Fig. 1A) and used for construction of the R. gelatinosus mutant lacking the puf operon. A 2.5-kb DNA fragment flanked by BamHI restriction sites and containing the 3Ј-region of pufM and the entire pufC of B. viridis was cloned from the genomic library of B. viridis DNA using a pUC119 plasmid as a cloning vector. This plasmid was named p9VP3 (Fig. 1B). The nucleotide sequence of pufC in the p9VP3 plasmid was confirmed to be identical to that previously reported for the B. viridis pufC (5). Both the genes for the M subunits of R. gelatinosus and B. viridis have a common SalI restriction site at the conserved 3Ј-regions. The region downstream of this SalI site in the insert DNA of the plasmid pGELPUF was removed and replaced by the DNA fragment containing the region downstream of the corresponding SalI site of the p9VP3 plasmid, in which the entire pufC of B. viridis was contained (Fig. 1B). The DNA fragment containing this "chimeric" puf operon was cloned into a pHSG298 plasmid. The p2GPVC-KF plasmid was thus obtained. A frameshift did not occur in this "chimeric" puf operon. The presumed pufM product derived from this plasmid was expected to have the N-terminal 292 amino acids of the R. gelatinosus M subunit and the C-terminal 34 amino acids from the B. viridis M subunit. DNA manipulation and hybridization were carried out according to a manual on molecular cloning (30) or according to instructions supplied by the enzyme manufacturers. Plasmid DNAs were introduced into R. gelatinosus cells by means of electroporation, according to the methods previously described (21).
Preparation of Membranes and Soluble Electron Donors-Cells in the late logarithmic growth phase were harvested by centrifugation at 7,200 ϫ g for 20 min, washed, and suspended in 10 mM MOPS-NaOH (pH 7.0). The cells were disrupted by passage through a French pressure cell at 1100 -1200 kg/cm 2 in the presence of a few grains of DNase I. The disrupted cell suspension was centrifuged at 28,000 ϫ g for 15 min to remove the debris and ultracentrifuged at 340,000 ϫ g for 20 min. The sediment was used as the membrane preparation. The cytochrome c 2 of B. viridis and HiPIP of R. gelatinosus were isolated from the remaining supernatant according to the methods previously reported (26). The protein content in the membrane was determined with a Protein Assay Kit (Bio-Rad), using bovine serum albumin (fraction V) as a standard protein. Reduced-minus-oxidized difference spectra were measured with a UV-3000 spectrophotometer (Shimadzu). Two millimolar (final concentration) of potassium ferricyanide or a few grains of sodium dithionite were added for the oxidation or the reduction of the membrane preparations.
SDS-PAGE-SDS-PAGE was carried out according to Laemmli (32). The proteins with a concentration of 5 mg/ml were denatured in 1% SDS by boiling for 1 min. Ten micrograms of protein was applied to each lane of the gel. After electrophoresis, the gel was treated with Coomassie Brilliant Blue using a Quick-CBB Kit (Wako, Japan) for staining the proteins or treated with TMBZ for detection of the hemes according to the method of Thomas et al. (33).
Isolation of the RC Complex of R. gelatinosus-The membranes prepared were suspended in a buffer containing 10 mM MOPS-NaOH (pH 7.0) to give an absorbance of 40 at 875 nm. The suspension was mixed with an equal volume of precooled 1.6% (v/v) ␤-D-octylthioglucoside and incubated on ice for 40 min with stirring. Subsequently, the suspension was applied on the top of a sucrose density gradient (from top to bottom, 10 -40%, w/v) containing 10 mM MOPS-NaOH (pH 7.0) and 0.05% Triton X-100 and ultracentrifuged at 146,000 ϫ g for 20 h. A brownish red band that formed at a sucrose density of about 25% was recovered as an RC fraction and concentrated with Centriflo CF25 membrane cones (Amicon). The RC fraction was diluted with a buffer containing 100 mM KCl, 20 mM MOPS-KOH (pH 7.0), and 0.05% Triton X-100.
Kinetic Measurements-Xenon flash-induced absorption changes were measured with a single beam spectrophotometer designed and assembled in our laboratory (34). The membranes were suspended in a 20 mM MOPS-KOH buffer (pH 7.0) containing 100 mM KCl, 1 mM sodium ascorbate, and 10 M DAD. The concentrations of the samples were adjusted to give an absorbance of 1 at 860 nm for the R. gelatinosus strain ⌬C, at 875 nm for other R. gelatinosus strains, and at 1012 nm for B. viridis. One micromolar valinomycin was added to the membrane preparation of the R. gelatinosus strain ⌬C to reduce the effect of the band shift of carotenoids responding to the membrane potential. The kinetics of an electron transfer from the soluble electron donor to the RC was measured in a buffer containing 5 mM MOPS-NaOH (pH 7.0). The R. gelatinosus HiPIP and the B. viridis cytochrome c 2 were added at the final concentrations of 1 M. One millimolar sodium ascorbate, 10 M DAD, and 0.03% Triton X-100 were also added.
Redox Titration-Redox titration of hemes in the RC-bound cytochrome c was carried out as described previously (35) using a double beam spectrophotometer UV-3000 (Shimadzu). The redox potential was monitored with an Ag/AgCl electrode (TOA) connected to an HM-40S voltmeter (TOA). The concentration of the sample was adjusted to give an absorbance of 2 at 875 nm for R. gelatinosus and at 1012 nm for B. viridis. The buffer for the measurements contained 100 mM KCl, 20 mM MOPS-KOH (pH 7.0), and 0.05% Triton X-100. The redox mediators used are as follows: 100 M potassium ferrocyanide, 10 M DAD, 100 M Fe-EDTA, 10 M phenazine methosulfate, 10 M vitamin K 3 , 10 M 2-hydroxy-1,4-naphthoquinone, and 3 M pyocyanine. The oxygen was eliminated by a continuous stream of nitrogen gas. At the beginning of the measurements, the suspension was reduced by the addition of a solution of sodium ascorbate or sodium dithionite. Subsequently, the redox potential was raised stepwise by additions of a solution of potassium ferricyanide.

Expression of B. viridis pufC Gene in Cells of R. gelatinosus-
The plasmid p⌬PUF2, which contains a kanamycin resistance gene and the R. gelatinosus puf operon disrupted by the insertion of an ampicillin resistance gene (Fig. 1A), was introduced into the cells of the R. gelatinosus strain IL144RL2. A strain showing resistance to ampicillin but sensitivity to kanamycin was picked up after successive cultivation under the pressure of ampicillin and named DP2. An absorption spectrum of the membrane prepared from the strain DP2 cells showed no accumulation of the RC-light-harvesting 1 complex (Fig. 2).
Southern hybridization, PCR, and DNA sequencing experiments for the genomic DNA isolated from the strain DP2 showed that the 3.7-kb NotI-NotI DNA region containing puf-BALMC was replaced by the ampicillin resistance gene (data not shown), probably due to a double cross-over recombination between the genomic DNA and the p⌬PUF2 plasmid. The strain DP2 could not grow photosynthetically.
The plasmid p2GPVC-KF, which contains the majority of the puf genes of R. gelatinosus and the entire pufC of B. viridis (Fig. 1), was introduced into the R. gelatinosus strain DP2 cells.
A new strain showing resistance to kanamycin was obtained and named VC-F. The VC-F cells were able to grow under anaerobic light conditions, although the growth rate was about 3 times slower than that of the strain IL144RL2. An absorption spectrum of the strain VC-F membrane was nearly identical with that of the strain IL144RL2 membrane (Fig. 2), suggesting that the strain VC-F recovered the ability to synthesize the RC-light-harvesting 1 complex without significant alterations in the environments of the photosynthetic pigments. The presence of puf genes in the strain VC-F cells was confirmed by Southern hybridization experiments. The p2GPVC-KF plasmid was detected in the cell lysate of the strain VC-F and seemed to be reproducible in the VC-F cells.
The synthesis of the RC-bound cytochrome subunit derived from the B. viridis pufC gene in the cells of the R. gelatinosus strain VC-F was examined by SDS-PAGE of the membranes, as shown in Fig. 3. The band patterns stained with Coomassie Brilliant Blue were almost identical among the membrane samples of the three strains of R. gelatinosus. Minor differences between the patterns lower than 14 kDa for the strain ⌬C and for two other strains, IL144RL2 and VC-F, may reflect the difference in the contents of the light-harvesting 2 complexes. In the gel stained for hemes, a peptide with a molecular mass of 43 kDa was detected in the membrane of the strain IL144RL2, which was assigned to the cytochrome subunit (36). No heme-stained peptides were observed in the membrane of the strain ⌬C, which lacks the cytochrome subunit (22). In the membrane of the strain VC-F, a heme-stained peptide was detected at the 39-kDa molecular mass, which coincided with that of the RC-bound cytochrome subunit of B. viridis. This result suggested that the RC-bound cytochrome subunit derived from B. viridis was expressed in the R. gelatinosus strain VC-F.
Redox and Spectroscopic Characteristics of Hemes in the Cytochrome Subunit- Fig. 4 shows the reduced-minus-oxidized difference spectrum in the ␣-absorption band region of the cytochromes c in the membrane of the strain VC-F, together with those of the strains IL144RL2 and ⌬C of R. gelatinosus and B. viridis. The difference spectrum of the strain IL144RL2 membrane, which has two low potential hemes that peaked at 551 nm and two high potential hemes that peaked at 555 nm in the cytochrome subunit, showed an absorption maximum at 552 nm, which was consistent with the results obtained from the wild type of R. gelatinosus shown in a previous study (36). In the strain ⌬C, the absorption bands derived from cytochromes were very small, as expected (21). The broad and small bands detected in the strain ⌬C membrane were probably due to the cytochrome bc 1 complex. The membrane of the strain VC-F, on the other hand, showed a different spectrum with an absorption maximum at 553 nm with a shoulder around 559 nm, which was similar to that obtained for the membrane of B. viridis.
Hemes contained in the cytochrome subunit of the R. gelatinosus strain VC-F were further characterized by redox titration (Fig. 5). Four redox components were detected when the peak heights of the ␣-absorption bands of the cytochromes c in the membrane preparation were plotted against the ambient potentials. When the data were fitted with Nernst equations, the redox midpoint potentials (E m values) of these components were estimated to be Ϫ60, ϩ32, ϩ320, and ϩ419 mV in the membrane and Ϫ60, ϩ32, ϩ310, and ϩ398 mV in the solubilized RC complex (Fig. 5). These values were consistent with those reported for the four hemes in the B. viridis cytochrome subunit (Table I). Fig. 6 shows the reduced-minus-oxidized difference spectra of the four components in the strain VC-F membrane. Four c-type hemes with different absorption maxima in the ␣-band regions were resolved. The heme with the highest E m value showed a narrow ␣-band peaked at 559 nm with a shoulder at near 552 nm. The heme with the second highest E m had a somewhat broad ␣-band peaked at 556 nm. The two low potential hemes showed narrow ␣-bands with peaks at 552 and 553 nm. These spectral and redox characteristics in the strain VC-F were almost identical with those reported for B. viridis (11). This indicated that the environments surrounding the hemes in the cytochrome subunit in the strain VC-F were almost the same as those in the original RC complex of B. viridis.
The redox and spectroscopic properties of the four hemes in the membrane preparations of the R. gelatinosus strain IL144RL2 are summarized in Table I. The values of the redox midpoint potentials in R. gelatinosus IL144RL2 were ϩ64 mV for the two low potential hemes peaking at 551 nm and ϩ281 and ϩ348 mV for the two high potential hemes peaking at 555 nm. These E m values were consistent with those for the R. gelatinosus strain IL144 (36) and for the R. gelatinosus strain 52 (20). Thus, the redox midpoint potentials of the four hemes determined in the strain VC-F and their spectra in the ␣-band region were clearly different from those in R. gelatinosus IL144RL2.
Flash-induced Redox Change of Hemes- Fig. 7 shows flashinduced absorption changes in the ␣-band region of the cytochromes c in the membranes prepared from the cells of R. gelatinosus strains and B. viridis. For these measurements, the two high potential hemes in the cytochrome subunit were reduced prior to the flash activation by the addition of 1 mM sodium ascorbate. In the membrane of the strain VC-F, rapid oxidation of the cytochrome was observed, as in the membranes of R. gelatinosus IL144RL2 and B. viridis. The flash-induced spectra derived from this rapid oxidation showed a peak at 556 nm in the membranes of the strain VC-F and B. viridis, indicating that the second high potential heme, c556 (heme-2), was oxidized by the flash. In the flash-induced absorption change of the cytochromes in the membrane of the strains IL144RL2, the major oxidized component showed a peak at 553 nm. This suggested that the low potential hemes peaked at 551 nm in the IL144RL2 cytochrome subunit were partially reduced prior to the flash activation possibly due to their relatively high redox potentials. No remarkable peaks were observed in the flash-induced spectrum in the membrane preparation of the strain ⌬C due to the lack of the cytochrome subunit. Fig. 8 shows the kinetics of the electron transfer from the soluble electron carrier proteins to the cytochrome subunit in the prepared membrane. When the R. gelatinosus HiPIP was added, re-reduction of the cytochrome was observed with a second order rate constant of 1.82 ϫ 10 Ϫ7 in the IL144RL2 membrane but not in the membranes of the strain VC-F and B. viridis. This suggested that HiPIP, which is the physiologically main electron donor to the oxidized cytochrome subunit in the R. gelatinosus wild-type cells, was not a good electron donor to the RC in the strain VC-F cells. When the B. viridis cytochrome c 2 was added to the membrane of the strain VC-F, re-reduction of the RC-bound cytochrome and concomitant oxidation of the cytochrome c 2 were observed with a second order rate constant of 7.75 ϫ 10 Ϫ6 , which was comparable with the value obtained from the measurement for the B. viridis membrane, 5.05 ϫ 10 Ϫ6 . The membrane of the strain IL144RL2 did not show an apparent electron transfer reaction with the B. viridis cytochrome c 2 .

DISCUSSION
In this study, the RC-bound cytochrome subunit of B. viridis was shown to be synthesized in the distantly related species of purple bacteria, R. gelatinosus. Measurements of spectra and flash-induced redox changes of the cytochromes c showed that the B. viridis cytochrome subunit was not only synthesized in R. gelatinosus but also functioned as the rapid electron donor to the photooxidized special pair. This means that the B. viridis cytochrome subunit can form a functional "chimeric" RC complex together with the R. gelatinosus LM core polypeptides.  This was somewhat surprising, since the amino acid sequence identities of the L, M, and cytochrome subunits between B. viridis and R. gelatinosus are only 66, 61, and 45%, respectively. It has been reported in the fine structure of the B. viridis RC that 30 amino acids in the cytochrome subunit are bound to the LM core via hydrogen bonds and salt bridges (10). These amino acids are indicated with the primary structure shown in Fig. 9. In the cytochrome subunit of R. gelatinosus, only 8 of these 30 amino acids are conserved, based on the comparison of the amino acid sequences. Some of the amino acid residues located near the binding residues are absent in the R. gelatinosus cytochrome subunit (Fig. 9). These observations suggest that the local protein structure contributing to the binding should be different between the cytochrome subunits of B. viridis and R. gelatinosus. It is known that the cytochrome subunit of R. gelatinosus is easily dissociated from the LM core by a moderate detergent treatment (36), although that of B. viridis is tightly bound to the core, possibly reflecting the structural difference. The cytochrome subunit of B. viridis seemed to be tightly bound to the R. gelatinosus LM core in the strain VC-F, since the RC complex with the cytochrome subunit was successfully isolated from the membranes of the strain VC-F under conditions in which the subunit was dissociated from the core complex in the R. gelatinosus wild type.
On the other hand, most of the amino acid residues connecting the cytochrome subunit, based on the fine structure of the B. viridis RC (10), were well conserved in the L (12 of 15 residues) and M (14 of 19 residues) subunits of R. gelatinosus (Fig. 9). The amino acid sequences around these residues also seemed to be well conserved. These findings suggest that the surfaces on the LM core that bind to the cytochrome subunit are similar in the two species and that the R. gelatinosus LM core can interact rather tightly with the B. viridis cytochrome subunit. In addition, the C-terminal residues of the M subunit may also contribute to the formation of the chimeric RC complex, since the C-terminal 34 amino acid residues after the 289th valine of the M subunit of the strain VC-F were derived from B. viridis. It has been suggested that the C terminus region of the M subunit is strongly associated with the cytochrome subunit in B. viridis as well as in other species (37). Thus, the cytochrome subunit of B. viridis in the strain VC-F would be closely associated with the "chimeric" M subunit.
It is known that the N-terminal 20 amino acid residues in the precursor form of the cytochrome subunit of B. viridis function as a signal peptide and are removed possibly after secretion for the periplasmic space (5). The N-terminal first amino acid of the mature form of the subunit is a cysteine residue, which is modified by two fatty acid molecules (38). The sequence LVAGC at positions 17-21 in the precursor protein of B. viridis may be responsible for this modification and for the cleavage by signal peptidase II (38). The cytochrome subunit of R. gelatinosus also has a similar motif, LLAGC, at positions 19 -23 (31). The post-translational modification shown in B. viridis presumably occurs in the cells of R. gelatinosus. Evidence for this comes from the results of SDS-PAGE, which showed that the mobility of a band corresponding to the B. viridis cytochrome subunit synthesized in the R. gelatinosus strain VC-F was identical with that in the B. viridis membrane, as shown in Fig. 3.
The redox midpoint potentials and the spectroscopic characteristics of four hemes in the cytochrome subunit of the strain VC-F were almost the same as those in the original B. viridis complex. This result suggests that the association with the R. gelatinosus LM core did not significantly affect the electrochemical properties of the hemes, except possibly for heme c559. Thus, the cytochrome subunit derived from the B. viridis gene probably holds its original structure and functions in the chimerical RC complex in the R. gelatinosus strains VC-F. The results of flash-induced spectrophotometric measurements in the membrane preparation of the R. gelatinosus strain VC-F indicated that the cytochrome subunit derived from B. viridis functioned as an efficient electron donor to the photooxidized special pair in the strain VC-F.
The rate of electron transfer from soluble electron carriers to the hemes in the cytochrome subunit in the strain VC-F was largely different from that in the wild-type strains of R. gelati-