©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of the Bacterial Core Light-harvesting Complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with Isolated - and -Polypeptides, Bacteriochlorophyll a, and Carotenoid (*)

(Received for publication, October 12, 1994; and in revised form, January 4, 1995)

Christine M. Davis Peggy L. Bustamante Paul A. Loach (§)

From the Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll a with isolated lightharvesting complex I (LHI) polypeptides of both Rhodobacter sphaeroides and Rhodospirillum rubrum. Reconstitution techniques first developed in this laboratory using the LHI polypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus reproduced bacteriochlorophyll a spectral properties characteristic of LHI complexes lacking carotenoids. In this study, carotenoids are supplied either as organic-solvent extracts of chromatophores or as thin-layer chromatography or high performance liquid chromatography-purified species. The resulting LHI complexes exhibit carotenoid and bacteriochlorophyll a spectral properties characteristic of native LHI complexes of carotenoid-containing bacteria. Absorption and circular dichroism spectra support the attainment of a native-like carotenoid environment in the reconstituted LHI complexes. For both R. sphaeroides- and R. rubrum-reconstituted systems, fluorescence excitation spectra reveal appropriate carotenoid to bacteriochlorophyll a energy-transfer efficiencies based on comparisons with the in vivo systems. In the case of R. rubrum reconstitutions, carotenoids afford protection from photodynamic degradation. Thus, carotenoids reconstituted into LHI exhibit spectral and functional characteristics associated with native pigments. Heterologous reconstitutions demonstrate the applicability of the developed assay in dissecting the molecular environment of carotenoids in light-harvesting complexes.


INTRODUCTION

Carotenoids are associated with all known photosynthetic organisms. In photosynthetic bacteria, the two roles ascribed to carotenoids are 1) harvesting of light energy not effectively absorbed by bacteriochlorophyll (BChl) (^1)and 2) protection of BChl from degradation in the presence of light and oxygen (Siefermann-Harms, 1987; Cogdell and Frank, 1987). The first of these functions involves the transfer of excited singlet state energy from the optically-forbidden 2A(g) state to the BChl Q(y) transition or from the 1B(u) state to the Q(x) transition (Thrash etal., 1979; DeCoster et al., 1992; Koyama et al., 1993). In the second function, carotenoids afford photoprotection by their capacity to quench triplet states of BChl, preventing singlet oxygen formation (Sistrom et al., 1956; Krinsky, 1979). Carotenoids in bacterial photosynthetic membranes are integral components of reaction center (RC) and light-harvesting (LH) complexes. Both photoprotective and light-harvesting capabilities have been demonstrated for RC- as well as LH-associated carotenoids (Koyama et al., 1990).

Each reaction center unit binds one carotenoid molecule while the carotenoid content of LH complexes varies among bacteria and type of LH. Rhodospirillum rubrum synthesizes only a core LH complex (B875 or LHI); organisms such as Rhodobacter sphaeroides contain an additional, peripheral LH termed B800-850 or LHII. The archetypal LH complex comprises two 6-kDa polypeptides termed alpha and beta, BChl, and carotenoid. LHI polypeptides bind two BChl and one or two carotenoids per alpha-beta pair, while LHII polypeptides bind three BChl and 1.5 carotenoid molecules per alpha-beta pair (Zuber and Brunisholz, 1991; Brunisholz and Zuber, 1992).

The protein environment exerts a profound effect on carotenoid-BChl energy transfer properties, as do carotenoid chemical structure and configuration (Noguchi et al., 1990). Historically, studies exploring carotenoid binding have employed photosynthetic bacterial strains of varying carotenoid compositions and carotenoidless mutant strains. Specificity of carotenoid binding has also been studied by incorporation of native or nonnative carotenoids into reaction centers (Boucher et al., 1977; Agalidis et al., 1980; Chadwick and Frank, 1986) or into LH complexes of carotenoidless strains (Davidson and Cogdell, 1981; Noguchi et al., 1990). In such reconstitution experiments, isolated complexes or lyophilized chromatophores were first incubated with carotenoid introduced in organic solvent. After sonication or stirring, excess carotenoid was removed by chromatography, centrifugation, or electrophoresis. Carotenoids incorporated in this manner demonstrated such native characteristics as efficient transfer of singlet excited state energy to BChl and BChl triplet state quenching. In addition, reconstituted carotenoids adopted conformations consistent with native carotenoids (Agalidis et al., 1980): all-trans in LH complexes and 15,15`-cis in reaction centers (Koyama et al., 1990). Despite the ability to reconstitute native and nonnative carotenoid with isolated RC and LHI complexes, little information has been acquired regarding the molecular interactions of carotenoids within these complexes.

The majority of spectroscopic and structural studies addressing carotenoid binding within LH complexes have focused on LHII. Unlike LHI, LHII complexes appear to incorporate carotenoids as necessary structural features (Klug and Drews, 1984; Iba et al., 1988; Zurdo et al., 1993). A notable difference between these two LH types concerns polarizability of bound carotenoids. The carotenoid environment in the LHII complex is thought to be highly polarizable due to nearby aromatic amino acid side chains or BChl molecules (Kakitani et al., 1982; Andersson et al., 1991; Richter et al., 1992; Koyama et al., 1993; Kuki et al., 1994). However, the environment of carotenoids in LHI of R. sphaeroides appears to be less polarizable (Kuki et al., 1994), consistent with the lack of electrochromism displayed by LHI carotenoids in isolated complexes (Goodwin and Jackson, 1993). While mutant strains of both R. sphaeroides (Griffiths et al., 1955) and R. rubrum (Cohen-Bazire and Stanier, 1958) blocked in colored carotenoid synthesis (containing only the highly saturated carotenoid phytoene) have been isolated, LHII is expressed only in low amounts in a phytoene background (Jones et al., 1992). Indeed, the LHII carotenoid environment appears to be quite different than that within the LHI complex.

During development of the methodology for preparing LHI structural subunits from R. rubrum and Rhodopseudomonas viridis, the addition of carotenoid to carotenoid-depleted systems resulted in partial restoration of wild-type spectra (Miller et al., 1987; Parkes-Loach et al., 1994). These results suggested that the LHI reconstitution techniques first developed using the alpha- and beta-polypeptides of R. rubrum (Parkes-Loach et al., 1988) and subsequently using LHI polypeptides of R. sphaeroides and Rhodobacter capsulatus (Loach et al., 1994) could be extended to include carotenoids.

The LHI reconstituted in vitro from isolated polypeptides and BChla exhibits spectroscopic properties nearly identical to the native carotenoidless LHI complex (Parkes-Loach et al., 1988; Loach et al., 1994). Above the critical micelle concentration of the mixed micelle, presumed to be near that of OG (0.60% (w/v), de Graça Miguel et al. (1989)), a subunit complex is formed, while reduction of the effective OG concentration by chilling or dilution results in LHI formation. Phospholipids and carotenoids, normally associated with native wild-type LHI are not required for in vitro complex formation. Preparation of the LHI complex, as well as a subunit form, from isolated components allows detailed molecular analyses of protein-pigment and protein-protein interactions (Parkes-Loach et al., 1990; Loach et al., 1994; Meadows et al., 1995). We report here the successful reconstitution of the alpha- and beta-polypeptides of both R. sphaeroides or R. rubrum with BChla and the other native pigment of LHI complexes, the carotenoid.


EXPERIMENTAL PROCEDURES

Materials

OG and Sephadex LH-60 were obtained from Sigma. The BChla(p) used in a few early experiments was also obtained from Sigma. Benzene, trifluoroacetic acid and hexafluoroacetone trihydrate were obtained from Aldrich. All other high purity, HPLC-grade solvents were purchased from Baxter Healthcare Corp., Burdick & Jackson Divison (Muskegon, MI). Anhydrous ethyl ether was obtained from Fisher. The LHII mutant strain of R. sphaeroides, PUC705-BA, was kindly provided by Dr. Samuel Kaplan of the University of Texas, Houston (Lee et al., 1989).

Bacterial Growth and Chromatophore Preparation

R. sphaeroides wild-type strain 2.4.1 and the LHII mutant PUC705-BA were grown in Sistrom's minimal medium (Leuking et al., 1978) under tungsten light. R. rubrum wild-type strain S1 was grown in modified Hutner's medium (Cohen-Bazire et al., 1957) under low intensity fluorescent lights. The cells were harvested in log phase after 2-3 days of growth at 25 °C, and chromatophores were prepared based on the procedure of Loach et al.(1963) with the following modifications. Cells were collected by centrifugation at 3,500 times g for 30 min in a Beckman J-6B centrifuge (JS-4.2 rotor), resuspended in 50 mM potassium phosphate buffer (pH 7.5), and pelleted at 10,000 times g (50.2 Ti rotor) in a Beckman L8-70 M ultracentrifuge. A whole cell suspension was sonicated for 12 min using a Branson cell disrupter 200 Sonifier at a power setting of 7 on a pulsed setting of 50% duration. After removal of cellular debris by a 38,000 times g, 30-min centrifugation, chromatophores were pelleted at 175,000 times g for 50 min in a 50 Ti rotor, washed with water, and lyophilized.

Isolation and Purification of alpha- and beta-Polypeptides

The LH polypeptides of R. rubrum were isolated as described in Parkes-Loach et al.(1988). R. sphaeroides polypeptides were isolated similarly, with the following modifications. Approximately 150 mg of PUC705-BA chromatophores were extracted with chloroform/methanol (1:1) containing 0.1 M ammonium acetate (Brunisholz et al., 1981, 1984; Theiler et al., 1984). The extract was loaded onto a Sephadex LH-60 column (127 times 2.5 cm), and the column was developed with the extraction solvent at a flow rate between 12 and 38 ml/h at 5 °C. If not sufficiently pure, the alpha- and beta-polypeptides were further purified by HPLC.

Samples of R. rubrum or R. sphaeroides polypeptides were separated by reverse-phase HPLC on a Waters system including two 501 pumps, a U6K injector, a 486 tunable absorbance detector, and an NEC Powermate SX Plus microcomputer. The mobile phases comprised 0.1% (v/v) aqueous trifluoroacetic acid (A) and acetonitrile/isopropyl alcohol (2:1), 0.1% (v/v) trifluoroacetic acid (B). Protein samples were injected in hexafluoroacetone trihydrate and resolved with a Perkin Elmer C(18) column (HCODS, 5 µm, 300 Å, 4.6 times 150 mm) preceded by an Alltech C(18) guard column (Macrosphere, 7 µm, 300 Å). The column was equilibrated with A:B (50:50), after which a linear gradient from 50 to 90% B was used to elute the polypeptides from the column at a flow rate of 0.7 ml/min with detection at 280 nm. Elution times for R. sphaeroides alpha and beta were 16.2 and 18.2 min, respectively; R. rubrum alpha and beta eluted at 26.9 and 16.0 min, respectively. The column was washed with 100% B prior to reequilibration with 50% B. Preparative scale HPLC employed a Waters Radial-Pak column (Delta-Pak, C(18), 300 Å, 15 µm, 8 times 100 mm) placed in an RCM 8 times 10 module. The gradient and solvents were identical to those described above. Proteins were chromatographed at a flow rate of 2.8 ml/min such that R. sphaeroides alpha and beta eluted at 21.6 and 24.0 min, respectively, while R. rubrum alpha and beta eluted at 34.7 and 21.2 min, respectively.

BChl Isolation

For most experiments, BChla was isolated in our laboratory and utilized unless otherwise noted. BChla, esterified with geranylgeraniol, was isolated from R. rubrum whole cells as described in Meadows et al.(1995). BChla(p), esterified with phytol, was isolated similarly from R. sphaeroides. An extinction coefficient of 55 mM cm at 777 nm in 4.5% (w/v) OG was used to calculate BChla concentrations in reconstitution experiments (Chang et al., 1990a).

Carotenoid Extraction and Purification

R. sphaeroides

All procedures were performed in dim or red light. A crude carotenoid extract was prepared by incubation of lyophilized wild-type or PUC705-BA chromatophores with petroleum ether (b.p. 30-60 °C) for several minutes prior to low speed centrifugation. The resulting supernatant, enriched in carotenoids, was concentrated by rotary evaporation. This procedure maximized the yield of carotenoids while excluding BChla from the extract.

Carotenoid samples in petroleum ether were spotted onto Analtech Silica GF (250 µm) TLC plates and developed with benzene/chloroform (1:1) (Cogdell et al., 1976). Preparative TLC separations were carried out on Whatman PLK5F Silica (1000 µm) plates. Carotenoid bands were eluted from the silica in ethyl ether. The two major pigmented bands (spheroidene (80%) and spheroidenone (20%)) were identified by their absorption spectra (Goodwin et al., 1955). Separation and identification of spheroidene and spheroidenone were also carried out by HPLC. A Beckman Ultrasphere ODS column (4.6 times 250 mm) was used with a Perkin-Elmer Series 4 liquid chromatograph. Samples were injected (for analytical and preparative purposes) in hexane or methanol and resolved using a linear gradient of 95:5 methanol/water to 95:5 methanol/hexane (DeCoster et al., 1992). Pure carotenoids were obtained in a 20% yield as calculated from the original absorbance of the petroleum ether extracts. The extinction coefficient of spheroidene in OG was determined relative to spheroidene in acetone. Using a value of 149 mM cm at 453 nm in acetone (Shneour, 1962; Goodwin et al., 1956), the extinction coefficient at 458 nm in 0.9% OG was determined to be 113 mM cm. The extinction coefficient of spheroidenone in OG, based on 120 mM cm at 472 nm in acetone, was calculated to be 90 mM cm at 469 nm.

R. rubrum

Carotenoids of R. rubrum were isolated and purified as above with the following modifications. Lyophilized chromatophores were extracted with benzene or petroleum ether. Carotenoids were resolved by TLC using 9:1 petroleum ether/acetone (van der Rest and Gingras, 1974). Spirilloxanthin was also purified by HPLC on the identical system described above, using solvents described in Lozano et al., 1989.

Reconstitution Procedure, R. sphaeroides and R. rubrum

The procedure described in Parkes-Loach et al.(1988) was modified as follows. For reconstitution experiments, 40-50 µg of each R. sphaeroides polypeptide was solubilized with 10 µl of hexafluoroacetone trihydrate followed by 0.5 ml of 4.5% (w/v) OG, with subsequent dilution to 0.90% OG. BChla was then added to this solution in a small volume of acetone. For experiments with the polypeptides of R. sphaeroides, BChla(p) was used, while BChla was used with R. rubrum polypeptides. At 0.90% OG, the BChla concentration was typically 2 µM, and the total protein concentration was 6 µM. The OG concentration was further reduced by successive additions of buffer until an optimal subunit absorbance was obtained, typically at 0.60% OG for R. sphaeroides and 0.73% OG for R. rubrum. The long wavelength LHI complex was formed by chilling overnight at 5 °C, although, in many cases, formation of this species was complete within 1 h. A Q(y) extinction coefficient of 119 mM cm (per BChl) was used for LHI quantification (Sturgis et al., 1988; Chang et al., 1990b). When carotenoids were included in the above assay, a small volume of acetone or petroleum ether was used to deliver the carotenoids into the solution of 0.90% OG after the addition of BChla(p) (to a carotenoid concentration approximately equivalent to the BChla concentration). Absorption and CD spectra were recorded with instrumentation previously reported (Parkes-Loach et al., 1988).

Photoprotection and Steady-state Fluorescence Measurements

To assess the photoprotective role of reconstituted carotenoids, samples were exposed to white light, and the decrease in LHI absorbance followed with time. Samples in 1-cm cuvettes were stirred for aeration and subjected to illumination. Actinic light from a tungsten bulb was passed through two focusing lenses and 2 cm of water before reaching the sample with an intensity of 1200 watts/m^2 as measured with a Yellow Springs-Kettering model 65 radiometer.

Fluorescence emission and excitation spectra were recorded using a Photon Technology International AlphaScan spectrofluorimeter. Slits were set at 10 nm. Second-order effects were reduced by placing a Corning 7-69 filter between the sample and the detector. Due to the decreased response of the Hamamatsu R928 phototube above 900 nm, LHI fluorescence was detected at 860-870 nm, corresponding to the leading edge of the emission maximum near 900 nm (Hunter et al., 1981).

Energy transfer efficiencies were obtained by measuring the light emitted from BChla relative to the light absorbed by the carotenoids in the visible region of the spectrum. Fluoresence excitation spectra were recorded in the region of carotenoid and BChla Q(x) absorption, measuring emission from BChla near 900 nm. For comparison to excitation spectra, absorption spectra were converted to a linear scale (1 - T), and the resulting fractional absorption (1 - T) times 100 was plotted. Excitation spectra were normalized to fractional absorption spectra in the region of the BChla Q(x) band near 590 nm, based on 100% efficient energy transfer from BChla (Q(x)) to BChla (Q(y)) (Goedheer, 1959). Efficiencies of energy transfer were calculated from the ratio of fluorescence excitation intensities to the fractional absorption intensities at several wavelengths.


RESULTS

The long wavelength complex formed by reconstitution of LHI alpha- and beta-polypeptides and BChla has been termed B873, reflecting the location of the BChla near IR absorption maximum. Because the actual wavelengths observed vary with the carotenoid content, a different nomenclature is adopted here. A ``reconstituted'' LHI refers to the complex formed from isolated protein and pigments, whereas ``native'' LHI refers to the complex as it exists in chromatophore membranes. Wild-type (WT) LHI complex denotes carotenoid-containing complexes formed either by reconstitution or as present in chromatophores. For example, the LHI of the R. rubrum carotenoidless strain G9 is termed a native, although not a wild-type, LHI complex.

Reconstitution of R. sphaeroides LHI

Absorption and CD characteristics of a typical reconstituted LHI are shown in Fig. 1. In the absorption spectrum (Fig. 1A), the long wavelength absorption band of BChla(p) (Q(y) transition) at 873 nm represents a hyperchromic red shift from unbound BChla(p), absorbing at 777 nm in OG. Upon LHI formation, the Soret band shifts to a slightly longer wavelength, and a 4-nm blue shift of the Q(x) band to 589 nm is evident. The near IR region of the CD spectrum (Fig. 1B) shows a conservative signal with a cross-over near the absorption maximum. Spectral features of the reconstituted complex concur with the properties of native LHI of R. sphaeroides (Bolt et al., 1981a; Kramer et al., 1984).


Figure 1: Absorption (A) and CD (B) spectra of reconstituted LHI of R. sphaeroides. A, LHI prepared with R. sphaeroides alpha- and beta-polypeptides and BChla(p) was chilled overnight at 5 °C in 50 mM phosphate buffer (pH 7.5) containing 5 mM MgSO(4) and 0.60% OG. At 0.60% OG, the alpha- and beta-polypeptide concentrations were 2.0 µM and 2.2 µM, respectively; the BChla(p) concentration was 1.5 µM. Absorbance at 873 nm was normalized to 0.1 cm. In addition to the Q(y) transition at 873 nm, the other absorption bands present are the Q(x) transition (589 nm) and the Soret band (375 nm) of BChla(p). B, the CD spectrum was recorded in a 2-cm path length, cylindrical cell placed in a water-cooled (0 °C) sample holder with the following data collection parameters: time constant = 4 s; slit = 160 µm (600-950-nm range) or spectral bandwidth = 2 nm (350-600 nm range); sensitivity = 2 millidegrees/cm; step resolution = 1 nm. The spectrum represents an average of four scans, scaled to correspond to an absorbance in a 2-cm cell of 0.1 at 873 nm. The above parameters apply to all CD spectra shown, unless otherwise indicated.



Whereas reconstituted LHI absorbs maximally at 873 nm, an absorption maximum of 875-880 nm is characteristic of R. sphaeroides native WT LHI (Fig. 2A). The CD spectrum of R. sphaeroides PUC705-BA chromatophores exhibits a peak and trough at 897 and 858 nm (Fig. 2B), while a hypsochromic shift of the corresponding features is evident in the reconstituted LHI CD spectrum (Fig. 1B). The Q(y) wavelength maxima of both the absorption and circular dichroism spectra of reconstituted LHI are characteristic of carotenoidless strains and wild-type chromatophores in which carotenoids have been extracted with organic solvent. A similar correlation between carotenoid content and LHI BChl absorption maxima is observed for other photosynthetic bacteria, e.g.R. rubrum wild-type compared with the carotenoidless strain, G9 (see Fig. 3of Chang et al., 1990a). Thus, it is evident that carotenoids influence the absorption and CD spectral properties of BChla in LHI complexes.


Figure 2: Absorption (A) and CD (B) spectra of R. sphaeroides PUC705-BA chromatophores. Apparent in the chromatophore spectrum of this mutant strain, lacking the LHII complex, is the near IR absorption due to LHI. Although the spectrum shown is representative, a range of near IR absorption maxima (875-880 nm) have been observed for LHI in whole cell and chromatophore preparations. The contribution of the RC to the absorption spectrum is visible at 800 nm; the RC contribution to the CD spectrum comprises a peak near 790 nm and a trough near 810 nm. The CD spectrum, recorded using the identical parameters as described in Fig. 1, was scaled to correspond to an absorbance of 0.1 at 877 nm.




Figure 3: Effect of carotenoid on optimization of subunit and LHI absorption. A, reconstitution of R. sphaeroides LHI without added carotenoid. Short dashed line, 0.90% OG; long dashed line, 0.70% OG; solidline, 0.60% OG. At 0.90% OG, the BChla(p) concentration was 1.9 µM; the alpha- and beta-polypeptide concentrations were 2.9 µM and 3.3 µM, respectively. The 0.70 and 0.60% OG spectra were normalized to the Q(x) absorption band of the 0.90% OG spectrum. At 0.90% OG, peak maxima are 778 and 822 nm; at 0.60% OG, the peak maximum is 825 nm. After chilling this sample overnight, complete conversion to an LHI with a near IR absorption maximum at 874 nm was observed (data not shown). B, reconstitution of LHI with added unpurified carotenoid extract. Short dashed line, 0.90% OG; long dashed line, 0.70% OG; solidline, 0.60% OG. At 0.90% OG, the component concentrations were as follows: 3.9 µM BChla(p); 2.4 µM carotenoid (spheroidene/spheroidenone 80:20); protein concentrations as in A. As above, the two spectra representing dilutions of the 0.90% OG solution were normalized to the Q(x) absorption band of the 0.90% OG spectrum. At 0.90% OG, peak maxima are 777 and 821 nm; at 0.60% OG, peak maxima are 781, 824, and 872 nm. Chilling this sample overnight resulted in a complex with a near IR absorption at 883 nm and carotenoid peaks red-shifted 16 nm from their absorption in 0.90% OG (data not shown).



Addition of Carotenoid Extract during Reconstitution of R. sphaeroides LHI: Formation of WT LHI

In an attempt to restore native WT properties to LHI, a crude preparation of carotenoids was added during reconstitution. Spheroidene, the principal carotenoid in anaerobically-grown R. sphaeroides cultures, was added to a solution of the alpha- and beta-polypeptides and BChla(p) in 0.90% OG. When the OG concentration was then optimized for LHI formation, the spectral changes shown in Fig. 3B and Fig. 4were observed. In the presence of carotenoid, a higher percentage of LHI forms at a given OG concentration when compared with a control reconstitution without carotenoid (Fig. 3A). For example, at 0.70% OG, the spectrum of the control reconstitution predominately exhibits absorption of the subunit complex (Fig. 3A), whereas in the presence of carotenoid, LHI absorption is also apparent (Fig. 3B). Although the ratios of the three species (unbound BChl, subunit complex, and LHI) varied among experiments, the propensity for LHI formation at higher OG concentrations was always evident in carotenoid-containing reconstitutions.


Figure 4: Absorption (A) and CD (B) spectra of R. sphaeroides reconstituted WT LHI prepared with unpurified carotenoid extract. Wt LHI was prepared with R. sphaeroides alpha- and beta-polypeptides, BChla(p) and a petroleum ether extract of chromatophores. A, LHI was formed by chilling overnight. At 0.60% OG, the concentrations of each component were as follows: 2.0 µM alpha, 2.2 µM beta, 1.2 µM BChla(p), and 0.6 µM carotenoid (spheroidene/spheroidenone 80:20). The absorption spectrum was normalized to an absorbance of 0.1 cm at 880 nm. B, the sample for the CD spectrum was prepared similarly. The OG concentration was 0.67%, with final concentrations of 2.2 µM alpha, 2.5 µM beta, 1.0 µM BChla(p) (Sigma), and 1.9 µM carotenoid. The CD spectrum was recorded and normalized to an absorbance of 0.1 at 880 nm as in Fig. 1.



Fig. 4A demonstrates that upon chilling the sample overnight to maximize LHI complex formation, a shift of the BChla(p) Q(y) band to a longer wavelength is evident (880 nm compared with 873 nm for the reconstitution without carotenoid, Fig. 1A). The carotenoid absorption, dominating the visible region of the spectrum, exhibits red shifts of 15-18 nm upon complex formation with the alpha- and beta-polypeptides and BChla(p) (see ``Discussion'' for further analysis of carotenoid band shifts). The peak wavelengths of both BChla(p) and carotenoid in the reconstituted WT LHI (Fig. 4A) closely match the absorption spectrum of native WT LHI (Fig. 2A).

The CD spectrum in Fig. 4B provides further evidence that the environment of BChla(p) is affected by the presence of carotenoid and that the carotenoid binding environment in the reconstituted system is quite similar to that of native WT LHI. The conservative nature of the BChla(p) CD in the near IR is preserved, while the maximum and minimum are red-shifted relative to LHI reconstituted without carotenoid. The carotenoid binding site imposes unique CD properties on the carotenoid such that the visible CD spectrum closely resembles the first derivative of the absorption spectrum in this region, contrasting sharply with the lack of CD displayed by carotenoid in organic solvent (Cogdell et al., 1976) or OG (data not shown). Moreover, the CD fine structure obtained for reconstituted carotenoids resembles the CD spectrum of carotenoids in chromatophores (Fig. 2B), strongly suggesting the attainment of a native carotenoid configuration in reconstituted WT LHI. Based solely on the yield of CD ellipticity for bound carotenoids, 50% of added carotenoids exist in a native conformation. When the criteria of energy transfer to BChl is considered (see below), the percentage of incorporation appears much higher. Thus, assimilation of carotenoid to form a reconstituted WT LHI in vitro simply requires mixing of the required components in a micellar solution rather than the extensive treatments (sonication, overnight incubations, etc.) previously reported for incorporation of carotenoids with light-harvesting complexes (Davidson and Cogdell, 1981; Lozano et al., 1990; Noguchi et al., 1990; Frank et al., 1993).

Isolation of Spheroidene and Addition of Pure Carotenoid to the alpha- and beta-Polypeptides of R. sphaeroides and BChla(p) to Form WT LHI

To show that the carotenoid was the only component of the petroleum ether extract required to explain the spectral observations, spheroidene was isolated from other extract components and used for reconstitution. The resulting LHI complex shown in Fig. 5A exhibits a BChla Q red shift, providing evidence of carotenoid incorporation. In addition, the carotenoid absorption shifts are similar to the absorption shifts observed with the crude carotenoid extract, although not as extensive (Fig. 4A). An excess of spheroidene was present in the particular experiment shown; the optimal (or minimal) amount of spheroidene needed for incorporation was not determined. In addition to the carotenoids of interest, potential contaminants of the chromatophore extract include lipids, sterols, other carotenoids, cis-trans isomers, and carotenoid degradation products (Davies, 1976). Although TLC purification successfully separated spheroidene and spheroidenone, copurification of contaminants or degradation products induced by the open air conditions of the procedure was not assessed, and therefore cannot be eliminated as possible factors in the slightly reduced incorporation of spheroidene. The lack of spectral resolution in the visible region of the spectrum probably results from a mixture of bound and unbound carotenoid molecules. Under conditions that stabilize LHI (low OG concentrations near 0.5%) adventitiously bound carotenoid would not separate using several gel-filtration resins.


Figure 5: Absorption (A) and CD (B) spectra of R. sphaeroides reconstituted WT LHI prepared with TLC-purified spheroidene. WT LHI was prepared with R. sphaeroides alpha- and beta-polypeptides, BChla(p) (Sigma), and TLC-purified spheroidene. The sample was diluted to 0.67% OG and then chilled to optimize LHI formation. The concentrations of the constituents were as follows: 2.2 µM alpha, 2.5 µM beta, 0.8 µM BChla(p), and 1.4 µM carotenoid. The spectra were recorded and normalized to an absorbance of 0.1 at 879 nm as in Fig. 1.



The near IR region of the CD spectrum shown in Fig. 5B resembles the spectrum of the carotenoid extract reconstitution in Fig. 4B. However, the peak and trough wavelengths of the BChla(p) near IR signal more closely approximate the wavelengths observed for the reconstitution without carotenoid (Fig. 1B) than with spheroidene added as an unpurified extract. Thus, in this case, reconstituted WT LHI exhibits a full red shift in the Q(y) absorption band with a lesser CD red shift. It is possible that we are unable to detect a red-shifted CD signal for the small population of LHI complexes fully incorporating spheroidene or that unbound spheroidene adversely affects the extent of incorporation. The fine structure observed in the visible region of the CD spectrum strongly resembles the CD features of WT chromatophores (Fig. 2B). Thus, despite the lack of significant red shift in the near IR CD, the visible region of the CD spectrum and the absorption spectrum suggest that a native-like carotenoid environment is adopted upon formation of the reconstituted WT LHI complex with pure spheroidene.

Further Analysis of the Observed Carotenoid Bandshifts and Induced CD Spectrum

A number of control experiments were necessary to demonstrate specificity of carotenoid interactions in the reconstituted system. First, the influence of OG on carotenoid absorption was examined. Pure, as well as unpurified, spheroidene exhibited red shifts on the order of 6 nm under subunit-forming conditions (dilution from 0.90 to 0.57% OG) as shown in Table 1. Despite such absorption changes, a CD signal was not detected for spheroidene in 0.57% OG in the concentration range used for reconstitution. In comparison, carotenoid band shifts of 9-15 nm were observed upon formation of reconstituted WT LHI, reproducing the carotenoid absorption maxima of R. sphaeroides PUC705-BA chromatophores, as listed in Table 1. Carotenoids in the presence of the alpha- and beta-polypeptides under reconstitution conditions, without BChla(p), also did not exhibit the band shifts and CD signal observed for reconstituted WT LHI (data not shown).



Formation of the R. sphaeroides Subunit Complex in the Presence of Carotenoid

The subunit form of the R. sphaeroides LHI complex can be reconstituted from isolated alpha- and beta-polypeptides, giving rise to a BChla(p) absorption maximum at 825 nm (Fig. 3A). A complex with identical absorption and CD properties can be formed with only the beta-polypeptide and BChla (Loach et al., 1994). Formation of LHI, on the other hand, requires the presence of both polypeptides. Reconstitution of the subunit complex with carotenoid in the presence of both the alpha- and beta- polypeptides was hindered by the fact that the equilibrium between unbound BChla(p), subunit, and LHI was greatly shifted toward the latter by the presence of spheroidene (Fig. 3B). Therefore, to investigate carotenoid interactions at the subunit-complex stage, subunit was prepared with only the beta-polypeptide to prevent further red shift to LHI. Under such conditions, carotenoid exerted little effect on BChla(p) absorption and CD spectra. Inspection of the carotenoid absorption and CD spectra revealed no evidence of incorporation. These results indicate a lack of detectable interaction between the beta-polypeptide and carotenoid at the level of subunit complex formation.

Spheroidenone Isolation and Reconstitution with the alpha- and beta-Polypeptides and BChla(p) of R. sphaeroides

The second major carotenoid synthesized by R. sphaeroides is spheroidenone, differing from spheroidene by the addition of a conjugated ketone near the methoxy end of the polyene. The ratio of the two carotenoids varies with growing conditions such that spheroidenone comprises 10% of the total carotenoid content of anaerobically grown cells and up to 98% under semiaerobic growth (Cogdell et al., 1981). As spheroidene/spheroidenone ratios in isolated LHI complexes have not been reported, the affinity of LHI complexes for spheroidenone is unknown. Therefore, the ability of purified spheroidenone to form a WT LHI complex was examined.

Spheroidenone purified by TLC or HPLC was tested in reconstitutions with alpha- and beta-polypeptides and BChla(p). The spectral effects of spheroidenone addition were markedly different from the results seen with spheroidene or unpurified carotenoid extract. The near IR absorption maximum of BChla(p) remained at 873 nm (data not shown). Despite the lack of a BChla(p) absorption red shift, the near IR region of the CD spectrum (with a peak and trough at 891 nm and 857 nm) resembled the spectrum of WT LHI reconstituted with carotenoid extract (Fig. 4B). A carotenoid absorption shift was not evident, although the absorption bands of spheroidenone are less distinct than those of spheroidene. The visible region of the CD spectrum exhibited a weak, broad signal attributed to spheroidenone (data not shown). The shape of the spheroidenone CD signal was similar to previously reported spectra of aerobically-grown cultures of R. capsulatus containing spheroidenone as the principal carotenoid (Bolt et al., 1981b).

Reconstitution of Carotenoids with the alpha- and beta-Polypeptides and BChla of R. rubrum: Formation of WT LHI

Initial experiments demonstrating reconstitution of R. rubrum WT LHI involved the addition of carotenoid extracts to the isolated subunit complex. (^2)An extension of these observations led to the reconstitution of carotenoids with individually-isolated LHI components in a manner similar to that described for the R. sphaeroides WT LHI.

A slightly different BChla Q(y) (max) behavior is observed for R. rubrum LHI reconstitutions as compared with similar experiments performed using R. sphaeroides polypeptides. Reconstituted LHI complexes formed with R. rubrum alpha- and beta-polypeptides and BChla exhibit near IR absorption maxima of 869 ± 3 nm (Parkes-Loach et al., 1988; Loach et al., 1994) compared with 873 nm for native LHI in R. rubrum G9 chromatophores. Addition of phospholipid vesicles during reconstitution can increase the BChla red shift to yield an absorption maximum of 875 nm (Bustamante and Loach, 1994).

The presence of spirilloxanthin, added as a petroleum ether extract of chromatophores, affected a red shift of the BChla absorption to 882 nm (Fig. 6A), which compares favorably to the BChl absorption of R. rubrum native WT LHI (881 nm; Fig. 7A). On the other hand, the peak locations and shape of the carotenoid absorption did not reproduce the spectrum of spirilloxanthin in chromatophores. The lack of fine structure of the reconstituted carotenoid absorption may result from the presence of a mixture of bound and unbound spirilloxanthin.


Figure 6: Absorption (A) and CD (B) spectra of R. rubrum WT LHI reconstituted with alpha- and beta-polypeptides, BChla and unpurified carotenoid extract of R. rubrum chromatophores. LHI was prepared with R. rubrum alpha- and beta-polypeptides, BChla, phospholipid vesicles, and spirilloxanthin (supplied as a petroleum ether extract of chromatophores). After chilling overnight in 0.63% OG to produce an optimal LHI absorption, the concentrations of the components were as follows: 1.8 µM each alpha and beta, 1.3 µM BChla, 73 µM phospholipids, and approximately 2.3 µM spirilloxanthin. The absorption peaks near 780 and 690 nm are due to uncomplexed BChla and a BChla degradation product, respectively. The single-scan CD spectrum (B) was recorded in a 2-cm pathlength cell using a chilled sample holder. The absorption and CD spectra were normalized to an absorbance of 0.1 at 882 nm.




Figure 7: Absorption (A) and CD (B) spectra of R. rubrum WT chromatophores. Chromatophores were prepared as described under ``Experimental Procedures.'' A, the absorption spectrum was normalized to an absorbance of 0.1 cm at 881 nm. B, the CD spectrum was recorded as described in Fig. 1such that the signal represents an average of four scans. In addition, the spectrum recorded in a 1-cm path length cell was normalized to a 2-cm path length. Phenazine methosulfate (10M) and ascorbate (10M) were added to maintain the RC primary electron donor in the reduced state.



The most striking effect of carotenoid addition upon the R. rubrum WT LHI reconstitution was the BChla CD spectrum; the long wavelength component of the BChla CD signal is red-shifted from the peak maximum of the absorption spectrum. The CD minimum at 891 nm shown in Fig. 6represents a 9-nm red shift from the near IR absorption maximum. In contrast, reconstituted LHI without carotenoid absorbing at 871 nm also exhibited a CD minimum at 871 nm (data not shown); a similar relationship holds for LHI reassociated from the isolated subunit complex (Bustamante and Loach, 1994). The BChla spectral properties of the reconstituted WT LHI are closely related to the native WT LHI spectra in Fig. 7B, where an 8-nm red shift of the R. rubrum WT chromatophore BChl CD signal relative to the absorption maximum is apparent. The CD spectra in Fig. 6and Fig. 7differ slightly in that the CD signal in the region of the BChla Soret transition is inverted with respect to the chromatophore CD spectrum and may be due to perturbations caused by unbound spirilloxanthin present in the reconstitution. Overall, as with the R. sphaeroides carotenoid reconstitution, the near IR absorption and CD spectra indicate that the reconstituted system reproduces the native BChla environment.

The interpretation of the carotenoid CD spectrum is complicated by the diminutive molar ellipticity exhibited by spirilloxanthin in a native WT LHI environment (Cogdell and Scheer, 1985). In comparing the spectra of reconstituted WT LHI and R. rubrum WT chromatophores, the reconstituted spirilloxanthin signal (Fig. 6B) appears to be split relative to the chromatophore CD spectrum (Fig. 7B). Although a broad, positive molar ellipticity in the visible CD is observed, the (max) occurs at a much shorter wavelength. These results contrast with the R. sphaeroides carotenoid reconstitution in which both the absorption bands and structured CD of the in vivo carotenoid were clearly reproduced. Successful reconstitution with spirilloxanthin was thus primarily supported by 1) the observed red shift in the BChla absorption and CD bands and 2) the enhanced molar ellipticity in the carotenoid region relative to the absence of a CD spectrum for spirilloxanthin in organic solvent or OG. A percentage of bound carotenoid could not be estimated due to the minimal fine structure of the carotenoid absorption and CD signals.

Substituting HPLC- or TLC-purified spirilloxanthin for unpurified carotenoid extract did not reproduce the above results. Whereas reconstituted WT LHI of R. rubrum exhibited appropriate spectral features in approximately 50% of the experiments using unpurified spirilloxanthin extract, reconstitutions with purified spirilloxanthin failed to satisfy all of the above requirements for WT LHI complex formation. Potential explanations for the lack of consistent activity of purified spirilloxanthin include lability upon purification (Polgár et al., 1944) or a requirement for a lipid component or other factor not yet identified.

Reconstitution of Carotenoids with Nonnative Polypeptides

The ability to incorporate carotenoid in the reconstituted LHI of two different bacteria enables reconstitution of polypeptides with nonnative carotenoids. Combining R. sphaeroides alpha- and beta-polypeptides and BChla with an unpurified carotenoid extract of R. rubrum demonstrated no evidence of carotenoid incorporation. In contrast, addition of unpurified spheroidene extract to the alpha- and beta-polypeptides of R. rubrum and BChla resulted in an LHI-type complex exhibiting BChla and carotenoid spectral shifts indicative of carotenoid incorporation (data not shown). A red shift of the near IR (max) of BChla to 883 nm was accompanied by a CD spectrum similar to that shown in Fig. 7B. The carotenoid absorption also did not exhibit a pronounced band shift. Detection of a small population of further red-shifted carotenoids was hindered by the excess of spheroidene present during reconstitution. The spheroidene CD signal, although inverted and reduced in rotational strength, displayed a fine structure similar in wavelength maxima and minima to spheroidene reconstituted with R. sphaeroides polypeptides (data not shown).

Photoprotective Role of Carotenoids in LHI of R. sphaeroides and R. rubrum

The ability of reconstituted carotenoids to afford photoprotection to LHI BChla was also examined. The expected photoprotection pattern was observed for chromatophores of R. rubrum wild-type and G9 strains (Fig. 8) and R. sphaeroides wild-type and a carotenoidless strain (data not shown). Carotenoid-containing chromatophores demonstrated a 2-fold slower degradation rate compared with carotenoidless chromatophores. Under illumination, reconstituted LHI of R. rubrum lost BChla absorbance at a faster rate than reconstituted WT LHI, suggesting a photoprotective effect of spirilloxanthin in the latter (Fig. 8).


Figure 8: Carotenoid photoprotection of R. rubrum LHI BChl. Photoprotection was measured as a decrease in LHI absorbance upon illumination, as described under ``Materials and Methods.'' 100% AbsorbanceRemaining corresponds to the LHI absorbance before illumination, in each case, this was near 0.1 cm. Time points were taken until the LHI absorbance reached approximately 20% of the starting absorbance. In addition to a decrease in BChl absorbance, each sample exhibited a concomitant blue shift in the LHI near IR absorption band. The points on this graph were not corrected for this peak-shift. times, R. rubrum wild-type chromatophores; bullet, R. rubrum LHI reconstituted with spirilloxanthin supplied as a petroleum ether extract of chromatophores; circle, G9 (carotenoidless) chromatophores; , R. rubrum LHI reconstituted without carotenoids.



The R. sphaeroides reconstitutions did not exhibit the expected correlation of carotenoid content with photoprotection. R. sphaeroides LHI reconstituted without carotenoid demonstrated much greater stability than R. rubrum LHI reconstituted with or without spirilloxanthin; the addition of unpurified spheroidene actually resulted in rapid loss of this stability. Furthermore, repetition of R. sphaeroides reconstitution measurements revealed a high variability in photodestruction rates.

Singlet Energy Transfer from Reconstituted Carotenoids to BChla

Restoration of the light-harvesting function of carotenoids was tested by measuring fluorescence excitation spectra of reconstituted WT LHI complexes. Although reconstituted carotenoids manifested the expected absorbance properties, demonstration of singlet energy transfer to BChla was essential to validate functional incorporation. If energy transfer proceeds by an electron-exchange mechanism (Dexter, 1953), the carotenoid and BChla molecules must be in close proximity, thereby restricting the number of functional binding sites and orientations. A number of successful incorporations of carotenoids into RC, LHII, or chromatophore preparations from mutant or carotenoid-depleted complexes have been reported (Agalidis et al., 1980; Davidson and Cogdell, 1981; Noguchi et al., 1990; Frank et al., 1993). In these studies, the ability of carotenoids to perform in a light-harvesting capacity was demonstrated.

The carotenoid to BChl energy transfer efficiencies of R. rubrum wild-type and R. sphaeroides PUC705-BA chromatophores were comparable with previously reported values of 30% (Goedheer, 1959) and 70-75% (quoted for isolated B875, Kramer et al.(1984) and Cogdell et al.(1992)). Incorporation of spheroidene with R. sphaeroides polypeptides and BChla(p) resulted in an LHI complex exhibiting energy transfer efficiencies quite similar to spheroidene associated with native LHI. From the spectra in Fig. 9, an efficiency of approximately 59-66% was calculated, comparing favorably with the calculated efficiency of 62-73% for PUC705-BA chromatophores (Fig. 9, inset).


Figure 9: Fractional absorption and fluorescence excitation spectra of R. sphaeroides reconstituted WT LHI. The absorption spectrum of R. sphaeroides reconstituted WT LHI (prepared with unpurified spheroidene) was converted to the linear scale fractional absorption (dashedline) for direct comparison to the fluorescence excitation spectrum after normalizing the two curves at the Q(x) band (590 nm) as described in the text. From the overlaid spectra, an efficiency of energy transfer from carotenoids to BChl was calculated. Using the graphics program, GRAPHER for Windows (Golden Software, Inc., Golden, CO), the fractional absorption spectrum was smoothed using a running average window = 3. Fluorescence was measured at 860 nm; the resultant excitation spectrum (solidline) represents the average of two scans. The peak wavelengths of the absorption spectrum are 500, 468, and 444 nm, and the peak wavelengths of the fluorescence excitation spectrum are 503 and 470 nm, corresponding to energy transfer efficiencies of 59.0% (503 nm) and 66.1% (470 nm). Inset, fractional absorption (dashedline) and fluorescence excitation (solidline) spectra of R. sphaeroides PUC705-BA chromatophores. The fractional absorption spectrum was smoothed with a running average window = 5. As above, the two-scan averaged fluorescence was detected at 860 nm. Absorption wavelengths are 507, 474, and 447 nm and fluorescence peak wavelengths are 506 and 470 nm. Calculated energy transfer efficiencies are 62.0% (506 nm) and 72.5% (470 nm).



The excitation spectrum of R. rubrum wild-type chromatophores reveals an efficiency of energy transfer from carotenoid to BChla of approximately 35% (Fig. 10A). Reconstitution of spirilloxanthin with the polypeptides and BChla of R. rubrum resulted in an excitation spectrum (Fig. 10B) resembling that of R. rubrum chromatophores, although the excitation peak locations were slightly blue-shifted. Comparing the excitation spectrum of the reconstituted system to the chromatophore excitation spectrum, an approximate energy transfer efficiency of 32% was calculated. In this particular experiment, the large contribution of unbound carotenoid to the absorption spectrum precluded a meaningful comparison of the fluorescence excitation spectrum to the fractional absorption spectrum.


Figure 10: Fractional absorption and fluorescence excitation spectra of (A) R. rubrum chromatophores and (B) R. rubrum reconstituted WT LHI. A, the fractional absorption spectrum of R. rubrum chromatophores (dashedline) was smoothed with a running average window = 5. Fluorescence (solidline) was measured at 868 nm and represents an average of three scans. The chromatophore sample contained a low concentration of Na(2)S(2)O(4) to allow detection of LH1 fluorescence (Loach, 1966; Godik and Borisov, 1979). The peak wavelengths of the absorption spectrum are 549, 513, and 485 nm. The fluorescence peak maxima are 549, 513, and 473 nm. The calculated energy transfer efficiencies for the longest to shortest transitions are 38.7, 34.1, and 32.4%. B, the fluorescence excitation spectrum of reconstituted WT LHI of R. rubrum incorporating the native carotenoid, spirilloxanthin, is shown. Spirilloxanthin was added during reconstitution as an unpurified benzene extract of R. rubrum WT chromatophores. The absorption and CD spectra of this sample are shown in Fig. 6. The excitation spectrum, with emission detected at 860 nm, was recorded as a single scan. An efficiency of energy transfer was calculated to be approximately 32% when the excitation spectrum was compared with the excitation spectrum of R. rubrum chromatophores in A.



Reconstitution of R. rubrum polypeptides and BChla with the nonnative carotenoid spheroidene provided spectral evidence for incorporation as discussed above. Functionality of the bound carotenoid was further supported by the fluorescence excitation spectrum shown in Fig. 11. The excitation spectrum most closely resembled that of R. sphaeroides reconstituted WT LHI, demonstrating energy transfer efficiencies in the range of 54-62%. As unbound carotenoid again contributed to the absorption spectrum, the efficiency was calculated by a relative comparison to R. sphaeroides chromatophore fluorescence. Thus, the carotenoid of R. sphaeroides, spheroidene, transfered energy in a reconstituted R. rubrum WT LHI with a higher efficiency than the native carotenoid, spirilloxanthin, and exhibited an efficiency of transfer indistinguishable from R. sphaeroides native WT LHI.


Figure 11: Fluorescence excitation spectra of reconstituted WT LHI of R. rubrum, incorporating spheroidene and R. sphaeroides PUC705-BA chromatophores. Shown is the fluorescence excitation spectrum (solidline) of the reconstitution combining the alpha- and beta-polypeptides and BChla of R. rubrum with the R. sphaeroides carotenoid, spheroidene. The concentrations of the constituents were as follows: 1.8 µM each alpha and beta, 1.0 µM BChla, 56 µM phospholipids, and 3.4 µM carotenoid (supplied as an unpurified petroleum ether extract of R. sphaeroides WT chromatophores). The absorption spectrum of the sample exhibited carotenoid maxima at 490, 460, and 436 nm and a BChla Q(y) maximum at 883 nm. The fluorescence excitation spectrum, detected at 860 nm, represents a single scan. The peak maxima of the excitation spectrum are 503 and 469 nm. Calculated energy efficiencies for these transitions are 54.2 and 61.7%, respectively. Efficiencies were calculated relative to the excitation spectrum of R. sphaeroides PUC705-BA chromatophores (dashedline) normalized to the fluorescence of the reconstituted WT LHI at the Q(x) band, due to the excess of unbound spheroidene contributing to the carotenoid absorption spectrum.




DISCUSSION

The criteria used to establish efficacious carotenoid reconstitution are as follows: 1) BChla Q(y) absorption shift further red by 7-14 nm than obtained for reconstituted LHI without carotenoid, 2) red shift of BChla Q(y) CD signal commensurate with absorption shift, 3) carotenoid absorption band red shifts of between 9 and 15 nm, 4) appearance of a native-like carotenoid CD signal, 5) efficient transfer of energy from carotenoid to BChla as measured by fluorescence excitation spectra, and 6) photoprotection of BChla by carotenoid. Among the experiments performed, each of these criteria was met with some variability. For example, observation of an appropriate CD spectrum was not always attendant to maximal carotenoid absorption band shifts. Nevertheless, the preparation of samples satisfying all of the above criteria provide a firm basis for the conclusions drawn.

Spectral Evidence of Carotenoid Incorporation

Spectral properties of BChla(p) and BChla were affected by the presence of carotenoid such that R. sphaeroides or R. rubrum reconstitutions performed with their own native carotenoids consistently resulted in formation of long wavelength species absorbing from 878 to 883 nm (Fig. 4A, 5A, and 6A). This criterion, being the most easily satisfied, was predicted from earlier experiments where extraction of carotenoids from membrane fragments caused blue shifts in the BChl absorption spectrum, mirroring carotenoidless mutant absorption maxima (Miller et al., 1987; Chang et al., 1990a). The near IR CD spectra reflected the spectral red shifts (Fig. 4B, 5B, and 6B). Wt LHI reconstituted from R. sphaeroides polypeptides and BChla(p) with pure spheroidene manifested spectral features similar to those observed with unpurified spheroidene, indicating that only purified polypeptides, BChl, and spheroidene were necessary for reconstitution of pigment-pigment or pigment-protein interactions to produce in vivo spectral characteristics (Fig. 5).

Carotenoid absorption maxima undergo pronounced red shifts upon formation of specific complexes with light-harvesting polypeptides and BChla. These 1B(u) energy shifts are thought to be induced by local charges, exciton interactions, or dispersion (induced-dipole) interactions (Andersson et al., 1991). Modeling of carotenoid absorption maxima in a variety of solvents has suggested that the R. sphaeroides carotenoid is located in a nonpolar environment with high polarizability (Andersson et al., 1991). The polarizability of the LHII carotenoid environment appears to be greater than that of LHI (Kuki et al., 1994), consistent with the observation that LHII-associated carotenoids absorb at longer wavelengths than carotenoids associated with LHI or reaction centers (Table 1; Broglie et al., 1980). The carotenoid spectrum of wild-type chromatophores represents a mixed population in which the LHI carotenoid components are a minority (Table 1). As for BChl absorption, R. sphaeroides PUC705-BA chromatophores serve as a more appropriate native comparison for carotenoids reconstituted into LHI.

While the extent of absorption red shifts reveals the sensitivity of the carotenoid to the surrounding media, the CD spectrum reflects this sensitivity to an even greater extent. The presence of a structured CD signal has been suggested to result from asymmetric binding to the protein and/or moderate exciton coupling with other pigments (Cogdell and Crofts, 1978). Carotenoid-carotenoid interactions have been proposed based on the observation that light-harvesting complexes containing two carotenoids per alpha-beta pair have strong CD signals compared with LH complexes with one bound carotenoid (Kramer et al., 1984). Moreover, that the LHI carotenoid CD spectrum resembles the first derivative of the absorption spectrum implicates degenerate exciton interaction (Lozano et al., 1990). The CD spectra of carotenoids associated with LHI, LHII, PUC705-BA chromatophores, and reconstituted WT LHI complex from R. sphaeroides resemble the first derivative of the absorption spectrum. The CD signals for R. rubrum native WT LHI (Dratz et al., 1967) and reconstituted WT LHI of R. rubrum mirror the absorption spectra, indicating differences in the binding sites between LHI complexes of R. sphaeroides and R. rubrum, or a difference in coupling between carotenoid molecules (Bolt et al., 1981a).

Energy Transfer

Native carotenoids of R. sphaeroides transfer excited singlet state energy to BChla with high efficiency (Table 2). The coupling of such high efficiencies with low carotenoid fluorescence quantum yields suggests that carotenoid and BChla are in close proximity. In model systems containing concentrated mixtures of carotenoid and chlorophyll molecules, energy transfer is not observed; correct proximity and orientation can only be afforded by covalent attachment of the two chromophores within model compounds or by the association with protein (Dirks et al., 1980; Gust and Moore, 1993).



For R. sphaeroides, the efficiency of singlet energy transfer in the reconstituted WT LHI, 59-66%, paralleled native WT LHI efficiencies. The measured efficiency of native LHI of R. sphaeroides PUC705-BA was consistent with reported values. Unbound carotenoids, contributing to the absorption spectrum, but not the fluorescence, would lower the calculated energy transfer efficiency. Since the efficiency of reconstituted carotenoids was so high, we can assume that unbound or adventitiously-bound carotenoids represented a small percentage (<10%) of the total carotenoids present. The conclusion that the majority of the carotenoids are uniquely bound is further supported by the comparable efficiencies calculated at several wavelengths across the carotenoid absorption.

Calculated energy transfer efficiencies for R. rubrum WT chromatophores compared well with the previously reported value of 30% for chromatophores (Goedheer, 1959). Our values were slightly higher; the greater definition of peaks in the absorption and fluorescence spectra shown here (Fig. 10) could indicate a more homogeneous carotenoid content relative to the cells used in the earlier study. Carotenoids of R. rubrum reconstituted WT LHI transfered excited singlet state energy to BChla with the same efficiency as in native WT LHI, despite slight differences in the fine structure of the fluorescence excitation spectra.

Although carotenoids reconstituted to form WT LHI of both R. sphaeroides and R. rubrum restored native fluorescence excitation spectra, the carotenoid CD spectrum was less reproducible. For example, the overall shape of the carotenoid CD signal for R. sphaeroides reconstituted WT LHI matched the native signal, but the relative amplitudes of the peaks and troughs varied, and the molar ellipticity of the signal was consistently lower than the native WT LHI carotenoid signal ( Fig. 2and Fig. 4). Thus, it appears that carotenoid function can be restored without fully reproducing the structural subtleties of the native carotenoid binding site.

Carotenoids also function in a photoprotective capacity to protect BChl from degradation in the simultaneous presence of light and oxygen (Griffiths et al., 1955). In vitro studies have shown that carotenoids incorporated into isolated reaction centers (Boucher et al., 1977) or a carotenoidless B850 complex (Davidson and Cogdell, 1981) also protect against this photodynamic reaction. The BChl absorption of WT R. rubrum chromatophores demonstrated enhanced stability relative to the G9 carotenoidless strain (Fig. 8). Following this trend, reconstituted WT LHI of R. rubrum exhibited greater stability than LHI reconstituted without carotenoid.

Hybrid Reconstitutions

The inability of spirilloxanthin to incorporate with R. sphaeroides polypeptides was not unexpected, based on previously published studies (see Table 2). Markedly different results were obtained for the converse experiment; spheroidene from R. sphaeroides was readily incorporated into reconstituted WT LHI with R. rubrum polypeptides and BChla. Carotenoid addition restored WT LHI BChla spectral properties, and the fluorescence excitation spectrum revealed an efficiency of energy transfer from spheroidene to BChla of approximately 58%. In contrast, native R. rubrum RC and LH complexes containing spirilloxanthin yielded only 30-35% efficient singlet energy transfer (Table 2). Despite such a high excitation energy transfer efficiency, the carotenoid absorption bands exhibited only a small red shift. The resultant carotenoid CD spectrum, although reduced in rotational strength, revealed a first derivative spheroidene-like spectrum.

Not surprisingly, a number of factors must be considered when discussing these energy transfer results. Carotenoid absorption properties are affected by the chemical structure and conformation of the carotenoid as well as by the protein environment. For example, the conjugation length affects the energy states of the carotenoid species (Noguchi et al., 1990; DeCoster et al., 1992). The two energy states of relevance to singlet energy transfer to BChl are the low energy, long lifetime 2A(g) state and the high energy, short-lived 1B(u) state. The locations of these energy states are currently the subject of intensive study (Cogdell et al., 1992; Gillbro et al., 1993; Frank et al., 1993). To some extent, the chemical structure can be of limited importance as exemplified by the number of different bacteria expressing a variety of carotenoid types yet yielding quite similar carotenoid to BChla energy transfer characteristics (Cogdell et al., 1981; van Grondelle et al., 1982; Angerhofer et al., 1986). The reconstitution assay described here provides ideal conditions to further characterize the relative contributions of chemical structure and protein on carotenoid binding in that WT LHI complexes are formed from only a minimum of pure components.

LHI Carotenoid Environment

Only a limited amount of information has been accumulated regarding carotenoid binding within LH complexes. Most investigations have dealt with LHII complexes due to their ease of purification relative to LHI. That the carotenoid must either be removed prior to LHI dissociation or is lost upon detergent solubilization has been repeatedly demonstrated (Miller et al., 1987; Visschers et al., 1992; Jirsakova and Reiss-Husson, 1993). The lack of demonstrable carotenoid binding upon formation of a subunit complex in this study is consistent with the suggestion that the spirilloxanthin binding site in R. rubrum LHI appears to be the result of interactions between multiple copies of the alpha- and/or beta-polypeptides (Gingras and Picorel, 1990).

Resonance Raman data have shown that LH carotenoids adopt an all-trans configuration relative to the 15,15`-cis configuration found for RC carotenoids (Lutz et al., 1978; Koyama et al., 1982; Iwata et al., 1985). Pronase sensitivity of R. capsulatus carotenoid light-induced absorption shifts (Symons and Swenson, 1983) and surface-enhanced resonance Raman spectra of R. sphaeroides and R. rubrum photosynthetic membranes have predicted that one end of the 40-Å-long all-trans carotenoid is close to the cytoplasmic surface of the membrane (Picorel et al., 1988, 1990). While the all-trans configuration restricts the possible orientations of the carotenoid in the membrane, structural subtleties have been revealed upon closer inspection of the bound carotenoid structure. For example, the carotenoid may exhibit a degree of curvature as opposed to a completely planar structure, as indicated by the crystal structure of beta-carotene (Sterling, 1964). Furthermore, resonance Raman data reveals vibrational modes consistent with chain distortions of carotenoids bound to LH complexes of several different bacteria (Noguchi et al., 1990). Linear dichroism measurements suggest that the average orientation of carotenoids in chromatophores is approximately 50° tilted relative to the membrane plane (Breton, 1974; Bolt and Sauer, 1979). The near in vivo-like BChl and carotenoid absorption band shifts and CD spectra along with the excitation spectra obtained in this study indicate that similar orientations between the carotenoid molecular axis and those of the alpha- and beta-polypeptides must also exist in reconstituted WT LHI complexes.

Conclusions

These results provide evidence that within the reconstituted WT LHI complex, native carotenoid binding is reproduced to give appropriate spectral and functional characteristics. The only components necessary are purified alpha- and beta-polypeptides, BChla, and carotenoid. Thus, this study represents the first reconsititution from individual components of a native-like LHI from a photosynthetic bacterium. Not only did native carotenoids transfer singlet energy to BChl in vitro, but heterologous combinations of carotenoids and polypeptides also proved instructive. In particular, the association of spheroidene with R. rubrum polypeptides yielded singlet energy transfer efficiencies reminiscent of spheroidene in a native environment. Such a comparison between two divergent bacteria such as R. rubrum and R. sphaeroides reveals quite similar BChl and carotenoid binding sites. Nevertheless, the LHI complexes of R. sphaeroides and R. rubrum are unique because of differences in BChl CD strength and pigment stoichiometry (Bolt et al., 1981a). The ability to reconstitute nonnative carotenoids allows a further analysis of the requirements for functional carotenoid binding.


FOOTNOTES

*
This work was supported by grant GM 11741 from the United States Public Health Service (to P. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Biochemistry, Molecular Biology, and Cell Biology, 2153 Sheridan Rd., Northwestern University, Evanston, IL, 60208-3500.

(^1)
The abbreviations used are: BChl, bacteriochlorophyll; BChla, bacteriochlorophyll a; BChla, bacteriochlorophyll a, esterified with geranylgeraniol, isolated from R. rubrum; BChla(p), bacteriochlorophyll a, esterified with phytol, isolated from R. sphaeroides; HPLC, high performance liquid chromatography; LH, light-harvesting complex; native LHI, light-harvesting complex I present in chromatophores of a carotenoidless mutant strain; native WT LHI, light-harvesting complex I present in chromatophores of a carotenoid-containing strain; OG, n-octyl beta-D-glucopyranoside; reconstituted LHI, light-harvesting I complex formed with alpha- and beta-polypeptides and BChla (also termed B875); reconstituted WT LHI, light-harvesting complex I formed from alpha- and beta-polypeptides, BChla, and carotenoid; RC, photosynthetic reaction center; TLC, thin-layer chromatography; WT, wild-type.

(^2)
Presented at the 18th Annual Midwest Photosynthesis Conference, October, 1992, Marshall, IN.


ACKNOWLEDGEMENTS

We thank Walter Fast for performing the initial experiments of carotenoid addition to the isolated subunit complex as well as individually isolated components of R. rubrum. We also thank Drs. Ruby and Robert MacDonald for the generous use of their spectrofluorimeter.


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