(Received for publication, April 10, 1997, and in revised form, June 5, 1997)
From the Institut Curie, Section de Recherche
UMR-CNRS 168 and LRC-CEA 8, 11 rue Pierre et Marie Curie, F-75231 Paris
Cedex, France and the ¶ Institut de Biologie Physico-Chimique,
CNRS UPR 9052 and University Paris-7, 13 rue Pierre et Marie Curie,
F-75005 Paris, France
The structure of the cytochrome b6 f complex has been investigated by electron microscopy and image analysis of thin three-dimensional crystals. Electron micrographs of negatively stained specimens were recorded and showed optical diffraction peaks to 10 Å resolution. A projection map was calculated at 8 Å resolution and showed the presence of cytochrome b6 f dimers. The extramembrane part of each monomer featured a C shape, with mean external diameter ~of 53 Å and an internal groove ~14 Å long and ~9 Å wide. Within each monomer, strong features were clearly resolved and tentatively attributed to some of the subunits of the cytochrome b6 f complex. The data are consistent with the Rieske iron-sulfur protein lying close to the monomer-monomer interface and the heme-bearing domain of cytochrome f far from it.
Cytochrome b6 f (plastoquinol:plastocyanin oxidoreductase) is an integral membrane protein complex that participates in electron transfer and generation of an electrochemical proton gradient in oxygenic photosynthesis. It is homologous to the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase) of the respiratory chains of the mitochondrion and many bacteria. The b6 f complexes from higher plants (1) and from the unicellular green alga Chlamydomonas reinhardtii (2) are highly similar and comprise four subunits with a molecular mass of >17 kDa. Three of them, cytochrome b6, cytochrome f, and the Rieske protein, contain redox prosthetic groups. The fourth, subunit IV, is involved together with cytochrome b6 and the Rieske protein in forming the oxidizing plastoquinol binding site Qo (1). There are at least three additional small hydrophobic polypeptides (~4 kDa). The number of transmembrane helices in the seven-subunit monomeric complex (105 kDa) is probably 11 (3, 4). A dimeric form is believed to be the native state both in higher plants (5) and in C. reinhardtii (6).
Spectroscopic, biochemical, genetic, and electron microscopy studies of cytochrome b6 f (reviewed in Ref. 1) have yielded only sparse information about the three-dimensional structure of this complex. Huang et al. (5) reported that negatively stained monomers and dimers of the b6 f complex from spinach both appeared as round particles with clefts with diameters of 77 ± 10 and 91 ± 9 Å, respectively. Boekema et al. (7) have reported, also from single particle analysis, an elongated shape of the complex from Synechocystis PCC 6803 with dimensions of 83 × 44 × 60 Å, which they interpreted as monomers. Freeze-fractured vesicles reconstituted with purified spinach b6 f featured particles 83 Å in diameter and 110 Å in height, which Mörschel and Staehelin interpreted as dimers (8), whereas reconstituted monomers and dimers from C. reinhardtii appeared as particles with diameters of ~80 and ~100-110 Å, respectively (6). Finally, Mosser et al. (9) obtained tubular crystals and two types of thin three-dimensional crystals of spinach b6 f. The projection map that was calculated did not give unambiguous limits of the molecule. Thus, these studies have not led so far to a clear description of the size and shape of the complex. Most interestingly, the structure of the cleaved extramembrane domain of turnip cytochrome f (10) and that of the catalytic domain of the mitochondrial Rieske protein (11) have both been solved to high resolution by x-ray crystallography. On the basis of these data, Link and Iwata have proposed a model for the association of cytochrome f with the photosynthetic Rieske protein (12). X-ray crystallography is expected to lead rapidly to a detailed structural model of the entire mitochondrial bc1 complex (13). However, due to the important differences between the two complexes (different subunit compositions, low sequence similarities of homologous subunits, presence of photosynthetic pigments in b6 f, sensitivity to antimycin limited to bc1, and possible functional differences), high resolution structural data on one complex might not be easily transposable to the other.
Understanding the functional mechanism of cytochrome b6 f hangs on the knowledge of its detailed structure. Growing well ordered two-dimensional crystals suitable for analysis by electron crystallography is one approach toward determining the structure of proteins at high resolution (14, 15). The yield, purity, and stability of the b6 f preparations from C. reinhardtii (2, 6) fulfill the prerequisites for crystallization attempts, whereas enzymatic activity, the presence of the Rieske protein, the spectral properties, and the dimeric state provide various checks on the native state of the complex (6, 16).
In the present article, we describe the crystallization of the b6 f complex from C. reinhardtii in very thin three-dimensional crystals. For this purpose, we have adapted and improved a reconstitution strategy that uses Bio-Beads as a detergent-removing agent and has been demonstrated successful for two-dimensional crystallization of different membrane proteins (17). Optimizing the pre-reconstitution conditions and combining the use of Bio-Beads with freeze-thaw cycles led to the formation of large and highly ordered thin three-dimensional crystals that diffract to better than 10 Å resolution in negative stain. After correction of lattice distortions, crystallographic analysis has yielded a projection map of cytochrome b6 f at 8 Å resolution. The map is discussed in the light of available evidence on the organization of the subunits in the complex.
SM2 Bio-Beads were obtained from Bio-Rad and di-C18:1-phosphatidylglycerol from Avanti Polars Lipids Inc. Sources for the other chemicals have been described in Ref. 2.
Purification of Cytochrome b6 fCytochrome
b6 f complex was purified from
C. reinhardtii thylakoid membranes in the presence of
6-O-(N-heptylcarbamoyl)-methyl--D-glucopyranoside (Hecameg)1 as described
previously (2). Briefly, the purification protocol comprises three
steps: selective solubilization from thylakoid membranes, sucrose
gradient sedimentation, and hydroxylapatite chromatography. Following
solubilization, all media are supplemented with egg phosphatidylcholine
to prevent the loss of the Rieske protein from the complex that follows
delipidation (2, 6).
Purified cytochrome
b6 f complex was resuspended in 20 mM Hecameg, 2 mM CaCl2, 0.3%
glycerol, 0.3 mM NaN3, 5.6 mM
-aminocaproic acid, 1.1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 245 mM
ammonium phosphate, 6.8 mM Tricine, pH 8.0, and
supplemented with a mixture of egg phosphatidylcholine and
di-C18:1-phosphatidylglycerol (1:1 to 1.4:1, w/w). The
protein concentration in the final reconstitution mixture was adjusted
to 0.5 g/liter and that of the lipid to a lipid/protein ratio of 0.2 w/w. The samples were preincubated overnight in the cold room under
gentle stirring and subsequently treated with 200 g/liter SM2 Bio-Beads
according to the batch procedure previously described (17). After 6-12
h of incubation with beads, the reconstituted material was pipetted off
and kept for 24 h at 4 °C before three cycles of freezing
(
190 °C) and thawing (37 °C). Aliquots were taken daily and
examined by electron microscopy.
Samples, negatively stained with 1% uranyl acetate, were observed on a Philips CM120 transmission electron microscope operating at 120 kV. Low dose electron micrographs were recorded at magnifications of 45,000× and 60,000×. The best images were selected by optical diffraction, and areas exhibiting strong coherent diffraction spots were digitized on a Leafscan 45 CCD-array microdensitometer with a 5 µm scan spot size. Areas ranging up to 3200 × 3200 pixels in size, corresponding to 0.34 × 0.34 µm at the specimen level, were subjected to analysis using the Spectra program package (18) and the MRC image processing system (19). A total of 14 crystalline areas from nine images were analyzed with unbending methods. The underfocus level was determined by multiple measurements on the Thon-rings from the Fourier transform of the raw image, and the reflections corrected for the overall transfer function. The reflections from the processed images were centered and averaged, utilizing spots up to IQ 7. An image scale factor and the reflection peak height were used as a weighing factor during averaging. A refinement step on the positions of the zeroes in the contrast transfer function was performed, using the preliminary average as a reference. An optimal position of the lattice extraction, centered around the best crystalline area of each image, was achieved by extracting a few sets of reflections and testing for phase quality. The data set was phase-minimized with a small step size and merged, in several rounds, to yield a new average. A cut-off selection of maximally 45 ° phase deviation from the real values, in addition to IQ 7 for the amplitudes, was applied for acceptance of a reflection prior to calculation of the map. An error weighing factor was included by multiplying the amplitudes by the cosine of the phase deviation. The symmetry was imposed, and the projection map was displayed using histogram equalization and the plot program Pluto. Equidistant line levels were generally employed to the maximum positive density (i.e. stain-excluding region) in the map.
Thin three-dimensional crystallization of cytochrome b6 f was achieved by reconstituting the solubilized complex into phospholipid bilayers containing egg phosphatidylcholine and an anionic unsaturated phospholipid, dioleoyl-phosphatidylglycerol, in the presence of calcium ions and protease inhibitors. Reconstitution was performed by removing detergent from the lipid-Hecameg-protein micellar solution by adsorption onto Bio-Beads SM2. Previous systematic studies have determined precisely the amount of beads to be added to remove the detergent initially present in about 6 h at 4 °C while avoiding protein adsorption and limiting lipid adsorption (17). Provided the amount of beads was carefully controlled, as well as the lipid to protein ratio, crystallization of cytochrome b6 f was reproducible. Another important parameter was the duration of the preincubation period before detergent removal, which had to be sufficiently long. Although the reason for this requirement is not definitely identified, it may be related to the time needed for full equilibration of the initial populations of detergent-lipid and detergent-lipid-protein micelles. Finally, the presence of calcium was found to be essential for crystal formation.
Following detergent removal, proteoliposomes with densely packed proteins were observed by electron microscopy. They tended to aggregate upon further incubation at 4 °C with concomittant formation of crystalline areas. Growth of large crystals from these aggregates was improved by treating the samples through freeze-thaw cycles. Possible explanations would be that such a treatment not only induces fusion of proteoliposomes but also creates some defects in the bilayer or some protein aggregation, which would favor crystal growth (20).
Samples were periodically checked by electron microscopy to monitor the
growth of the crystals. The crystals presented a smooth homogeneous and
continuous gray appearance (Fig.
1A) and were always composed
of a stack of lamellae. The best time to collect them was generally
between the third and the seventh day after three freeze-thaw cycles.
After that, the crystals increased inhomogeneously in thickness and
sometimes started to deteriorate.
Our belief that the crystals observed in the reconstituted preparations are formed by the b6 f in its native state is based on several lines of evidence. Neither visible absorption spectra nor SDS-polyacrylamide gel electrophoresis patterns changed during the 3-day to 1-week period required for crystallization. Two good indices of the native state of the complex are its dimeric nature and the stability of the spectrum of the b6 f-associated chlorophyll a; indeed, loss of the Rieske protein, which is the first step in the degradation of the complex (6), is always accompanied by a red shift of the visible absorption peak of the chlorophyll (16) and almost invariably followed by monomerization (6).
Electron Microscopy and Image ProcessingThe crystals (Fig.
1A) generally grew up to 5 µm in one direction (along the
b direction) and up to 1.5 µm in the other (along the
a direction). In some cases, they reached up to 10 µm 3 µm. At high magnification, the array appeared clearly and spread over
the whole crystal (Fig. 1B). Although the crystals presented in this study are actually stacks of lamellae, they were selected such
that they presented a small number of layers and were of extremely good
crystallinity. The coherence of the lattice was confirmed by optical
diffraction analysis of low dose negatives, which revealed sharp,
coherent peaks out to 10 Å over the whole electron micrographs. This
also strongly suggests that the different layers are in perfect
register, making the stacks equivalent to very thin three-dimensional
crystals. The diffraction pattern corresponded to a rectangular lattice
with parameters a = 175 Å, b = 68 Å,
and
= 90 °. Systematic extinctions were observed for
[h(odd),0] and [0,k(odd)].
Fourteen areas were selected by optical diffraction from nine of the best images and digitized, and the lattice distortions were corrected. Calculated phases indicated that the crystals belonged to plane group p22121. In this space group, rows of molecules alternatively face up and down with respect to the membrane plane. The results of the R value test for this symmetry are shown in Table I for four resolution ranges, indicating the high quality of the phases to 8 Å resolution.
|
Fig. 2A shows the Fourier
transform of the best distortion-corrected images. Reflections with IQ
values of 4 are visible out to 8 Å resolution. Fig. 2B
shows the phase deviations of reflections from the expected values of
zero or for all images. Excluding the phases of reflections having
IQ values larger than 5, the root mean square phase error is about
16° at 8 Å resolution. Because plane group
p22121 gave the best
phase residual, it was used to calculate projection maps to 20 and 8 Å resolution (Fig. 3, A and
B).
The 20 Å density map reveals the dimeric organization of the b6 f complex. The dimer features an elongated S shape, ~88 Å long and ~53 Å wide. The monomer has a C-like shape with two major domains denoted X and Y. The 8 Å resolution map reveals within the extramembrane part of each monomer a ring of densities surrounding a deep groove (G). The external diameter of the monomer is ~53 Å, whereas the internal groove is ~14 Å long and ~9 Å wide.
The main goal of this study was to generate a projection map of cytochrome b6 f to provide information about the localization of its different subunits. The purity and reproducibility of the b6 f preparations from C. reinhardtii facilitated the identification of conditions favoring the growth of large and coherent thin three-dimensional crystals suitable for structural analysis by electron microscopy. In addition to the usual parameters that affect the formation and quality of crystals, we believe that two key factors in the success of our procedure are the rapid and total removal of detergent by Bio-Beads (see also Ref. 17) and the use of freeze-thaw cycles to increase the size and ordering of the crystals (see also Ref. 20).
The excellent quality of the crystals allowed us to record low dose images of negatively stained specimens that diffracted out to 10 Å resolution prior to the correction of lattice distortions and to better than 8 Å following it. The IQ plot of reflection intensities (Fig. 2A) and the crowding of calculated phases around the values of 0 ° and 180 ° expected from the p22121 lattice symmetry (Fig. 2B) are indicative of the quality of the data. Such a high resolution has never been reported before for negatively stained crystals of any membrane protein. The multi-layered nature of the crystals cannot by itself account for this result, because similar resolutions have already been observed with negatively stained single-layered crystals such as those of annexin V (21) and of subunit B of cholera toxin.2 Thus, our observations suggest that the resolution attainable following negative staining may be higher than usually accepted (~15 Å).
The projection maps show a dimeric organization of cytochrome
b6 f, in keeping with biochemical
determinations on the solubilized complex (6). Each monomer presents a
C-like shape, ~53 Å in diameter, covering a total area of about
2,000 Å2. By comparison with the dimensions of the
transmembrane regions in bacteriorhodopsin (14) or cytochrome
c oxidase (22), this area significantly exceeds that needed
to accommodate 11 transmembrane -helices per monomer. In the 20 Å projection map, each monomer features two main domains (labeled
X and Y in Fig. 3), surrounding a deep central
groove (G in Fig. 3). Domain X, which is near the 2-fold
axis of symmetry, is less bulky than domain Y, suggesting that much of
the extramembrane mass of the complex lies away from this axis. The
dimensions and overall appearance of cytochrome b6 f at 20 Å resolution are not
dissimilar to those of the subcomplex of Neurospora crassa
cytochromes b and c1 (lacking the
Rieske and core proteins), except that in the latter case domain X
appeared stronger than domain Y (23), at variance with the
b6 f map. A three-dimensional
reconstruction of bovine heart mitochondrial bc1
at 16 Å resolution has recently been calculated from electron
micrographs of frozen tubes (24). It shows, protruding into the
intermembrane space, four proteic masses per dimer, reaching into the
solvent and delineating a relatively empty space around the 2-fold axis
of symmetry. A depression about the C2 axis is also apparent in the
preliminary x-ray map of beef heart bc1
(13).
The projection of the monomer obtained does not resemble any of the three possible projections (Y, Z, and L shapes) previously observed with spinach b6 f (9). Differences in the conditions of crystal formation may explain these dissimilarities. Whereas low calcium concentration (0.5 mM), Hecameg, and Bio-Beads were used to crystallize the complex from C. reinhardtii, comparatively high calcium concentration (10 mM), octylglucoside, and dialysis were employed for the crystallization of the spinach complex. These differences may have led to the monomerization of the complex from spinach, explaining why no tight dimers were observed.
The location of the stain in the case of membrane proteins is not perfectly understood. It seems to vary from one protein to another, but it is usually accepted that the hydrophobic membrane core is largely stain-excluding. Therefore, we made the assumption that the structural information obtained, including the presence of the groove, primarily concerns the extramembrane parts of the b6 f complex. Positive densities should be due mainly to the cytochrome f extramembrane domain (28 kDa; residues 1-250), the Rieske protein (19 kDa), the main loops of cytochrome b6 (11 kDa; residues 1-33, 58-82, and 140-181) and subunit IV (8 kDa; residues 215-249 and 272-308), and the extramembrane extensions of the three 4-kDa subunits (~1 kDa each). However, because of the smallness of 4-kDa subunits' extramembrane extensions, their contribution to the projection map will not be discussed in the following.
The projection map at 8 Å resolution (Fig. 3A) confirms the
C shape of the monomer. Each monomer features four domains of variable
importance (II > I III > IV), which can be
tentatively allocated to some of the
b6 f subunits. The two major
stain-excluding regions, I and II, are well individualized, indicating
that they correspond to two independent proteic masses. Taking into
account their respective importance (II > I) and the fact that
the extramembrane part of cytochrome f and the Rieske
protein fold into autonomous domains, one may tentatively assign
density II to the largest of these subunits, the cytochrome
f, whereas domain I would correspond to the Rieske protein.
The other densities (domains III and IV), which include that close to
the 2-fold symmetry axis, would be due to the extramembrane loops of
cytochrome b6 and subunit IV. However, in this
case, because the mass of the loops is small compared with the whole
mass of these two subunits, it would be premature to assign individual
subunits to specific features at this stage of the structural
study.
Domain IV appears to be separated from domain III by a gap and appears
to connect more closely to domain I. This suggests that the
extramembrane regions of the two monomers may partially engage into one
another close to the symmetry axis, as proposed in Fig.
4. Moreover, the continuity observed
between the two densities IV of the dimer suggests that the subunits
that contribute to it tightly interact through their extramembrane
parts. It should also be stressed that domain IV appears small to fit
either subunit IV or cytochrome b6. However,
this domain is rather flat, and it is reasonable to assume that the
subunit that contributes to it may extend under the Rieske protein
while contributing in a negligible way to domain I.
This general arrangement would be consistent with biochemical data that
have shown that cytochrome b6 can be easily
cross-linked to cytochrome b6 and subunit IV to
subunit IV within a dimer, indicating that regions of both subunits
come close to the symmetry axis (25, 26). On the contrary, homologous
cross-linking between the large extramembrane domains is almost never
observed. The effects of mutations on the functionality and sensitivity
to inhibitors of site Qo (reviewed in Ref. 27) and on the
strength of the Rieske protein's association with the complex (27, 28)
suggest that at least part of the second (c-d)
extramembrane loop of cytochrome b6 and of the
first loop of subunit IV is located close to the Rieske protein. The
allocation of the Rieske protein to density I and of parts of
cytochrome b6 and subunit IV to less intense features near the monomer-monomer interface would imply that site Qo, which is formed by these three subunits, might lie
relatively close to the 2-fold axis of the dimer. Such a location would
be consistent with the close apposition of the two
bL hemes in the bc1
dimer, as revealed by x-ray diffraction (13). The relative proximity of
the two Qo sites and the two bL
hemes within the bc1 and
b6 dimers opens up interesting vistas
regarding the possibility of a functional cooperation between the two
monomers (cf. Ref. 13). It is also worth noting that removal
of the Rieske protein is generally, even though not always, accompanied
by the monomerization of the complex and vice versa (6). The proposed positioning of the Rieske protein close to the monomer-monomer interface suggests a possible origin for the frequent correlation between these two events: destabilization could result from the loss of
interactions between the Rieske protein of one monomer and neighboring
subunit(s) of its partner.
In conclusion, our results provide the first crystallographic information on the structure of the cytochrome b6 f complex in its intact form. The 8 Å projection map of the enzyme has permitted proposals to be made regarding the location cytochrome f and the Rieske protein in the b6 f dimer. In future work, subunit assignment will be further examined using the same approach to crystallization associated with labeling methods or with the removal of specific subunits (cf. Ref. 6). Obtaining highly ordered crystals opens the way to the determination of a three-dimensional model of cytochrome b6 f using cryo-electron microscopy. Establishing a three-dimensional model of the complex, although facing the problem of analyzing multilayered crystals, can be attempted by appropriate treatment of the crystallographic data and/or by modifications of the crystallization protocol favoring the growth of monolayered crystals. It should be stressed that even a moderate resolution three-dimensional map would be extremely useful in helping to delineate structural differences between the bc1 and b6 f complexes.
We are extremely grateful to D. Picot (Institut de Biologie Physico Chimigue, Paris) for useful discussions.