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Originally published In Press as doi:10.1074/jbc.M302753200 on April 28, 2003

J. Biol. Chem., Vol. 278, Issue 26, 23678-23685, June 27, 2003
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A Reaction Center-Light-harvesting 1 Complex (RC-LH1) from a Rhodospirillum rubrum Mutant with Altered Esterifying Pigments

CHARACTERIZATION BY OPTICAL SPECTROSCOPY AND CRYO-ELECTRON MICROSCOPY*

Pu Qian, Hugh A. Addlesee {ddagger}, Alexander V. Ruban, Peiyi Wang, Per A. Bullough and C. Neil Hunter §

From the Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom

Received for publication, March 18, 2003 , and in revised form, April 28, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Introduction of the bchP gene from Rhodobacter sphaeroides encoding geranylgeranyl reductase into Rhodospirillum rubrum alters the esterification of the bacteriochlorophylls so that phytol is used instead of geranylgeraniol. The resulting transconjugant strain of Rs. rubrum grows photosynthetically, showing that phytolated Bchla can substitute for the native pigment in both the reaction center (RC) and the light-harvesting 1 (LH1) complexes. This genetic manipulation perturbs the native carotenoid biosynthetic pathway; several biosynthetic intermediates are assembled into the core complex and are capable of energy transfer to the bacteriochlorophylls. RC-LH1 complexes containing phytolated Bchla were analyzed by low temperature absorption and fluorescence spectroscopy and circular dichroism. These show that phytolated Bchls can assemble in vivo into the photosynthetic apparatus of Rs. rubrum and that the newly introduced phytol tail provokes small perturbations to the Bchls within their binding sites in the LH1 complex. The RC-LH1 core complex was purified from membranes and reconstituted into well ordered two-dimensional crystals with a p4212 space group. A projection map calculated to 9 Å shows clearly that the LH1 ring from the mutant is composed of 16 subunits that surround the reaction center and that the diameter of this complex is in close agreement with that of the wild-type LH1 complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Most photosynthetic organisms use chlorophylls for the capture of light energy and its conversion into a useful form of cellular energy. This molecule consists of two main components, a magnesium-porphyrin macrocyle and a "tail," which is usually phytol, a C-20 isoprenoid (1). The combination of these components constitutes the terminal step of the bacteriochlorophyll biosynthetic pathway, which is catalyzed by the BchG enzyme in the photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides (2, 3). The phytol tail is ~30% of the total molecular mass of (bacterio)chlorophyll, and, apart from being an important determinant of the hydrophobicity of the Bchl1 molecule, it plays an essential role in the assembly and stability of photosynthetic complexes. This was apparent from the early discovery that mutations in bchG abolish the assembly of the bacterial photosynthetic apparatus (4). However, the roles of the various esterifying alcohols used in bacterial photosynthesis, which include phytol, geranylgeraniol, and farnesol (1), are not understood. In this study a molecular genetic approach has been used to alter the esterification of the Bchls in Rhodospirillum rubrum so that BchlaP is used instead of BchlaGG.

High resolution crystallographic data of photosynthetic reaction centers have revealed detailed information on the conformation of the phytol tails of bacteriochlorophyll and bacteriopheophytin molecules within this complex (5, 6). The conformations of the phytol tails differ between the active and inactive branches of the electron-transferring pigment system in the reaction center, although the significance of this is unclear. In the bacterial light-harvesting LH2 complex, attention has been drawn to the way in which the phytol tails of the B800 and B850 BChls intertwine; such interactions have been proposed to play an important role in establishing fast energy transfer within these complexes by controlling the orientation of the transition dipoles of the Bchl molecule (7).

Phytol is the most common esterifying alcohol of chlorophylls and Bchls (8–10). In this respect, Rhodospirillum rubrum differs from other purple bacteria such as Rb. sphaeroides and Rb. capsulatus by employing the less saturated C20 isoprenoid alcohol geranylgeraniol (GG) instead of phytol (11). BchlaGG is a biosynthetic precursor of BchlaP, and the product of a single gene is responsible for the three steps (Fig. 1A) necessary for reducing BchlaGG to BchlaP (10, 12, 13). This gene, bchP, encodes the enzyme BchlaGG reductase and resides among other photosynthesis-related genes in a photosynthesis gene cluster (12, 14, 15). Homologs of bchP have been found in cyanobacteria and plants, wherein the gene product has been identified as the catalyst for the reduction of the isoprenoid moiety of chlorophyll (Chl) (16, 17).



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  		FIG. 1.
A, proposed biosynthetic pathway of the terminal hydrogenation of bacteriochlorophyll based on the chlorophyll biosynthesis in green plants (43). DHGG, dihydro-GG; THGG, tetrahydro-GG and BchlaP. B, HPLC profile of acetone-methanol extracts from whole cells of wild-type Rs. Rubrum (i), wild-type Rb. sphaeroides (ii), and transconjugant Rs. rubrum containing the bchP gene from Rb. sphaeroides (iii). The peaks labeled on the HPLC traces (shown by arrows) correspond to BchlaGG (1), BchlaDHGG (2), BchlaTHGG (3), and BchlaP (4). The detector was set at 375 nm.

 

Previous work has indicated that BchlaGG permits assembly of both LH1 and LH2 in bchP mutants of Rb. capsulatus and Rb. sphaeroides (10, 13). However, LH2 is severely affected by this change, whereas LH1 is not. As already noted, Rs. rubrum uses BchlaGG and can be thought of as a naturally occurring BchP mutant (18). Recently it has been shown that normal BchP function can be restored to Rs. rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol (18). In this new strain of Rs. rubrum, the new phytolated pigments have been assembled into a reaction center-light-harvesting 1 complex (RC-LH1), but there are no structural or spectroscopic data available for this new reaction center-antenna complex.

The LH1 light-harvesting complex of Rs. rubrum is the best characterized of any such complex, with a projection map at 8.5-Å resolution generated from cryo-electron microscopy studies (19). More recently, cryo-electron microscopy has also revealed the projection structure of the RC-LHI from Rs. rubrum at 8.5 Å, which shows a single reaction center surrounded by 16 LH1 subunits in a ring of ~115 Å diameter (20). The availability of a transconjugant strain of Rs. rubrum containing phytolated Bchls (18) therefore provides the first opportunity to see how the provision of a different C20 alcohol moiety influences the in vivo assembly, structure, and function of this light-harvesting LH1 complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Growth of Rs. rubrum and Preparation of Membranes—Cells of Rs. rubrum containing the bchP expression plasmid pSK1bchP (13) were grown photosynthetically at low light intensity (3 watts/m2) at 20 °C in M22+ medium in the presence of 250 ng/ml doxycycline (18). Cells were disrupted, and cell-free intracytoplasmic membrane fractions were isolated as described previously (21). Absorbance spectra were recorded on a Cary 500 spectrophotometer.

HPLC Analysis of Pigments—Carotenoid pigments were initially extracted into acetone/methanol, 2:1 (v/v), and subsequently transferred into ether by the method of Britton and Riesen (22). The samples were then evaporated to dryness under N2, and the pigments were dissolved in ethyl acetate/acetonitrile/water, 10:9:1 (v/v/v). Carotenoids were separated by HPLC on a Spherisorb ODS2 column (4.6{phi} x 250 mm) using a protocol provided by Professor A. Young (Liverpool John Moores University, United Kingdom). The flow rate was 1 ml/min, with an initial solvent composition of 20% ethyl acetate, 72% acetonitrile, and 8% water, changing over the course of 6 min to 60% ethyl acetate, 36% acetonitrile, and 4% water. This solvent composition was maintained for 15 min, during which time the carotenoids eluted. Elution of carotenoids was monitored using a Waters 996 photodiode array detector scanning from 270 to 600 nm every 2 s. Chromatograms at 447 nm were derived from the accumulated absorbance scans using the Millenium software (Waters). Carotenoids were identified by their retention times on the column and their absorbance spectra.

Purification of the RC-LH1 Complex—Cells of Rs. rubrum containing pSK1bchP were disrupted using a French press at a pressure of 1450 kg/cm2. The cell extract was applied to the top of a sucrose gradient (15–40% sucrose in Tris-HCl buffer, pH 7.9) and spun at 100,000 x g for 4 h at 4 °C. The clear membrane band just above the 40% sucrose layer was collected, and its protein concentration was determined by the BCA method (Pierce and Warriner Ltd., Chester, England). The detergent DHPC (23) was added to membranes dropwise over 15 min with gentle stirring, with the ratio of 3 mmol of DHPC per microgram of protein in membrane. The solution, after equilibrium for another 30 min, was centrifuged for 1 h at 150,000 x g to remove undissolved material and then purified with a DEAE anion exchange column (20 {phi} x 70 mm). The column was eluted with a gradient of buffer A (10 mM Tris, 1 mM EDTA, and 3 mM DHPC, pH 7.9) and buffer B (300 mM NaCl in buffer A). The main peak, which contains the RC-LH1 complex, corresponds to 250 mM NaCl. The best fractions judged from the 880:280 nm absorbance ratio (>2.0) were concentrated and loaded on a gel filtration column (Hiload 16/60, Superdex 200 preparative grade). The column was washed by buffer C (50 mM NaCl in Buffer A). Fractions with an 880:280 nm absorbance ratio of more than 2.2 were pooled and used for crystallization.

Two-dimensional Crystallization—The purified RC-LH1 sample was concentrated to ~1 mg/ml protein and then mixed with one of the lipids DOPC, POPC, or POPC-DMPC (4:1; w/w) in a lipid-to-protein ratio from 0.6 to 1.6 (w/w). The final sample, which was used for dialysis, was adjusted to 100 µl with a protein concentration of 0.5 mg/ml. The detergent was removed slowly by dialysis against a continuously flowing buffer (10 mM HEPES, 100 mM NaCl, and 0.01% NaN3, pH 7.5). The temperature was controlled as described in Walz et al. (24). After 64 h of dialysis, samples were collected and stored at 4 °C before use.

Electron Microscopy and Image Processing—The quality of the two-dimensional crystals was checked by negative staining with 0.75% (w/v) uranyl formate. Electron micrographs were taken with a 1024 x 1024 CCD camera (Gatan) attached to a Philips CM100 transmission electron microscope operated at 100 kV with a nominal magnification of 28,500x. For cryo-electron microscopy, two-dimensional crystals were adsorbed onto a 16 nm-thick carbon film mounted on a molybdenum grid. The grid was washed twice with distilled water followed by 1% glucose. Electron micrographs were taken with a Philips CM200 FEG microscope operated at a 200-kV acceleration voltage with the sample holder (Oxford Instruments) cooled by liquid nitrogen. Low-dose images, about 5–10 e–/Å2 at the specimen, were recorded at a nominal magnification of 50000x on Kodak SO163 film and developed with Kodak D19 for 12 min. Crystalline areas on the film were checked roughly by optical diffraction, and the well ordered areas were digitized with a Zeiss SCAI scanner using a step size of 7 µm per pixel. The MRC image processing program package version 2000 was used for data processing (25, 26).

Low Temperature Absorption—The purified RC-LH1 sample was mixed with 80% glycerol solution in 10 mM Tris-HCl, pH 8.0, to a final concentration of OD880 = 0.4–0.6 and 60% glycerol. The sample was cooled down slowly under nitrogen gas using an OptistatDN LN-2 cooled bath cryostat (Oxford Instruments) coupled with a temperature detector. After the temperature had stabilized, the cryostat was placed in a Cary 500 UV-Vis-NIR spectrophotometer. Spectra were recorded with a resolution of 0.2 nm. Room temperature spectra of samples were recorded with the same instrument.

Fluorescence and CD Spectroscopy—Fluorescence emission and excitation spectra of RC-LH1 complexes were measured using a SPEX Fluorolog FL3–22 spectrophotometer (Jobin-Yvon) equipped with xenon excitation and a CCD detector. The sample preparation and cooling procedure were the same as for the absorbance measurements. For excitation spectra, the 425–625 nm excitation range covered the carotenoid and Bchla Qx absorption region; detection of fluorescence emission was at 925 and 917 nm for wild-type and mutant complexes of Rs. rubrum, respectively.

CD spectra were recorded on a Jasco J810 spectropolarimeter at room temperature using a 5-mm path length cell with resolution of 0.5 nm. The absorbance of each sample was adjusted to 1.0 in a 1-cm path length at the maximum in the near infrared region.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Verification of BchlaP in the Transconjugant Strain of Rs. rubrum Containing bchP—HPLC traces of acetone-methanol extracts from whole cells are shown in Fig. 1B. Under the conditions used, BchlaGG (Fig. 1B, section i) had a retention time of ~18.4 min compared with 20.7 min for the BchlaP control (Fig. 1B, section ii). The HPLC profile of Bchls from the transconjugant Rs. rubrum (Fig. 1B, section iii) clearly indicates that introducing the bchP gene from Rb. sphaeroides into Rs. rubrum replaces the native BchlaGG with the phytolated pigment.

Alteration of BchlaGG to BchlaP Has a Marked Effect on the Carotenoid Composition—The carotenoids in the wild-type Rs. rubrum follow the spirilloxanthin branch of the biosynthetic pathway (Fig. 2A). HPLC analysis of the carotenoids present in the strain of Rs. rubrum, which contained the "empty" pRKSK1 expression plasmid as a control, showed that spirilloxanthin is the major component as expected, along with minor amounts of 3,4-didehydrorhodopin, rhodovibrin, and anhydrorhodovibrin (Fig. 2B, section i). It was noticed that, under photosynthetic conditions, cultures of Rs. rubrum [pSK1bchP] were distinctly red/brown in appearance, in contrast to the normal bright red color found in wild-type or pRKSK1-containing Rs. rubrum. Consistent with this change in color, Rs. rubrum [pSK1bchP] had a significantly altered carotenoid content (Fig. 2B, section ii). Spirilloxanthin was present but did not constitute a significantly greater proportion of the total carotenoid content than many of the other pigments present. The most abundant carotenoid was one of the previously mentioned spirilloxanthin precursors, putatively identified as rhodovibrin; the other carotenoids present appear to include rhodopin, 3,4-didehydrorhodopin, and anhydrorhodovibrin.



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  		FIG. 2.
A, probable pathway of carotenoid biosynthesis in Rs. rubrum. This linear scheme does not depict a cryptic branch of the pathway in Rs. rubrum, which became apparent in a mutant lacking the rhodopin 3,4-desaturase (44). B, carotenoid analysis by HPLC. The traces correspond to extracts from the wild type Rs. rubrum control containing the empty pRKSK1 expression plasmid (i) and the transconjugant Rs. rubrum mutant containing the bchP gene from Rb. sphaeroides (ii). Labeled peaks (shown by arrows) are rhodovibrin (1), a neurosporene-type pigment (2), rhodopin (3), spirilloxanthin (4), 3,4-didehydrorhodopin (5), and anhydrorhodovibrin (6). The detector was set at 447 nm.

 

Spectroscopic Studies of Membranes Containing the BchlaP LH1-RC Mutant Complex from Rs. rubrum—Absorbance spectra of the bacteriochlorophylls within bacterial LH1 complexes are sensitive to the aggregation state and environment of these pigments (21, 27) and therefore provide essential information on the effects of altering the esterifying pigments within the LH1-RC complexes studied. Low temperature absorbance spectroscopy revealed differences between RC-LH1 core complexes purified from wild-type and transconjugant strains; the Qy absorption maximum of the newly phytolated Bchla molecules within LH1 is slightly blue-shifted in comparison with the native complex containing BchlaGG. At room temperature, this shift is about 2 nm (876 nm to 874 nm; data not shown) and it extends to ~4 nm (from 894 to 888 nm, see Fig. 3) at 77 K, although the Qx maximum is maintained at these different temperatures. In the visible region, the different carotenoid composition of the mutant apparent from the HPLC analysis in Fig. 2 is manifested as an altered series of absorbance bands. For the purified wild-type complex, the bands at 457 (shoulder), 485, 517, 547, and 568 (shoulder) nm mainly reflect the presence of spirilloxanthin, whereas the increased number of well resolved bands in this region for the mutant at 433, 457 (shoulder), 487, 505, 529, and 549 nm arises from the spirilloxanthin precursor rhodovibrin together with rhodopin, 3,4-didehydrorhodopin, and anhydrorhodovibrin. The absorbance spectrum of the membranes containing the mutant complex is also shown in Fig. 3 for comparison; it demonstrates that the purified complex has retained the features of the membranes from which it was prepared with regard to both the carotenoid and bacteriochlorophyll absorbance bands in the visible and near infrared regions of the spectrum, respectively.



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  		FIG. 3.
Absorbance spectra at 77 K of RC-LH1 core complexes of mutant and wild type strains of Rs. rubrum. Dot-dash line, membranes used as the source of mutant complex containing BchlaP; solid line, RC-LH1 complex containing BchlaP; dotted line, wild-type Rs. rubrum.

 

The blue shift in absorbance for the mutant is reflected in the fluorescence emission spectra. Fluorescence emission spectra of the purified RC-LH1 core complexes recorded at 77K showed a clear difference in emission maximum, with a blue shift of 8 nm in the BchlaP mutant relative to the control (Fig. 4A). To observe any energy transfer from carotenoids to Bchls in the RC-LH1 core complex containing BchlaP, an excitation spectrum of the carotenoid region was recorded (Fig. 4B) with detection of emission from the Bchls. The excitation spectra were normalized to the Bchl Qx band to provide an indication of the relative efficiencies of the energy transfer process. For the wild-type control, three main excitation peaks are observed at 481, 515, and 549 nm in approximate agreement with the absorbance spectra, therefore reflecting the dominance of spirilloxanthin in terms of carotenoid composition. An efficiency of ~36% can be estimated, which is in reasonable agreement with the figure of ~35% obtained by Duysens (28) and cited in Ref. 29. In contrast, the excitation spectrum of the BchlaP mutant shows six well defined peaks at 432, 457, 487, 503, 529, and 549 nm, reflecting the presence of several carotenoids in this mutant such as rhodovibrin, rhodopin, 3,4-didehydrorhodopin, and anhydrorhodovibrin.



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  		FIG. 4.
Fluorescence spectroscopy at 77 K of RC-LH1 core complexes purified from mutant and wild type strains of Rs. rubrum. A, normalized emission spectra from complexes excited in the visible region at 505 nm. Solid line, RC-LH1 complex containing BchlaP; dotted line, wild-type Rs. rubrum. B, excitation spectrum with detection of emission at 926 nm for the wild-type complex and 918 nm for the mutant complex (A). The excitation spectra were normalized at the Bchl Qx band at ~590 nm. Lines as for A.

 

CD spectroscopy can provide a very sensitive indicator of alterations in the aggregation state and orientation of bacterial LH complexes (for examples, see Refs. 30–33). Near-IR CD spectra of intact membranes containing wild-type and mutant complexes were therefore recorded at room temperature and are presented in Fig. 5B. The corresponding absorbance spectra are shown in Fig. 5A, in which the shift to the blue noted for the purified BchlaP complex in Fig. 3 relative to the WT is also seen for the membrane sample; this time, the shift is 4 nm. Similarly, the CD spectrum of membranes containing the BchlaP RC-LH1 complex was blue-shifted in comparison with the WT. The differences between the CD spectra are the zero crossing points and the peak intensities. The zero crossing point for the WT complex corresponds exactly to the absorbance maximum, whereas in the case of the BchlaP mutant this zero crossing point is blue shifted by 2 nm with respect to absorbance maximum. The peak intensity ratios at 870/897 nm for the mutant and 865/892 for the wild type are similar, and, overall, the shapes of the two spectra differ only slightly.



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  		FIG. 5.
A comparison of CD spectra recorded on membranes from the WT and mutant strains of Rs. rubrum. Three scans were averaged for each spectrum; these spectra have been normalized to the same near infrared absorbance at ~880 nm. Solid line, RC-LH1 complex containing BchlaP; dotted line, wild-type Rs. rubrum.

 

Two-dimensional Crystallization of the RC-LH1 Complex Containing BchlaP—Recent progress in the preparation of RC-LH1 crystals from Rs. rubrum has allowed the projection structure of this complex to be determined at 8.5-Å resolution (20). This shows a single reaction center surrounded by 16 LH1 subunits in a ring of ~115-Å diameter. Within each LH1 subunit, densities for the {alpha}- and {beta}-polypeptide chains are clearly resolved. The availability of a mutant of Rs. rubrum provided the opportunity to see if alteration of the Bchl tail influences the structure and organization of this complex.

The RC-LH1 complex was purified from membranes prepared from the BchlaP-synthesizing strain of Rs. rubrum and used in crystallization trials. The best quality two-dimensional crystals were formed with a DOPC/protein (w/w) ratio of 1.0 after 64 h of dialysis; the typical size of a vesicular crystal was about 1600 x 1600 nm (Fig. 6). The quality of crystal was judged by negative stain electron microscopy with more than four orders of calculated diffraction. All individual images showed that the mutant RC-LH1 core complex forms a two-dimensional crystal with a square lattice and p4212 space group symmetry.



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  		FIG. 6.
Electron micrograph of negatively stained two-dimensional crystal of the RC-LH1 complex containing BchlaP from Rs. rubrum. The crystal was reconstituted with lipid DOPC at a lipid-to-protein ration of 1.0 by continuous flow dialysis for 64 h. The scale bar represents 100 nm. The inset shows a computer-generated diffraction pattern from the electron micrograph.

 

The crystals were embedded in glucose and found to be of sufficient quality to use for cryo-electron microscopy; the contrast transfer function (CTF) plot of a Fourier transform of a typical image is shown in Fig. 7. The phase residuals in resolution shells are summarized in Table I.



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  		FIG. 7.
The CTF plot of a Fourier transform of a typical image of an RC-LH1 crystal embedded in glucose. The circles represent the zero transitions of the CTF, and the boxed numbers indicate the IQ values of the individual reflections (25).

 

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  		TABLE I
Mean phase residuals in resolution shells for the merged image with p4212 symmetry

 

Fig. 8A shows a projection map displayed with p1 symmetry, within which densities forming two concentric rings can be seen, representing membrane-spanning {alpha}- and {beta}-helices. The densities in the middle are contributed by helices in the reaction center. Three individual unbent, CTF-corrected lattices were merged and p4212 symmetry applied, giving the image in Fig. 8B. A high-resolution map (Fig. 8C) has been calculated with a resolution of 9 Å, and it is clear enough to show that the RC sits in the middle of the ring surrounded by a closed ring of LH1. Rotational power spectrum analysis of the density from Fig. 8B shows a strong 16-fold component far above the noise level (Fig. 8D). The lattice parameters a and b, calculated from high-resolution images, are 168 ± 0.5Å, which corresponds to a molecule diameter of 116 Å. This diameter of the BchlaP LH1 ring is in close agreement with the figure of 115 Å for the wild-type LH1 complex, also determined by cryo-electron microscopy (20).



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  		FIG. 8.
Electron cryo-microscopy of glucose-embedded two-dimensional crystals of RC-LH1 complexes containing BchlaP. A, projection map displayed with p1 symmetry. The densities forming two concentric rings represent membrane-spanning {alpha} and {beta}-helices. The densities in the middle are contributed by helices in the reaction center. B, merged projection map from three individual, unbent, CTF-corrected lattices and with p4212 symmetry applied. C, the 16-fold rotationally filtered image extracted from B. D, the rotational power spectrum for one ring of density of merged images using the calculation method in Crowther and Amos (45).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Alteration of Pigment Synthesis in a Transconjugant Strain of Rs. rubrum Containing the bchP Gene from Rb. sphaeroides and the Effect on Bchls and Carotenoids—We recently demonstrated that the BchP enzyme of Rb. sphaeroides converts BpheaGG to BpheaP as well as BchlaGG to BchlaP, whereas BchP of Rs. rubrum is restricted to the former of these activities (18). As such, Rs. rubrum is a naturally occurring bchP mutant. In addition, it was shown that introducing the Rb. sphaeroides bchP gene into Rs. rubrum generates a novel transconjugant strain in which the Bchls are esterified with phytol. Given that the LH1 antenna complex of Rs. rubrum has been heavily studied using structural, spectroscopic, and reconstitution techniques (for example, Refs. 19, 20, 29, and 34–36), we decided to investigate this new Rs. rubrum core complex containing BchlaP.

It was a surprise to find that an altered carotenoid composition had accompanied the change from BchlaGG to BchlaP. The transconjugant strain had fallen short of completing the carotenoid biosynthetic pathway to spirilloxanthin (see Fig. 2A). The close match between the absorbance spectra of the membrane and the purified complex for the mutant in the 400–600 nm region (Fig. 3) suggests that the carotenoid composition of the membranes (Fig. 2B) is also reflected in the purified complex. The BchlaP-synthesizing transconjugant strain accumulated several biosynthetic intermediates, among them rhodovibrin and rhodopin. The reason for this is unclear; one possibility is a linkage between the carotenoid and (bacterio)chlorophyll biosynthetic pathways at the level of GGPP. In the former case, two molecules of GGPP can condense to form the C-40 carotenoid phytoene (37), and, in the second case, GGPP (either "free" GGPP or attached to the (B)chlide macrocycle as (B)chlaGG) can be reduced to form phytol pyrophosphate or (B)chlaP (13, 17). The Rb. sphaeroides GGPP reductase, when introduced into Rs. rubrum, could therefore deplete a common pool of GGPP, thus depriving the cell of the GGPP molecules necessary to maintain normal functioning of its carotenoid biosynthetic pathway. A second possibility is that the linkage between the type of Bchl tail and the carotenoid reflects structural factors imposed by the presence of a less rigid phytol tail, which, in turn, influences the type of carotenoid that can be efficiently assembled within the mutant complex.

The assembly of the carotenoid biosynthetic intermediates rhodovibrin, rhodopin, 3,4-didehydrorhodopin, and anhydrorhodovibrin into the RC-LH1 complex did take place, as judged by the appearance of a series of new peaks in both the absorbance and excitation spectra of purified complexes in Figs. 3 and 4B. The excitation spectra also reveal that these new carotenoids are at least partially efficient in transferring energy to the Bchls within LH1. In fact, the native carotenoid spirilloxanthin is itself only 36% efficient in this respect; such inefficiency is the consequence of the relatively high number (13) of conjugated C=C bonds in spirilloxanthin (38), which lowers the S1 state of the carotenoid relative to the S1 state of the Bchl Qy transition (39).

Spectroscopic Properties of the Membrane-bound Mutant RC-LH1 Complex from Rs. rubrum Containing BchlaP—Phytol tails are predicted to be less rigid than GG tails, so the conversion of BchlaGG to BchlaP in the RC-LH1 complex might be expected to change the ways in which these tails pack and interact with the LH polypeptides and carotenoids. For example, it has been observed that the phytol tails of the Bchls bound to the {alpha}-polypeptides are nearly fully extended and make close contacts with the carotenoids in the LH2 complex of Rhodopseudomonas acidophila (7). Consequently, the conversion of BchlaGG to BchlaP in the RC-LH1 complex could alter the mutual orientation of pairs of antenna-bound Bchl molecules in the LH1 ring, with attendant effects on the absorbance, fluorescence emission, and CD spectra.

As noted previously (21) the absorbance spectra of membranes, which contain RC-LH1 complexes in an aggregated form, are shifted to the red in both mutant and wild-type when compared with purified complexes (882/876 nm for the wild type; 877/874 nm for the mutant). Upon re-aggregation to form two-dimensional crystals, the spectrum of the purified RC-LH1 of Rs. rubrum complex shifts back to the red by a few nanometers (21). Thus, this reversible shift could provide a simple indication of the aggregation state of the complex. Similar shifts to the blue and back again to the red have been seen for membranes, purified monomeric complexes, and the two-dimensional crystals of the LH2 complex from Rb. sphaeroides (24), which suggests that this is a general property of bacterial LH complexes. In the present work, the extent of the blue shift provoked by liberation of complexes from the membrane by detergent treatment is 6 nm for the wild-type and only 3 nm for the mutant, which might indicate that, in its native membrane environment, the mutant RC-LH1 complex containing BchlaP is in a less aggregated form than the wild-type complex.

The CD spectra of the membrane-bound mutant and wild-type RC-LH1 complexes were measured to provide information on the complexes in their native environment (Fig. 5B). The spectra are very similar, showing a non-conservative spectrum arising mainly from the LH1 Bchls. The differences between the spectra are the zero crossing points and the peak intensities, with the zero crossing point of the BchlaP mutant shifted 2 nm to the blue, over and above the 5 nm shift in the absorbance spectra. The effects of these shifts are that the zero crossing point for the WT complex corresponds exactly to the absorbance maximum, whereas in the case of the BchlaP-containing mutant, this zero crossing point is blue shifted by 2 nm with respect to absorbance maximum. The significance of CD zero crossing/absorption shifts has been discussed and modeled extensively by Koolhaas et al. (30), in the context of the LH2 complex. They conclude that the CD/absorption red shift seen for LH2 arises from an energy mismatch between the {alpha}- and {beta}-bound B850 Bchls. It is also interesting that dimeric LH systems have been observed to exhibit a CD/absorption blue shift (31, 32). We suggest that the appearance of a CD/absorption blue shift in the BchlaP-containing mutant is a consequence of the new phytol tail exerting an influence on the relative distances and orientations of the Bchls, simultaneously decreasing the energy mismatch between the {alpha}- and {beta}-bound B875 Bchls and increasing their dimeric character. We note, however, that the CD spectra of LH complexes are extremely sensitive to small changes in the angles made by Qy transition moments between paired Bchls (31, 33), so our data indicate that there are no large-scale changes to the geometry of the Bchls in the mutant. One reason for expecting significant changes was the work of Parkes-Loach et al. (35), who compared the CD spectra of LH1 complexes from Rs. rubrum reconstituted from purified Bchl and polypeptide components. This approach made it possible to compare the effects of using different pigments, in this case BchlaP and BchlaGG. Their CD spectra showed that BchlaP was incapable of restoring the normal CD spectrum seen using BchlaGG, and an inverted spectrum was obtained. Similarly, in vitro experiments with RC complexes reconstituted with BchlaP showed that this pigment cannot substitute for BchlaGG in Rs. rubrum (8). Our results, using a genetic approach to alter pigment composition, show that BchlaP does substitute for BchlaGG in vivo, in both the LH1 and RC complexes. Bollivar et al. (10) working in the other direction by substituting native BchlaP with BchlaGG in Rb. capsulatus, concluded that both the light-harvesting and photochemical functions of the LH1 and RC complexes were unaffected. It should be borne in mind that both carotenoids and reaction centers are absent from the reconstitution work in Parkes-Loach et al. (35). Possibly one or both of these components, as well as LH1-RC-specific assembly factors (40–42), act as additional constraints on the way in which Bchl geometries are assembled within LH1 in vivo.

The Structure of the RC-LH1 Complex Containing BchlaP— The LH1 and RC-LH1 complexes of Rs. rubrum have repeatedly demonstrated a tendency to form well ordered two-dimensional crystals in vitro (19) using carotenoid-less B820 dimers or carotenoid-containing RC-LH1 complexes (20) as starting material. The present work on the BchlaP complex further emphasizes the suitability of the Rs. rubrum RC-LH1 complex for two-dimensional structural studies. This complex was examined using purification and crystallization approaches similar to those used successfully for the wild-type RC-LH1 complex from Rs. rubrum (20). This recent cryo-electron microscopy study revealed the projection structure of this complex at 8.5-Å resolution and showed that it consists of a single reaction center surrounded by 16 LH1 subunits in a ring of ~115 Å diameter. The availability of a transconjugant strain of Rs. rubrum containing phytolated Bchls (18) provided an opportunity to see if the provision of a different C20 alcohol moiety had affected the in vivo assembly, structure, and function of this light-harvesting LH1 complex.

The data show that this mutant complex (diameter, 116 Å) has an architecture very similar to that of the native complex (diameter, 115 Å; Ref. 20). One striking finding for the wild-type complex was the occurrence of both tetragonal and orthorhombic crystal forms comprising ordered arrays of circular and elliptical complexes, respectively. This reveals a degree of flexibility in the LH1 ring, which might be important for its function in terms of allowing reduced quinone to leave the reaction center. The fact that 20 individual images of the crystals from the BchlaP mutant show only the tetragonal form does not allow us to draw any conclusions in terms of any effects of BchlaP on the flexibility of the LH1 ring. However, because the mutant grows photosynthetically, it can be assumed that BchlaP does not impair the assembly or function of RC complexes in Rs. rubrum and that quinone transfer from the RC can still proceed.


   FOOTNOTES
 
* This research work was supported by grants from the Biotechnology and Biological Sciences Research Council of the United Kingdom and the Joint Infrastructure Fund. 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. Back

{ddagger} Present address: Crusade Laboratories, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF. Back

§ To whom correspondence should be addressed. Tel.: 0114-222-4191; Fax: 0114-222-2711; E-mail: c.n.hunter{at}sheffield.ac.uk

1 The abbreviations used are: Bchl, bacteriochlorophyll; BchlaGG, bacteriochlorophyll a esterified with geranylgeraniol; BchlaDHGG, bacteriochlorophyll a esterified with dihydrogeranylgeraniol; BchlaTHGG, bacteriochlorophyll a esterified with tetrahydrogeranylgeraniol; BchlaP, bacteriochlorophyll a esterified with phytol; CTF, contrast transfer function; CD, circular dichroism; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; GGPP, geranylgeranyl pyrophosphate; HPLC, high pressure liquid chromatography; LH1, light-harvesting 1; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PPP, Phytol pyrophosphate; RC, reaction center. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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