On the Presence and Role of a Molecule of Chlorophyll a in the Cytochrome bf Complex*

(Received for publication, December 31, 1996, and in revised form, May 22, 1997)

Yves Pierre Dagger , Cécile Breyton Dagger , Yves Lemoine §, Bruno Robert , Claudie Vernotte par and Jean-Luc Popot Dagger **

From the Dagger  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 par  CNRS UPR 9061, F-91198 Gif Sur Yvette, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Highly purified preparations of cytochrome bf 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.


INTRODUCTION

Cytochrome bf, 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/bf complex (see Refs. 8 and 9, and Table I). It was no surprise, therefore, that traces of Chl should be found in purified bf 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).

Table I. Prosthetic group composition (mol/mol ratios) of thylakoid membranes and cytochrome b6 f complexes purified from either wild-type or LDS C. reinhardtii strains

Most determinations were performed on two different wild-type preparations. All other pigments (antheraxanthin, lutein-5,6-epoxide, zeaxanthin, alpha -carotene, beta -carotene-5,6-epoxide) were either absent or present in trace amounts (<= 1% of total pigment mass).

WT
LDS
Membranes Cyt b6 f Membranes Cyt b6 f

Chla/Cytf ~900a 0.78 -1.1 NDb 0.33
 beta -carotene/Cytf 21 -33 0.26 -0.38 NDb 0.036
PQ/Cytf 13 0.05 -0.07 NDb 0.14
 beta -carotene/Chla 0.035 -0.05 0.23 -0.48 1.01 0.16
Chlb/Chla 0.37 -0.48 0.09 -0.10 0.35 0.45
Lutein/Chla 0.09 -0.11 0.06 -0.08 1 1.10
Neoxanthin/Chla 0.09 -0.11 0.01 0.36 0.50
Violaxanthin/Chla 0.08 -0.09 0.02 0.06 0.71

a Mixture of Chlb and Chla.
b Concentration of Cyt f not determined.

In the present article, we present further evidence in support of our earlier conclusion that the native bf 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 bf complex;2 (ii) the rate of exchange of bf-associated Chla for free [3H]Chla is extremely slow; and (iii) Chla is bound to the bf complex at a single, specific site. Putative functional roles for a chlorophyll in the bf complex are examined and some of them are experimentally ruled out.


EXPERIMENTAL PROCEDURES

Materials

Decylplastoquinone (C10-PQ), Tricine, egg yolk L-alpha -phosphatidylcholine (PC), phenylmethylsulfonyl fluoride (PMSF), epsilon -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.

Media

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 epsilon -aminocaproic acid).

Strains and Growth Conditions

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 epsilon -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.

Preparative and Analytical Techniques

Cytochrome bf 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 epsilon 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 epsilon 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).


Fig. 2. Visible absorption spectrum of chlorophyll a under various experimental conditions. Panel A, (solid line) visible spectra of purified bf in TMK buffer containing either 0.2 or 5 mM LM; (dotted line) visible spectrum of free Chla in TMK buffer + 5 mM LM, using Joliot-type spectrophotometer, [Cytfsime  0.2 µM. Panel B, effects of various perturbations on the spectrum of bf-bound Chla; purified bf complex (~0.25 µM in TMK buffer containing 0.2 mM LM) (1); same sample ~2 h after [LM] was brought up to 5 mM (2); same sample as in 1 after ~2 h incubation at 50 °C (3); same sample as in 1 after ~2 h incubation at room temperature in the presence of 8 M urea and ~1 mM LM (4); purified bf reconstituted into egg PC vesicles according to ref. (26) (5); same sample as in 5 after ~90 min incubation at 50 °C, using Joliot-type spectrophotometer (6). Panel C, spectra of Chla bound to native (dotted line) and to Rieske-depleted (solid line) cytochrome bf dimer in AP-HP buffer (Ref. 26; see "Results"), using Kontron Uvikon 930 spectrophotometer, [Cytfsime  9.8 µM and 6.4 µM, respectively. In the three panels, spectra have been arbitrarily displaced vertically.
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Fig. 3. Fluorescence excitation and emission spectra of bf-bound chlorophyll a. Panel A, absorption spectra of free Chla in AP-HP buffer (a) and of purified bf complex in the same buffer without addition (b) and after addition of dithionite (c), using Varian Cary 2300 spectrophotometer. Panel B, fluorescence emission spectra at 77 K of purified bf complex in AP-HP buffer without addition (a) and after 1 h incubation in 100 mM HG in the same buffer (b), and emission spectrum of free Chla in AP-HP buffer (c); excitation light at 440 nm. Panel C, fluorescence excitation spectra at 77 K of purified bf complex without addition (open circle ) and following addition of dithionite (+); emission was measured at 673 nm.
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Fig. 6. Visible absorption spectra of bf complexes purified from wild-type and LDS C. reinhardtii strains. Cytochrome bf was purified from WT and LDS strains as described under "Experimental Procedures," and the preparations were analyzed for Cytf (left), following addition of ascorbate, and for Chl (right). Spectra were recorded on a Kontron Uvikon 930 spectrophotometer normalized to the same absorbance at 554 nm and vertically displaced.
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Pigment Analysis

Pigments were extracted from thylakoid membranes or from cytochrome bf 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.

Preparation of [3H]Chlorophyll a

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.


Fig. 1. Purification of [3H]chlorophyll a. C. reinhardtii cells were grown on [3H]acetate, thylakoid membranes were purified, and pigments were extracted and separated by thin-layer chromatography. The Chla fraction was further purified by reversed-phase HPLC on a Zorbax-ODS column. The peak eluting at 20.8 min was collected and used for [3H]Chla exchange experiments (see Fig. 5 and Tables III, IV).
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Purification of Cytochrome bf 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 bf 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).


Fig. 5. Migration of free and protein-bound chlorophyll upon fractionation of a solubilization supernatant on a sucrose gradient. A 500-µl sample of C. reinhardtii thylakoid membranes (3 g/liter chlorophyll) was solubilized by addition of an equal volume of HG 50 mM containing 0.074 g/liter [3H]Chla (6.5 × 107 cpm/ml). After 10 min of centrifugation in a TL100 ultracentrifuge, the supernatant was layered on top of a sucrose gradient in TMK-HP buffer and centrifuged for 24 h (see "Experimental Procedures"). Fractions 1-8 were analyzed for total and radioactive Chl. Fractions 13-15 were pooled, and cytochrome bf was purified by HA chromatography (Table III, experiment 1).
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Determination of the Rate of Exchange of bf-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 bf 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 bf-bound Chl. The brownish band containing the complex was collected, and the specific radioactivity of bf-bound Chl was determined and compared with that of the free Chl present at the top of the gradient.

Fluorescence Measurements

Low-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 Spectroscopy

Resonance Raman spectra were recorded with a U1000 Raman spectrometer equipped with a charge-coupled device camera (Jobin-Yvon, France) on pellets of oxidized cytochrome bf 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 bf-bound Chlorophyll a

Purified 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.


RESULTS

Highly Purified Preparations of bf Complex from C. reinhardtii Contain One Molecule of Chlorophyll a/Cytochrome f

The bf 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 bf-associated Chla (epsilon 668 = 75,000 M-1 · cm-1; cf. "Experimental Procedures") and an extinction coefficient epsilon 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 bf 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 bf (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 bf 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 bf-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 bf-associated Chlorophyll a Is Affected by Its Interactions with the Complex

Exposing 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 bf complex was in its detergent-solubilized state or reconstituted into lipid vesicles (Fig. 2B).

The spectrum of Chla in bf 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 bf 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 bf-associated Chlorophyll

Interactions of the Chl with its environment in the bf complex were further examined by low temperature fluorescence measurements. Cytochrome bf exhibits, in the Soret region, several absorption bands due to the Chl, the carotenoids, and cytochromes f and b, 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 bf 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 bf 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 bf 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 bf complex results in a redox-independent quenching of its fluorescence (by a factor of ~4), which is partially relieved following detergent treatment.

Table II. Compared fluorescence intensities of free and b6 f-bound Chla


Chlorophyll a Cytochrome b6 f
Fresh After freeze/thaw +100 mM Hecameg

Relative fluorescence intensity at 77 K 100 25 ± 3 39 ± 4 51 ± 5

Chlorophyll a Is Bound to the bf Complex at a Specific Site

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 bf. This laser line is located on the red side of the Soret electronic transition of Chla, more than 1500 cm-1 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 b. 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).


Fig. 4. Resonance Raman spectrum of bf-bound chlorophyll a. Spectrum was recorded at 77 K on a pellet of purified bf complex oxidized with potassium ferricyanide. Illumination wavelength was 441.6 nm.
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In the resonance Raman spectrum of the bf-bound Chl (Fig. 4), the band arising from the methine bridge stretching modes is observed at 1606 cm-1, 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 cm-1 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 bf complex interacts with such a strong ligand.

Chlorophyll a Does Not Become Artifactually Bound to the Complex during Solubilization

The existence of a single Chla binding site per bf 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 bf 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 bf 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 bf. Should one accept the hypothesis that free Chla becomes artifactually bound to the bf 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 bf from membranes solubilized using detergent micelles preloaded with 3H-labeled Chla and compared the specific radioactivity of free versus bf-associated Chl. In a first experiment (experiment 1 in Table III), the regular protocol for bf 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 bf 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 bf 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 bf 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 bf-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.

Table III. Specific radioactivity of free and b6 f-bound Chl following solubilization of thylakoid membranes in the presence of [3H]Chla


Experiment number Purification protocol Chl/Cyt f ratio in purified b6 f Chlorophyll specific activity
Top of gradient Purified b6 f Specific activity (bound)/specific activity (free)

mol/mol cpm/pmol
1 Solubilize/gradient/HA 1.0   65 6.7 0.10
2 |Solubilize/gradient/HA 0.9 |61 0.49 0.008
3 |Solubilize/HA/gradient/HA 1.25 |61a 0.27 0.004

a Determined in experiment number 2.

Rate of Exchange of Bound and Free Chlorophyll

In keeping with these observations, the rate of exchange between bf-bound Chl and free Chl was found to be very slow. Purified bf 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 bf-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).

Table IV. Exchange of bound for free Chl upon incubation of purified b6 f complex with [3H]Chla


Incubation time
0 1 5 10

days
Chl/Cyt. f molar ratio in purified b6 f 1.1 0.9 0.9 1,3
Specific activity of Chl at the top of the gradient (cpm/pmol)  (119)a 200 210 241
Specific activity of Chl in purified b6 f (cpm/pmol)  12 27 43 72
Percent exchange (10%) 13 20 30

a The reason for which the specific radioactivity of free Chlalpha determined at time zereo was off by a factor of ~2 has not been identified.

Cytochrome b6f Accumulates in a Chlorophyll-poor C. reinhardtii Mutant

Accumulation of the bf 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/beta -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 bf (Fig. 6). No cytochrome bf accumulated, on the other hand, in mutant strain Delta Gid (kindly provided by P. Bennoun, IBPC, Paris) in which Chl synthesis is totally blocked (not shown).

Chlorophyll a Is Not Essential to in Vitro Electron Transfer by Cytochrome bf

To examine whether Chl plays a role in the electron-transfer function of cytochrome bf, 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 bf complex (13). As shown in Fig. 7, bleaching up to 70% of the Chla in a bf preparation had no effect on its PQH2-plastocyanin oxidoreductase activity.


Fig. 7. Oxidoreductase activity of the bf complex as a function of chlorophyll a bleaching. Purified bf complex was irradiated with red light (lambda >620 nm) at 4 °C. Chla absorbance at 667 nm (black-square) and PQH2-plastocyanin oxidoreductase activity (+) are normalized to their initial value.
[View Larger Version of this Image (13K GIF file)]


DISCUSSION

Our conclusion that a molecule of chlorophyll a is a genuine component of the cytochrome bf 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 bf 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 bf-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 bf 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 bf 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 bf preparation experience an identical, specific environment; and (v) there is spectroscopic evidence for the presence of Chla in the bf complex in vivo (32; see below).

While these observations leave little doubt that the native bf 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 bf 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 bf 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 bf reconstituted into lipid vesicles. Also compatible with a localization close to Q0 is the recent observation that chlorophyll comigrates with cytochrome b 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 bf. 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 bf complex raises the question of its eventual function. The plastoquinol-plastocyanin oxidoreductase activity of the bf 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 bf for electron transfer to the Chl to be contemplated. The spectrum of the bf-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 possibilities---none of which, admittedly, is very compelling---and tried to rule out some of them.

Light Protection

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 Transfer

This 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 bf 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 bf Assembly

In this hypothesis, binding of Chl would regulate the stability of the complex, preventing accumulation of bf 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 bf 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/bf dimer is not necessary to its accumulation. We have not, on the other hand, observed any accumulation of bf in mutant Delta 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.

A Structural Role

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 bf 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.

An Evolutionary Relic

One possible view would be that some of the small subunits of the bf 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 bf subunits PetG and PetM and the alpha  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 bf 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 bf Chl-binding site and of the arrangement of the two Chl in the bf 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.


FOOTNOTES

*   This work was supported by the Centre National de la Recherche Scientifique, Paris-7 University, The Collège de France, the Commissariat à l'Energie Atomique, and by Grant BIO2-CT93-0076 from the European Economic Community (to J.-L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence should be addressed. Fax: 33-1-40-46-83-31.
1   The abbreviations and trivial names used are: PQH2, plastoquinol; HG, Hecameg (6-O-(N-heptylcarbamoyl)-methyl-alpha -D-glycopyranoside); AP-HP buffer, ammonium phosphate/Hecameg/phosphatidylcholine buffer; Chl, chlorophyll; Chla, chlorophyll a; Cytf, cytochrome f; HA, hydroxylapatite; HPLC, high-pressure liquid chromatography; LM, laurylmaltoside (dodecyl-beta -D-maltoside); PC, L-alpha -phosphatidylcholine; PMSF, phenylmethylsulfonyl fluoride; TAP, Tris acetate/phosphate growth medium; TMK, Tris/magnesium/potassium buffer; TMK-HP, TMK buffer supplemented with HG and egg PC; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type.
2   This observation has been reported in preliminary form in Ref. 14.
3   P. Bennoun, personal communication.
4   R. Barbato, personal communication.
5   J.-L. Popot, unpublished observations.

ACKNOWLEDGEMENTS

We particularly thank P. Bennoun for providing C. reinhardtii LDS and Delta 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.


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