Electron transfer by domain movement in cytochrome bc1

Zhaolei Zhangac, Lishar Huangab, Vladimir M. Shulmeister, Young-In Chib, Kyeong Kyu Kimb, Li-Wei Hungc, Antony R. Croftsd, Edward A Berrya, and Sung-Hou Kimabc

E. O. Lawrence Berkeley National Laboratorya,

Department of Chemistryb, and the Graduate Group of Biophysicsc, University of California, Berkeley, CA, USA and Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, IL, USAd

This paper is dedicated to the memory of our friend and colleague Vladimir M. Shulmeister, who was a key member on the project until his untimely death on September 27, 1995.

Overall shape of the cytochrome bc1 complex dimer
Inhibitor binding sites
Arrangement of the protein domains in the intermembrane region
Structure of cytochrome c1
Structure and location of the Rieske ironsulfur protein
The extrinsic domain of the ironsulfur protein is loosely attached and functionally swapped between monomers.
Two conformations of the Rieske protein: Dynamic domain mediated electron transfer
Feasibility of electron transfer from the ironsulfur protein in the distal conformation to cytochrome c1.
Model for electron transfer through the high potential chain.
Figure Legends

X-ray crystallographic analysis of multiple crystal forms of the cytochrome bc1 complex from three different species reveals two different locations for the extrinsic domain of the ironsulfur protein (ISP). One location is close to the putative quinol oxidation site, allowing reduction of the ISP by ubiquinol, and the other site is close enough to cytochrome c1 to allow rapid oxidation of the ISP by the cytochrome. However, neither location would allow both reactions to proceed at a kinetically competent rate. The reaction mechanism thus must involve a movement of the extrinsic domain of ISP, a mechanism not observed before for electron transport within redox protein complexes.

The energy conversion of the biosphere is achieved predominantly through respiration and photosynthesis, and represents a flux several orders of magnitude greater than all anthropogenic energy usage. The underlying mechanism involves the coupling of electron transfer along a chain of redox or photoredox enzymes to proton translocation across an organellar membrane in which those redox components are embedded. This gives rise to an electrochemical proton gradient across that membrane, which can be coupled to energy-requiring processes including synthesis of ATP--a principle first proposed by Peter Mitchell in his chemiosmotic hypothesis (1).

The central component of the electron transfer chain in mitochondria and in many aerobic or photosynthetic bacteria is a complex of membrane proteins known as the cytochrome bc1 complex, or ubiquinol:cytochrome c oxidoreductase (E.C. This enzyme complex catalyzes electron transfer from ubiquinol to a soluble cytochrome c, coupled to translocation of 2 H+ across the inner mitochondrial membrane per quinol oxidized (2, 3, 4). The complex isolated from beef heart consists of 11 different polypeptides (5, 6) with a total molecular mass of 240 kDa (Table 1). There are four redox centers: two hemes bH and bL of cytochrome b, one heme of cytochrome c1, and one ironsulfur cluster of the Rieske protein. A mechanism accounting quantitatively for the proton translocation coupled to electron transport by this enzyme is a version of the "protonmotive Q cycle" of Peter Mitchell (3, 4). The mechanism also explains the pattern of inhibition by the ubiquinone analogs, antimycin, stigmatellin, UHDBT, myxothiazol, and MOA-stilbene, which react specifically at one or the other of the two catalytic sites at which quinone is processed (3, 4).

Until recently only a low resolution structure for the complex from Neurospora crassa has been available from electron microscopy of two-dimensional crystals (7). In 1991 Yue et al. reported 3-D crystallization of the bc1 complex from beef mitochondria in a tetragonal space group (8). Two other groups independently reported crystallization of the beef complex in different space groups in the following year (9, 10). Recently Yu et al. (11) and Xia et al. (12) reported a partial structure of the complex from the tetragonal crystals. In this structure, the extrinsic domain of the Rieske protein was too disordered to be traced, and cytochrome c1 was only partially traced.

In the meantime we have obtained other crystal forms (13) from other species, including one from chicken heart mitochondria which diffracts to 3.0 Å resolution (Table 2). With these crystals we determined the structure of the complex, including the functionally important Rieske ironsulfur protein and cytochrome c1. We were also able to assign three additional subunits (subunits 8, 10, and 11) that were not assigned before (12).

A comparison of our structures with and without various inhibitors reveals that the ironsulfur cluster-containing extrinsic domain of the Rieske protein assumes one of two conformations in the complexes. In one conformation the ironsulfur cluster of the Rieske protein is close to its electron acceptor, the heme of cytochrome c1, but far from the presumed binding site of its electron donor, ubiquinol, in cytochrome b. In the other conformation the ironsulfur cluster is closer to cytochrome b, and farther from cytochrome c1. This latter conformation is similar to that found in the tetragonal beef crystals of Xia et al. (12).

The binding sites for two Qo site inhibitors, stigmatellin and myxothiazol, and the Qi site inhibitor, antimycin, have been located. The two Qo site inhibitors bind in overlapping but not identical sites.

Taken together our two conformations for the ironsulfur protein and three positions for ubiquinone analog inhibitors are compatible with all the reactions proposed by the Q-cycle mechanism for electron transfer coupled to proton translocation. However, no one structure alone would be competent. We therefore propose that the reaction mechanism for electron transfer in the cytochrome bc1 complex requires a dramatic conformational change involving movement of the ironsulfur protein.

Overall shape of the cytochrome bc1 complex dimer

In all crystals of the complex from three sources (Table 1) the bc1 complex is present as a dimer (Figure. 1a), in which two monomers are related by a two-fold axis running vertically in the plane of the paper. The protein extends from the membrane 79 Å into the matrix space and 31 Å into the inter-membrane region on either side of a transmembrane region 40 Å thick, giving a total length of 150 Å perpendicular to the membrane. The overall shape of the dimer is similar to that described for the beef complex by Yu et al. (11) and Xia et al. (12), but considerably more protein has been modeled in the inter-membrane domain. We have located subunits 1 through 8 and 10 in the electron density of the chicken crystal. We assign subunit 10 to the transmembrane helix labeled N1 by Xia et al (12), based on good correlation between side chains observed in our chicken map with the sequence of this subunit from beef. Subunit 11 seems not to be present in our preparation of the chicken enzyme, but is present in the beef and rabbit enzymes. It probably corresponds to the transmembrane helix labeled N2 by Xia et al., because this helix is present in the three crystal forms from the beef and rabbit enzymes and not in the chicken crystals. However the resolution of our mammal crystals is not high enough to confirm this by side chain correlation with the sequence. Subunit 9 has not been assigned yet, and is also missing from the structure of reference 12. This subunit is the pre-sequence (14) of the Rieske protein, which gets cleaved off by a matrix processing protease, so it is likely that at least its cleavage site is on the matrix side of the membrane. We also see densities at a number of sites in the transmembrane portion which we attribute to ubiquinone, detergents, and phospholipids. These have not been modeled yet.

In the transmembrane domain the helices of the dimer fall into two clearly separated, packed bundles. We arbitrarily divide the dimer so that one monomer corresponds to one packed bundle of helices in the transmembrane region.

Inhibitor binding sites

The inhibitors antimycin, myxothiazol, and stigmatellin, when present in stoichiometric excess during crystallization, resulted in electron density increases that could be interpreted as due to the bound inhibitors. The general positions of the antimycin, stigmatellin, and myxothiazol binding sites are similar to those inferred from the figures in references 11 and 12. Although the limited resolution does not allow detailed atomic model-building, we have constructed speculative models consistent with the electron density. These inhibitor binding sites, and especially the Qo site, are the target of active drug design efforts to produce environmentally safe and effective plant protection fungicides for agricultural use (15-17).

The antimycin site. Based on its mode of inhibition, antimycin is thought to bind at the Qi site postulated in the Q-cycle mechanism, where ubiquinone is reduced by electrons from cytochrome b with uptake of protons from the matrix space (resulting in proton translocation when ubiquinol is subsequently oxidized at the Qo site with proton release to the external medium). The antimycin site (Figure 2a) was near the high potential heme of cytochrome b, in a cavity surrounded by the heme, the transmembrane helices A, D, and E, and the amphipathic surface helix a, with possible protonic connection to the matrix phase via or around conserved histidine H202. The close approach of the aromatic ring of the inhibitor to the heme was expected from the effect of antimycin on the alpha absorption peak of the high potential heme and the fluorescence quenching of antimycin when specifically bound at this site (18). Residues F221, D21, and T194 are also close enough to contact the inhibitor. One of the heme propionates is in van der Waals contact with the inhibitor and curves around to form an ion pair with R101. The conformation of this propionate, which differs from that depicted for the tetragonal beef crystals (12), is the same in the absence of antimycin.

The stigmatellin site. The stigmatellin binding pocket (Figure 2b) is formed by the C-terminal end of helix C, the helix cd1, the ef linker (including the highly conserved -PEWY- sequence and the helix ef), and the N-terminal end of helix F. Residues P271, F275, and M125 of cytochrome b and H161 of the Rieske protein, which has moved from its position in the native crystal (see below), are near the inhibitor. Residues 126-129 of helix C, and 140-147 of the linker cd, are also close by. In the native crystals Y279 passes through the region where we have modeled the stigmatellin head group, but in the stigmatellin-complexed crystal Y279 has moved and is interacting with R283 and with the Rieske backbone around C160.

The myxothiazol site. Myxothiazol (not shown) binds in roughly the same place as stigmatellin, but displaced a little toward the center of the membrane and toward the low potential b heme. It is also close to P271, but where stigmatellin reaches outward from P271 toward the Rieske protein, myxothiazol and MOA-stilbene reach toward Y132 and F129 in helix C, in the vicinity of the low potential heme. This may be the site from which electron transfer from the ubisemiquinone to the cytochrome bL heme occurs.

Arrangement of the protein domains in the intermembrane region

Figure 3 shows a slab including the extrinsic domains in the inter-membrane region of the chicken complex. The two cytochrome c1 molecules (purple) contact each other through loops which surround an empty area around the two-fold axis. Subunit 8 (the "hinge protein for formation of the cytochrome c1-c complex" of ref. 19, light blue) and the external ends of subunits 7 (dark blue) and 10 (green) interact with cytochrome c1 on the side away from the dimer interface. The hinge protein consists of a bent hairpin held by two internal disulfide bonds.

Structure of cytochrome c1

Cytochrome c1 is one of the three redox-active proteins in the cytochrome bc1 complex, but is incomplete in the structure of the beef complex described by Xia et al. (12). This subunit is well ordered in our chicken crystals and the entire polypeptide could be traced. Its extrinsic domain forms a wedge-like structure containing the heme, with a C-terminal transmembrane anchor adjacent to helix E of cytochrome b. Figure 4 compares the backbone folding pattern of cytochrome c1 and mitochondrial cytochrome c, a member of Ambler's Class I cytochromes c (20). Cytochrome c has five helical segments, labeled 1- 5. Three helices ( 1, 3, and 5), which are conserved in Class I cytochromes in general, are present in cytochrome c1 and occupy the same positions relative to each other and to the heme. They are colored alike and labeled on both molecules in Figure 4. Conserved aromatic residues involved in interaction between 1 and 5 (F10 and Y97 in mitochondrial cytochrome) are present as Y33 and F189, respectively. The tripeptide PNL starting at residue 30 is conserved in mitochondrial cytochromes c. The proline carbonyl accepts a hydrogen bond from Nd of the histidine heme ligand and the leucine provides hydrophobic environment for the heme ring. This aligns with the tripeptide PDL starting at residue 111 of cytochrome c1. It is conserved in all cytochromes c1 except that of Rhodobacter sphaeroides, which, barring a sequencing error, has ADL. These similarities justify inclusion of cytochrome c1 in the family of class I cytochromes.

Mitochondrial cytochromes c have the pyrrole C corner of the heme exposed at the "front" face, where electron transfer is believed to take place. This corner is also exposed in our cytochrome c1 structure. The exposed C corner of the heme is surrounded by three regions of the protein, consisting of residues 36-41 (corresponding to cytochrome c 13-18, "fingerprint" region), the side-chain of Y95 and residues 104-106 (helix 2' , no corresponding residues in cyt c), and 158-163 (containing the heme ligand M160 and corresponding to cytochrome c 77-82).

Major differences between cytochromes c and c1 are the result of additions or deletions in loop regions. For example, bovine cytochrome c1 has an N-terminal extension of 24 residues before helix 1, compared to 1 residue in bovine cytochrome c. In cytochrome c1 this region interacts with subunit 8, the hinge protein. After helix 1 and the "fingerprint" CXYCH heme-binding stretch, which are similar in the two cytochromes, cytochrome c1 has a long insertion (residues 52-109) between residues 18 and 29 of cytochrome c. This expanded loop includes a region implicated for cytochrome c binding (21) and the dimer contact with cytochrome c in the other monomer seen in Figure 3. Another insertion is found between the methionine heme ligand and helix 5, the six residues 81-86 in cytochrome c correspond to 18 (161-178) in the c1 cytochromes. This region has also been implicated in cytochrome c binding (22). The end of helix 5 is the C-terminus of cytochrome c but the transmembrane helix a6' continues after in cytochrome c1.

There is a second exposure of the heme on the A-D edge: the long loop corresponding to residues 41-58 in Tuna cytochrome c, which is present in cytochromes c and c2, is absent in cytochrome c1. This results in exposure of the heme propionates to the surface. As described below this edge is within electron transfer distance of the ironsulfur cluster in some crystals, suggesting this is the pathway for reduction by the ironsulfur protein.

Structure and location of the Rieske ironsulfur protein

Another of the three functionally important redox-active subunits of the cytochrome bc1 complex, the Rieske ironsulfur protein, is missing in the structure of the tetragonal beef crystal (12). Electron densities in the region of the globular extrinsic domain of this protein in our crystals are weaker than those in the rest of the structure, but clearly present and recognizable (Figure 5). The backbone density is completely connected only when contoured at 1 level or lower, whereas the cytochrome b backbone in the transmembrane helices was continuous even when contoured at 3 . However, the density was good enough to unambiguously locate the known structure of the soluble domain of the Rieske protein (23).

As predicted from hydropathy plots and molecular engineering results (24, 25), the ironsulfur protein has a membrane-spanning helical segment near the N-terminus. This was removed by proteolysis in preparing the soluble domain for structure determination (23). However, the electron density in our map (Figure 5a) continues where the model stops and connects to a transmembrane helix. The transmembrane helix is well ordered.

The N-terminal 24 residues are on the matrix side, and interact with subunit 1. Residues 25 to 62 form a transmembrane helix, in close proximity with the transmembrane helices of subunit 10 and cytochrome c1 (and, in the mammal crystals, the putative subunit 11). The transmembrane helix is slightly curved and highly slanted. It passes through the membrane at an angle of about 32deg. to the two-fold axis, assumed perpendicular to the membrane. This high degree of tilt accounts for the length of the transmembrane helix (37 residues), which had led to suggestions of two transmembrane helices for the Rieske protein (25).

Residues 60-66 are in close contact with both cytochrome b subunits in the dimer, while residues 67-73 provide a flexible "tether" connecting the extrinsic domain of the Rieske protein to its transmembrane helix. Figure 5b shows a close-up of the ironsulfur cluster region of the Rieske protein. Two histidine ligands, residues 141 and 161, are clearly evident as bulges in the density at the tip of the protein. The iron atoms (oblong orange net) are not individually resolved in this map.

The extrinsic domain of the ironsulfur protein is loosely attached and functionally swapped between monomers.

Except for the transmembrane helix, only residues 141-143 of the ironsulfur protein (one of the two loops which enclose the cluster) make contact with cytochrome b in the native chicken crystals. This contact, depicted in Figure 5c, seems to involve interaction of L142 and G143 of the Rieske protein of one monomer with T265 and L263 of cytochrome b of the other monomer.

The extrinsic domain of the ironsulfur protein has no contacts with the other extrinsic domains within a monomer in the native chicken crystals (Figure 3). But the ironsulfur cluster is close to the heme of cytochrome c1 of the other monomer within the complex dimer. As described below this is likely to be the pathway for electron transfer between the ironsulfur protein and cytochrome c1. Taking monomers to be as defined above based on the transmembrane region, the ironsulfur cluster is in a position to transfer electrons with cytochromes b and c1 of the other monomer.

The small number of contacts with the rest of the dimer probably accounts for the poor order of the Rieske extrinsic domain, and suggests that the domain is mobile. This mobility is somewhat restricted in one monomer of the chicken crystals and in the beef and rabbit hexagonal crystals by inter-dimeric crystal contacts involving the extrinsic domain of the Rieske protein. Xia and coworkers (12) also concluded that the ironsulfur protein is highly mobile. They suggested that mobility may be required for function, based on poor order of this domain in their crystals and the large distance between the cluster and the heme of cytochrome c1.

Two conformations of the Rieske protein: Dynamic domain mediated electron transfer

While the distances between the six heme iron peaks of the dimer were the same within experimental error in all four crystal forms, the distance from the iron-sulfur cluster to any heme varied by up to 5 Å in the different native crystals. Based on published distances between iron centers, the ironsulfur cluster in the tetragonal beef crystals (11, 12) is in a markedly different position than in any of our native crystals (Table 3). But when the chicken cytochrome bc1 complex was treated with a saturating amount of the inhibitor stigmatellin before crystallization, we found the extrinsic domain of the ironsulfur protein at a location different from that in native crystals, and the ironsulfur cluster at a location similar to that reported for the tetragonal beef crystals (12). This movement can be simply and dramatically demonstrated using Bivoet difference maps constructed from diffraction data collected with X-ray wavelength near the iron absorption edge. Due to anomalous scattering by iron, the peaks in such maps indicate positions of irons in the complex: 3 heme irons of the cytochromes and the ironsulfur cluster of the Rieske protein. Such maps are shown in Figure 6, in the absence (a) or presence (b) of stigmatellin. The peak labeled Fe2S2 increases in intensity and moves closer to the hemes of cytochrome b in the presence of stigmatellin. We call this the proximal conformation of the Rieske protein, and the conformation in our native crystals the distal conformation. The relative position of the ironsulfur cluster in the chicken crystals with stigmatellin is 16 Å from the position in the native chicken crystals, and 20 Å from that in the beef hexagonal crystals.

A stereo view of the two conformations of the Rieske protein in context of the entire bc1 complex dimer and in isolation with cytochrome b and the heme of cytochrome c1 are shown in Figures 1b and c. The two locations of the extrinsic domain of the Rieske protein are related by a rotation of 57deg. about an axis passing near residues 93 and 182 of the protein, perpendicular to the plane of the picture in Figure 1c. The transmembrane helix and matrix-side portion are unchanged in the presence of stigmatellin. The coil consisting of residues 68-73 is stretched out in the presence of stigmatellin, allowing this end of the soluble domain to move farther from the membrane as the Fe2S2 cluster on the other end moves closer. In a crystal with both stigmatellin and antimycin bound, the ironsulfur position was essentially the same as that with only stigmatellin. In crystals with only antimycin or myxothiazol bound the ironsulfur position was similar to that in the crystals with no inhibitors.

In the proximal conformation the ironsulfur cluster of the Rieske protein is in H-bond distance of the occupant of the Qo site - stigmatellin in our crystals (Figure 2b) but by inference the electron donor ubiquinol in vivo - and in the distal conformation the ironsulfur cluster is close to its electron acceptor, the heme of cytochrome c1. This suggests that the reaction mechanism for electron transfer in the cytochrome bc1 complex requires this dramatic conformational change involving movement of the extrinsic domain of the ironsulfur protein.

Feasibility of electron transfer from the ironsulfur protein in the distal conformation to cytochrome c1. In the native chicken crystals the second loop of the Rieske protein enclosing the cluster (residues around H161) faces toward cytochrome c1, approaching the heme propionates and residues 106 and 145 of cytochrome c1. There is, however, no electron density contact with cytochrome c1 in the chicken crystals when contoured at the 1.0 level.

As shown in Table 3, the Rieske protein is closer to cytochrome c1 in our two beef crystals. In these crystals there is electron density contact at the 2 level between the Rieske protein around C160 (which forms a disulfide bond holding the cluster-binding loops together) and cytochrome c1 around G107 (between helix 2' and the heme-bracing proline P111). This very likely represents the configuration of the ironsulfur protein during electron transfer to the cytochrome. H161 of the Rieske protein, which provides one of the ligands to the Fe2S2 cluster, is 4.0 Å from an oxygen atom of heme propionate D and 8.2 Å from the edge of the heme -bonded system at the C3D atom. From this distance (8.2 Å) we can calculate an approximate rate of electron transfer from the ironsulfur protein to cytochrome c1 of 4.8 - 80. x 106 s-1, assuming nonadiabatic electron tunneling with a reorganization energy of 0.7 to 1.0 electron volts and G near zero (26). This is significantly faster than measured rates for this reaction (27) so if the protein spends a small fraction of time in this conformation it could account for the rate. In the native chicken crystals this distance is 14.4 Å, which would give a rate of 1.8 - 15. x 103s-1) with the same assumptions. In the crystal with stigmatellin, or the tetragonal beef crystals (12) the shortest distance from the cluster or its ligands to the heme tetrapyrrole ring is 27 Å, giving with the same assumptions a rate of 10-4 s-1 and making it very unlikely that the enzyme could function in this single conformation.

Model for electron transfer through the high potential chain.

Figure 7 shows a ribbon diagram of the extrinsic domains of the Rieske ironsulfur protein and cytochrome c1, as well as cytochrome c bound to cytochrome c1 at a hypothetical site and orientation. The position of the ironsulfur protein is that from the chicken crystals (in the beef crystals it is about 4 Å closer to cytochrome c1). This diagram illustrates the possibility of electron transfer into cytochrome c1 via the D propionate and out of cytochrome c1 via the C corner of the heme to cytochrome c. The distance between the two cytochromes is 10.2 Å, measured between atoms C2C of each heme (the closest approach of the -bonded systems). Assuming G near zero and reorganization energy in the range 0.7 to 1.0 gives electron transfer rates in the range of 0.6-5.1 x 106 s-1.


Purification and crystallization

The cytochrome bc1 complex was purified from different vertebrate heart tissues essentially as described for the potato complex (28). Mitochondria were prepared by the method of Smith (29) and solubilized using the detergent dodecyl maltoside. The complex was isolated from the extract by chromatography on DEAE Sepharose CL6B and further purified by size exclusion chromatography on Sepharose CL6B (13). The protein was concentrated to around 200 uM by ultrafiltration through an Amicon YM-100 membrane, precrystallized by mixing with 100 mM KMES pH 6.5 and 10% PEG-4000, and redissolved in 20 mM K-MOPS 7.5, 20 g/L n-octyl--D-glucopyranoside, and 100 mM NaCl. Aliquots (5-20 l) were mixed with an equal volume of precipitant containing 20 mM KMES pH 6.7, 75 mM NaCl, 10% glycerol, and 6% PEG 4000; and subjected to vapor diffusion against 30% glycerol.

To co-crystallize the bc1 complex with the high-affinity inhibitors antimycin, myxothiazol, or stigmatellin, the inhibitor was added from an ethanolic solution (the final ethanol concentration was below 1% v/v) in a 1.5-2.0-fold molar ratio to the pooled fractions from the final column at a protein concentration of 5-10 M before concentrating and precrystallizing as above.

Cryogenic data collection and reduction

After crystallization was complete (five to thirty days after setup), 20 ul of cryoprotectant containing 10 mM K-MES pH 6.7, 10 mM n-octyl--D-glucopyranoside, 25% glycerol, and 10% PEG 4000 was added to the solution containing the crystals from chicken complex, and the reservoir was changed to 35% glycerol for further concentration of glycerol and PEG without increasing ionic strength. After this equilibration or in some cases after further soaking in cryoprotectant consisting of 30% glycerol, K-MES, and n-octyl--D-glucopyranoside, crystals were frozen in liquid ethane or nitrogen, or in the cryogenic stream, and data were collected at 70deg.-100deg. K. A suitable procedure for flash-freezing the beef and rabbit crystals has not yet been developed. Diffraction data were processed by the programs DENZO and SCALEPACK (30).

Structure determination

The chicken crystals were phased by isomorphous replacement and the resulting electron density was used to phase the other crystal forms by molecular replacement. Heavy atom derivatives were first analyzed using XtalView (31). The RAVE package (32) was used for molecular averaging, map skewing, and rotation-translation operator improvement. The CCP4 package (33) was used for final heavy atom refinement and phase calculation (program MLPHARE), and for finding molecular replacement solutions (programs ALMN and TFFC). The phases were improved and extended to the resolution limit of the data by multi-crystal and noncrystallographic symmetry averaging. During the phase improvement and extension process, correlation coefficients between the calculated electron density map of the Rieske protein and our experimental electron density, which were monitored as a measure of the improvement of the maps, increased to 80-85% in the different data sets. The coefficient between subunits 1 and 2 increased to 40-48%.

Model building was done with the program O (34), and the structures were illustrated using this program or Molscript (35) and Raster3D (36).

All subunits of the bc1 complexes of vertebrates are expressed in the cytoplasm, except cytochrome b. Cytochrome b, which is expressed in mitochondria, has sequence identity of 74% between the chicken and beef proteins. Since amino acid sequences of chicken subunits of the complex have not been reported, we have used the sequences of the beef proteins for the remainder in our model building. Cytochrome c, a cytoplasmically expressed mitochondrial protein, has 89.5% identity between chicken and beef. Myosin light chain two of chicken is 91% identical to that from human or mouse.

Location of iron centers from anomalous data

Anomalous data at wavelength near the iron K absorption edge (7131 eV) were collected for native and stigmatellin-containing crystals, and Bivoet difference maps with coefficients of (F+ - F-) and improved experimental phases retarded by 90deg. were made to locate the iron centers.

Electron density map calculations

The electron density maps were calculated using coefficients of (2Fo-Fc) e-ic, where the Fo values are from the experimentally determined intensities but the Fc and c are calculated from the previous map after multiple crystal averaging. In the case of unobserved reflections, Fo was replaced by Fc as recommended (37), resulting in coefficients of Fc e-ic for those terms. This "fill-in" procedure was used both during averaging and, unless otherwise noted, in making the final maps used in the figures.

Correspondence and requests for materials to: Dr. Edward A. Berry and Prof. Sung-Hou Kim, Calvin Laboratory # 5230, University of California, Berkeley, Berkeley, CA 94720-5230, U.S.A. Atomic coordinates of chicken cytochrome bc1 complex have been deposited in the Brookhaven Protein Database for release in May 1998 (entry XXXX for the native chicken structure and YYYY for the stigmatellin+antimycin inhibited chicken structure).


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20. Ambler, R.P. The Structure and Classification of Cytochromes c in From Cyclotrons To Cytochromes (eds. N.O. Kaplan & A. Robinson), pp. 263-280 (Academic Press, New York, 1982), and Sequence Variability in Bacterial Cytochromes c, Biochim. Biophys. Acta 1058, 42-47, (1991)

21. Stonehuerner, J., O'Brien, P., Geren, L., Millett, F., Steidl, J., Yu, L., & Yu, C.-A. Identification of the Binding Site on Cytochrome c1 for Cytochrome c. J. Biol. Chem. 260, 5392-5398 (1985).

22. Broger, C., Salardi, S. & Azzi, A. Interaction between Isolated Cytochrome c1 and cytochrome c. Eur. J. Biochim. 131, 349-352 (1983).

23. Iwata, S., Saynovits, M., Link, Th. A. & Michel, H. Structure of a water soluble fragment of the 'Rieske' iron-sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5Å resolution. Structure 4, 567-579 (1996).

24. Van Doren, S. R., Yun, C.-H., Crofts, A. R. & Gennis, R. Assembly of the Rieske iron- sulfur subunit of the cytochrome bc1 complex in Escherichia coli and Rhodobacter sphaeroides membranes independent of the cytochrome b and c1 subunits. Biochemistry 32, 628-636 (1993).

25. Link, T. A., Schägger, H. & von Jagow, G. Structural analysis of the bc1 complex from beef heart mitochondria by the sidedness hydropathy plot and by comparison with other bc complexes. in Cytochrome Systems: Molecular Biology and Bioenergetics (eds. Papa, S., Chance, B. & Ernster, L.) 289-301 (Plenum Press, New York, 1987).

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27. Crofts, A.R. and Wang, Z. Photosynthesis Research 22, 69-87 (1989)

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Acknowledgments: We thank Thomas Link and his co-workers for providing coordinates for the soluble domain of the Rieske ironsulfur protein before their release from the Protein Data Bank, Henry Bellamy for performing the XAFS scan and for advice on MAD data collection, Sangjin Hong for preparing coordinate files for the inhibitors, and Liang Tong and Dave Schuller for advice on molecular averaging. This investigation was supported by NIH (grants to EAB and ARC) and by the Office of Biosciences and Environmental Research, U.S. Department of Energy (grant to Sung-Hou Kim). The work was partially done at SSRL which is operated by the Department of Energy, Division of Chemical/Material Sciences. The SSRL Biotechnology Program is supported by the National Institutes of Health Biomedical Resource Technology Program, Division of Research Resources.


Table 1. Subunits of bovine heart cytochrome bc1 complex

Subunit                          Residues        MW                    
1 Core 1                   446                   49132                 
2 Core 2                   439                   46471                 
3 Cytochrome b             379                   42592                 
4 Cytochrome c1            241                   27288                 
5 Rieske Fe-S              196                   21611                 
6 13.4 kDa                 110                   13347                 
7 "Q-binding"              81                    9590                  
8 c1 "hinge"               78                    9170                  
9 Fe-S preseq.             78                    7956                  
10 c1-assoc.               62                    7198                  
11 6.4 kDa                 56                    6363                  
Apo-bc1 complex            2166                  240718                
Fe2S2                                            76                    
Heme c1                                          616                   
Heme bH                                          616                   
Heme bL                                          616                   
Prosthetic groups                                2024                  
Holo-bc1 complex                                 242,742 Da            

Table 2. Parameters of crystal forms used in the structure determination
                                                              #AU   Mono                     
Protein            Space Group   Unit Cell                   cell   AU      Å3/Da    Dmax1   
Beef bc1                          a b c                                              
 Hex bipyramid        P6522      217  217  378  90deg.        12      1     5.20     3.8     
                                 90deg.  120deg.                                             
 Monoclinic2           P21       118  178  200  90deg.         2      2     4.16     3.7     
                                 106deg.  90deg.                                             
Rabbit bc1                                                                                   
 Hex bipyramid        P6522      212  212  354  90deg.        12      1     4.60     3.5     
                                 90deg.  120deg.                                             
Chicken bc1                                                                                  
 Orthorhombic        P212121     176  184  242  90deg.         4      2     4.04     3.0     
                                 90deg.  90deg.                                              
 frozen-             P212121     169  181  239  90deg.         4      2     3.76     2.9     
                                 90deg.  90deg.                                              
1. AU = asymmetric unit.  Mono = monomer.  Dmax = d-spacing of recorded indexable                           
reflection with highest resolution.                                                                         
2. The monoclinic beef crystals were mistakenly reported as centered orthorhombic                           
(C2221)  in ref. 13                                                                                         

Table 3. Distances between the Rieske ironsulfur cluster and cytochromes c1 and bL
                                       Distance (Å) from  Fe2S2       Designation:   
                                              cluster to:            (proximal/dist  
Crystal                                  heme bL     heme c1       From heme bL )  
beef P4122 (from ref 13)                   27.0      31.0             proximal     
chicken P212121 (+stigmatellin)            26.4      31.6             proximal     
chicken P212121                            34.3      21.3              distal      
beef P6522                                 34.9      17.2              distal      
beef P21                                   35.1      17.5              distal      
rabbit P6522                               35.5      19.1              distal      

Iron peaks located as peaks in electron density calculated from averaged experimental phases improved and extended by molecular averaging, except for chicken P212121 in the absence of inhibitor, in which case Bivoet difference amplitudes were used with improved experimental phases retarded by 90deg..

Table 4. Structure determination statistics.

(a) Diffraction data for chicken crystals. Each line corresponds to one dataset collected from a single native or derivatized crystal of chicken cytochrome bc1.

                                   Unique      Complete-nes              X-Ray1  
                                                  s (%)                          
              dmin    No. Refln   Refl            (I > 1      Rmerge    Source   
                                                  sigma)        (%)              
chn21         3.60     279,119      70,363     76.3 (58.1)     18.6       RA     
chc01         3.10     556,456      123,869    91.6 (80.5)     10.2      SSRL    
chm           3.01     569,255      141,427    96.6 (74.2)     16.2      SSRL    
chb           2.95     433,902      131,641    81.7 (51.2)     27.8       BNL    
PIP2          3.50     292,339      86,221     91.8 (76.3)     10.8      SSRL    
NSDMA3        3.90     425,028      67,109     99.7 (79.0)     12.4       RA     
TML024        3.50     203,105      61,380     65.2 (53.5)     17.1       RA     
TML034        4.30     110,201      38,103     74.4 (50.6)     21.6       RA     
HPDL5         4.00     129,187      48,715     78.4 (54.0)     20.2       RA     
Iridium6      3.50     177,303      68,522     71.7 (43.5)     13.4       RA     
TMLssrl4      3.50     160,826      61,128     65.6 (43.9)     19.9      SSRL    
HPDLssrl5     3.15     350,204      92,367     71.6 (60.2)     19.3      SSRL    
1. X-ray sources and wavelengths are indicated by:  RA- rotating anode                          
(1.54 Å). SSRL- Stanford Synchrotron Radiation Laboratory BL7-1 (1.08 Å).                       
BNL- Brookhaven National Laboratory X-12b (1.006 Å).                                            

(b) Isomorphous phase determination

Derivatives    Rderi   No. of   Rc            Phasing power in Resolution shell (Å)                               
                 (%)   sites          12.56   8.77   6.74   5.47   4.61   3.98   3.50   Total   
PIP2            13.2     16    0.83    0.88   1.01   1.12   1.12   0.94   0.97   0.86    0.97   
NSDMA3          15.2     16    0.88    0.68   0.61   0.76   0.96   0.93   0.96   0.97    0.89   
TML024          21.5     23    0.79    1.92   1.81   1.94   1.76   1.24   1.05   0.99    1.32   
TML034          28.1     23    0.71    1.84   1.67   1.88   1.67   1.38   -      -       1.66   
HPDL5           15.3     4     0.89    1.04   0.94   0.98   0.96   0.70   0.64   -       0.82   
Iridium6        20.6     11    0.93    0.49   0.50   0.66   0.84   0.72   0.64   0.66    0.67   
Figure of Merit                          0.25   0.58   0.62   0.56   0.48   0.41   0.29    0.46   
2. Ethylenediamine platinum iodide)2                                                                                        
3. N- (5-Nitrosalicyl)- (S-decylmercuri)6-aminothiophenol, a putative antimycin analog                                      
4. Trimethyl lead acetate (different concentrations and soaking times)                                                      
5. Hexaphenyl di-lead                                                                                                       
6. Iridium carbonyl                                                                                                         

Figure Legends

Figure 1. Stereoview ribbon diagrams of the bc1 complex. (a) The native chicken bc1 dimer. The molecular two-fold axis runs vertically between the two monomers. The key for the color coding of each subunit is given in the inset. The presumed membrane bilayer is represented by a gray band. (b) Two conformations of the Rieske protein in one monomer shown in context of the entire dimer. One conformation found in our native chicken crystal (yellow) is superimposed on the other conformation (blue) from crystals grown in the presence of stigmatellin (green). The hemes (red) of cytochrome c1 and cytochrome b as well as two positions of the ironsulfur clusters of the Rieske proteins are shown in orange and green. (c) Isolated close-up of the two conformations of the Rieske protein in contact with cytochrome b with associated hemes (red), stigmatellin (green) and antimycin (purple). The isolated heme of cytochrome c1 (red) is also shown. The rotation axis relating the two positions of the Rieske protein is indicated (white cross).

Figure 2. Inhibitor binding sites. The electron density maps are from crystals containing the inhibitors. (a) antimycin binding site, electron density map contoured at 0.7 . (b) stigmatellin binding site, density contoured at 0.8 (blue) and 5.0 (orange) for the ironsulfur cluster. The backbone of cytochrome b is in red and that of the ironsulfur protein is in magenta.

Figure 3. Structure of the inter-membrane (external surface) domains of the chicken bc1 complex. Viewed from within the membrane, with the transmembrane helices truncated at approximately the membrane surface. Ball and stick models represent the heme of cyt c1, the Rieske ironsulfur cluster, and the disulfide cysteines of subunit 8.

Figure 4. The structure of cytochrome c1 compared to cytochrome c. Top: ribbon diagram of mitochondrial cytochrome c with the open corner of C pyrrole of the heme facing the viewer, and the heme propionates directed downward. Bottom: our current structure of cytochrome c1, rotated to put the common features between the two cytochromes in the same orientation. Corresponding segments of each cytochrome are drawn with the same color. Helices labeled 1, 3, and 5 correspond to similarly labeled helices in cytochrome c, while those labeled 2* and 6* have no counterpart in cytochrome c.

Figure 5. The Rieske ironsulfur protein. The electron density maps are from improved experimental phases. The atomic model of the soluble domain of the ironsulfur protein is from the coordinates of the protein database entry 1RIE (28), positioned as described in the text. (a) a slab through the protein including the cluster and the connection to the transmembrane helix (b) a closeup of the electron density around the ironsulfur cluster of the Rieske protein. The maps are contoured at 1 (blue) and 5 (orange). (c) contact of the Rieske protein (in the heme b distal conformation) with cytochrome b. The model of cytochrome b (red C backbone plus ball-and-stick models for residues W142 and L263 through P266) is from our coordinates. The maps are contoured as in a. (d) interface between cytochrome c1 and the Rieske protein. The electron density map is from merged data of two beef hexagonal crystals, extending to 4.5 Å, phased using experimental phases improved by density modification. The fill-in procedure was omitted in calculating the map, which was contoured at 2 (blue) and 5 (orange). The cytochrome c1 model at right is from our chicken bc1 model, positioned with the same operator used to position the main part of the bc1 complex in these beef crystals.

Figure 6. Relative positions of the redox centers in the two different conformations of the bc1 complex dimer. a. iron centers revealed by Bivoet difference maps near the iron edge. b. Schematic drawing representing the cofactors. Left: from a native crystal, with the ironsulfur cluster of the Rieske protein in distal position from the low-potential heme of cytochrome b. Right: from a crystal with bound stigmatellin, with the cluster in proximal position.

Figure 7. Electron pathway through cytochrome c1 in a hypothetical complex of the bc1 complex with cytochrome c. The ribbon diagram shows the backbones of cytochrome c1, cytochrome c (both with the same color scheme as in Figure 4), and the Rieske protein (yellow). The hemes, the ironsulfur cluster, and surrounding residues are drawn as ball-and-stick models. The balls representing the ironsulfur cluster (red & green) are enlarged for visibility. The position of the Rieske protein relative to cyt. c1 is from the beef hexagonal crystals.