TY - JOUR
AU - Cogdell, Richard J.
AB - Abstract The reaction center (RC) and the core (RC-LH1) complex were isolated and purified from Rhodobium marinum; together with the LH1 complex [Meckenstock et al. (1992a)FEBS Lett. 311: 128], a complete set of RC, LH1 and RC-LH1 from the same wild-type strain of a purple photosynthetic bacterium can therefore now be made. Comparison of the BChl a/BPhe a ratio (determined by HPLC) between the RC and the RC-LH1 complexes lead us to the determination of the number of BChls in the LH1 ring to be 32.06±2.90, indicating that the LH1 ring from Rh. marinum consists of 16 αβ subunits. (Received June 23, 2000; Accepted September 26, 2000). Introduction Over the last 15 years, the structures of reaction centers (RC) from Rhodopseudomonas viridis (Deisenhofer et al. 1985) and Rhodobacter sphaeroides (Allen et al. 1987, Arnoux et al. 1995) as well as those of LH2 antenna complexes from Rhodopseudomonas acidophila (McDermott et al. 1995) and Rhodospirillum molischianum (Koepke et al. 1996) have been determined. Based on these structures, the detailed mechanisms of the electron-transfer reactions in the RCs (Deisenhofer et al. 1985) as well as the carotenoid (Car)-to-bacteriochlorophyll (BChl) and BChl-to-BChl energy-transfer reactions in the LH2 antenna complexes have been investigated (Koyama et al. 1996, Cogdell et al. 1996, Hu and Schulten 1997, Sundström et al. 1999, Cogdell et al. 1999). We are now in the position to begin to study, in detail, how these processes occur in the higher-order assemblies of the photosynthetic super-molecular complexes in vivo. Probably, the simplest organisms to begin with are those bacteria which only contain the reaction center-LH1 antenna (RC-LH1) complexes. In order to fully characterize the structure and the function of a particular RC-LH1 assembly, we need to have, for comparison, the individual RC and LH1 complexes together with the RC-LH1 complex all from the same strain. The LH1 ring of the detergent-solubilized RC-LH1 complex forms a ‘ring’ like structure around the RC. However, the size of this ring and whether it is actually complete in the natural form are both controversial at this time. Two different stoichiometries have been proposed for the number ofαβ -apoprotein subunits (n) in the LH1 ring, namely 12 and 16 (Cogdell et al. 1999). In the case of the LH1 complex from Rhodobium marinum (previously called Rp. marina), n = 12 was proposed for the LH1 ring based on the determination of its molecular weight by gel-filtration chromatography (Meckenstock et al. 1992a). The resolution of electron microscopy (26 Å) was not high enough to directly determine the number of αβ subunits (Meckenstock et al. 1992b). On the other hand, n was indirectly determined by determining the number of BChl molecules in the LH1 ring (Francke and Amesz 1995); since it is well documented that the RC contains four bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes), and the RC-LH1 is supposed to contain 2n+4 BChls and 2 BPhes, n can be determined by the measurement of the BChl/BPhe ratio in those complexes. The application of this technique to Rh. marinum and Rs. rubrum lead the above authors to propose n = 12 as a most probable value. Here, the ratio of the extinction coefficients, BChl a/Bphe a = 1.33±0.11 at their Qy absorption maximum, which had been determined for the extract from the RC of Rb. sphaeroides assuming the BChl a/BPhe a ratio of 4 : 2, was used for other organisms as well. Recently, the same method was applied to the intact cells of purple bacteria (Akiyama et al. 1999). When the molar ratio of BChl/BPhe = 2 in the RC and the number of BChls in LH1 = 32 were assumed, they were classified into three main groups in terms of the LH1/RC ratio, i.e. 3/4 (A. rubrum and Rps. viridis), 1/1 (Rs. rubrum) and 4/3 (Rh. marinum and Rvi. gelatinosus). On the other hand, when the LH1/RC ratio of 1/1 was assumed, the number of BChls in the LH1 complex varied from 20 to 44 depending on the organism. Those results lead the authors to propose various organizations of the LH1 and RC complexes in the photosynthetic membranes (see Fig. 2 of Akiyama et al. 1999). Electron microscopy with higher resolution provided more definitive information concerning n in the case of LH1 complex from Rs. rubrum S1. A 8.5 Å projection map clearly showed a ring of 16 αβ subunits (Karrasch et al. 1995). In the case of the LH1-only and RC-LH1-only mutants of Rb. sphaeroides containing no PufX, a 25 Å projection map showed that the LH1 ring has approximately the same mean diameter as that from Rs. rubrum (~90 Å); therefore, n = 15–17 was proposed (Walz et al. 1998). Very recently, native tubular membranes were purified from Rb. sphaeroides RCLH10, a mutant which contains only the RC-LH1 complex (Jungas et al. 1999). A 20 Å projection map was interpreted to show that a dimeric, S-shaped RC-LH1 complex between which a cytochrome bc1 complex was located. Most importantly, this organization of the RC-LH1 complex, consisting of two open C-shaped RC-LH1 structures would ensure efficient exchange of ubihydroquinone and ubiquinone between the RC and the cytochrome bc 1 complex. Independently, it was shown that a single PufX protein is involved in the above dimerization of the RC-LH1 complex (Francia et al. 1999). Taking all the above circumstances into consideration, we set the following objectives in the present investigation before starting application of fast laser spectroscopies to study the energy-transfer and electron-transfer reactions in the RC-LH1 complex: (a) to obtain a well-defined, highly purified RC-LH1 preparation together with those of the RC and the LH1 for comparison, and (b) to precisely determine the number of BChls in the LH1 ring attached to the RC, which is one of the key parameters in the analysis of the excited-state dynamics. In order to achieve these objectives, we have taken the following strategies: (1) Selection of the system. We wanted to make the system to be studied as simple as possible. First, we tried to choose a purple bacterium having only the RC and LH1 complexes. Second, even in such an organism, either the RC : LH1 stoichiometry or the size of the LH1 ring (when 1 : 1 stoichiometry is assumed) can vary from one organism to another in the intact cell membranes (Akiyama et al. 1999). Therefore, we avoided the cell membrane. Third, we confined ourselves dealing with, at the present stage, a ring-shaped detergent-solubilized RC-LH1 complex. This is an interesting model, even if the native RC-LH1 complex is actually S-shaped. Fourth, we carefully chose a strain from which we would be able to obtain a set of preparations, i.e. the RC, the LH1 and the RC-LH1 complexes. Since the LH1 complex had already been prepared from Rh. marinum (Meckenstock et al. 1992a), and since a general method of preparation for the RC-LH1 complex had been described (Dawkins et al. 1988), we chose this organism and tried to establish the methods of preparation for both the RC and the RC-LH1 complexes. (2) Selection of the method. It would be ideal if the structure of the RC-LH1 complex had been determined by means of X-ray diffraction or electron diffraction. Before those results become available, we thought most useful the above conventional method, i.e. to determine by HPLC the BChl/BPhe molar ratios in the extracts from the RC and RC-LH1 complexes and to use the ratio in the RC as an internal standard. Even when any additional peptide is tightly bound to the RC-LH1 complex (Cogdell et al. 1996), we can easily circumvent its contribution using this technique. Even in the absence of such a protein, direct quantitative analysis of the BChl content in a unit RC-LH1 complex is not straightforward, because we are unable to determine the amounts of water and ions attached to the α and β subunits (Meckenstock et al. 1992a, Meckenstock et al. 1992b), although their amino acid sequences have been determined (Brunisholz et al. 1989). (3) Complete extraction of BChl and BPhe from intact complexes. We have developed a new method using a DE52 column to completely extract BChl and BPhe, at low temperature and in a short period time, so that we can avoid degradation of the pigments and pigment-protein complexes. (4) Precise determination of the number of BChls. The ε values of BChl and BPhe (or their ratio) estimated at the detection wavelength of HPLC analysis can cause serious error. However, in the present determination of the BChl/BPhe molar ratio, we used a pair of the RC and the RC-LH1 complexes from the same strain, and therefore, we did not need to use any ε value at all. Materials and Methods Isolation and purification of the RC Rhodobium marinum DSM 2698 was cultured anaerobically at 30°C for 4–6 d at a light intensity of ~3,400 lux in the DSM-27 Rhodospirillaceae medium which was supplemented with 3% NaCl (Imhoff 1983). The cells were washed 2–3 times with a 100 mM Tris-HCl (pH 8.0) buffer, and disrupted twice by using a French press (1,450 kg cm–2) after addition of a few grains of both DNase and MgCl2. The chromatophore membranes (25,000–200,000×g fraction) were collected by centrifugation at 4°C. The RC was isolated by means of mild solubilization of the chromatophores at a low detergent concentration followed by sequential purification steps consisting of DEAE ion-exchange chromatography, sucrose density-gradient centrifugation, and the same ion-exchange chromatography. The chromatophore membranes (OD880=100 cm–1) in 100 mM sodium phosphate (pH 7.5) were solubilized at 4°C with 0.3% (v/v) LDAO for 2 h in the dark. After ×3 dilution, the suspension was subjected to centrifugation for 1 h at 200,000×g. This set of processes was repeated until the RC component appears. The supernatant was dialyzed overnight at 4°C against 20 mM Tris-HCl (pH 8.0) buffer (hereafter we call this‘ Tris buffer’) containing 0.05% LDAO. Crude RC thus obtained was then purified by the use of an ion-exchange column (30 mmϕ × 100 mm) which had been packed with DEAE-cellulose (DE52, Whatman) and equilibrated with the ‘Tris buffer’ containing 0.1% LDAO (‘eluent’). The crude RC was applied to the column, washed with the above eluent without and then with 50 mM NaCl to remove free pigments. The salt concentration was then increased to 100–150 mM NaCl to remove the LH1 component. Finally, the remaining RC component was quickly eluted with the eluent containing 250 mM or 300 mM NaCl. The RC-containing fraction (A280/A800 <1.45) was collected, dialyzed against the ‘Tris buffer’ containing 0.05% LDAO, and then, concentrated to OD800 = 5 cm–1. The purified RC was then subjected to sucrose density-gradient centrifugation (0.3, 0.6, and 1.2 M sucrose, 200,000×g) at 4°C for 20 h. The main RC band in the 0.3–0.6 M sucrose layer was collected, and then subjected to the same procedure of DEAE ion-exchange column chromatography described above. The RC component thus obtained exhibited the A280/A800 ratio of 1.1–1.2; it was stored at –20°C under nitrogen atmosphere until required. Preparation of the RC-LH1 and LH1 complexes The RC-LH1 complex was prepared according to the method described previously (Dawkins et al. 1988). Specifically, the chromatophore membranes (OD880 = 50 cm–1) were solubilized with 1% LDAO for 15 min at room temperature in the dark. The suspension diluted ×2 was centrifuged (12,000×g), and the resultant supernatant was loaded onto a stepwise sucrose density-gradient (0.3, 0.6 and 1.2 M) and centrifugated again (200,000×g) at 4°C for 15 h. The RC-LH1 component appearing between the 0.3 and 0.6 M sucrose layers was collected, dialyzed at 4°C against 20 mM Tris-HCl (pH 8.0) containing 0.05% LDAO, concentrated, and stored at –20°C under nitrogen atmosphere. The LH1 complex was prepared as described previously (Law and Cogdell 1998). SDS-PAGE and measurement of electronic absorption spectra The RC (OD800 = 3 cm–1), LH1 or RC-LH1 complex (OD880= 10 cm–1) was mixed, in the ratio of 1 : 1, with 400 mM Tris-HCl buffer (pH 6.8) containing 2% SDS and 10 mM dithiothreitol, and heated in boiling water for 3 min. PAGE was performed by the method of Laemmli (1970) using 15% polyacrylamide gel and a Mini-gel System (M&S, Osaka, Japan). The size of the gel was 55 mm long, 85 mm wide and 1 mm thick. Electrophoresis was run, at room temperature, in the constant-voltage mode (100 V). Photographs were taken after destaining the gel with an aqueous solution containing 20% ethanol and 10% acetic acid. The electronic-absorption spectra of the pigment–protein complexes were recorded, at room temperature, by using a Hitachi U-2000 double-beam spectrophotometer. Pigment analysis and determination of the BChl a/BPhe a ratios in the RC and the RC-LH1 complex The BChl a/BPhe a ratio was determined for the RC or the RC-LH1 complex as follows. The RC (OD800 = 3 cm–1, 1 ml) or the RC-LH1 complex (OD880 = 10 cm–1, 0.5 ml) was loaded, in the dark, onto a 6 mmϕ × 30 mm DEAE column (DE 52, Whatman) that had been equilibrated with 20 mM Tris-HCl (pH 8.0). Then, distilled water (10 ml) was added to the column in order to completely bind the pigment–protein complex, and nitrogen gas was purged through the column which was placed on ice to completely remove the water. An ice-cooled, acetone/methanol (1 : 1 v/v) mixture was passed through the column to continuously extract the pigments until the extract completely loses its absorption in the entire region of 200–900 nm. The pigment component was subjected to HPLC analysis after evaporating the solvent and re-dissolving the residue into the eluent. The HPLC conditions for the BChl a and BPhe a components were as follows: column, 4.6 mmϕ × 250 mm packed with silica gel (Lichrosorb Si-60, 5 µm, Merck); eluent, a mixture of n-hexane, 2-propanol and methanol (50 : 1 : 3 v/v/v); flow rate, 0.7 ml min–1; and detection, 750 nm (actually, the entire spectral region of 300–800 nm was monitored by a 2D detector, Waters 996). Results Characterization of the RC, LH1 and RC-LH1 components The RC Fig. 1a shows the electronic-absorption spectrum of the RC. The relative intensity of the absorbance due to aromatic amino acids representing protein concentration versus the peak due to the absorption of the accessory BChls, i.e. A280/A800 = 1.1, indicates the purity of this RC preparation. For a pure RC preparation from Rb. sphaeroides, this ratio has been reported to be 1.2 (Clayton and Wang 1971). The vibrational structures of the Car, 1Bu+ absorptions are slightly distorted due to the overlap of the 538 nm, Qx absorption of Bphe a. The shape of the ~370 nm absorption is ascribable to an overlap of the Soret absorptions of the accessory and the special-pair BChls and BPhes. Their Qy absorptions appear distinctively at 800, 863 and 752 nm, respectively. Fig. 2 shows the electronic-absorption spectra of the RC preparation in (a) the reduced and (b) the oxidized forms (produced by addition of ascorbate and ferricyanide, respectively), and (c) the oxidized minus reduced difference spectrum. The bleaching of the 864 nm absorption of the special-pair BChls as well as the blue-shift of the 800 nm absorption of the accessory BChls produce a difference spectrum that is very similar to that seen in other purple-bacterial reaction centers (Sauer and Austin 1978, Feher 1971). Fig. 3 (lane 2) illustrates the result of SDS-PAGE of the RC. It shows that the RC consists of the H, M and L (28, 24 and 21 kDa) sununits; this RC preparation contains no cytochrome subunit. The LH1 complex Fig. 1b shows the electronic-absorption spectrum of the LH1 complex. It exhibits a 278 nm peak and a doublet around 320 nm that are ascribable to the aromatic amino-acid sidechains of the α and β peptides. The A278/A880 is 0.42, which is very close to that reported previously, i.e. 0.43 (Meckenstock et al. 1992a). The sharp and clear Soret, Qx and Qy absorptions of BChl appear at 377, 588 and 880 nm. SDS-PAGE analysis of the LH1 complex (Fig. 3, lane 3) shows the presence of a pair of bands with apparent mol wt of 9.5 and 10 kDa which are ascribable to the α and β subunits, respectively. The RC-LH1 complex Fig. 1c shows the electronic-absorption spectrum of the RC-LH1 complex. It is a sum of the absorption spectra of the RC (Fig. 1a) and the LH1 complex (Fig. 1b), although the contribution of the latter is of course much stronger than that of the former. The A276/A880 ratio of this preparation is 0.44. SDS-PAGE analysis of this complex (Fig. 3, lane 4) shows the presence of bands due to the H, M and L subunits of the RC and those due to the α and β subunits of the LH1 complex. The origin of ~14 kDa band is not clear; since it is not completely reproducible, it may be due to aggregated peptides. Identification of the sidechain of BChl and determination of the number of BChl molecules in the RC-LH1 complex Fig. 4 shows the HPLC elution profiles of the extracts from the cells of (a) Rh. marinum, (b) Rb. sphaeroides R26.1 and (c) Rs. rubrum. It is well documented that Rb. sphaeroides contains BChl a having the phytol sidechain and that Rs. rubrum contains BChl a having the geranyl-geraniol sidechain (Scheer 1991). Therefore, it is clear that the cells of Rh. marinum contain a single BChl a species having the phytol sidechain. In other words, the RC and the LH1 complex from Rh. marinum bind the same type of BChl a species. Fig. 4 also shows the HPLC elution profiles of the extracts from (d) the RC, (e) the RC-LH1 and (f) LH1 complexes. Since it is known that the RC should contain 4 BChls and 2 BPhes, the ratio (x) of the integrated intensities of BChl (ABRC) versus that of BPhe (AHRC), in the elution profile of the RC, is given by (1) whereε Band εH indicate the extinction coefficients of BChl a and BPhe a at the detection wavelength (750 nm). If we assume that the LH1 complex is an oligomer of the αβ-peptide pair, each of which binds two BChls, then the ratio (y) of the BChl a versus BPhe a integrated intensities, in the elution profile of the RC-LH1 complex, can be given by (2) Then, Eqs. (1) and (2) lead to the number of the BChl pairs or the αβ subunits (n) in the LH1 ring (3) Table 1 lists the observed values of the BChl a/BPhe a ratio of the integrated intensities for the RC (x) and the RC-LH1 complex (y). By the use of the averaged values and the standard deviations, the value of n can be calculated to be 16.03±1.45. Thus, we conclude that the LH1 ring of Rh. marinum contains 32 BChl molecules and 16 αβ subunits. Discussion We have been able to prepare the RC, LH1 and RC-LH1 complexes from the same purple bacterium, Rh. marinum. Further, we have shown that all the BChl a molecules in this organism have the phytol sidechain. These results have allowed us to determine the number of BChls in the LH1 ring more straightforwardly without using the ε values of BChl a and BPhe a, because all we needed to assume was (1) that the number of BChl a and BPhe a molecules in the RC complex are 4 and 2 respectively, and (2) that the number of BChl a molecules in the LH1 ring is 2n. We modified the extraction and HPLC procedures of Francke and Amesz (1995) so that we could detect all the BChl a and BPhe a molecules in those pigment–protein complexes. (1) The RC or RC-LH1 complex was adsorbed completely onto the ion-exchange column, and dried with nitrogen gas at ~4°C to facilitate efficient extraction and to avoid degradation of the pigments and pigment–protein complexes. (2) Complete extraction was confirmed by measuring the electronic absorption of the extract in the entire UV to near-infrared region. (3) The amount of methanol in the eluent was increased (from 1.9 to 5.6%) to avoid possible adsorption of the pigments to the column. Our results strongly support the conclusion that the number of αβ subunits in the LH1 ring, at least in the isolated RC-LH1 complex from Rh. marinum, is 16. This value is in complete agreement with that of LH1 complex isolated from Rs. rubrum S1 which was determined by electron diffraction (Karrasch et al. 1995). Finally, discrepancy in the number of BChls in the LH1 ring in the RC-LH1 core complex from Rh. marinum between 25.3±2.9 determined by Francke and Amesz (1995) and 32.06±2.90 determined in the present study needs to be discussed. Both results are based on basically the same method, and it is unlikely such a large difference originates from the ε values of BChl a and BPhe a used in the former. The most important difference resides in the sample; the former study used the intact or crude chromatophere membrane, whereas the latter study used a detergent-solubilized RC-LH1 (core) complex. There remains a possibility that the S-shaped dimeric core complex (n = 12, for example) as a major component in the intact membrane (Jungas et al. 1999), and that it transforms into the ring-shaped monomeric core complex (n = 16) after solubilization with detergent (Francia et al. 1999). Actually, we observed two components after sucrose density-gradient centrifugation, i.e. the upper major component and the lower minor component. In the present investigation, we collected and characterized the upper component. Therefore, analysis of the lower component is most important at the present stage, because it may solve this apparent contradiction. Acknowledgements This work has been supported by grants from The Science Research Fund and Japan Society for the Promotion of Science. 3 Corresponding author: E-mail, ykomyama@kwansei.ac.jp; Fax, +81-798-51-0914. View largeDownload slide Fig. 1 Electronic-absorption spectra of (a) the RC, (b) the LH1 complex and (c) the RC-LH1 complex from Rh. marinum. View largeDownload slide Fig. 1 Electronic-absorption spectra of (a) the RC, (b) the LH1 complex and (c) the RC-LH1 complex from Rh. marinum. View largeDownload slide Fig. 2 Redox activity of the RC from Rh. marinum. The RC that was (a) reduced with ascorbate, (b) oxidized with ferricyanide, and (c) the difference spectrum of oxidized minus reduced. View largeDownload slide Fig. 2 Redox activity of the RC from Rh. marinum. The RC that was (a) reduced with ascorbate, (b) oxidized with ferricyanide, and (c) the difference spectrum of oxidized minus reduced. View largeDownload slide Fig. 3 The results of SDS-PAGE for the marker proteins (lane 1), including phosphorylase b (Mr = 94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and α-lactalbumin (14,400); the RC (lane 2); the LH1 complex (lane 3); and RC-LH1 complex (lane 4) from Rh. marinum. View largeDownload slide Fig. 3 The results of SDS-PAGE for the marker proteins (lane 1), including phosphorylase b (Mr = 94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and α-lactalbumin (14,400); the RC (lane 2); the LH1 complex (lane 3); and RC-LH1 complex (lane 4) from Rh. marinum. View largeDownload slide Fig. 4 HPLC elution profiles for BPhe a and BChl a extracted from the cell of (a) Rh. marinum, (b) Rb. sphaeroides R26.1 and (c) Rs. rubrum, and those from (d) the RC, (e) the LH1 complex and (f) the RC-LH1 complex all from Rh. marinum. View largeDownload slide Fig. 4 HPLC elution profiles for BPhe a and BChl a extracted from the cell of (a) Rh. marinum, (b) Rb. sphaeroides R26.1 and (c) Rs. rubrum, and those from (d) the RC, (e) the LH1 complex and (f) the RC-LH1 complex all from Rh. marinum. Table 1 The ratio of areas under the BChl a/BPhe a peaks in the HPLC elution profiles of the extracts from the RC and RC-LH1 complex detected at 750 nm Complex  Ratio  Average ± SD  RC  x = 1.396, 1.176, 1.387, 1.271, 1.412  1.329±0.103  RC-LH1  y = 10.41, 11.90, 11.51, 12.64, 11.25, 12.43, 12.64, 12.20  12.00±0.97  Complex  Ratio  Average ± SD  RC  x = 1.396, 1.176, 1.387, 1.271, 1.412  1.329±0.103  RC-LH1  y = 10.41, 11.90, 11.51, 12.64, 11.25, 12.43, 12.64, 12.20  12.00±0.97  SD: standard deviation. 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TI - Isolation and Purification of the Reaction Center (RC) and the Core (RC-LH1) Complex from Rhodobium marinum: the LH1 Ring of the Detergent-Solubilized Core Complex Contains 32 Bacteriochlorophylls
JO - Plant and Cell Physiology
DO - 10.1093/pcp/pcd068
DA - 2000-12-15
UR - https://www.deepdyve.com/lp/oxford-university-press/isolation-and-purification-of-the-reaction-center-rc-and-the-core-rc-46uYW5eeJe
SP - 1347
EP - 1353
VL - 41
IS - 12
DP - DeepDyve
ER -