Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.65 A resolution: cofactors and protein-cofactor interactions

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1 Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.65 A resolution: cofactors and protein-cofactor interactions Ulrich Ermler, Gunter Fritzsch, Susan K Buchanant and Hartmut Michel* Max-Planck-lnstitut fr Biophysik, Heinrich-Hoffmann-Str. 7, D Frankfurt/Main, Germany Background: Photosynthetic reaction centres (RCs) catalyze light-driven electron transport across photosynthetic membranes. The photosynthetic bacterium Rhodobacter sphaeroides is often used for studies of RCs, and three groups have determined the structure of its reaction centre. There are discrepancies between these structures, however, and to resolve these we have determined the structure to higher resolution than before, using a new crystal form. Results: The new structure provides a more detailed description of the Rb. sphaeroides RC, and allows us to compare it with the structure of the RC from Rhodopseudomonas viridis. We find no evidence to support most of the published differences in cofactor binding between the RCs from Rps. viridis and Rb. sphaeroides. Generally, the mode of cofactor binding is conserved, particularly along the electron transfer pathway. Substantial differences are only found at ring V of one bacteriochlorophyll of the 'special pair' and for the secondary quinone, QB. A water chain with a length of about 23 A including 14 water molecules extends from the QB to the cytoplasmic side of the RC. Conclusions: The cofactor arrangement and the mode of binding to the protein seem to be very similar among the non-sulphur bacterial photosynthetic RCs. The functional role of the displaced QB molecule, which might be present as quinol, rather than quinone, is not yet clear. The newly discovered water chain to the QB binding site suggests a pathway for the protonation of the secondary quinone QB- Structure 15 October 1994, 2: Key words: bacteriochlorophyll, electron transfer, membrane protein, photosynthesis, proton transfer Introduction The first steps of the photosynthetic conversion of light to energy take place in photosynthetic membranes of various bacteria and organelles. Light is absorbed first by light-harvesting antenna complexes and the energy is then transferred to the so-called photosynthetic reaction centres (RCs), where the primary charge separation and the subsequent electron transfer across the photosynthetic membranes occur with a quantum yield of 100%. The photosynthetic RCs are complexes containing several integral membrane proteins and a number of cofactors. Best characterized are those from the purple photosynthetic bacteria (see [1-3] for reviews). In general, they consist of three protein subunits, which are called H (heavy), M (medium) and L (light) according to their apparent molecular weights as determined by sodium dodecyl sulphate gel electrophoresis. The L and M subunits bind the photosynthetic pigments in a nearly symmetrical manner. The pigments are; one carotenoid, four bacteriochlorophylls of the a and b type, two bacteriopheophytins (a and b), one ubiquinone (or menaquinone) QA (as the so-called primary quinone), one non-haem iron, and another ubiquinone QB (as secondary quinone). Some RCs from purple bacteria contain a fourth protein subunit, a cytochrome c with four covalently bound haem groups. Examples include the RC from Rhodopseudomonas viridis, which possesses bacteriochlorophylls and bacteriopheophytins of the b type, and menaquinone as QA. The RC from Rhodobacter sphaeroides contains bacteriochlorophylls and bacteriopheophytins of the a type, and a ubiquinone as QA. It does not possess a tightly bound cytochrome subunit. The RCs of the purple bacteria Rps. viridis and Rb. sphaeroides were the first membrane-protein complexes of which the crystal structure was determined. Initially the structure of the RC from Rps. viridis was solved by the multiple isomorphous replacement method [4,5] and refined to a final R-factor of 19.3% up to a resolution of 2.3 A [6]. The structure of the RC from Rb. sphaeroides has been determined by molecular replacement by three different groups. All three groups used an orthorhombic crystal form (with slightly different cell dimensions) for structure determination [7-9]. The structures were refined to a resolution of around 3 A with crystallographic R-factors between 20% and 22%. Although the overall structure of the Rb. sphaeroides RC is very similar to that of the Rps. viridis RC, a number of significant differences in cofactor binding have been reported [10-12]. However, it is difficult to assess the importance of these, as there are also differences between the published structures of the Rb. sphaeroides RC. Recently a trigonal crystal form has been obtained which diffracts X-rays to a resolution of at least 2.65 A [13]. Structure determination and refinement to a crystallographic R-factor of 18.6% has now been completed. In this report we describe the cofactor geometry and the *Corresponding author. t Present address: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas , USA. Current Biology Ltd ISSN

2 926 Structure 1994, Vol 2 No 10 protein-cofactor interactions and compare them with equivalent features in the Rps. viridis RC. There are no discrepancies between the various published RC structures with respect to the overall architecture of the L, M and H subunits. This result is in agreement with the sequence homologies of 59% for the L subunits, 50% for the M subunits and 39% for the H subunits [14,15]. The L and M subunits, which possess five membrane-spanning helices each, bind the pigments and form the core of the RC in a quasi-symmetrical manner. Two bacteriochlorophyll molecules (DA, DB) form the primary electron donor, which is also called the 'special pair' due to the close, symmetrical association of DA and DB (we use here the nomenclature proposed by Hoff [16]). Starting from the special pair at the periplasmic side of the membrane, two branches (A and B) of cofactors extend through the protein to the cytoplasmic side. Each consists of one 'accessory' bacteriochlorophyll (BA, BB), one bacteriopheophytin (A 4)B), and one quinone (QA' QB)' (The subscript A indicates the active branch of pigments, which is used to transfer electrons across the membrane, and which is more closely associated with the L subunit.) The electron transfer starts from the special pair. It is likely that BA is the first electron acceptor [17]. However, this is still a matter of debate [18]. The electron is then transferred to A and thereafter to QA. The final electron transfer step, from QA to QB occurs parallel to the membrane close to the cytoplasmic surface. During or after a second electron transfer to the secondary quinone QB' this two-electron accepting cofactor binds two protons. A number of amino acids which seem to be involved in the proton transfer to QB have been identified in mutated RCs [19-21]. Results and discussion Quality of the atomic model Fig. 1 shows part of the electron density map together with the atomic model. The quality of the electron density is excellent for a nominal 2.65 A resolution map. This is probably due to a finer sampling of the Fourier transform as a consequence of the low crystal packing density. The volume to mass ratio, V [22] is 5.3 A 3 Da-1 for the trigonal crystal form, compared with A 3 Da-' for the orthorhombic crystal forms [23-25] and 2.6 A 3 Da -, the average for crystals of water soluble proteins. A total of unique reflections were used in the determination of the structure of the Rb. sphaeroides RC in the new trigonal crystal form, compared with [26], [12] or [9] in the orthorhombic forms. In addition, the crystallographic R-factor (18.6%) and the deviations from standard geometry, 0.14 A for bond lengths and 1.60 for bond angles, are considerably better for the atomic model presented here. We can now hope to resolve some of the discrepancies between the RCs from Rps. viridis and Rb. sphaeroides as well as those between the published structures of the Rb. sphaeroides RC [12]. This is Fig FoI-IFcl electron density map at 2.65 A resolution showing density for the DA bacteriochlorophyll a molecule. The map was contoured at l a level. important, since efficiency, speed and unidirectionality of the light-driven electron transport depend on the finetuning of the interactions of the cofactors with the protein matrix. Cofactor arrangement The arrangement of the cofactors (Fig. 2) along the two branches of the RC is highly conserved in the two purple bacteria [26-28]. The head groups of the cofactors superimpose well except for QB Small differences of the cofactor arrangement are difficult to discuss because they are dependent on the coordinate errors and the points chosen as fixed points to make the alignment. The distances between the centres of mass of the chromophoric rings (Fig. 3) are less dependent on errors. They show clearly that the relative cofactor positions deviate less than 1 A between the two RCs (except for QB)' The arrangement of the bacteriochlorophyll monomers DA and DB of the special pair may indicate minute differences. The overlapping area of the two pyrrol rings I of DA and DB is slightly larger in the Rps. viridis RC

3 Rhodobacter sphaeroides reaction centre Ermler et al X _ (7.4) (10.8) " (10.5) ) Fig. 2. Image of the superpositioned RC cofactors from Rb. sphaeroides (red) and Rps. viridis (green). Regions of exact superposition in the projected image plane appear yellow ) than in the Rb. sphaeroides RC. Furthermore, the minimal distance between the pyrrol rings I of DA and DB is 3.4 A in Rb. sphaeroides and 3.2 A in Rps. viridis. The bacteriochlorophyll BA, the bacteriopheophytins 4 A B and the quinone QA are positioned nearly identically (Fig. 3). The bacteriochlorophyll B B in the Rb. sphaeroides RC is located closer to the periplasmic side than in the Rps. viridis RC. Consequently, the distance between the centres of mass of BB and DB is 0.6 A shorter, that of DB and B 0.5 A larger (Fig. 3). The Q position is shifted substantially and is placed about 3.5 A further away from QA than was observed in the other RC structures from Rps. viridis and Rb. sphaeroides. The deviation angles from the two-fold axis for the cofactors of the A and the B branches agree significantly in both RCs. An optimal superposition of the porphyrin rings in the Rb. sphaeroides RC is achieved by a rotation of between the monomers DA and DB of the special pair, of between the accessory bacteriochlorophylls and of between the bacteriopheophytins. The corresponding values in the Rps. viridis RC are 179.7, and [6]. Large conformational differences (Fig. 2) between the RCs of Rb. sphaeroides and Rps. viridis are found for the phytyl tails of the cofactors, particularly along the B branch. The phytyl tails in both branches are also arranged rather differently, presumably due to the additional carotenoid molecule attached to the B branch [6]. The flexibility of the porphyrin head groups, described by the temperature factors, is 11 A 2 higher along the B branch than along the A branch in the Rb. sphaeroides RC. In the Rps. viridis RC the corresponding temperature factor is only 3 A 2 higher. Using all atoms of the porphyrin cofactors, the average temperature factor along the B branch is 18 A 2 higher than along the A branch in the Rb. sphaeroides RC. In the Rps. viridis RC, this difference is 15 A 2. Fig. 3. Distances (in A) between the cofactors in the RCs of Rb. sphaeroides and of Rps. viridis (in brackets) using only the porphyrin and the quinone rings for the centre of mass calculations. Cofactor conformation and protein-cofactor interactions The special pair In order to simplify the comparisons, the Rb. sphaeroides amino acid residues were labelled according to the Rps. viridis sequence. The special pair is located between the L and the M subunits close to the periplasmic side. It is kept in its position by residues of the C, D, E and CD helices. Small deviations from the planarity of the porphyrin rings are observed but with the exception of the special pair we have not analyzed these further due to the limited resolution. Substantial conformational differences at the special pair are found at rings V. They are discussed below. Both central Mg 2 + ions of the DA and DB molecules bind strongly to the NE2 atoms of HisL173 and HisM200 with distances of 2.3 A and 2.2 A, respectively. In order to simplify the comparisons, the Rb. sphaeroides amino acid residues were labelled according to the Rps. viridis sequence. In contrast to published results [10], the ring I keto carbonyl oxygen of DA is hydrogen bonded to HisL168 (3.2 A distant) as in the RC from Rps. viridis (where the distance is 2.8 A). The symmetry-related amino acid residue near the D B molecule is a phenylalanine (a tyrosine in the Rps. viridis RC) incapable of forming a hydrogen bond to the ring I keto carbonyl group. The electron density slightly favours the conformation where the carbonyl oxygen of D is oriented away from the Mg 2 + ion of DA. However, the electron density at 2.65 A resolution also allows a 180 rotation of the ring I keto carbonyl group. In this conformation the ring I carbonyl oxygen of D B might form weak contacts to the

4 928 Structure 1994, Vol 2 No 10 Mg 2 + ion of DA (about 3.5 A away) and to the hydroxyl group of TyrM208 (about 3.7 A away). The ring V of the DA bacteriochlorophyll in the Rb. sphaeroides RC is bent towards helix C. The analogous ring in the Rps. viridis RC is bent in the opposite direction, towards helix D (Fig. 4). Conformational differences linked to those of ring V are observed at ring III, at ring IV and at the beginning of the phytyl tail. The different bending of ring V results in a maximal distance of 5.3 A between the ester methyl groups of ring V. In contrast to ring V of the DA bacteriochlorophyll itself, the polar groups of ring V in the Rb. sphaeroides RC are directed towards helix D (Fig. 4). The ester carbonyl oxygen interacts with the sulfhydryl group of CysL247 (3.7 A away) whereas the keto carbonyl oxygen forms an energetically unfavourable contact to the side chain of MetL248. The hydroxyl group of SerL244 does not interact with the ester carbonyl oxygen of ring V (in contrast to [12]) but with the peptide carbonyl oxygen of AlaL240. Since CysL247 and MetL248 are replaced by the smaller glycine and threonine residues in the Rps. viridis RC, ring V turns towards these residues of helix D to form a hydrogen bond between the ester carbonyl oxygen and the hydroxyl group of ThrL248. Furthermore, ring V is oriented towards helix D because MetL127 of helix C and the ethylidene group of BA occupies the space on the other side of ring V. In the Rb. sphaeroides RC the change of MetL127 to alanine and the more favourable conformation of the ethyl group of BA allows the conformational change of ring V towards helix C (Fig. 4). The largest conformational differences at the DB bacteriochlorophyll of the two superimposed RCs are again observed at ring V, but the deviations of about 1.0 A for the ester methyl group are significantly smaller than those for the DA bacteriochlorophyll. A few amino acid changes around ring V, notably GlyM278 and LeuM154 in the Rb. sphaeroides RC (which are replaced by alanine and phenylalanine, respectively, in the Rps. viridis RC) and a small shift of helix E are responsible for the small rotation of ring V. Neither the ester nor the keto carbonyl oxygen atom of ring V from the DB bacterio-chlorophyll molecule undergo polar interactions with the polypeptide chain. Accessory bacteriochlorophylls The monomeric or accessory bacteriochlorophylls BA and BB are located between the special pair and the bacteriopheophytins with van der Waals contacts to both. Residues of the helices B, C, D and CD form the polypeptide scaffold of the binding site. Although the intermediate position and the favourable redox properties of the BA bacteriochlorophyll suggest a direct role as intermediate electron acceptor, its role is still under discussion [17,18]. Significant conformational differences between the Rps. viridis and Rb. sphaeroides RCs are only found at the side groups of ring II, and these are caused by the chemical difference between the ethyl group of bacteriochlorophyll a and the ethylidene group of bacteriochlorophyll b (Fig. 4). The polar interactions of the protein matrix with the accessory bacteriochlorophylls are symmetrical and correspond to those in the Rps. viridis RC. The Mg 2+ ions of the BA and B B molecules coordinate with the NE2 atoms of HisL153 (2.3 A away) and HisM180 (2.2 A away). The No1 atom of HisL153 is hydrogen bonded to the peptide carbonyl oxygen of GlyL149 and IleL150. This kind of interaction between the Mg 2 + and HisL153 was not observed in one of the published Rb. sphaeroides RC structures [10]. As in the Rps. viridis RC the keto carbonyl oxygen atoms of both rings V are hydrogen bonded via a water molecule, to HisL173 and HisM200, respectively. In contrast to earlier studies on the orthorhombic crystal forms [10,12], the hydroxyl group of SerL178 is 4.3 A away from the ester carbonyl oxygen of the BA bacteriochlorophyll, too far to form a hydrogen bond. Bacteriopheophytins The bacteriopheophytins A and 4 B are surrounded by residues of the transmembrane helices D, E, B and C. Their conformations are identical in the two RCs, except for the side groups of rings II, where the differences are due to the chemical difference between bacteriopheophytin a and b. The asymmetric hydrogenbond pattern between 0A and B corresponds to the observations in the Rps. viridis RC [29]. The indole nitrogens of TrpL100 and TrpM127 form hydrogen bonds with the ester carbonyl oxygens of rings V with Fig. 4. Stereoview around ring V of the D molecule in the RCs of Rb. sp'aeroides (carbon atoms drawn in yellow, oxygen atoms in red, nitrogen atoms in blue, sulphur atoms in magenta, helices C and D shown as blue coils) and Rps. viridis (green). The large structural differences at ring V are induced by several amino acid exchanges and especially by the different conformations of the ethyl and the ethylidene groups of bacteriochlorophyll a and b.

5 Rhodobacter sphaeroides reaction centre Ermler et al. 929 distances of 2.9 A and 2.8 A, respectively. The carboxyl group of GluL104, which is probably in the protonated state, forms a hydrogen bond to the ring V carbonyl (2.7 A) of 4 A. No such hydrogen bond is found for qb. However, functional studies of site-specific mutants have clearly shown that other amino acids in position L104 scarcely influence the dynamics and direction of the electron transfer [30]. Along the B branch of the RC, the residue corresponding to GluL104 is ThrM131. In contrast to the results derived from the orthorhombic crystal forms [10,12], the hydroxyl group of ThrM131 is 3.9 A away from the ring V carbonyl group and is therefore unable to form a hydrogen bond. The conformations of the residues involved in hydrogen bond formation are very similar in both species. Primary quinone The primary quinone binding site is located on the A branch between the bacteriopheophytin A and the cytoplasmic side of the RC, which is formed primarily by hydrophobic residues of the M subunit. The structural difference between the menaquinone in the Rps. viridis RC and the ubiquinone in the Rb. sphaeroides RC causes only minor rearrangements of the QA binding site (Fig. 5). The side chain of MetM260 (replaced by isoleucine in the Rps. viridis RC) evades the methoxy group of the ubiquinone and is directed towards AlaM243. The more bulky valine in the Rps. viridis RC would prevent the observed movement of MetM260. Both quinone oxygens interact with the protein surrounding it in the same way as reported previously for the RC from Rps. viridis [29] (Fig. 5). The proximal quinone oxygen is hydrogen bonded to the side chain of HisM217 (distance 3.2 A), the distal quinone oxygen to the peptide nitrogen of AlaM258 (2.8 A). The different bond lengths may be consistent with the finding that one carbonyl group is dominant for QA binding [31]. Although fixed water molecules are not found in the QA binding pocket, several water molecules between the cytoplasm and QA indicate solvent access to the QA binding site. The closest neighbouring water molecule is 4.5 A away from QA and connected to a solvent cluster inside the H subunit. The positions of the water molecules are remarkably well conserved in the structure of the Rps. viridis RC. Secondary quinone The binding site of the secondary quinone QB formed by the residues of the D, E and DE helices of the L subunit, is located at the B branch close to the H subunit.the temperature factor of the secondary quinone is very high (75 A 2 ) suggesting that the binding site is only partly occupied. Although the electron density of the QB molecule is not well formed, its position appears to be reliably determined. The major difference between the RC structure based on the trigonal crystal form and the other RC structures (both from Rps. viridis and Rb. sphaeroides) is a 5 A displacement of the position of the QB molecule (Fig. 2). The QB molecule in the Rps. viridis RC is deeply buried in the QB binding pocket. In the Rb. sphaeroides RC structure described here, QB is bound at the more hydrophobic entrance of the QB binding pocket, in a manner similar to the QB inhibitors of the triazine class in the Rps. viridis RC [29]. The space in front of the QB molecule is filled by water molecules partly seen in the electron density map of the Rb. sphaeroides RC. The hydrogen bond pattern of the different bound QB molecules is consequently changed. In the structure of Rb. sphaeroides RC described here, only the distal ubiquinone carbonyl oxygen can form polar interactions with the protein environment - namely with the peptide nitrogen of IleL224 (about 2.8 A away) and the peptide carbonyl oxygen of TyrL222 (about 3.1 A away). The proximal ubiquinone oxygen is not in contact with a hydrogen bond donor (Fig. 6). Both methoxy oxygen atoms are in contact with polar groups of the polypeptide chain. The distal oxygen interacts with the peptide nitrogen of IleL224, the proximal oxygen interacts with HisL190 via a water molecule. In the Rps. viridis RC both quinone and methoxy oxygen atoms are hydrogen bonded to the protein matrix. Despite the different QB binding positions, the profile of the pocket is conserved in all characterized RC structures. Only the side chain of PheL216 (Fig. 6), positioned within van der Waals bonding distance of QB rotates significantly to improve the aromatic interactions with the different bound quinones. The residues Fig. 5. Stereoview of the primary quinone QA and the surrounding protein residues of the aligned RC structures from Rb. sphaeroides (colour scheme as for Fig. 4) and Rps. viridis (green). Helices D' and E' are shown as blue coils, hydrogen bonds between the quinone carbonyl oxygens and the protein matrix as red dotted lines. Despite the fact that QA is a menaquinone in the Rps. viridis RC and a ubiquinone in Rb. sphaeroides RC, the profile of the pocket, the position of the quinone ring and the hydrogenbond pattern are nearly identical.

6 930 Structure 1994, Vol 2 No 10 Fig. 6. Stereoview of the secondary quinone QB and the surrounding protein residues in the aligned Rb. sphaeroides and Rps. viridis RCs. The putative polar interactions between the distal quinone carbonyl oxygen and the polypeptide chain are shown as red dotted lines. The image shows the conserved QB binding pocket with the two different QB positions interacting with different residues of the polypeptide chain. The colour scheme is as in Fig. 4. GluL212, AspL213 (AsnL213 in Rps. viridis) and SerL223, which seem to be involved in the proton transfer to QB [19-21], possess similar conformations in both RCs (Fig. 6) but their distances to the QB binding position described here are greatly increased. We think that this RC structure may contain quinol and not quinone as QB. Though we can not prove it at present, it is possible that ascorbate present during the isolation of the RC may have led to the double reduction and protonation of QB upon light absorption. The increased overlapping area between the QB ring and the benzyl ring of PheL216 and the interaction of the distal carbonyl oxygen of QB and the peptide oxygen of TyrL222 additionally support the assumption that QB is present as quinol. Experiments to test this hypothesis are under way. Non-haem iron Whether the non-haem iron ion plays a role in the electron transfer from QA is still unknown. However, it seems to have a function in stabilizing the tertiary structure of the RC [32]. It is ligated to residues of the D, E and DE helices of the L and the M subunits, and lies close to the H subunit between QA and QB' Its ligands are the Ne2 atoms of the histidines L190 (2.1 A distant), L230 (2.1 A), M217 (2.1 A) and M264 (2.1 A) from four different transmembrane helices and the two side chain oxygens of GluM232 (2.2 A and 2.3 A away). These ligands form a distorted octahedron which was described for the Rps. viridis RC [5] and in one of the Rb. sphaeroides RC structures [11]. The RCs from Rb. sphaeroides and Rps. viridis can be superimposed well, with a root mean square deviation (rmsd) of less than 0.5 A in the region around the iron ion. This conservation of the structure underlines the importance of the non-haem iron ion. Spheroidene The carotenoid molecule (Fig. 7) is bound at the B branch and interacts with residues of the helices A, B, C and the connective segment between helices C and D. The two ends of the spheroidene molecule are chemically similar but not identical (Fig. 7). The small differences are detectable in the electron density map (Fig. 8). Consequently the orientation of the spheroidene molecule can be determined reliably. The residues that are in contact with the carotenoid are predominantly aromatic and hydrophobic. Five tryptophan and eight phenylalanine residues are found within a radius of 5 A (Fig. 7). Polar atoms of the polypeptide chain are only observed near the centre of the polyene chain, not near the polar head group of the spheroidene molecule [9,10]. Spheroidene is attached to the M subunit in a kinked conformation perpendicular to the membrane-spanning helices. Its change in direction is caused by a cis bond in position 13 or 15 (Fig. 8). A definite assignment has to await refinement at higher resolution. A 13,14-cis bond would contradict the results obtained by Raman and NMR spectroscopy [9,33] which indicate a 15,15'-cis bond. The cis bond is oriented parallel to the porphyrin plane of the accessory bacteriochlorophyll B B and within van der Waals distance (3.7 A) of it. The interaction between BB and spheroidene might be important for the binding affinity, because removal of B B is accompanied by loss of the carotenoid [34]. The distance between the carotenoid at the cis bond and the bacteriochlorophyll ring of DB is approximately 10 A.

7 Rhodobacter sphaeroides reaction centre Ermler et al. 931 Fig. 7. Stereoview of the spheroidene molecule, the surrounding aromatic residues within 5 A, the B and the D B molecule. The colour scheme is as in Fig. 4. The aromatic residues (five tryptophans, eight phenylalanines, one tyrosine and one histidine) are predominantly found at the ends of the carotenoid. The carotenoid protects the Rb. sphaeroides RC against photo-oxidation by quenching the triplet state of the special pair. Its proximity to BB suggests that the energy transfer from the special pair is achieved via BB' as proposed from spectroscopic measurements [35]. The chemically different carotenoid 1,2-dihydro-neurosporene in the RC of Rps. viridis [6] possesses a similar conformation and position, and the deviation of the molecule around the cis bond is in the range of 0.8 A. In contrast to spheroidene (Fig. 8) no electron density was found at the ends of the 1,2-dihydroneurosporene molecule. Electron transfer The unidirectional charge transfer is due to asymmetric features of the A and B branches. The chemical nature of the cofactors is identical at both branches. However, the sequence homology between the L and the M subunits is only 31%. The overall asymmetric features derived from the structure of the Rps. viridis RC [6] are maintained in the Rb. sphaeroides RC presented here, namely the different conformations of the phytyl and isoprenoid chains, the higher temperature factors of the cofactors along the M subunit and the deviations of the cofactor arrangement from the two-fold symmetry. Additionally, specific protein-cofactor interactions (and therefore the asymmetry between the branches) are also highly conserved for the accessory bacteriochlorophylls, the bacteriopheophytins, QA and the non-haem iron (Table 1). This contradicts a number of hydrogen bond assignments reported for the orthorhombic crystal forms of the Rb. sphaeroides RC [10,12]. Significant structural differences between both RCs are observed at the special pair, especially in the pattern of the hydrogen bonds to the carbonyl oxygens of the bacteriochlorophylls (Table 1) and in the deviations from planarity of the bacteriochlorophyll macromolecules. These structural changes induce a different charge distribution between the monomers of the special pair which does not affect the preference of the A branch for electron transfer as postulated previously [36]. The large displacement of the QB molecule is at present not understood and must be further investigated. Fig Fol-IFcl electron density map (contoured at la level) for the spheroidene molecule, showing the orientation of the asymmetric polyene chain. The resolution of the electron density map is not sufficiently high to distinguish between a cis bond in position 13 (giving the structure shown in yellow) or 15 (in red). The electron transfer between the chromophoric cofactors is influenced by specific amino acid residues. When the model is inspected for putative electron transfer pathways TyrM208, located between the special pair, the BA and the 4 A molecules (Fig. 9), is immediately noticeable. The residue is found in the same conformation and nearly at the same position in both RCs. Furthermore, the symmetry-related residue at the B branch is PheL181. Site-specific mutants reveal that the speed, but not the unidirectionality of the electron transfer is influenced by the residue at position M208 [37-39]. As depicted in Fig. 9, some of the hydrophobic amino acid residues in the vicinity of TyrM208 are not conserved. AlaL240 in Rb. sphaeroides is changed to isoleucine in Rps. viridis, ValL241 to phenylalanine, PheL180 to leucine and LeuM207 to alanine. However, the net change of the four neighbouring residues is only one methyl group (from valine to isoleucine) providing a very similar hydrophobic environment for TyrM208. TrpM250 is located between ~A and QA and is within van der Waals contact distance of both. It is well conserved among all purple bacteria and found in a

8 932 Structure 1994, Vol 2 No 10 Table 1. Specific interactions between apoprotein and the cofactors in the RCs of Rb. sphaeroides and Rps. viridis. Rhodobacter sphaeroides Rhodopseudomonas viridis Cofactor Atom of Atom of DH...A Atom of Atom of DH...A cofactor apoprotein distance (A) cofactor apoprotein distance (A) DA Mg 2 + HisL173-NE2 2.3 Mg 2 + HisL173-NE2 2.2 Ring-I keto-o HisL168-NE2 3.2 Ring- keto-o HisL168-NE2 2.8 Ring-V keto-o CysL247-SG 3.7 Ring-V keto-o ThrL248-OG1 2.6 DB Mg 2 + HisM200-NE2 2.2 Mg 2+ HisM200-NE2 1.9 Ring-I keto-o TyrM195-OH 3.0 BA Mg 2 + HisL153-NE2 2.3 Mg 2+ HisL153-NE2 2.2 Ring-V keto-o w59a 2.7 Ring-V keto-o w302a 3.0 BB Mg 2 + HisM180-NE2 2.2 Mg 2+ HisM180-NE2 2.1 Ring-V keto-o w62b 2.9 Ring-V keto-o w304b 2.7 (DA Ring-V ester-o TrpL100-NE1 2.9 Ring-V ester-o TrpL100-NE1 3.0 Ring-V keto-o GluL104-OE1 2.7 Ring-V keto-o GluL104-OE1 2.7 ( B Ring-V ester-o TrpM127-NE1 2.8 Ring-V ester-o TrpM127-NE1 2.7 QA Proximal keto-o HisM217-ND1 3.2 Proximal keto-o HisM217-ND1 3.1 Distal keto-o AlaM258-N 2.8 Distal keto-o AlaM258-N 3.1 QB Proximal keto-o HisL190-ND1 (2.7) Distal keto-o llel224-n (2. 8 )d Distal keto-o SerL223-OG (2.7) Distal keto-o TyrL222-0 (3.1) Distal keto-o GlyL225-N (2.7) Proximal methoxy-o w65c (3.0) Proximal methoxy-o HisL190-ND1 (3.0) Distal methoxy-o llel224-n (2.9) Distal methoxy-o AlaL226-N (2.9) Non-haem Fe 2+ HisL190-NE2 2.1 Fe 2 + HisL190-NE2 2.0 iron Fe 2+ HisL230-NE2 2.1 Fe 2 + HisL230-NE2 2.4 Fe 2 + HisM217-NE2 2.1 Fe 2+ HisM217-NE2 2.1 Fe 2 + HisM264-NE2 2.1 Fe 2 + HisM264-NE2 2.0 Fe 2 + GCluM232-OE1 2.2 Fe 2 + GluM232-OE1 2.2 Fe 2 + GluM232-OE2 2.3 Fe 2 + GluM232-OE2 2.0 adistance to HisM200 ND 1 is 2.8 A for the Rb. sphaeroides RC and 3.0 A for the Rps. viridis RC. bdistance to HisL173-ND1 2.5 A for the Rb. sphaeroides RC and 2.4 A for the Rps. viridis RC. CDistance to HisL190-ND1 is 3.4A. dthe values in parentheses are of lower accuracy. Fig. 9. Stereoview around TyrM208 showing the region that provides the primary electron transfer from the special pair to the bacteriopheophytin 4A in the RCs of Rb. sphaeroides (colour scheme as in Fig. 4) and Rps. viridis (green). The position and the conformation of TyrM208 involved in the electron transfer process is very similar in both RCs. All surrounding residues are hydrophobic and exchanged amino acids compensate each other with respect to their sizes. similar conformation in the Rb. sphaeroides and Rps. viridis RCs (Fig. 5). Its indole nitrogen forms a hydrogen bond with the hydroxyl group of ThrM220, which was not observed in one of the earlier Rb. sphaeroides RC structures [12]. Several site-specific mutants at position M250 confirmed its proposed role for electron transfer and established its extraordinary importance for the binding of QA [40,41]. Only the aromatic residues phenylalanine and tyrosine can replace the tryptophan at position M250, only to a certain extent. PheM249, in van der Waals contact with TrpM250, might enhance the aromatic system involved in the binding of QA MetM216, not conserved in the RC of Rps. viridis, is a neighbour of TrpM250 and QA (Fig. 5) and is a candidate to explain spectral and functional differences between the two RCs.

9 Rhodobacter sphaeroides reaction centre Ermler et al. 933 Proton transfer The pathways of proton transfer to the secondary quinone QB are currently investigated predominantly by analysis of site-specific mutants. Based on these investigations it was suggested [1], that the first proton to QB is transferred via AspL213 and SerL223, the second proton via AspL213 and GluL212. The QB binding site is deeply buried in the protein complex. The question arises of how the protons pass from the cytoplasm through the protein to the QB molecule. The protons may move along a chain of proton donors and acceptors connected by hydrogen bonds. Using this hypothesis, several proton transport channels can be postulated from the inspection of the structure. In a recent report [42] putative water binding positions were simulated and empty space was filled up with solvent using one of the earlier Rb. sphaeroides RC structures. In the structure of the trigonal crystal form of Rb. sphaeroides, a water chain with a length of about 23 A including 14 water molecules was found. The fixed water molecules, mostly within hydrogen bonding distance of their neighbours, are located in the electron density map from the QB binding site to the cytoplasm across the H subunit (Fig. 10). The chain starts from the proximal ether oxygen of QB and ends at water w124 (Fig. 11), directly accessible to the bulk solvent outside the protein. An alternative path branches off from solvent molecule wll and proceeds via water w39 and w57 to AspL210 and from there to the outside of the RC (Fig. 11). The average temperature factor of the fixed water molecules and the surrounding amino acids are comparable (29 A 2 and 25 A 2 ). In the Rps. viridis RC the water chain is interrupted and shorter. QB binds in place of waters w65 and w66 and the bulk solvent is already accessible to the region where in the Rb. sphaeroides RC water w93 is situated. The interruption between water w55 and w127 is caused by a conformational change of the side chain of GluM234, presumably due to the replacement of ArgH73 and ArgH121 in the Rb. sphaeroides RC by glycine and alanine in the Rps. viridis RC. Waters wll and w135 mimic the two carboxyl oxygens of GluM234 in the Rps. viridis RC. The fixed water molecules are in extensive contact with amino acid residues (Fig. 11). A qualitative analysis of the Rb. sphaeroides RC indicates that negative charges dominate in the QB binding pocket and along the water chain, where carboxyl side chains of three glutamic acids interact with water molecules. No preference for positively or negatively charged residues could be identified close to the protein surface at the cytoplasmic end of the water chain. Whether an electrostatic potential difference exists between the surface of the H subunit and QB must be analyzed by electrostatic calculations. The dominance of acidic residues along the water chain provides a suitable environment for transporting a Fig. 10. Image of the water chain extending from the Q binding site to the cytoplasm across the H subunit in the Rb. sphaeroides RC. The water molecules were painted as blue spheres with a radius of 1.5 A, the QB molecule is in red and the protein is shown in ribbon representation with the L subunit in green, the M subunit in turquoise and the H subunit in yellow. positive charge. A mutual repulsion of the negative charges is prevented by the formation of several salt bridges to positively charged residues. Two clusters of salt bridged acidic and basic residues are placed around the water chain and can be seen in Fig. 11. The first cluster consists of GluM230, ArgH181, AspH174, LysH133 and GluH177 (3.9 A away from water w55), the second cluster consists of GluH125, ArgM231 and GluH235. At least some of these charged residues between 13 A and 18 A away from QB are functionally relevant, as indicated by studies of photosynthetically competent revertants of the inactive mutant AspL213-Ala in the Rb. sphaeroides RC [43]. The Rb. capsulatus RC was inactivated by the double mutation of AspL213 and GluL212 to two alanines [44]. An exchange of ArgM231 for leucine in the Rb. capsulatus [44] and of ArgM231 for cysteine in Rb. sphaeroides RC [43] restore the photosynthetic activity to a level similar to that of the wild-type. ArgM231 forms a salt bridge to GluH235 and GluH125, which directly interacts with water w93 and w135 of the water chain. However, the many fixed water molecules observed in the region around the water chain may solely be a consequence of the increased electric field density caused by a large number of charged residues. Although the

10 934 Structure 1994, Vol 2 No 10 O-CH3 Fig. 11. Schematic drawing of the water chain and the surrounding protein matrix in the Rb. sphaeroides RC: the water molecules are in oval boxes, the polar residues interacting with the water molecules and several charged residues forming salt bridge clusters are in rectangular boxes. Broken lines indicate hydrogen bonding, the distances between donors and acceptors are given in A. Two other relevant atomic distances are drawn as solid lines and the distances are labelled with brackets. unbroken row of water molecules over a length of 23 A is striking, there is no evidence at present about its use for proton transfer. It will be interesting to see whether this 'water channel' can be interrupted by site-directed mutagenesis, and what the effect of this on protonation of QB might be. Biological implications The conversion of light energy to chemical energy in photosynthesis is, at least from an energetic point of view, the most important biological process on earth. The primary photosynthetic charge separation and the build up of a proton 3.0 ( )... A.. ND1 His " OE1 GluM OE2 GluM GluL OE [1 NHI Asp 213 0NLeu M233 NH2 2.5 OD2 :3.0 ASPH174 OD1 (51) OD2 OE1 (3.9) LysH133 NZW ' 32 OD1 :3.3 0 :2..N" O NE N.' NLeu M233I Ar' GM '. 3.1 OE EGluH235 OE.. NE ArgH73 3.s.) 5 NE Arg H121-!3.4 NH1.' N -ArgH120 NH2 2. -o o.. gradient across the periplasmic membrane are catalyzed by the photosynthetic reaction centres (RCs) (in conjunction with the cytochrome-bc 1 complex). The electron and proton transport mediated by protein-bound cofactors have been investigated intensively by various experimental and theoretical methods to determine the energetics, the dynamics and the pathway of this process. Here we report the structure of the RC from Rhodobacter sphaeroides, the organism that is widely used for spectroscopic and site-directed mutagenesis studies of the RC. The quality of the electron density obtained from the trigonal crystal form that we used is sufficient to identify hydrogen bonds and bound water molecules with reasonable confidence. As the structure of the reaction centre from the purple bacterium Rhodopseudomonas viridis has been solved to 2.3 A, we were also able to compare the RCs of the two bacteria. In general, the mode of pigment binding and the asymmetry between the A and the B branch of the RC are conserved. The ligation of the bacteriochlorophyll magnesium atoms and of the non-haem iron atom is identical, and the hydrogen bonding pattern between the cofactors and the polypeptide chain is maintained except for the 'special pair' (the primary electron donor of the RC) and the displaced ubiquinone (QB) molecule. Visible minor structural differences between the two reaction centres may now be used to explain the differences seen in spectroscopic experiments. A striking feature of our structure is an unbroken chain of fixed water molecules from QB to the cytoplasm. It may be used for proton transfer to the reduced quinone molecule QB. It will be interesting to see what happens if this water chain is blocked by site-directed mutagenesis, which is still difficult to perform in Rps. viridis, but which is straightforward in Rb. sphaeroides. In general, the newly discovered and analyzed trigonal crystal form should also be of great help for studying the structures of site-specific mutants. Materials and methods Crystallization The preparation and crystallization of the RC from Rb. sphaeroides were performed as described [13]. Crystals were grown using the sitting drop method with 1 M potassium phosphate, ph 6.5 or 7.0, 3% heptane-1,2,3-triol, 1% dioxane, 0.1% N-lauryl-N,N-dimethylamine-N-oxide (LDAO) and about 100 lim (10.6 mg ml-') protein in the drop and 1.5 M potassium phosphate in the reservoir. (With the heptane-1,2,3- triol now available from Fluka, a concentration of about 1.5% is more favourable for the growth of trigonal crystals.) The crystals obtained belong to the trigonal space group P with unit cell dimensions of A, A, A. They diffract X-rays to a resolution of 2.65 A.

11 Rhodobacter sphaeroides reaction centre Ermler et al. 935 Structure determination and refinement The structure was determined by the molecular replacement method using coordinates from an orthorhombic crystal form of the Rb. sphaeroides RC as model [26] and native data to 3.8 A resolution measured on a MAR-Research imaging plate detector connected to a Rigaku RU200 X-ray generator. The rotation search performed with the X-PLOR and MERLOT program packages [45,46] resulted in a unique 4 peak with the Euler angles 01=0, 02=50 and 03=600. The translation search using X-PLOR established P and not P as the space group. The highest peak was obtained in P at 10or with fractional translation parameters of x=0.495, y=0.653 and z= The RC model was positioned into the unit cell and an initial R-factor of 47.3% from 10.0 A to 3.8 A was calculated. The structure was refined using a native data set to a resolution of 2.65 A collected at the EMBL outstation at the Deutsches Elektronen Synchrotron (DESY) from one single crystal as described [13]. The Rym of the data was determined to be 10.6% and their completeness to be 89.3% with unique reflections (86.7% of them above 2r). The completeness was 82.5% and the I/a ratio was 2.8 within the A resolution shell. Positional refinement using molecular dynamics combined with the energy minimization option and temperature factor refinement were performed within X-PLOR [47]. After 12 rounds of refinement and alternate manual inspections with the graphics program O [48] the refinement converged at a crystallographic R-factor of 18.6% ( A). The root mean square deviations (rmsd's) from the ideal values were 0.14 A for bond lengths and 1.60 for bond angles using geometric parameters for the polypeptide chain of Engh and Huber [49] and for the cofactors of Treutlein et al. [50], and C Roy D Lancaster (personal communication). The coordinate error was estimated to be 0.3 A from a Luzzati plot [51]. Water molecules were included when the 12F o - F c I and the Fo I -IF I electron density map showed a roughly spherical peak larger than 1 and 3(r in hydrogen bond distance to a polar atom of the protein or a cofactor. The final model contains 6461 protein atoms, four bacteriochlorophylls a, two bacteriopheophytins a, two ubiquinones, one non-haem iron, one carotenoid, one phosphate, nine detergent and 160 solvent molecules. The electron density is reliable for the head group of the cofactors and the neighbouring amino acid residues, but at the end of the phytyl and isoprenoid tails the map becomes poorer, in particular at BB and QB. The average temperature factors are 34.7 A 2 for the whole structure, 33.5 A 2 for the polypeptide, 34.3 A 2 for the cofactors and 36.1 A 2 for the water molecules. Superposition The RCs from Rb. sphaeroides and Rps. viridis were superimposed with an algorithm of Kabsch [52] using only backbone atoms of the L and M subunits which deviate less than 1 A from the calculated superposition matrix. The rmsd of 0.5 A for 1627 atoms is very small, since only structurally equivalent residues of the two RCs were taken into account. The coordinates have been deposited in the Brookhaven Protein Data Bank. Acknowledgements: We thank Drs G Feher and M Schiffer for supplying the RC coordinates of the orthorhombic crystal forms, C Roy D Lancaster for discussion and reading the manuscript, and Lothar Germeroth for help in data collection. We acknowledge help in use of the facility at DESY by the staff of the EMBL outstation. This work was supported by the Max-Planck- Gesellschaft and the Fonds der Chemischen Industrie. References 1. Okamura, M.Y. & Feher, G. (1992). Proton transfer in reaction centres from photosynthetic bacteria. Annu. Rev. 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