(Received for publication, October 12, 1994; and in revised form, January 4, 1995)
From the
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.
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) ()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
state to the BChl Q
transition or from the 1B
state to the Q
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
and
, BChl, and carotenoid. LHI polypeptides bind two BChl
and one or two carotenoids per
-
pair, while LHII
polypeptides bind three BChl and 1.5 carotenoid molecules per
-
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 - and
-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 - and
-polypeptides of both R.
sphaeroides or R. rubrum with BChla and the other native
pigment of LHI complexes, the carotenoid.
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 column (HCODS, 5 µm,
300 Å, 4.6
150 mm) preceded by an Alltech C
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
and
were 16.2 and 18.2 min, respectively; R. rubrum
and
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
, 300 Å, 15 µm, 8
100 mm) placed in an RCM 8
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
and
eluted at 21.6 and 24.0 min,
respectively, while R. rubrum
and
eluted at 34.7
and 21.2 min, respectively.
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 (4.6 250 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.
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 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)
100
was plotted. Excitation spectra were normalized to fractional
absorption spectra in the region of the BChla Q
band near
590 nm, based on 100% efficient energy transfer from BChla
(Q
) to BChla (Q
) (Goedheer, 1959). Efficiencies
of energy transfer were calculated from the ratio of fluorescence
excitation intensities to the fractional absorption intensities at
several wavelengths.
The long wavelength complex formed by reconstitution of LHI
- and
-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.
Figure 1:
Absorption (A) and CD (B) spectra of reconstituted LHI of R. sphaeroides. A, LHI prepared with R. sphaeroides - and
-polypeptides and BChla
was chilled overnight at 5
°C in 50 mM phosphate buffer (pH 7.5) containing 5 mM MgSO
and 0.60% OG. At 0.60% OG, the
- and
-polypeptide concentrations were 2.0 µM and 2.2
µM, respectively; the BChla
concentration was
1.5 µM. Absorbance at 873 nm was normalized to 0.1
cm
. In addition to the Q
transition at
873 nm, the other absorption bands present are the Q
transition (589 nm) and the Soret band (375 nm) of
BChla
. 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 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 concentration was 1.9 µM; the
- and
-polypeptide concentrations were 2.9 µM and 3.3 µM, respectively. The 0.70 and 0.60% OG
spectra were normalized to the Q
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
; 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
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).
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 - and
-polypeptides, BChla
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
, 2.2
µM
, 1.2 µM BChla
, 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
, 2.5 µM
, 1.0 µM BChla
(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 Q
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
- and
-polypeptides and BChla
(see
``Discussion'' for further analysis of carotenoid band
shifts). The peak wavelengths of both BChla
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 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
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).
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 - and
-polypeptides, BChla
(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
, 2.5
µM
, 0.8 µM BChla
, 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 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
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.
Spheroidenone purified by TLC or HPLC was tested in reconstitutions
with - and
-polypeptides and BChla
. 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
remained at 873 nm
(data not shown). Despite the lack of a BChla
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).
A slightly
different BChla Q
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
- and
-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
- and
-polypeptides, BChla
and unpurified
carotenoid extract of R. rubrum chromatophores. LHI was
prepared with R. rubrum
- and
-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
and
, 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 (10
M)
and ascorbate (10
M) 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 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.
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.
, R.
rubrum wild-type chromatophores;
, R. rubrum LHI
reconstituted with spirilloxanthin supplied as a petroleum ether
extract of chromatophores;
, 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.
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 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 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
NaS
O
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 - and
-polypeptides and
BChla
of R. rubrum with the R. sphaeroides carotenoid, spheroidene. The concentrations of the constituents
were as follows: 1.8 µM each
and
, 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
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
band, due to the excess of unbound spheroidene
contributing to the carotenoid absorption
spectrum.
The criteria used to establish efficacious carotenoid
reconstitution are as follows: 1) BChla Q absorption shift
further red by 7-14 nm than obtained for reconstituted LHI
without carotenoid, 2) red shift of BChla Q
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.
Carotenoid absorption
maxima undergo pronounced red shifts upon formation of specific
complexes with light-harvesting polypeptides and BChla. These 1B 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 -
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).
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.
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 state and the high energy,
short-lived 1B
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.
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 -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
- and
-polypeptides must also exist in
reconstituted WT LHI complexes.