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INTRODUCTION |
Elucidation of the structural basis of energy transduction in the
cytochrome bc complexes is proceeding rapidly. High
resolution structure data from x-ray diffraction have been obtained for
the 11 subunits of the integral cytochrome bc1
complex from the bovine respiratory chain (1-3). For the cytochrome
b6f complex, atomic structures (<2.0-Å
resolution) have been obtained for the lumen side domains of cytochrome
f (4-5) and the Rieske iron-sulfur protein (6). It is clear
that along with many similarities between the
b6f and bc1 complexes
(7), there are significant differences in structure, now most obvious
in the comparison of the structures of cytochromes f and
c1 (Refs. 2 and 3 versus 4). The
presence of stoichiometrically bound pigment molecules in the
cytochrome b6f complex, discussed in the present
work, is another contrasting aspect.
The electronic connection between the reducing and oxidizing sides of
photosystems II and I, provided by the cytochrome
b6f complex, through which the proton
electrochemical potential is generated, is traditionally considered
part of the "dark" reactions of oxygenic photosynthesis (8). Thus,
the finding of a molecule of the light-absorbing pigment, chlorophyll
a, to be present at an approximate 1:1 stoichiometry in
active cytochrome b6f complex after exhaustive
chromatography, was unexpected (9). In addition to the unit
stoichiometry, a specific binding site for the chlorophyll molecule in
the complex was implied by the dichroism of the bound chlorophyll
a in oriented monomeric b6f complex
isolated without the Rieske protein from the cyanobacterium,
Synechocystis sp. PCC 6803 (10). A specific binding site of
a chlorophyll a in the complex from the green alga,
Chlamydomonas reinhardtii, was implied by spectroscopic
identification of a specific hydrogen-bonding mode of the chlorophyll
and slow rates of exchange of the b6f-bound Chl1 with
3[H]Chl in detergent micelles (11).
The conclusion that the chlorophyll a molecule is bound at a
specific site in the b6f complex raises the
questions of where the two chlorophyll molecules per dimer are located
and the functional role of the pigment molecules in the complex. It has
been proposed that the function of the chlorophyll a may be
to stabilize the dimeric form of the b6f complex
(12). The presence of a chlorophyll molecule also implies that there
should be a neighboring carotenoid to quench the chlorophyll triplet
state that, otherwise, will inevitably lead to photodamage through
formation of singlet oxygen (13). It is reported in the present study
that
-carotene is present in the cytochrome
b6f complex of the cyanobacterium,
Mastigocladus laminosus, at a unit stoichiometry relative to
cytochrome f, approximately equivalent to that of the bound
chlorophyll a. The Chl a in the M. laminosus b6f complex, compared with the complex
isolated from C. reinhardtii with half the
-carotene
content, is twice as resistant to bleaching by actinic light, which was
found to be O2-dependent.
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MATERIALS AND METHODS |
Isolation and Purification of Cytochrome b6f
Complex
The b6f complex was purified from spinach
chloroplasts and C. reinhardtii according to the procedures
described previously (9) and from cyanobacteria as described (14). The
differences in the latter procedure are the use of a propyl-agarose
column before the sucrose gradient and of dodecylmaltoside in the
sucrose gradient step.
Determination of Chlorophyll and Carotenoid Concentration
Pigment Extraction--
Pigments were extracted with 80%
acetone, 20% methanol at 4 °C under dim light (15), vortexed (1 min), sonicated (2 min), and centrifuged at 5,000 × g
(10 min). The supernatant was decanted and filtered through a Millex
SLCR L04 NS (Millipore) 0.5-µm filter. A second extraction was
carried out with 80% acetone, 20% methanol to ensure complete
extraction. Because of the prominence of the spectrum of the bound
chlorophyll a,
mM = 79 for the absorbance peak at 669 nm (16), the efficiency of extraction based on
the absorbance of first and second extracts could be determined to
be > 98%. The second extract was colorless by visual inspection.
HPLC Separation
The pigments were resolved on
HPLC following procedures in (17), using a Microsorb-MV C-18 column
(Rainin Instruments ODS (4.6 mm, inner diameter × 25 cm)), and
eluted isocratically (0--
4 min) with acetonitrile/Tris-HCl (0.1 M, pH 8.0), 80:3 (v/v), and then by a 4.5-min linear
gradient to methanol:hexane, 4:1 (v/v). All solvents were filtered
through GH-Polypro 0.2-µm filters (Gelman Sciences). Sample
injections were 20 µl, the flow-rate for all separations was 2 ml/min, and the eluent was monitored at 440 nm.
Pigment Identification and Calibration--
Pigments were
identified by UV-visible spectrophotometry and mass spectrometry. For
visible spectra, chlorophyll a and
-carotene fractions
were collected from the HPLC column, dried under an N2
stream, the fractions resolubilized in methanol and hexane, respectively, and their absorption spectra recorded on a Cary 3 UV-visible spectrophotometer. The pigment concentrations were calculated from the visible spectra using the extinction coefficients for chlorophyll a,
mM
(A665-A750) = 71.4 in methanol (16), and for
-carotene,
mM
(445 nm) = 134 (18). The pigment content was quantitated by measurement
of the area under the peaks in reverse-phase HPLC corresponding to
addition of known amounts of pigment standards, and the standard
samples were diluted into 80-20% acetone-methanol. The two separated
components from the HPLC column were collected, dried under a nitrogen
stream, redissolved in methanol (Chl a) and
hexane (carotenoids), and their concentrations determined from their
visible spectra. The two methods agreed within approximately 3%.
Mass Spectroscopy
Mass spectra were obtained at the Nebraska Center for Mass
Spectrometry by high resolution fast atom bombardment using Micromass AutoSpec double focusing-liquid secondary ion mass spectrometry. Neat
samples were dissolved in 1-2 µl of matrix (3-nitrobenzyl alcohol
doped with LiI) on the probe tip. Mass calibration was achieved by
using ions of known mass present in a saturated solution of CsI in
glycerol. The putative chlorophyll a sample in the
3-nitrobenzyl alcohol matrix produced a mixture of M+ and
(M-H)+ ions with a mass/charge ratio = 892 and 893, respectively. The mass of M+, determined with high
precision, was 892.5421 Da and that for the putative
-carotene
sample was determined to be 536.4371.
Determination of Cytochrome Concentration
The cytochrome content was determined from difference spectra of
the cytochrome b6f complex, ~0.1 mg/ml in 30 mM Tris-HCl, pH 7.5, 20 mM NaCl, 30 mM
-D-octyl glucoside, using a Cary 3 UV-visible spectrophotometer with a measuring beam half-bandwidth of
2.0 nm. Ferricyanide, ascorbate, and dithionite were used as redox
agents. A reduced minus oxidized 
mM = 26 mM
1 cm
1 (19) of cytochrome
f at 554 nm, relative to the isosbestic wavelengths at 534, 544, and 561 nm, was used to determine cytochrome concentrations.
Fluorescence Excitation and Energy Transfer Spectra
Room temperature fluorescence excitation and emission spectra of
the chlorophyll a in the M. laminosus cytochrome
b6f complex, at a cytochrome f
concentration of 0.3-1 µM in 30 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 25 mM
-D-octyl glucoside, were measured with an
SLM-Aminco 8000 spectrofluorimeter thermostatted at 20 °C. The
excitation and emission wavelengths, for measurements of emission and
excitation spectra, respectively, were 440 and 676 nm, with
bandwidths of 4 and 8 nm, respectively.
Photobleaching of Chl a in the cyt b6f Complex
A stirred 1-ml cyt b6f sample, ~1
µM cyt f in 25 mM Tris-HCl, pH
7.5, 1 mM MgCl2, 0.02%
-D-dodecyl maltoside, was illuminated with 1.6 × 106 µE/m2 of white light filtered through a
12.5-cm 1% CuSO4 solution as a heat filter. 0.2 ml of
sample were taken at different time intervals and diluted 5-fold into
the same buffer. The chlorophyll a visible spectrum was
measured with a Cary 3 UV-visible spectrophotometer. The photobleaching
was measured in the presence and relative absence of air, in the latter
case by de-aerating the stirred sample on ice for 2 h with
N2 gas, adding the b6f complex to
the degassed buffer with a syringe, and then flushing the air space
above the sample with N2 gas before and during illumination.
Circular Dichroism Spectra
Circular dichroism (CD) spectra were measured on a JASCO J600
spectropolarimeter. Spectral parameters were as follows: sampling, 0.2-nm wavelength increments; time constant, 2 s; spectral
half-bandwidth, 2 nm; optical path length, 1 cm; spectra, average of 4 scans after subtraction of the buffer blank. Cyt
b6f (6 µM) from spinach thylakoid membranes was suspended in 20 mM MOPS, pH 7.2, 50 mM NaCl, and 20 mM
n-octyl-
-D-glucoside.
 |
RESULTS |
Characterization of the Cytochrome b6f
Complex--
The cytochrome b6f complex
contains the four "large" petA-D gene products, cytochrome
f, cyt b6, the Rieske protein, and
subunit IV, seen in the first dimension SDS-PAGE (9, 20) and three "small" polypeptides, the petG, M, and L hydrophobic polypeptides (21-24), separated on SDS-PAGE in the second dimension after blue native gel electrophoresis (8). The turnover of the chloroplast b6f complex used in the present work was
approximately 100, 150, and 450 electrons transferred from
decyl-plastoquinol to plastocyanin-ferricyanide per cyt f
per second at 25 and 28 °C, respectively, for the complex from
spinach chloroplasts (9), C. reinhardtii and the
thermophilic cyanobacterium, M. laminosus.
The ratio of f to b heme in the complex from
spinach chloroplasts (Fig. 1A)
C. reinhardtii or M. laminosus (not shown) was determined from the amplitudes in the cytochrome
-band region of the
chemical difference spectra, ascorbate minus ferricyanide for cyt
f (Fig. 1A, a) and dithionite minus
ascorbate for cytochrome b6 (Fig. 1A,
b), whose ratio was 1:2. The concentration of cytochrome f and the complex were determined using the extinction
coefficient,
mM = 26 (19). Although it has
not been separately determined, we infer from the ratio of amplitudes
and from the known structural requirement for two
b hemes in bc complexes (1-3) that
their average extinction coefficient is also approximately 24 mM
1 cm
1 (19).

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Fig. 1.
Room temperature absorbance spectra of
cytochrome b6f from spinach chloroplasts.
A, difference spectra of cytochrome
b6f complex (~1 µM in 30 mM Tris-HCl, pH 7.5, 20 mM NaCl, 30 mM octyl glucoside) showing amplitude of cyt f
(a) and b6 (b) obtained as
described under "Materials and Methods." Measuring beam
half-bandwidth, 2.0 nm. Ferricyanide (0.1 mM) and excess
dry ascorbate and dithionite were used, respectively, as oxidant and
reductants. B, a, absolute visible absorbance
spectrum of the cyt b6f complex, 1 µM cyt f. The spectral bands associated with
the presence of carotenoid and chlorophyll a are marked.
B, b, inset, room temperature
fluorescence spectra of M. laminosus cytochrome
b6f complex. The concentration of
b6f complex was 0.3 µM in 30 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 0.2% -D-dodecyl
maltoside. The excitation wavelength was 440 nm with half-bandwidths 4 and 8 nm, respectively, for excitation and emission. The putative
excitation band in the region of -carotene absorbance is marked
( ).
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Pigment Content of the Cytochrome b6f Complex--
The
presence of carotenoid, as well as the chlorophyll a already
noted and characterized (9-11, 25), can be seen in the absolute
absorbance spectrum of the complex from spinach chloroplasts (Fig.
1B). An essentially identical spectrum was obtained for the
complex isolated from M. laminosus (not shown). Extraction of all pigments from the b6f complex was carried
out using 80% acetone, 20% methanol at 4 °C under dim light. Total
extraction (>98%) was determined as described under "Materials and
Methods." The pigments from the chloroplast (Fig.
2A), M. laminosus
(Fig. 2B), and C. reinhardtii (not shown)
b6f complex were separated into two major
components by reverse phase HPLC in hexane:methanol, 4:1, detected by
their absorbance at 440 nm, whose visible and mass spectra are
diagnostic of chlorophyll a and
-carotene. The visible
absorbance spectra of the two HPLC peaks from the chloroplast b6f complex (Fig.
3) are characteristic, respectively, of
-carotene (Fig. 3A; Ref. 26) and chlorophyll a
(Fig. 3B). Essentially identical spectra were obtained for
the HPLC-separated (Fig. 2B) pigment components of M. laminosus (not shown). Through the features of the major and minor
peak positions at 434 and 473 nm, the spectrum for
-carotene (Fig.
3A) most closely resembles a 9-cis or
15-cis
-carotene (26). The mass of the isolated pigments
was determined by fast atom bombardment ionization mass spectrometry to
be 892.5421 and 536.4371, respectively (cf. "Materials and
Methods"), for the two pigment components shown to be separated by
reverse phase HPLC in Fig. 2A. The calculated molecular
masses of chlorophyll a
(C55H72N4O5Mg) and
-carotene (C40H56) are 892.96 and 536.85, leading to an absolute assignment of chlorophyll a and
-carotene for the two isolated pigments.

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Fig. 2.
HPLC separation of pigments from cyt
b6f complex as described under "Materials and
Methods." A, from spinach chloroplasts; B,
from M. laminosus. The ratios of the areas of peak
1 to peak 2 in panels A and B are
0.64:1 and 0.68:1, respectively.
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Fig. 3.
Room temperature absorption spectra of
HPLC-purified (A) -carotene dissolved in 100% hexane
and (B) chlorophyll a dissolved in 100%
methanol, both pigments obtained from spinach cytochrome
b6f complex. The spectrum (A)
is characteristic of 9-cis or 15-cis- -carotene
(26).
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The concentration of the pigment components separated by HPLC was
determined using the areas under the peaks, from addition of known
concentrations of pigment standards, and by collecting and drying each
of the two components, redissolving them in methanol (Chl) and hexane
(
-carotene), and determining their spectra, checked through the
extinction coefficients (
mM
(A665-A750)) of 71.4 mM
1 for chlorophyll a in
methanol and 134 mM
1 cm
1 at 445 nm for
-carotene in 100% hexane. Using the differential extinction
coefficient, 
mM = 26 mM
1 cm
1, for cytochrome
f, the stoichiometry of the chlorophyll
a and
-carotene relative to cytochrome f is
0.97 ± 0.13 and 0.77 ± 0.07, respectively, in the
chloroplast complex (Table 1, spinach chloroplasts), 1.65 ± 0.3 and 1.02 ± 0.1 in that from M. laminosus (Table 1),
and 1.37 ± 0.3 and 0.55 ± 0.08 in C. reinhardtii. Recalculation of the data of Huang et al.
(9) and Pierre et al. (11), using the recently redetermined

mM = 26 mM
1
cm
1 for cyt f yields values of
the Chl a/cyt f ratio of 1-1.2 and 1.1-1.6,
respectively. This eliminates any problem of the significance of a
stoichiometry smaller than 1:1 (25), but raises the possibility of
either a second bound chlorophyll a or a small amount of
nonspecifically bound chlorophyll. The latter is a crucial issue in
studies of the fluorescence properties of the specifically bound
chlorophyll a. Recalculation of the
-carotene/cyt
f stoichiometry previously reported (11) using the cyt
f extinction coefficient determined previously (19) yields a
value of 0.38-0.55, close to the value found in the present study.
Possible Inter-pigment Interactions--
Because the fluorescence
yield and lifetime of
-carotene are small (27), these two pigments
have to be located within a small distance of each other for energy
transfer from
-carotene to the chlorophyll a to occur.
The excitation spectrum for chlorophyll a fluorescence at
676 nm from the b6f complex shows a broad band of small amplitude centered near 490 nm (Fig. 1B,
inset, spectrum (b)). This band is absent in a
solution mixture of chlorophyll a and
-carotene (not
shown). Such a band was absent in the excitation spectrum for
chlorophyll a fluorescence at low temperature (77 K) for the
complex from the green alga C. reinhardtii (11). In the
latter work, the issue of a carotenoid excitation band was not
specifically addressed because the carotenoid content was found to be
less than stoichiometric.
Photobleaching of the Chlorophyll a, Dependence on
-Carotene
Content--
The existence of three preparations from different
sources that have different contents and stoichiometries of
-carotene to cytochrome f creates the possibility of
testing the hypothesis that one of the functions of the
-carotene,
as in the photosynthetic antenna proteins, is to protect the
chlorophyll a from photodamage. Such damage can arise from
production of chemically reactive singlet oxygen because of the
interaction of ground state triplet oxygen with the excited state (*)
chlorophyll triplet (13) shown.
-carotene (Car) can serve as an alternative acceptor of the
excited state chlorophyll triplet, preventing the production of excited
singlet oxygen,
1O2*.
Significant photobleaching of the Chl a in the C. reinhardtii b6f complex could be observed after a
10-min exposure to heat-filtered white light with an intensity of
2.7 × 103 µE/m2 s (Fig.
4A, b, dashed
spectrum, compared with the spectrum of unilluminated sample, Fig.
4A, a, solid curve). The residual
spectrum in the 400-450 nm region (Fig. 4A, b,
dashed spectrum) arises mostly from the Soret bands of
cytochrome f and the two hemes of cytochrome
b6 and explains why the amplitude of the
-carotene near 500 nm appears relatively small compared with that in
the Soret region arising from one-two chlorophylls and three hemes from
cytochromes b6 and f (Fig.
1B).

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Fig. 4.
Bleaching of Chl a in the
cytochrome b6f complex as a function of
incident light energy. A, visible absorption spectra of
Chl a in the cytochrome b6f complex
of C. reinhardtii, solid line, before
(a) and after (b) illumination for 10 min with
1.6 × 106 µE/m2 of white light filtered
through 12.5 cm of 1% CaSO4 solution as a heat filter.
B, stability of the Chl a molecule in the
cytochrome b6f complex as a function of
illumination energy with heat-filtered white light at an intensity of
2.7 × 103 µE/m2 s, after which the
residual absorbance of the Chl 676-nm band was 92, 70, and 46%.
b6f complex from M. laminosus
(a), spinach chloroplasts (b), and C. reinhardtii (c).
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The time course of the bleaching of the Chl a in C. reinhardtii is shown (Fig. 4B, curve c).
Also shown in Fig. 4B is the time course of the bleaching of
the Chl a in the b6f complex of M. laminosus (curve a) and spinach chloroplasts
(curve b). It can be seen that the bleaching of Chl
a in the M. laminosus complex is very small. The
residual absorbance of the 676 nm Chl a band, after the
10-min illumination (1.7 × 106 µE/m2)
was 92 ± 2%, 70 ± 5%, and 46 ± 10%
(n = 3), respectively, for the complex from M. laminosus, spinach chloroplasts, and C. reinhardtii (Fig. 4B). The relative magnitude of this residual
absorbance for the Chl a from the b6f
complex from these three sources was 1.0, 0.76, 0.50, almost
proportional to the respective stoichiometries of
-carotene to
cytochrome f (Table I). The
bleaching of the Chl a band in the C. reinhardtii
complex was reduced substantially, the residual absorbance increasing
from 47 ± 8 to 74 ± 4% (n = 3) when the
sample was equilibrated with N2 instead of air before exposure to the actinic light (Fig. 4B,
, linked by
arrow, showing the change in photobleaching of the
b6f complex from C. reinhardtii).
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Table I
Pigment content (mol/mol) of purified cytochrome b6f complex
from spinach chloroplasts, M. laminosus, and Chlamydomonas reinhardtii
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Circular Dichroism Spectra Show
-Carotene Is Bound in an
Asymmetric Environment--
Carotenoids in a symmetric environment in
organic solvent show no optical activity (26). CD spectra of
-carotene in the spinach chloroplast b6f
complex have minima at 430, 457, and 485 nm (Fig.
5), reasonably close to the absorbance
peaks at 422, 451, and 478 nm for
-carotene in 100% hexane (Fig.
3A). In a somewhat more polar solvent, 80% acetone, these
peaks are red-shifted to 429, 456, and 483 nm, very close to the
extrema in the CD spectrum. The CD spectra are indicative of an
asymmetric binding environment of the
-carotene in the complex. The
ratio of the ellipticity peak of the
-carotene at 457 nm to that of
the Chl a at 670 nm is 4.6:1. This ratio in the CP43
light-harvesting complex, which contains chlorophyll a and
-carotene in a ratio of 4:1, is 1:1 (28). It is concluded that all
of the bound
-carotene is bound in the complex in an asymmetric
environment.

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Fig. 5.
Circular dichroism spectrum of the cyt
b6f complex at room temperature. Cyt
b6f complex from spinach chloroplasts was in 20 mM MOPS, pH 7.2, 50 mM NaCl, 20 mM
octyl glucoside corresponding to an absorbance of 0.7 for Chl
a at 672 nm.
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 |
DISCUSSION |
Function of b6f-bound Pigments--
Carotenoids can
have multiple roles in light-harvesting and oxygen-related reactions of
photosynthesis (29, 30): (a) light harvesting via energy
transfer to chlorophyll, (b) photoprotection via quenching
of chlorophyll excited triplet states, (c) excess excited
state energy dissipation, (d) singlet oxygen scavenging, and
(e) structure stabilization (31). A possible function of
-carotene as a radical-trapping antioxidant that can limit the damage induced by oxy-radical generating systems (32) seems unlikely
for a stoichiometrically bound
-carotene because it would involve
chemical modification of the carotene. For chlorophylls, the range of
possibilities for function is smaller. They function, most obviously
(i), in light harvesting, (ii) they could conceivably influence
pathways of electron transfer because of their aromaticity, and (iii),
in addition, they could participate in structure stabilization. The
latter function would involve the long hydrophobic phytyl chain as well
as the bulky ring moiety.
Function related to light-harvesting seems unlikely for the pigments in
the b6f complex. Although this possibility has
been proposed (33) in the context of "super-complexes" that could be formed with the PSI reaction center (34), (a) the
fractional contribution of the b6f-bound pigment
to the total antenna function of such a super-complex is small;
(b) the lack of inhibition of electron transfer through the
b6f complex caused by photobleaching of the
chlorophyll in the C. reinhardtii b6f complex
implies that the chlorophyll does not facilitate a rate-limiting step
of electron transfer (11); and (c) a light energy transfer
function of
-carotene to chlorophyll is either small, as shown in
the present work (Fig. 1B, inset), or absent
(11).
Thus, one is led to think of a function for the
b6f-bound
-carotene in quenching of the
chlorophyll excited triplet state, and protection against the formation
of single O2, and in structure stabilization. The latter
function for the long chain
-carotene and the Chl a with
its long phytyl chain is suggested by the role for
-carotene in
stabilization of the assembly of the D1 protein into a functional
photosystem II complex (31) and of the LHCI polypeptides (35) and, as
well, the proposal that bound chlorophyll a prevents
dissociation of the cytochrome b6f dimer into
monomers (12).
Evidence for a role of the
-carotene in protection against
oxygen-mediated damage presumably involving formation of singlet O2 from the chlorophyll-excited triplet state is provided
in the present work. The extent of the residual absorbance of the
chlorophyll a molecule in the cytochrome
b6f complex after photobleaching (Figs. 4,
A and B) correlates well with the stoichiometry
of
-carotene relative to cytochrome f (Fig.
4B, a-c,
-carotene:cyt
f = 1.0, 0.77, 0.55), and is markedly dependent upon
the ambient O2 concentration (Fig. 4B). One
-carotene per monomer of b6f complex confers
virtually complete protection against photodamage to the 1-2 Chl
a molecules bound per monomer. It is inferred that the lower
content of
-carotene in the complexes isolated from spinach
chloroplast and C. reinhardtii is an artifact of preparation.
The mechanism of protection by
-carotene is inferred to be triplet
transfer from the excited state of the chlorophyll to the
-carotene.
However, triplet transfer to carotenoid was not found in the
b6f complex of the cyanobacterium,
Synechocystis sp. PCC 6803 (36). From the precedents of loss
of chlorophyll upon monomerization or dissociation of the Rieske
protein (12) and of diminished content of
-carotene in the C. reinhardtii complex (Table I), it is possible that the
measurements of triplet transfer to
-carotene in Peterman et
al. (36) were made difficult by a lowered content of
-carotene
and chlorophyll a that is a consequence of the loss of the
Rieske protein in this preparation, which results in complete loss of
activity. Preliminary experiments designed to detect by EPR the
presence of a
-carotene triplet in the b6f
complex following laser excitation of the chlorophyll a did
not reveal any component that could be unambiguously assigned to a
carotenoid triplet with a decay time in the expected 0.5-10 µs time
range.2
Location of b6f-bound Pigments--
The bound
chlorophyll a molecule is inferred to be located close to
the pathway of electron and/or proton transfer to cytochrome f, from the correspondence of the rates of reduction of
cytochrome f and the rise time of an electrochromic band
shift of a chlorophyll a (37). This is consistent with the
tendency of the chlorophyll molecules in the LHCII protein structure to
be localized near the membrane interfacial domain (38). The LHCII
structure also provides a precedent for glutamate residues serving as
chlorophyll ligands. The carboxylate residue on the lumen side of the
small hydrophobic petG, L, and M polypeptides could provide such a
residue as a ligand for the b6f-bound
chlorophyll. These polypeptides are uniquely present, along with the
bound pigments, in the cytochrome b6f compared
with the bc1 complex. It has been proposed that
the Chl-specific binding site is associated with the cytochrome
b6 polypeptide (39). However, because the
stoichiometry of this association was approximately only 10%, the
association with cyt b6 might be a consequence
of preferential and adventitious binding of the chlorophyll with the
most hydrophobic polypeptide of the complex. From the present studies
on the role of
-carotene in protecting the Chl a in the
b6f complex from
O2-dependent photobleaching, it is inferred
that the
-carotene is positioned close to the chlorophyll
a and the membrane polar interface (Fig.
6).

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Fig. 6.
Schematic of cytochrome b6f
complex, assuming a total of 12 transmembrane helices/monomer (4, cyt b6 (pink); 3, suIV
(yellow); 1, cyt f (green); 1, Rieske iron-sulfur protein (red); pet G, M, and L
(brown)). Approximate shapes and sizes of lumen-side
extrinsic domains of cyt f and Rieske [2Fe-2S] protein are
from solved structures (4, 6). A quinone, chlorophyll, and carotenoid
are shown in the bilayer region. The position of the chlorophyll
a shown in the excited triplet state that is believed to be
quenched by -carotene and the -carotene ring near the interface
is based on the precedent for the position of Chl a
molecules in LHCII (38) and kinetic correlation of an electrochromic
shift of the Chl a with cyt f reduction
(37).
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Differences between b6f and bc1 Complex,
Significance of Absence of Pigments in the Photosynthetic
bc1 Complex--
The cytochrome b6f
complex is similar in many respects to the bc1
complex of the mitochondrial respiratory chain and photosynthetic bacteria, to which it is known to be related in many structure-function aspects. The possibility of major differences between the complexes has
recently become clear with the discovery that cytochromes f
and c1 are completely dissimilar proteins, and
provide an excellent example of divergent functional evolution in
membrane proteins.
A major role for oxygen scavenging in oxygenic photosynthetic membranes
would explain the apparent absence of pigments in the cytochrome
bc1 complex of mitochondria and photosynthetic bacteria. Singlet oxygen, which readily causes oxidative damage at
conjugated bonds, e.g. unsaturated fatty acids and aromatic amino acids (40), can be readily produced by the reaction of ground
state triplet oxygen with the excited triplet state of many dyes and
pigments. It is produced in PSII, presumably by oxygen quenching of the
triplet-excited state of the reaction center, 3P680* (41).
-carotene is known to be an extremely efficient quencher of singlet
oxygen, able to quench at a rate that is diffusion-controlled (40). Two
molecules of
-carotene per monomer can quench singlet oxygen in
isolated photosystem II reaction centers (41).
We infer that the oxygen produced in the PSII region of the membrane
will readily diffuse because it is highly soluble in the non-polar
membrane and must be present, perhaps at a somewhat lower
concentration, in the region of other thylakoid membrane-bound protein
complexes (Fig. 6). Here it can also inflict similar molecular damage
to lipids and protein. The range of action of the
1O2 depends upon its lifetime and diffusion
rate in the membrane. The lifetime of 1O2 in
dioxane, whose dielectric constant (
= 2.2) is similar to that of
the lipid bilayer core, is 30 µs and is not very dependent on solvent
polarity (40). If the viscosity of the relatively fluid photosynthetic
membrane is assumed to be at the lower end of the range of viscosities,
0.5-5 poise, of biological membranes (42), the effective diffusion
constant of O2 in the membrane would be
~10
7 cm2 s
1. The root mean
square distance of 1O2 diffusion from its site
of generation in PSII, where the O2 concentration is
highest, is then ~350 Å. This distance is larger than the average
distance between the PSII reaction center and the
b6f complex, which has been estimated to be 200 Å (43).
The data in the present study imply that one function of
-carotene
in the b6f complex is to prevent generation of
singlet O2 from the photoexcited Chl a in the
complex. The role of the chlorophyll itself, presumably structural, is
still unclear. It is suggested that it is also needed to exert a
protective role against the toxicity of photosynthetically produced
O2 toward the cytochrome b6f
complex. One may suggest that all protein complexes within an effective
diffusion distance of PSII in the oxygenic photosynthetic
membrane may require such protection.
The bound chlorophyll and
-carotene may be "evolutionary relics"
that resulted from the appearance of reaction centers prior to that of
quinol oxidoreductases (11). However, there is no report of bound
bacteriochlorophyll or carotenoid in the cyt bc1 complex isolated from photosynthetic bacteria. Therefore, it seems likely that these "relics" are utilized functionally in the
oxygenic photosynthetic membrane. In the case of carotenoids, these
pigment molecules are synthesized not only to assist in light
harvesting but also as a "bonus" (44) to cope with the problem of
oxygen toxicity and structure stabilization in both the
light-dependent and "dark" integral membrane protein
complexes in the membranes of oxygenic photosynthesis.