(Received for publication, December 31, 1996, and in revised form, May 22, 1997)
From the Institut de Biologie Physico-Chimique and
Paris-7 University, CNRS UPR 9052, 13 rue Pierre et Marie Curie,
F-75005 Paris, France, the § Université de Lille and
the Ecole Normale Supérieure, CNRS URA 1810, 44 rue d'Ulm,
F-75005 Paris, France, the ¶ Commissariat à l'Energie
Atomique, CNRS URA 2096, Centre d'Études de Saclay, F-91191 Gif
Sur Yvette, France, and the
CNRS UPR 9061, F-91198 Gif Sur
Yvette, France
Highly purified preparations of cytochrome b6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed.
Cytochrome b6 f, the central complex in the photosynthetic electron transfer chain, receives from plastoquinol (PQH2)1 electrons stripped from water by photosystem II reaction centers and transfers them to plastocyanin, the electron donor to photosystem I. In the process, part of the electron free energy drop is transduced into a proton electrochemical potential gradient (1-5). A homologous complex, cytochrome bc1, carries out an equivalent function in the respiratory chains of mitochondria and many prokaryotes (6, 7). Chlorophyll molecules are part of the two photosystem reaction centers and the associated antenna complexes. In the photosynthetic electron transfer chain, there are 500-1000 chlorophyll (Chl) molecules/b6 f complex (see Refs. 8 and 9, and Table I). It was no surprise, therefore, that traces of Chl should be found in purified b6 f preparations (see e.g. Ref. 10), and they have long been regarded as contaminants. In recent years, however, several reports have noted that, in very pure preparations, the amount of Chl tends to be close to 1/cytochrome f (Cytf) (11-14), raising the intriguing possibility that its presence might not be adventitious (12, 14).
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In the present article, we present further evidence in support of our earlier conclusion that the native b6 f complex from Chlamydomonas reinhardtii comprises 1 molecule of chlorophyll a (Chla) per monomer as an authentic component (14). Namely: (i) free [3H]Chla added to C. reinhardtii thylakoid membranes at the time of solubilization does not associate with the b6 f complex;2 (ii) the rate of exchange of b6 f-associated Chla for free [3H]Chla is extremely slow; and (iii) Chla is bound to the b6 f complex at a single, specific site. Putative functional roles for a chlorophyll in the b6 f complex are examined and some of them are experimentally ruled out.
Decylplastoquinone (C10-PQ), Tricine,
egg yolk L--phosphatidylcholine (PC),
phenylmethylsulfonyl fluoride (PMSF),
-aminocaproic acid,
benzamidine, and sucrose were purchased from Sigma; acetone Uvasol was
from Merck; sodium dodecyl sulfate (SDS) was from Pierce; Hecameg (HG)
was from Vegatec (Villejuif, France); hydroxylapatite (HA) was
from Bio-Rad; dithiothreitol was from Boehringer Mannheim; 3,3
,5,5
-tetramethylbenzidine was from Fluka Chemie AG; urea was from
Tebu; [3H]acetic acid was from ICN; and Aqualuma was from
Packard Instruments.
TMK buffer contained 20 mM Tricine-NaOH,
pH 8.0, 3 mM MgCl2, 3 mM KCl.
TMK-HP buffer contained TMK buffer supplemented with 20 mM
HG and 0.1 g/liter egg PC. AP-HP buffer contained 400 mM ammonium phosphate, pH 8.0, 20 mM HG, 0.1 g/liter egg PC,
protease inhibitors (200 µM PMSF, 1 mM
benzamidine, 5 mM -aminocaproic acid).
Wild-type strain (WT) and
mutant strain LDS (lacking chlorophyll synthesis when grown in the
dark) were kindly provided by J. Girard-Bascou and P. Bennoun (CNRS UPR
9072, Institut de Biologie Physico-Chimique). C. reinhardtii
was grown in Tris acetate-phosphate medium (TAP) (15) at 25 °C under
an illumination of 300-400 lux (WT) or in the dark (LDS) on a rotary
shaker until stationary phase (~107 cells/ml). Cells were
harvested at 5,000 × g for 10 min. Thylakoid membranes
were prepared as described previously (WT, Ref. 16; LDS, Ref. 17),
resuspended in 10 mM Tricine-NaOH, pH 8.0, containing protease inhibitors (200 µM PMSF, 1 mM
benzamidine, 5 mM -aminocaproic acid), and stored at
80 °C. The final concentration of WT membranes was adjusted at 3 mg of Chl/ml. The concentration of LDS etioplast membranes was
estimated from their optical density at 460 nm.
Cytochrome
b6 f complex was purified and
analyzed, and its PQH2-plastocyanin oxidoreductase activity
was determined as described previously (13). UV-visible absorbance
spectra were recorded either on a Kontron Uvikon 930, a Varian Cary
2300, or a Joliot-type homemade spectrophotometer (18), as specified in
the legends to Figs. 2, 3, and 6. Cytf concentrations were determined from the ascorbate-reduced minus ferricyanide-oxidized spectra, using 554 = 18,000 M
1·cm
1 (19);
A554 was measured by reference to a line joining
isosbestic points at 545 and 575 nm. Chl concentrations were determined
from the absorption at 668 nm, using
668 = 75,000 M
1·cm
1 (20; we checked that
the extinction coefficient of Chla bound to the complex is
identical to that in acetone).
Pigment Analysis
Pigments were extracted from thylakoid
membranes or from cytochrome b6 f
preparations by 10 volumes of ice-cold 100% acetone under vigorous
stirring. Precipitated proteins were spun down at 5,000 × g for 10 min. The supernatant was collected, evaporated to
dryness in a glass flask under a flow of N2 and stored at
80 °C. Pigments were first separated by chromatography on
thin-layer silica gel plates according to Eichenberger and Grob (21).
After methanol extraction from the silica powder, each fraction was further purified by reversed-phase HPLC on a Zorbax-ODS column (Rockland Technologies, Inc.; 4.6 × 250 mm, 5 µm granulometry). Elution proceeded in the following three phases: (i) during 8 min,
0.1% methylene chloride in acetonitrile/methanol (70:30 v/v); (ii)
during 4 min, a 0.1-40% (v/v) gradient of methylene chloride in the
same solvent mixture; and (iii) a constant concentration of 40%
methylene chloride in the same mixture. The absorption spectrum of the
eluted fractions was continuously monitored with a Hewlett-Packard 1040 A diode array detector (wavelength range 230-600 nm). The detector
response was calibrated with standards, using extinction coefficients
given by Lichtenthaler (22) for pheophytins and chlorophylls, by
Britton (23) for carotenoids, and by Barr and Crane (24) for
quinones.
Wild-type
C. reinhardtii cells were grown in TAP medium under standard
conditions until stationary phase, diluted 10 times into 200 ml of TAP
medium containing 3.7 GBq of sodium [3H]acetate, and
further grown under about 1000 lux until stationary phase. Cells were
harvested, thylakoid membranes were prepared, and
3H-labeled pigments were separated as described above
(Fig. 1). Their specific activity was
determined by liquid scintillation counting in Aqualuma in a LS1801
counter (Beckman) and spectrometry.
Purification of Cytochrome b6 f Solubilized in the Presence of Radioactive Chlorophyll
A 500-µl sample of C. reinhardtii thylakoid membranes (containing 1.5 mg Chl) in 10 mM Tricine-NaOH buffer, pH 8.0, plus protease inhibitors,
was solubilized by addition of an equal volume of HG 50 mM
in 2× concentrated TMK buffer supplemented with 37 µg of
[3H]Chla (3.25 × 107 cpm).
After 15 min of incubation at 4 °C in the dark and 10 min of
centrifugation at 80,000 rpm (160,000 × g) in the TLA
100.3 rotor of a TL100 ultracentrifuge (Beckman), the supernatant was layered on top of an 11-ml 10-30% (w/w) sucrose gradient in TMK-HP buffer and centrifuged for 24 h at 40,000 rpm (270,000 × g) in the SW41 rotor of an L8 ultracentrifuge (Beckman).
Fractions of 400 µl were collected. The top fractions were analyzed
for Chl and radioactivity. The fractions containing the
b6 f complex were pooled, and the
complex was purified by HA chromatography as described (13). The
specific activity of free Chl was taken to be that of the Chl present
in the uppermost three fractions of the gradient (Fig. 5).
Determination of the Rate of Exchange of b6 f-bound for Free 3H-labeled Chlorophyll
An acetone solution containing 2 nmol of [3H]Chla (~480,000 cpm) was evaporated to dryness under N2. The [3H]Chla was redissolved in 0.5 ml of a 5 µM solution of purified b6 f complex in AP-HP buffer and incubated in the dark under N2 at 4 °C. At time intervals, 0.1-ml aliquots were layered onto 2-ml 10-30% (w/w) sucrose gradients in TMK-HP buffer and centrifuged for 3 h at 55,000 rpm (260,000 × g) in the TLS 55 rotor of a TL100 ultracentrifuge (Beckman) to separate free from b6 f-bound Chl. The brownish band containing the complex was collected, and the specific radioactivity of b6 f-bound Chl was determined and compared with that of the free Chl present at the top of the gradient.
Fluorescence MeasurementsLow-temperature (77 K) fluorescence spectra were recorded on a homemade instrument (see Ref. 25). The exciting beam and the fluorescence emission were passed through a Y-shaped light guide. The sample (~5 µM in AP-HP buffer) was placed in a flat quartz cuvette (0.1-mm light path), immersed in liquid nitrogen, and held at the common end of the guide. The exciting beam wavelength was selected by a monochromator, with a bandwidth set at 3 nm for excitation spectra and 12 nm for emission spectra. The fluorescence emitted by the sample was monitored by a photomultiplier through a monochromator with a bandwidth of 12 nm for excitation spectra and 3 nm for emission spectra. Spectra were recorded as uncorrected responses of the photomultiplier.
Resonance Raman SpectroscopyResonance Raman spectra were recorded with a U1000 Raman spectrometer equipped with a charge-coupled device camera (Jobin-Yvon, France) on pellets of oxidized cytochrome b6 f obtained by ultracentrifugation after exposure to 1.5 mM ferricyanide and dilution under the critical micellar concentration of HG. The 441.6 nm excitation light (less than 15 milliwatts on the sample) was provided by a HeCd continuous laser (Model 4270N, Liconix, CA). To prevent photodegradation of Chla during the experiments, samples were cooled at 77 K in a gas flow cryostat (TBT, France).
Photobleaching of b6 f-bound Chlorophyll aPurified b6 f complex in AP-HP buffer was diluted 10 times into 20 mM HG, 0.1 g/liter PC, to a final concentration of 0.5 µM Cytf, 40 mM AP. A 300-µl sample was irradiated at 4 °C under gentle stirring by the two light beams produced by a KL 1500 lamp (Schott; power set at 3). The white light was filtered by a red Wratten low-pass filter No. 92 (Kodak; cut-off wavelength 620 nm) and by 1-cm plastic cuvettes filled with water.
The b6 f
complex from C. reinhardtii contains seven subunits in
stoichiometric ratio and four identified redox carriers, one
c-type heme, two b-type hemes, and a [2Fe-2S]
cluster (13). In addition to the three cytochromes, UV-visible spectra
of even the most highly purified preparations reveal the presence of
carotenoids (absorbance peaks at ~460 and 483 nm) and of
Chla (peak at 667-668 nm) (13). Within experimental
accuracy, the visible spectrum of the Chl does not depend on the redox
state of the complex (Ref. 13; see Fig. 3A). Using the
in situ extinction coefficient of b6 f-associated Chla
(668 = 75,000 M
1 · cm
1; cf.
"Experimental Procedures") and an extinction coefficient
554 = 18,000 M
1 · cm
1 for
Cytf (19), the Chla/Cytf ratio was
found to be 0.93 ± 0.18 (mean ± S.D. over 26 preparations).
Chemical analysis confirmed that
b6 f preparations contain
essentially pure Chla; Chlb, which makes up to
~30% of Chl in thylakoid membranes from WT C. reinhardtii, represents less than 10% of Chl in
purified b6 f (Table
I). Carotenoids are present in
substoichiometric ratio with respect to Chla, while other
pigments and quinones either are totally absent or are present in trace
amounts (Table I).
The approximate 1:1 molar ratio of Chla to Cytf, the excess of Chla over Chlb, as compared with thylakoid membranes, and the retention of Chla throughout the purification procedure suggest that there exists, on the b6 f complex, one binding site with high affinity and specificity for Chla. However, the average stoichiometry is somewhat smaller than 1:1, and its variation from one preparation to the next tends to be larger than the uncertainty on the measurements would lead one to expect. Several factors may explain the dispersion of the data. (i) Traces of Chl collected from the sucrose gradient and incompletely washed from the hydroxylapatite column may contaminate some preparations; (ii) the b6 f-associated Chl is easily bleached (see below); and (iii) exposure of the complex to detergent micelles tends to release the Chl (26).
The Spectrum of the b6 f-associated Chlorophyll a Is Affected by Its Interactions with the ComplexExposing the complex to an excess of laurylmaltoside (LM) micelles induced a bathochromic shift of the Chla peak by ~2 nm, from 667-668 to 669-670 nm (Fig. 2A). Similar shifts were observed following denaturing treatments, such as heating the preparation at 50 °C or adding 8 M urea, and occurred whether the b6 f complex was in its detergent-solubilized state or reconstituted into lipid vesicles (Fig. 2B).
The spectrum of Chla in b6 f preparations treated with an excess of detergent resembles that of pure Chla dissolved in LM micelles (Fig. 2A), suggesting that this treatment releases Chl from the complex. Delipidation by detergents indeed induces dissociation of the complex into chlorophyll-free monomers (26). However, closer examination reveals that the spectral shift actually precedes Chl dissociation. We show elsewhere that mild treatment of the complex with detergent first generates a dimeric form that has lost the Rieske protein and retains the Chl, while a harsher treatment is required for the complex to release the Chl and break down into monomers (26). Analysis of the visible spectrum of the Chl bound to purified, Rieske-depleted b6 f dimer revealed a red-shifted (and broadened) absorption peak (Fig. 2C), indicating that the environment of the Chl has been affected even though it is still bound to the complex and co-purifies with it (26).
Fluorescence Characteristics of b6 f-associated ChlorophyllInteractions of the Chl with its environment in the b6 f complex were further examined by low temperature fluorescence measurements. Cytochrome b6 f exhibits, in the Soret region, several absorption bands due to the Chl, the carotenoids, and cytochromes f and b6 , the latter bands being modulated by the redox potential (Ref. 13 and Fig. 3A). The possible occurrence of energy transfer between hemes and Chla was examined by analyzing the fluorescence characteristics of the Chl under various redox conditions. At 77 K, excitation at 440 nm in the Chl band of cytochrome b6 f produced an emission of fluorescence with a maximum at 673 nm (Fig. 3B). Excitation spectra of the fluorescence emitted at 673 nm and emission spectra of the fluorescence excited at 440 nm were recorded in the presence or absence of 5 mM ascorbate or ~5 mM sodium dithionite. The effects of these additions on the redox state of the cytochromes were checked by absorption spectroscopy (Fig. 3A). Regardless of the addition, there was no significant change in the fluorescence excitation and emission spectra (Fig. 3C, and data not shown).
Incubation at room temperature for 1 h with 100 mM HG, which is known to induce partial dissociation of the cytochrome b6 f complex (26), did not modify the shape of the fluorescence spectra, but enhanced fluorescence intensity by a factor of ~2 (Fig. 3B). A more limited increase in fluorescence intensity was observed after freezing and thawing the b6 f solution (Table II). A comparison of these fluorescence intensities with that of free Chla in the same buffer is shown in Fig. 3B and Table II. These observations indicate that association of Chla with the b6 f complex results in a redox-independent quenching of its fluorescence (by a factor of ~4), which is partially relieved following detergent treatment.
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The mode of binding of Chla to the
protein was further investigated using resonance Raman spectroscopy. To
detect selective contributions from the Chl molecules present in the
samples, experiments were performed at 441.6 nm excitation wavelength
on oxidized cytochrome b6 f. This
laser line is located on the red side of the Soret electronic
transition of Chla, more than 1500 cm1 away
from the Soret band of the oxidized cytochromes (~413 nm). As
expected, resonance Raman spectra recorded under these conditions led
to barely detectable signals from cytochrome
b6 . Nevertheless, under these conditions of
excitation, intense contributions typical of carotenoid molecules
partially masked the middle frequency modes of Chla (data
not shown). Analysis, therefore, was focused on the high-frequency
region of the spectrum (Fig. 4). Below
1600 cm
1, resonance Raman spectra of Chla
molecules typically feature an intense band at ~1550
cm
1, which has been attributed to complex vibrational
modes of the chlorin ring (27). Between ~1600 and 1710 cm
1, two or three bands may be observed: (i) a band
between 1595 and 1615 cm
1, arising from the stretching
modes of the methine bridges of the molecule (27), the frequency of
which depends on the conformation of the chlorin ring (28) and is thus
sensitive to the coordination state of the central Mg2+ ion
(27); (ii) a band at ~1620 cm
1, arising from the
stretching mode of the conjugated vinyl group in position
C2 (29), and often just appearing as a weak shoulder on the
high frequency side of the methine stretching band (29); and (iii)
between 1640 and 1710 cm
1, bands arising from the
stretching modes of the conjugated 9-keto carbonyl group, the frequency
of which is extremely sensitive to the H-bonding state and to the
environment of this group (27, 30).
In the resonance Raman spectrum of the
b6 f-bound Chl (Fig. 4), the band
arising from the methine bridge stretching modes is observed at 1606 cm1, and is, as expected, asymmetric, because of the
presence of the weak contribution of the vinyl stretching modes. At
higher frequencies, a single band is observed, at a frequency of 1676 cm
1. The full width at half-maximum of these two bands
(14 and ~11 cm
1, respectively) is similar to that
observed in resonance Raman spectra of isolated, monomeric
Chla molecules (~12 cm
1; see Ref. 27). Since
the frequency of the 9-keto carbonyl mode is extremely sensitive to the
environment of this group, this indicates that the binding sites of all
Chla molecules share very similar, if not identical
physicochemical properties. The frequency of this band is as high as
1696 cm
1 when the keto group is free from intermolecular
interactions (27, 30). A 1676 cm
1 frequency unambiguously
indicates that this group is involved in a medium-strength hydrogen
bond.
The 1606 cm1 frequency observed for the methine bridge
stretching mode is somewhat ambiguous with respect to the liganding of
the Mg2+ ion. It could originate either from a
5-coordinated Chla molecule with an unusually planar
conjugated system or from a 6-coordinated molecule slightly distorted
by its proteic environment (28, 31). Imidazole side chains of histidine
residues are known to be particularly strong ligands for the central
magnesium atom of (bacterio)chlorophyll molecules. In most of the
well-documented cases where a magnesium atom interacts with a histidine
residue, the methine bridge band is observed at 1612-1615
cm
1 at low temperature (see Ref. 31, and references
therein). A 1606 cm
1 frequency makes it extremely
unlikely that the Chla of the
b6 f complex interacts with such a
strong ligand.
The existence of a single Chla binding site per b6 f complex does not in itself exclude the possibility that this site is normally empty, or occupied by another ligand, and that Chla binding occurs artifactually when the thylakoid membrane is disrupted by the detergent. Two indirect arguments militate against this view, but do not strictly rule it out. (i) The presence of Chl in b6 f complex preparations has been reported in several species, which implies evolutionary conservation of the site; and (ii) it is not clear where a Chl molecule artifactually picked up by the b6 f complex would originate from since very little Chl is actually set free by the solubilization process. Most of the Chl found in the supernatant from HG solubilization indeed is associated to proteins. Upon fractionating the supernatant on a sucrose gradient, it entered the gradient while free radiolabeled Chl added as a tracer stayed at the top (Fig. 5). Those few Chl molecules that did not enter the gradient, and were presumably free in detergent micelles, represented less than one-tenth of the amount associated with the b6 f. Should one accept the hypothesis that free Chla becomes artifactually bound to the b6 f complex upon solubilization, one would have to contend with the curious coincidence that the solubilization process releases only the amount of Chl that is needed to saturate the complex, and no more.
To more directly rule out this possibility, we have purified C. reinhardtii b6 f from membranes solubilized using detergent micelles preloaded with 3H-labeled Chla and compared the specific radioactivity of free versus b6 f-associated Chl. In a first experiment (experiment 1 in Table III), the regular protocol for b6 f purification (13) was followed, except for the addition of trace amounts of [3H]Chla to the HG stock solution used to solubilize the membranes (see "Experimental Procedures"). The specific radioactivity of the Chl associated with purified b6 f was found to be ~10-fold lower than that of the free Chl collected from the top of the gradient. In a second set of experiments, we checked on the possibility that [3H]Chla initially bound to cytochrome b6 f upon solubilization might back-exchange with the unlabeled Chla bound to antenna proteins that comigrate with the complex during the first hours of the centrifugation, thus lowering the final specific activity. Immediately following solubilization, half of the sample was adsorbed onto a HA column, washed, and eluted as described (13). This resulted in the rapid removal (within ~15 min) of most of the antenna proteins. This sample (experiment 3) was then layered on a sucrose gradient along with the other half of the supernatant (experiment 2), and the two samples were purified by ultracentrifugation and HA chromatography. As shown in Table III, early separation of the freshly solubilized b6 f from Chl-containing proteins does not increase the specific activity of the Chl molecule in the final purified complex; it actually decreases it, because this protocol prevents the limited exchange of b6 f-bound for free Chl that can take place at the top of the gradient during the first hours of centrifugation. The different specific activities of b6 f-bound Chl in experiments 1 and 2 probably stem from a higher level of contamination in experiment 1 by free, radioactive Chl.
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In keeping with these observations, the rate of exchange between b6 f-bound Chl and free Chl was found to be very slow. Purified b6 f was incubated at 4 °C with [3H]Chla in AP-HP buffer. At intervals, aliquots were removed and free Chl separated from the complex by sucrose gradient centrifugation. The specific radioactivity of b6 f-bound Chl increased slowly, until after 10 days it reached approximately one-third that of the free Chl recovered from the top of the gradient (Table IV).
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Accumulation of the
b6 f complex was examined in
C. reinhardtii LDS mutant. When grown in the dark, cells
from this strain etiolate due to the almost complete shutdown of Chl
synthesis.3 The Chl content
of thylakoid membranes purified from dark-grown LDS cells was severely
depleted, as reflected in the fact that the
Chla/-carotene ratio dropped by a factor of 20-30 (Table I). Such membranes contained almost no photosystem I, photosystem II,
and light-harvesting complex proteins (not shown), but they did contain
cytochrome b6 f
(Fig. 6). No cytochrome
b6 f accumulated, on the other hand,
in mutant strain
Gid (kindly provided by P. Bennoun, IBPC, Paris) in
which Chl synthesis is totally blocked (not shown).
To examine whether Chl plays a role
in the electron-transfer function of cytochrome
b6 f, purified samples were exposed to red light, and their enzymatic activity was followed as a function of time, in parallel with the bleaching of Chla. We have
shown previously that, under our experimental conditions, the rate of electron transfer from PQH2 to plastocyanin is limited by
the rate of Cytf/plastocyanin collisions and is strictly
proportional to the concentration of active
b6 f complex (13). As shown in
Fig. 7, bleaching up to 70% of the
Chla in a b6 f preparation had no effect on its PQH2-plastocyanin
oxidoreductase activity.
Our conclusion that a molecule of chlorophyll a is a genuine component of the cytochrome b6 f complex is based on a central observation and backed by a number of corroborative experiments. The most direct observation is the lack of radioactivity in b6 f complex preparations that have been solubilized and purified in the presence of [3H]Chla. Depending on the exact way the experiment was performed, the radioactivity of the Chl associated with the complex varied between ~10 and <1% that of the free Chl present in the supernatant, demonstrating that b6 f-bound Chl does not originate from free Chl artifactually picked up by the complex. Our data rule out the (far-fetched) possibility that free, radioactive Chla becomes initially bound upon solubilization but is replaced in the course of purification by back-exchange with the non-radioactive Chla present on the small fraction of antenna proteins co-solubilized with the b6 f complex.
More circumstantial evidence runs as follows: (i) the mole ratio of Chla to Cytf in highly purified preparations is always close to 1:1 (0.93 ± 0.18 over 26 preparations); (ii) very little Chl is actually set free by the solubilization process (<10% of that recovered in the b6 f complex); (iii) spectral changes upon mild treatment of the complex with detergents show that the Chl, while still bound to the complex, is sensitive to its conformational state and/or to the presence of the Rieske subunit; (iv) resonance Raman data indicate that all Chl molecules in a b6 f preparation experience an identical, specific environment; and (v) there is spectroscopic evidence for the presence of Chla in the b6 f complex in vivo (32; see below).
While these observations leave little doubt that the native b6 f dimer contains two molecules of Chla, they raise a number of questions. One of them is how the complex is protected from oxidation by the triplet state of Chla generated upon illumination. From this point of view, it is probably significant that purified b6 f preparations also contain carotenoids, albeit at a substoichiometric level. Experiments are in progress to determine their origin and the extent of protection they confer against photooxidation.
Another question is the localization of the chlorophyll a molecule in the complex and the identity of the subunit(s) it interacts with. Spectroscopic evidence indicates that a molecule of Chla is located close to the Q0 site in vivo in both C. reinhardtii and Chlorella sorokiniana (32). This conclusion is based on the observation of a Chl spectral shift that correlates with proton release at this site. Our observations indicate that, while the Chla molecule remains bound to cytochrome b6 f dimer depleted of the Rieske protein (26), its spectrum shifts to the red (this work). The latter effect is compatible with a localization of the Chl close to Q0, where it would become exposed to a more polar environment upon removal of the Rieske protein. It cannot be excluded that part of the red shift of the Chl spectrum is a consequence of the delipidation that is used to trigger the dissociation of the Rieske protein (26), but it is notable that a similar shift also occurred following heat treatment of purified b6 f reconstituted into lipid vesicles. Also compatible with a localization close to Q0 is the recent observation that chlorophyll comigrates with cytochrome b6 upon SDS-polyacrylamide gel electrophoresis analysis of Synechocystis PCC 6803 thylakoid membranes.4 We have not been able, unfortunately, to corroborate this experiment using purified C. reinhardtii b6 f. Resonance Raman data indicate that the 9-keto group of the Chl is, in the native complex, involved in a medium strength hydrogen bond. They are more ambiguous in regard to the number of ligands to the Mg2+ ion, but they do not support 5-fold liganding with a strong electron donor such as histidine as the fifth ligand. Experiments are in progress to determine the orientation of the chlorin ring with respect to the membrane plane and the accessibility of the Chl from the lipid and aqueous phases.
More fundamentally, the presence of Chla in the
b6 f complex raises the question of
its eventual function. The plastoquinol-plastocyanin oxidoreductase
activity of the b6 f complex is not
driven by the energy of light, and the redox potentials of either
ground state or excited state Chla (33) are too different
from those of either of the redox carriers in the
b6 f for electron transfer to the
Chl to be contemplated. The spectrum of the
b6 f-associated Chl, indeed, does
not depend on the oxidized or reduced state of the cytochromes. The
homologous ubiquinol-cytochrome c oxidoreductase, the
cytochrome bc1 complex, does not require any Chl
for its function. We have examined a number of possibilitiesnone of
which, admittedly, is very compelling
and tried to rule out some of
them.
A role of the Chl in deactivating the hemes appears a priori unlikely, given the high efficiency of quenching of heme fluorescence by the iron atom. Indeed, fluorescence energy transfer measurements show no change of the excitation spectrum of the Chl upon heme reduction.
Facilitation of Electron TransferThis also is not a priori very likely given that conjugated double bond systems such as that of the chlorin ring are thought to hamper rather than facilitate electron tunneling (34). Bleaching of the b6 f Chl with red light resulted in no loss of the PQH2-plastocyanin oxidoreductase activity. It should be noted, however, that this experiment rules out a complete shut-down of electron transfer upon Chla bleaching, but not a slowing down, since electron tunneling is not the rate-limiting step under our experimental conditions. This point is under more detailed examination.
Regulation of b6 f AssemblyIn this hypothesis,
binding of Chl would regulate the stability of the complex, preventing
accumulation of b6 f in the absence
of light-energy transduction. This idea holds little appeal given that
the complex accumulates in cells of mutant LDS grown in the dark, even
though their low content of Chl prevents accumulation of both reaction
centers and light-harvesting complexes. The substoichiometric ratio of
Chl to cytochrome f in
b6 f preparations purified from
etioplasts of this mutant suggests that, while the complex may bind Chl
even under these conditions, a full complement of 2 Chl/b6 f dimer is not necessary to
its accumulation. We have not, on the other hand, observed any
accumulation of b6 f in mutant
Gid, which does not synthesize any Chl at all. Whether this is a
direct or an indirect consequence of the absence of Chl is not
known.
There are precedents for prosthetic groups
that serve no catalytic function or that are used in atypical manners.
The inactive electron transfer branch in purple bacteria reaction
centers appears to fulfill a primarily or purely structural role (see
e.g. Refs. 35 and 36). Pyridoxal 5-phosphate, whose role in
enzymatic catalysis usually depends on Schiff base formation between
its aldehyde function and amino acid amino groups, is used by glycogen phosphorylase in a totally different way. Its 5
-phosphate group serves
as a proton donor-acceptor shuttle while the Schiff base that
associates it to the protein can be reduced without loss of activity
(see Refs. 37 and 38). Chla being freely available in
thylakoid membranes could have been recruited by the
b6 f either as a mere building block
in the assembly of the complex or in a catalytic function not
necessarily related to its usual roles as a light harvester and an
exciton or electron carrier. The hypothesis of a purely structural
role, of course, is very difficult to rule out. In the present case, it
is made particularly unappealing by the fact that incorporation of
Chla implies the simultaneous development of photoprotecting
devices, such as the additional recruitment of carotenoids.
One possible view would be that some of
the small subunits of the b6 f
complex, which have no equivalent in bc1
complexes, originated from Chl-binding proteins that were recruited by
the complex at a late stage in evolution and that have lost most, but
not all, of their Chl-binding sites. There is indeed a very low level
of sequence identity between b6 f
subunits PetG and PetM and the subunit of purple bacteria
light-harvesting proteins.5
There are several arguments against this hypothesis, among which are
the following. (i) Chloroplasts originate from cyanobacteria with quite
different light-harvesting systems (however, see Ref. 39); (ii) in
Synechocystis PCC 6803, Chl has been observed4
to be associated with apocytochrome b6, even
though (iii) the genome of Synechocystis PCC6803 contains
genes homologous to petG and PetM (40).
An alternative view would hold that Chla, similar to the inactive electron transfer branch in reaction centers, is a relic from an earlier evolutionary stage of the b6 f complex. This could be understood, for instance, if quinol oxidoreductases and reaction centers shared a common, photochemically active ancestor. Such a hypothesis is relatively straightforward if, as is often thought, photosynthesis predates respiration (see e.g. Refs. 36 and 41). It becomes more involved if, as proposed by some recent evolutionary schemes, reaction centers evolved in the context of electron transfer chains where b-type cytochromes already operated (7, 39, 42, 43). Further studies of the b6 f Chl-binding site and of the arrangement of the two Chl in the b6 f dimer, examination of the distribution of bf/bc-associated (bacterio)chlorophylls among phyla, and comparison of the upcoming three-dimensional structure of mitochondrial bc1 complex with those of reaction centers should contribute to shedding light on this puzzling question.
We particularly thank P. Bennoun for
providing C. reinhardtii LDS and Gid mutant strains, P. Joliot for constant support and interest and for many stimulating
discussions, R. Barbato for communication of data prior to publication,
and A. Verméglio, W. Nitschke, D. Picot, D. Waltz, P. Mathis,
W. A. Cramer, and M. Saraste for discussions and exchange of
information.