Crystallographic Refinement at 2.3 A Resolution and Refined Model of the Photosynthetic Reaction Centre from Rhodopseudomonas viridis

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1 J. MoL BioL (1995) 246, JMB O Crystallographic Refinement at 2.3 A Resolution and Refined Model of the Photosynthetic Reaction Centre from Rhodopseudomonas viridis Johann Deisenhofer, Otto Epp, Irmgard Sinning and Hartmut Michel Max-Planck-Institu t fiir Biochemie, D Martinsried, F.R.G. The atomic model of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis has been refined to an R-value of at 2.3,~ resolution. The refined model contains 10,288 non-hydrogen atoms; 10,045 of these have well defined electron densit}~ A Luzzati-plot indicates an average co-ordinate error of 0.26 ~. During refinement, the positions of a partially ordered carotenoid, a unibiquinone in the partially occupied QB site, a detergent molecule, seven putative sulphate ions, and 201 water molecules were found. More than half of these waters are bound at interfaces betweeno protein subunits and therefore contribute significantly to subunit interactions. Water molecules also play important structural and probably functional roles in the environment of some of the cofactors. Two water molecules form hydrogen bonds to the accessory bacteriochlorophylls and to the protein in the vicinity of the special pair of bacteriophylls, the primary electron donor. A group of about 10 water molecules is bound near the binding site of the secondary quinone QD. These waters are likely to participate in the transfer of protons to the doubly reduced QB. Keyzoords: Crystal structure; membrane protein; photosynthetic reaction centre; subunit interactions; cofactor interactions Introduction Photosynt.hetic reaction centres (RCs) are complexes of proteins and co-factors, embedded in biological membranes, which execute the primary steps in the conversion of light energy into chemical energy The RC from the photosynthetic purple Present addresses: J. Deisenhofer, Howard Hughes Medical Institute and Department of Biochemistr~ University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX , U.S.A.; I. Sinning, European Molecular Biology Laborator~ Meyerhofstrasse 1, D Heidelberg, F.R.G.; H. Michel, Max-Planck-lnstitut fiir Biophysik, Abteilung Molekulare Membranbiochemie, Heinrich-Hoffman-Str. 7, D Frankfurt/Main, F.R.G. Abbreviations used: BChl-b, bacteriochlorophyll b; BPh-b, bacteriopheophytin b; DESY, Deutsches, Elektronen-Synchrotron; FFT, fast Fourier transform; Fob,, F~,k, observed and calculated structure factor, respectively; H-bond, hydrogen bond; LDAO, N;N-dimethyl dodecylamine oxide; m.i.r., multiple isomorphous replacement; RC, photosynthetic reaction centre; r.m.s., root mean square; Rps. viridis, Rhodopseudomonas viridis; Rb. sphaeroides, Rhodobacter sphaeroides. bacterium Rhodopseudomonas viridis consists of four protein subunits and 14 co-factors (Thornber et al., 1980). The protein subunits are called cytochrome, M, L, and H; their amino acid sequences were derived from the corresponding gene sequences (Michel et al., 1985, 1986b; Weyer et al., 1987a). With 336 amino acid residues, and Mr=37,400, the cytochrome has the longest polypeptide chain, followed by subunits M (323 residues, Mr = 35,902), L (273 residues, Mr = 30,571), and H (258 residues, Mr = 28,345). Four of the co-factors are haem groups, covalently attached via thioether linkages to the cytochrome subunit. The remaining co-factors are four BChl-b, two BPh-b, two quinones (one menaquinone-9, one ubiquinone-9), a ferrous nonhaem iron ion, and a carotenoid; they are associated with the subunits L and M. The RC from Rps. viridis was the first integral membrane protein complex for which well ordered three-dimensional crystals suitable for X-ray structure analysis were obtained (Michel, 1982, 1983). This analysis was started at 3 A resolution and continued at 2.3 fk resolution (Deisenhofer et al., 1984, 1985a; Deisenhofer & Michel, 1989). Its result was an atomic model that showed the arrangement of proteins and co-factors, the interactions between them, and remarkable and unexpected symmetries /95/ $08.00/ Academic Press Limite~d

2 430 Rps. viridis Reaction Center in the RC structure (Deisenhofer et al., 1984, 1985a; Michel et al., 1986a; Deisenhofer & Michel, 1989). Spectroscopic experiments with RCs from Rps. viridis and from Rhodobacter sphaeroides (for references, see Michel-Beyerle, 1985,1990; Shuvalov et al., 1986; Kirmaier & Holten, 1987; Breton & Vermeglio, 1988, 1992; Friesner & Won, 1989; Bixon et al., 1992), combined with structural information has led to the following view on the RC's function. A closely associated pair of BChl-b (Norris et al., 1971) near the periplasmic membrane surface, also called the "special pair", is the starting point of a series of electron transfer reactions. Absorption of a photon or energy transfer from the light-harvesting antenna complex causes the transition of the pair to its first excited singlet state. At room temperature, the photoexcited pair reduces one of the BPh-b within about 3 ps. From the BPh-b, the electron moves on to the primary quinone acceptor QA (menaquinone-9 in Rps. v.iridis) within about 200 ps, and to the secondary quinone acceptor, QB (ubiquinone-9 in Rps. viridis), within 17 to 25 ~xs (Leibl & Breton, 1991; Mathis et al., 1992). Both quinones are bound near the, cytoplasmic membrane surface QB accepts two electrons from two successive charge separation events, and two protons from the cytoplasm during or after the second charge separation; as a quinol it dissociates from the RC. The Q~ binding site is re-populated by a quinone from the pool in the membrane. The bound cytochrome reduces the photo-oxidized primary donor It obtains electrons via the water-soluble cytochrome c2 from the cytochrome b/c, complex, which oxidizes the quinol and simultaneously translocates 3-4 protons to the periplasmic side of the membrane (for a review, see Trumpower, 1990). Thus, during the primary photosynthetic processes in Rps. viridis, light drives a cyclic electron transport that results in the generation and maintenance of an electrochemical proton gradient across the membrane; this gradient drives the synthesis of ATP, and transport processes against concentration gradients through the membrane. The electron transfer reactions within the RC have several remarkable properties: Only one of two approximately symmetric branches of co-factors originating at the special pair is used for electron transfer; this finding has raised both functional and evolutionary questions. The quantum yield for quinone reduction is very close to unity The electron transfer from the special pair to QA is faster at temperatures near absolute zero than at room temperature (for a review, see Friesner & Won, 1989). The involvement of one of the accessory BChl-b in the reduction of the BPh-b, has been a matter of debate. Recent experiments indicate a direct role for it in the electron transfer reactions as the very first electron acceptor (Holzapfel et al., 1989, 1990); earlier experiments suggested a more indirect role (Martin et al., 1986; Breton et al., 198.6). Similarly surprising was the sequence of the individual redox midpoint potentials of the four haems in the cytochrome subunit. Using different experimental techniques, four research groups found that the midpoint potentials of the haems alternate between high and low along the linear chain leading from the special pair to the tip of the cytochrome (Shopes et al., 1987; Alegria & Dutton, 1987; Dracheva et al., 1988; Fritzsch et al., 1989; Vermeglio et al., 1989; Shinkarev et al., 1990). A recent theoretical investigation could reproduce the experimental findings (Gunner & Honig, 1990, 1991). RCs from some other purple photosynthetic bacteria lack the bound cytochrome; most of these contain BChl-a and BPh-a instead of BChl-b and BPh-b. Nevertheless, because of homologous amino acid sequences (B61anger et al., 1988), their structure must be very similar to that of the RC from Rps. viridis, as was shown directly for crystals of the RC from Rb. sphaeroides. The crystal structures could be solved by molecular replacement methods using part of the Rps. viridis model (Chang et al., 1986; Allen et al., 1986; Ermler et al., 1992). The model of the RC from Rps. viridis also helped to better understand the structure of the RC of photosystem II from chloroplasts and cyanobacteria (Michel & Deisenhofer, 1986, 1988; Trebst, 1986, 1987). The symmetric structure of the RC from purple bacteria, and marginal sequence homologies between the D1 and D2 polypeptides of photosystern II and the subunits L and M of purple bacteria, which included amino acid residues crucial for function and folding of these subunits, led to the proposal that the RC of photosystem II is composed of the D1 and D2 proteins. The isolation of a photosystem II RC complex consisting of D1, D2, and cytochrome bs5~ was convincing evidence for the correctness of this proposal (Nanba & Satoh, 1987). In this paper we report the crystallographic refinement at 2.3 A resolution of the RC from Rps. viridis, and describe details of the refined model. We focus our report mainly on those structural features that were not covered in previous publications (Deisenhofer et al., 1984, 1985a; Michel et al., 1986a; Deisenhofer & Michel, 1989). Results Course of refinement and quality of the model Table 1 lists the diffraction data sets and Table 2 summarizes the course of the refinement of the Table 1 Intensity data sets used for refinement Data set 2.9 ]~ 2.3.~ Unique reflections with 1 > 2a(l) R.~,.t Completeness,,~ to 2~9 A 3.0 to 2.9 A 84.7% 50.3% 87.5% 76.5% ** to 2.3 ~, 2.4 to 2.3/k % 49.2% t R.,~<. = Zak~Z, ll~s.,- (t~s)ll~,,e,l,,,.

3 Rps. viridis Reaction Center 431 Table 2 Course of refinement No. of Number r.m.s deviation from cycles Resol. of ideal values Step (pos./b) Method1" (,~) R-value:~ atoms Bonds (A) Bond angles (o) Start /0 JL /0 JL /0 JL /1 JL /1 JL /1 JL /1 J L /2 JL /1 JL /1 JL /2 JL /2 JL / 1 J L / 6 T /13 T /10 T fjl: Jack & Levitt (1978), T: Tronrud et al. (1987). ::1: R = Y,,. IIF,,~I- IF..,..Ii/T.,.IF,,~.I. RC model. The final model consists of 10,288 atoms; the positions of 243 df these atoms are undefined due to disorder in the crystal (see below). The R-value for 10,045 model atoms and 95,762 unique reflections between 20/~ and 2.3 A resolution is The r.m.s, deviation of bond lengths and bond angles from ideal values is A and 2.75, respectivel~ The overall B-value is 22.5,&2 with individual B-values ranging from 3 d2 (lower limit used) to -70 dj. The r.m.s, deviation of B-values of bonded atom pairs is 6.8 ~2. The average error in atomic co-ordinates, as estimated with the method of Luzzati (1952), is 0.26,~. Figure 1 shows a Ramachandran diagram for the protein sub~nits of the RC. With very few exceptions the backbone conformation ranges within the energetically favourable region. Leucine L165, part of the loop connecting helices LCD and LD, is the only residue in a disallowed region. Close scrutiny of this and other exceptions did not indicate alternative backbone conformations that were compatible with the electron density maps. The diagram documents the high helix content of the protein subunits. Scale factor and AB for the solvent contribution to the calculated structure factors, described in Methods, converged at 0.3 e/a 3, and 63.~2 during refinement. The scale factor falls slightly below the electron density expected for aqueous solutions (0.37 e/,~3); the AB damps the effects of the sharp edges of the solvent model On the high resolution terms of the Fourier transform. The limited resolution of the diffraction data did not allow us to distinguish between carbons, n,itrogens, and oxygens. The orientation of branched groups, such as the acetyl groups of BChl-b, or the amide groups of asparagine and glutamine residues, were chosen so that the polar atoms can have favourable interactions with their environment. The refinement did not unveil any serious errors in the starting model; it confirmed the overall structure of the RC as well as the nature of the cofactor-protein interactions reported previously (Deisenhofer et al., 1984, 1985a; Michel et al., 1986a). Additions to the model In total, 1650 atoms were added to the model during refinement. The majority of these, 1328, belonged to amino acid side-chains of the cytochrome subunit which were added after the sequence had been determined (Weyer et al., 1987a). The improvement of the phases during refinement also led to the discovery of ordered constituents of the RC that could not be seen in the m.i.r, map. Carotenoid Electron density for a long, curved chain molecule appeared during early steps of refinement. This density feature was interpreted as part of the carotenoid 1,2-dihydroneurosporene that had been. found in the RC by chemical analysis (Sinning, 1988). Figure 2 shows the density of a 2FOB, -- Fc.,~ map after refinement step 8 (before the carotenoid was added to the model), together with the final carotenoid model. O~ Contrary to the situation at the QA site, the m.i.r. map did not show well defined electron density in the Q, binding pocket, but the density of His L190, the iron ligand that forms part of the QB pocket, appeared somewhat enlarged (Deisenhofer et al., 1984). An Fob,- F~,jc map calculated during refinement step 14 had weak but significant density for a flat object with the size and shape of a ubiquinone head group inside the Q~ binding pocket; an

4 432 Rps. viridis Reaction Center o p t i m u m fit of the m o d e l to the d e n s i t y s i m u l t a n e o u s l y led to f a v o u r a b l e i n t e r a c t i o n s b e t w e e n Q~, a n d the p r o t e i n. A u b i q u i n o n e - 1 m o d e l w i t h an e s t i m a t e d o c c u p a n c y of 0.3 w a s t h e r e f o r e a d d e d to the RC m o d e l. N o s i g n i f i c a n t e l e c t r o n d e n s i t y w a s f o u n d for the q u i n o n e ' s tail. F i g u r e 3 s h o w s the F.,B, - F,.,~, m a p t o g e t h e r w i t h the r e f i n e d Q. m o d e l. A l o w o c c u p a n c y of the Q. site w a s e x p e c t e d b e c a u s e a l a r g e p e r c e n t a g e of Q, is u s u a l l y lost d u r i n g p r e p a r a t i o n a n d c r y s t a l l i z a t i o n of the Rps. v M d i s RC (Gast et al., 1985). Detergent M o s t of the d e t e r g e n t in the RC c r y s t a l s a p p e a r s d i s o r d e r e d ( D e i s e n h o f e r et al., 1985a). H o w e v e r, t h e r e is at least one e x c e p t i o n : A n F,,,~ - F,,,~, m a p after r e f i n e m e n t s t e p 12 s h o w e d e l e c t r o n d e n s i t y for 180 " ~!ii!iii iii i ~D eto 0-45 LEU 165~ ~~.~.~'?.~NI.i~~ ~:~:~1 ~!,4~~. ~ i ~ ' ~ ~ ~i~.-.!'~:-~.~~:~:i<::;i:j:~:g,-~l :i.'.:.::::.~:::::.'::::~.'.::,~:::1 :::::::::::::::::::::::::::::::::: ' -,B I 6 45 Phi (degrees)! 9O Plot statistics Residuesin most favouredregions [A,B,L] Residues in additionalallowedregions [a,b,l,p] Residues in generouslyallowedregions [-a,-b,-1,-p] Residues in disallowedregions I 89.6% Numberof non-glycineand non-pralineresidues Number of end-residues(excl.gly and Pro) Number of glycineresidues (shown as triangles) Numberof praline residues % Total number of residues l % 0.3% 0.1% Figure 1. Ramachandran diagram of tile protein subunits. Regions allowed for L-amino acids are shaded, with the most favoured regions appearing darkest. Glycine residues are marked with triangles, all other amino acids are marked with squares. The only non-glycine residue in an "outside" region is leucine L165. This diagram was produced with the program PROCHECK (Laskowski et al., 1993).

5 Rps. viridis Reaction Center 433 Figure 2. Stereo pair: refined model of the carotenoid 1,2-dihydroneurosporene with 2Fo~- Fc~k density of step 8 at 2.9 resolution, contoured at 0.2 e/al In our interpretation the density coincides with the ordered central part of the carotenoid molecule; both ends are disordered The map was calculated before the carotenoid was added to the model an LDAO molecule The perfect fit of model and density; and the suitable protein environment leave little doubt about this interpretation. The head group of the LDAO is at the edge, and the hydrocarbon chain points to the interior of the detergent region determined by low resolution neutron diffraction experiments (Roth et al., 1989) A second LDAO molecule included in the model represents a much more tentative interpretation since the associated electron density accounts only for a nine-carbon chain It is located near the periplasmic membrane surface at an interface of subunits L, M, and H. \ / ', $ / z'" Solvent Ordered solvent was included in the model in the form of 201 water molecules, and seven sulphate ions. These assignments, based on electron density maps and local chemistry are tentative. Some objects identified as water or sulphate ma34 for example, be ordered parts of larger, partially disordered compounds Most of the putative waters were found associated with the peripheral subunits, and with the subunits L and M near the interfaces to the peripheral subunits. 108 of these water molecules are located at the periplasmic side, 88 on the cytoplasmic side of the membrane. Five water molecules are bound in the interior of the L-M complex The putative sulphate ions are exclusively located at the cytoplasmic side of the membrane, mostly near the interface of L-M with the H-subunit. cis-prolines, Figure 3. Stereo pair: refh~ed Qu model with Fob, - F~, density of step 14 at 2.3 A resolution, contoured at 0.1 e/a2. Atom types: C, large empty circles; O small double circles The map was calculated before the Q~ was added to the model. In total, six peptide units preceding proline residues adopt the cis conformation. The cytochrome subunit contains three (residues C6, C153, C330), subunit H contains two (residues H42, H79), and subunit M contains one cis-proline (residue M48); none is found in subunit L. Thus, the cis-proline residues are distributed equally between the periplasmic and the cytoplasmic sides of the membrane

6 434 Rps. viridis Reaction Center Disorder The model contains atomic co-ordinates for 243 atoms which are disordered in the crystal to an extent that they are not represented by significant electron density They were kept as part of the model if they belonged to a partially ordered group or occurred within a polypeptide chain, but were given zero occupancies, so that they did not contribute to the X-ray part of the least-squares refinement. Among the protein subunits the fraction of disordered atoms is lowest in subunits L and M (13 and 26 atoms, respectively). All of them belong to side-chains; these are five Lys (L72, L205, M31, M298, M323), three Ile (L17, L269, M104), three Phe (L271, M59, M85), Asp L202, Gln M22, and Arg M136. Only one of them (Phe M59) is located well inside the transmembrane region. Similarly, the transmembrane segment of subunit H does not contain disordered side-chains. The largest region of disorder in this subunit is seven complete residues (H47 to H53) with 49 atoms. The remaining 57 disordered atoms are again in side-chains: five Glu, four Lys, two Gln, two Arg, one His, one Phe, and one Thr. Residues C333 to C336 at the C terminus of the cytochrome subunit are completely disordered; these residues are not included in the model. Side-chain disorder affects 54 atoms in nine Lys, two Arg, two Glu, two Gln, and one Thr. The latter residue, Thr C159, is the only case where electron density for the complete side-chain is missing. Within the set of prosthetic groups, disorder is found in the phytyl chains of BChl-b Be (13 atoms), and of BPh-b ~ (seven atoms). Only six isoprenoid units of the side-chain of QA have defined positions. In its.tentative positioning, the carotenoid molecule appears to have a fixed central part, and disorder at the ends (nine and five atoms, respectively). Unidentified features in the electron density maps No satisfactory model interpretation could be found for a number of electron density features in the Fot~ - Fc,~c or 2Fob~ -- F~ maps at the end of refinement. Most of these features are elongated, and most probably represent partially ordered molecules from the crystallization solution, e.g. heptane-l,2,3-triol, detergent, or clusters of water molecules. The Refined Model Overview Overall views of the refined structure of the RC complex in two different orientations as shown in Figur.e 4 are very similar to those of the structure at 3/k resolution reported previously (Deisenhofer et al., 1985a). In its longest.dimension the complex measures about 130 A; the maximum width perpendicular to that direction is about 70/~.. The closely associated subunits L and M, together with the bound BChl, BPh, quinones, non-haem iron, and carotenoid form the central part of the RC. The most prominent structural features of each of the central subunits are five membrane-spanning helices. Both the polypeptide backbones of subunits L and M, and the attached co-factors display a high degree of local 2-fold symmetry, with the symmetry axis oriented perpendicular to the membrane plane (Paillotin et al., 1979; Breton, 1985a,b; Nabedryk et al., 1985). O11 either side of the membrane spanning region the L-M complex forms a flat surface parallel to the membrane surface. A peripheral subunit is attached to each of these surfaces. The cytochrome with its four bound haem groups binds at the periplasmic side and the globular domain of the H-subunit at the cytoplasmic side of the membrane; we call these contact surfaces of the L-M complex C-contact surface, and H-contact surface, respectivelf The H-subunit contributes a single membrane-spanning helix. As can be seen from Figure 4, neither the cytochrome nor the H-subunit obey the local symmetry found in the central part of the RC; the cytochrome has internal local symmetry of its own (see below). With the exception of the carotenoid, the co-factors in the L-M complex are arranged in two approximately symmetric branches, each consisting of two BChl-b, one BPh-b, and one quinone. These branches originate at the special pair of closely associated BChl-b located near the periplasmic membrane surface on the symmetry axis, and lead through the membrane to the cytoplasmic side; they are called A- and B-branch. The non-haem iron is bound near the cytoplasmic membrane surface, between the quinones near the symmetry axis. The carotenoid is associated with the BChl-b of the B-branch. The surface of the RC complex is highly hydrophobic in a 30 A wide band around its centre including the transmembrane helices of the subunits M, L, and H, and parts of the co-factors (Deisenhofer & Michel, 1989; Michel & Deisenhofer, 1990). The detergent in the crystal covers this hydrophobic surface (Roth et al., 1989). Charged amino acid residues are totally absent, polar residues and bound water molecules are very rare in this central region (Deisenhofer & Michel, 1989; Michel & Deisenhofer, 1990). Charges on the membrane surface segments of subunits L and M are distributed such that they counteract the electrostatic membrane potential (Michel & Deisenhofer, 1987; Deisenhofer & Michel, 1989). The structure of subunits L and M Table 3 summarizes the local 2-fold symmetry of subunits L and M (Deisenhofer et al., 1985a) and lists the structurally equivalent segments. In total, 216 {x-carbon atoms from subunit M are superimposable onto corresponding ones of subunit L (Rossmann & Argos, 1975) with an r.m.s, distance of 1.22 ~. This value is very low in view of only 26% sequence

7 Rps. viridis Reaction Center 435,0 aej ] ob~".',...-". i jj,t~! ': "'- :~ :' -I,( i ~'~., gg? "Vd" N_~ :.. j r (a) (b) Figure 4. Overall views of the refined RC model; (a) broad view, (b) side view. The central 2-fold symmetry axis runs vertical in the planes of the Figures; both views are projections parallel to the membrane. The protein subunits are shown as ribbons; the cofactors, the ordered LDAO molecule, and 6 putative sulphate ions are shown as ball models. Ribbon colours: cytochrome green, L-subunit brown, M-subunit blue, H-subunit pink. Ball colours: carbon atoms white or grey (LDAO), nitrogen atoms blue, oxygen atoms red, sulphurs yellow, iron and magnesium light turquoise. (a), (b) were produced with the program Ribbons (Carson, 1991). identity (percentage based on the length of subunit L). The p o l y p e p t i d e chain segments that do not o b e y the local 2-fold s y m m e t r y are located at the Table 3 Local 2-fold symmetry of subunits L and M Transformation: Rotation matrix: R o t a t i o n in p o l a r angles-t: T = 43.0, = 39.5, K = - 179,5 T r a n s l a t i o n vector: 129.2, , (A) T r a n s l a t i o n c o m p o n e n t a l o n g r o t a t i o n axis: 0.4 (A) r.m.s, d e v i a t i o n o f 216 e q u i v a l e n t C A a t o m s a f t e r r o t a t i o n : 1.22 Equivalent segments Length (residues) M ) L M ) L M ~ L M ~ L M ~ L t ~: angle between rotation axis and y-axis, : angle between projection of rotation axis on xz plane and x-axis, ~c:rotation angle. m e m b r a n e surfaces or outside the m e m b r a n e (see Figures 9 and 10). Helices (mostly s-helices), the p r e d o m i n a n t elements of secondary structure in subunits L and M, are listed in Table 4. The t r a n s m e m b r a n e helices, called A, B, C, D, and E, alone constitute 46% and 39% of the structures of subunits L and M, respectively. The segments connecting these helices, and t h e N-terminal and C-terminal segments contain shorter helical regions, notably helices CD and DE. The segments with similar folding of both subunits include all the t r a n s m e m b r a n e helices, and the complete connections b e t w e e n helices B and C, and C and D. Differences in chain conformation are consequences of insertions in the M subunit (or deletions in the L-subunit): 20 residues in the N-terminal segment, seven residues in the A-B connection, forming a short helix, seven residues in the D-E connection, forming another short helix, and 16 residues at the C terminus which are in close contact with the surface of the cytochrome. Most of the non-local polar interactions (defined as H-bonds and salt bridges b e t w e e n residues separated by at least five residues in the amino acid

8 436 Rps. viridis Reaction Center Table 4 Helical se~unents in subunits L and M Segment flength) Helix Subunit L Subunit M Trensmembrane A L33-L53 (21) B L84-L111 (28) C L116-L139 (24) D L171-L198 (28) E L226-L249 (24) M52-M76 (25) MIll-M137 (27) M1&3-M166 (24) M198-M223 (26) M260-M284 (25) Periplasmic -- M81-M87 (7) CD L152-L162 (11) M179-M190 (12) C-terminal L259-L267 (9) M292-M298 (7) Cytoplasmic -- M232-M237 (6) DE. L209-L220 (12) M241-M254 (14) Total number of residues sequence) between the various structural segments within each subunit occur at or near the membrane surfaces. Table 5 shows a list of these non-local polair interactions within subunits L and M. Although the transmembrane helices of subunits L and M contain 74 residues with polar side-chains, only seven inter-helix H-bonds, all listed in Table 5, are formed between those residues. There are at most two H-bonds between any pair of helices (helices LD and LE). H-bonding between small polar side-chains and carbonyl oxygens in positions i and i- 4, respectivel}~ within helices involves 11 of the 20 serine residues, seven of the eight threonine residues, and four of the six cysteine residues in the transmembrane helices of subunits L and M; such H-bonds in transmembrane (z-helices had been predicted by Gray & Matthews (1984). Charged and polar residues are particularly frequent at the ends of the helices LB and MB; they make important contributions to intra- and inter-subunit interactions (see below). The remaining polar residues in transmembrane helices either interact with protein segments at the membrane surfaces or with the cofactors. The transmembrane helices A, B, and D of subunits L and M are almost perfectly straight a-helices. The E-helices in both subunits are smoothly curved. The most distorted transmembrane helices in L and M are the C-helices; Figure 5 shows the C-helix of subunit L. Both C-helices have kinks of more than 30 eight residues from their C-terminal ends, at residues L132 and M159, respectivel~ The carbonyl oxygen atoms of these residues are not involved in H-bonds; presumably, this is in part caused by the presence of proline residues at positions L136 and M163. The C-helix of the L-subunit has an additional proline nine residues from its N-terminal end at position L124. This proline does not cause a large change in direction of helix LC. The carbonyl oxygens of residues L 119 and L120 form H-bonds to-the peptide nitrogens of residues L122 and L123, respectivel~ in a pattern characteristic for a 310-helix. Remarkab134 the a-helical H-bonding pattern is interrupted in an equivalent position also in the C-helix of the M-subunit. The carbonyl oxygen of residue M146 is not involved in an H-bond, even though there is no proline in this part of the MC helix. The C-helices in subunits L and M of the RC from Rps. viridis show that proline residues in a (x-helices and the presence of significant kinks in those helices are correlated, but that this correlation is by no means perfect; proline residues may also cause only minor local distortions of regular helix geometry The structure of subunit H The structure of subunit H can be divided into three distinct segments, a membrane-spanning segment between residues H1 and H40, a surface segment between residues H41 and H106, and a globular segment formed by the remainder of the subunit. Within the membrane-spanning segment, residues H12 to H35 form a helix, which starts as a regular a-helix, but becomes distorted near the cytoplasmic membrane surface. Here, the helix diameter increases, and three hydrogen bonds typical for a n-helix exist (peptide N-H of residues H32 to H34 with C---O of H27 to H29, respectively); the carbonyl oxygen of residue H30 does not participate in the helical hydrogen bonding system. Near the membrane-spanning segment the sequence of the H-subunit consists of seven consecutive charged residues (H33 to H39 with the sequence Arg-Arg-Glu-Asp-Arg-Arg-Glu). Following the membrane-spanning segment, about 65 residues of the H-subunit are mostly in contact with the surface of the L-M complex. Residues H47 to H53 are disordered, presumably due to the absence of such a contact. The remainder of the surface segment forms a short helix (residues H56 to H60), and two two-stranded antiparallel ~-sheets (residues H66 to H69 with H75 to H78, and residues H90 to H92 with H101 to H103). The major secondary structure elements of the globular region of subunit H belong to an extended system of antiparallel and parallel ~-sheets, and an a-helix (residue s H232 to H248). Two short pieces of helix are formed by residues H107 to Hl10, and H215 to H218. The globular region is shown schematically in Figure 6. Due to the distinct right-handed twist of the ~-sheets, the strands form a pocket or channel; hydrophobic amino acid side-chains point towards its interior. The structure of the cytochrome subunit Folding of the polypeptide chain The model of the cytochrome subunit includes 332 of the 336 residues in the amino acid sequence (Weyer et al:, 1987a); four residues at the C terminus are disordered. The predominant elements of secondary structure are a-helices, listed in Table 6. As in

9 m Rps. viridis Reaction Center 437 subunits L and M, 13-sheets are very rare in the cytochrome; only very short antiparallel segments are found (residues C8 to C12 with C19 to C23, C146 with C302, and C248 with C260). The structure of the cytochrome subunit is best described as consisting of five segments: The N-terminal segment (segment 1: residues C1 to C66), the first haem-binding segment with the binding sites of haems 1 and 2 (segment 2: residues C67 to C142), a connecting segment (segment 3: residues C143 to C225), the second haem-binding segment with the binding sites of haems 3 and 4 (segment 4: residues C226 to C315), and the C-terminal segment (segment 5: residues C316 to C336). Segments 2 and 4, and their attached haem groups are related to one another by local 2-fold symmetry (Deisenhofer et al., 1985a); the equivalent C~-atoms and the transformation superimposing them are listed in Table 7. Despite this structural similarit34 only the cysteines and histidines attached to the haems are identical in the sequences of segments 2 and 4 (Weyer et al., 1987a). Segments 1, 3, and 5 do not obey the local.symmetry Within the first 24 residues of the N-terminal segment, residues C8 to C12 and C19 to C23 form two strands of antiparallel 13-sheet that follows the C-contact surface of the L-subunit (see also below). The amino-terminal cysteine residue extends over the edge of that surface, so that the diglyceride moiety linked to the sulphur (Weyer et al., 1987b) can point towards the membrane interior. Most of the diglyceride is disordered in the crystal; the 2Fob, - F~c maps and the Fob, - Fo~c maps calculated at the end of the refinement show only short traces of electron density continuing from the sulphur. Residues C25 to C50 follow a course approximately perpendicular to the C-contact surface leading to the tip of the cytochrome subunit; residues C25 to C34 within that region form an (x-helix. The side-chains of residues Thr C12, Lys C31, Arg C34, Asp C35, Tyr Table 5 Long range't H-bonds:~ within subunits L and M A. L-subunit Cytoplasmic membrane surface: Connected Donor Acceptor segments Arg L12 NE - OD2 Asp L20 Arg L12 NH2 OD1 Asp L20 Arg L12 NH2 OD2 Asp L20 Gly L13 N - OD1 Asp L23 Val L31 N - O Phe L24 Gly L32 N - O Phe L22 Leu L16 N - OE2 Gin L106 B Arg L103 NE O Phe L30 B Arg L103 NH2 - O Phe L30 B Arg L109 NE - O Gly L14 B Arg L109 NH2 - O Thr L15 B Lys Ll10 NZ - O Val Lll B Lys Ll10 NZ - O Gly L13 B Lys Ll10 NZ - OD2 Asp L23 B Lys L207 N - O Val L197 D Set L223 OG - OD1 Asn L213 DE Tyr L222 N - O Phe L216 DE Periplasmic membrane surface: Donor Acceptor Connected segments DE DE DE Ser L65 OG - OG Ser L152 AB - CD Ile L66 N - O Tyr L148 AB - CD Ser L141 OG - O Leu L71 AB - CD Trp L142 N - O Gly L74 AB - CD Trp L142 NE1 - O Leu L75 AB - CD Tyr L148 N - O Ile L66 AB - CD Ala L78 N - OE1 Gin L87 AB - B Gin L87 NE2 - O Gly L76 AB - B Trp L86 NE1 - O Phe L146 B - CD Thr L90 OG1 - OH Tyr L148 B - CD Gin L132 NE2 - O Phe L146 C - CD Gin L132 NE2 - OH Tyr L148 C - CD Phe L146 N - OE1 Gin L132 C - CD Arg L135 NE - O Ser L251 C - CT Arg L135 NH2 - O Ser L251 C - CT Asn L170. ND2 - O Gly L247 CD - E Trp L259 N - O Tyr L164 CD - CT Trp L263 NE1 - O Tyr L169 CD - CT Trp L259 NE1 - OG Ser L251 CT - CT Trp L272 NE1 - O Trp L266 CT - CT continued overleaf

10 438 Rps. viridis Reaction Center Table 5 continued Long ranger H-bonds:~ within subunits L and M Residues in transmembrane helices: Connected Donor Acceptor segments Ser L37 OG - OG Ser L99 A - B Ser L175 OG - OG 1 Tin- L243 D - E Ser L176 OG - O Ile L240 D - E B. M-subunit Cytoplasmic membrane surface: Connected Donor Acceptor segments Thr M8 N - O Lys M40 Thr M8 OG1 - O Lys M40 Gin Mll NE2 - O Tyr M36 Arg M28 NH1 - O Val M19 Arg M28 NH2 - O Val M19 Val M29 N - O Ile M49 Phe M33 N - O lie M46 - Ser M35 N - O Ala M44 Ile M46 N - O Phe M33 lle M49 N - O Val M29 Leu M51 N - O Asp M27 Glu M22 N - O Ala M137 B Arg M134 NH2 - O Trp M23 B Arg M130 NH1 - O Tyr M50 B Arg M130 NH2 - O Tyr M50 B Trp M250 NE1 - OG1 Thr M220 DE Arg M251 NH1 - OD1 Asn M257 DE Periplasmic membrane surface: Connected Donor Acceptor segments Gly M92 N - OE1 Glu M76 AB - AB Lys M97 N - OD1 Asp M109 AB - AB Ala M98 N - OD2 Asp M109 AB - AB Tyr M100 OH - OD2 Asp M109 AB - AB Leu M93 N - O Phe M175 AB - CD Trp Ml13 NE1 - O Val M173 AB - CD Trp M169 NE1 - O Met M102 AB - CD Phe M175 N - O Leu M93 AB - CD Trp M178 N - O Phe M89 AB - CD His M162 NE2 - OE1 Glu M171 CD - CD Trp M183 NE1 - O Glu M171 CD - CD Tyr M191 OH - OE1 Glu M171 CD - CD Phe M194 N - OG Ser M188 CD - CD Arg M190 NH1 - OD2 Asp M290 CD - CT Trp M292 N - O Tyr M191 CD - CT Tyr M293 N - O Gly M192 CD - CT Trp M292 NE1 - OG1 Thr M285 CT - CT Residues in transmembrane helices: Connected Donor Acceptor segments Ser M123 O G - O Ala M58 A - B Trp M128 NE1 - O Ala M145 B - C Trp M268 NE1 - O His M143 C - E Ser M203 OG - O Val M274 D - E -I" At least 5 residues between donor and acceptor. :~ Donor-acceptor distance less than 3.5 ~, angle (donor-h-acceptor) greater than 120. : N-terminal segment; CT: C-terminal segment; A: transmembrane helix A; AB: connection between transmembrane helices A and B, etc. DE DE C38, as well as those of.arg C72, Thr C131, Arg C137, Arg C321, and Tyr C325 anchor the N-terminal segment to the remainder of the cytochrome. Most of the polar interactions of these residues are H-bonds between the side-chains and carbonyl oxygen atoms; the only side-chain interactions are those of Asp C35 with Arg C137, and Tyr C38 with Thr C131. Residues C51 to C66 approach the binding site of haem I with

11 Rps. viridis Reaction Center 439 L o L124 tl Li36 i J i / R LI~4 0 LI3~ l % i + ) -LII8 Figure 5. Stereo pair: the C-helix of subunit L with proline residues Ll18, L124 and L136. Hydrogen bonds, inferred from donor-acceptor distances less than 3.5 A, are indicated as broken lines. Also shown is one water molecule (large circle) forming a hydrogen bond to one of the carbonyl oxygen atoms (double circles) near proline L136. residues C56 to C62 being in direct contact with this haem group (see below). The two haem binding segments are connected by the chain segment of section 3 (residues C143 to C225) which, apart from two short helices, contains little regular secondary structure (see Table 6). This connecting segment comprises about 60% of the interface of the cytochrome subunit with the C-contact surface of subunit M. It also forms the small contact of the cytochrome with the H-subunit (see below). Residues C144 to C149, C179, C193, C194, C199, and C201 to C206 form polar interactions with residues in section 4. The side-chain of Arg C/~02, and the peptide nitrogen atoms of Val C203 and Val C204 interact with the propionic acids of haem 3. The short C-terminal segment (residues C316 to C336) leads from the binding site of~aem 4, along part of the N-terminal segment, to the end of the cytochrome chain near haem 2. The haem environments The haems and the haem-binding segments of the polypeptide chain make up the core of the cytochrome structure; they represent the part of the cytochrome with the lowest B-values. Each haem-.binding site consists of an {z-helix that runs parallel to the haem plane (see Table 6), followed by a loop, and by the haem attachment site with the sequence Cys-X-X-Cys-His. Each cysteine residue forms a thioether bond to the haem group; the histidine is a ligand to the haem iron. Methionine residues C74, Cl10, and C233 in the helices are the sixth iron ligands for haems 1, 2, and 3, respectively; the helix associated with haem 4 has a glycine residue (C270) instead of methionine in the equivalent position. The sixth iron ligand of haem 4 is histidine C124, which is located in the loop region of the haem 2 binding site. The individual redox midpoint potentials (Era) of the four haems in the cytochrome subunit follow the sequence low, high, high, low for haems 1 to 4, respective134 or high, low, high, low if the haems are ordered with increasing distance from the special pair (Shopes et al., 1987; Alegria & Dutton, 1987; Dracheva et al., 1988; Fritzsch et al., 1989; Vermeglio et al., 1989; Shinkarev et al., 1990). Haem 3, the closest to the special pair, has the highest Em (370 mv); haem 4, the second from the special pair, has an Em of 10 mv, haem 2, the third from P has Em= 300 mv, and haem 1, the most distant from P has Em= - 60 inv. A recent theoretical investigation could reproduce these experimental values with remarkable accurac~ and quantify the contributions of the protein environment and haem arrangement (Gunner & Honig, 1990, 1991). This distribution of redox properties is contrary to the internal local symmetry of the cytochrome subunit, which equivalences haems I and 3, and also haems 2 and 4. The rationale for this arrangement is unknown. Even though the four haem-binding sites follow the same structural scheme, the detailed conformation and length of the polypeptide chain in each site, as well as the local symmetry make it more appropriate to group them in pairs. In the binding sites of haems I and 3 the iron ligands in the helices and the first haem-binding cysteine residues are separated by only 12 and 10 residues, respectively The helices and loops of the binding sites of haems 2 and 4 are much longer: 21 and 34 residues, respectivel~ connect the ligand (or, in the case of haem 4, the residue Gly C270) and the first haem-binding cysteine. Figure 7 shows a superposition of the structural segments 2 and 4, in which the differences in chain lengths are clearly demonstrated. Figure 8 shows the arrangement of segments 2 and 4 in the cytochrome molecule, viewed approximately along the local 2-fold axis. The fact that the loop region of the haem 4 binding site is 13 residues longer than that of the haem 2 binding site (see Figures 7, 8) has interesting consequences for the environments of haems 2 and 4. The propionic acid groups of haem 2 are almost completely shielded from solvent by loop residues from the haem 4 binding site. Pro C301 appears to play an especially important role in preventing propionic acid D from forming an H-bond with the

12 440 Rps. viridis Reaction Center ~ 60 Figure 6. Stereo pair: polypeptide backbone model of the globular domain of the subunit H (residues H120 to H258). Hydrogen bonds (donor-acceptor distances less than 3.5.~) are indicated by broken lines. protein, and forcing it to be close to propionic acid A of haem 2. This may keep propionic acid D protonated and thus provide an explanation for the high redox midpoint potential of haem 2 (Gunner & Honig, 1990, 1991). Table 8 lists bonds and polar interactions between the haems and the protein or localized water molecules. Haem 1 is bound via thioether bridges to Cys C87 and Cys C90; the fifth and sixth iron ligands are Met C74 and His C91. Its propionic acid groups are H-bonded to the peptide NH-groups of residues C57, C58, C61, C62; there is a salt bridge between the atom O2D of the haem to NH1 of Arg C108. With Tyr C56, Phe C70, and Tyr C104 three aromatic residues form a major part of the haem cavity; further major haem contacting residues are Leu C71, and Leu C96. Haem I has a large accessible surface along the edge formed by pyrrole rings A and B. Table 6 HeUcal structures in the cytochrome subunit Haem associated helices: C67--C81 (haem 1) C102-C120 (haem 2) C224-C239 (haem 3) C262-C280 (haem 4) Other helices longer than 4 residues: C25-C34 (N-terminal segment) C132--C136 (haem 2 attachment site) C172--C177 C189-C193 C305--C309 (haem 4 attachment site) In contrast to haem 1, haem 2 has a very small static accessible surface area. It is linked to the protein via Cys132 and Cys135; the iron ligands are Met Cl10, and His C136. Only the propionic acid A has polar interactions with the protein (H-bonds between O1A and OH groups of Tyr C89, and Tyr C102); the same propionic acid is also in salt bridge distance to Arg C293. As mentioned above, propionic acid D does not form H-bonds with the protein or water; if it were protonated, it could be H-bonded to the neighbouring propionic acid A. The binding cavity for haem 2 does not contain aromatic residues; major contributors are Val C106, Pro C142, and Pro C301. Table 7 Internal symmetry of the cytochrome subunit Transformation: Rotation matrix: Rotation in polar angles: ~P = 116.8, ~ = , ~c = Translation vector: 159.5, 88.3, (~) Translation component along rotation axis: 0.3 (,~) r.m.s, deviation of 65 equivalent CA atoms after rotation: 0.93.~ Equivalent segments Length (residues) C226-C240 -o C67--C81 15 C243-C256 ~ C86-C99 14 C261--C ) C101-C C301-C315 ~ C128-C142 15

13 Rps. viridis Reaction Center 441 Figure 7. Stereo pair: superposition of the 2 haem-binding segments of the cytochrome subunit. Thick lines: C~-atoms of residues C67 to C142, side-chains of the thioether links and ligands to haems 1 and 2 (residues Met C74, Cys C87, Cys C90, His C91, and residues Met C110, Cys C132, Cys C135, His C136, respectively), and haems 1 and 2. Thin lines: C~-atoms of residues C226 to C315, side-chains of the thioether links and ligands to haems 3 and 4 (residues Met C233, Cys C244, Cys C247, Hid C248, and residues His C124 (labelled 'HIS'), Cys C305, Cys C308, His C309, respectively), and haems 3 and 4. Haem 3 is closest to the special pair. It is linked to the protein via Cys C244 and Cys C247; the iron ligands are Met C233, and His C248. Of all the four haems, the propionic acid groups of haem 3 form the most salt bridges and H-bonds: two to peptide NH (C203, C204), three to guanidinium groups of Arg C202, C272, one to NE1 of Trp C268, and four to firmly bound water molecules; residues Tyr L162, Leu L165, and Ile M189 form the "bottom" of the haem 3 binding pocket; other major contributors to the pocket walls are Arg C202, Ile C236, Phe C253 and Arg C264. The side-chain of Arg C264 is stretched out parallel to" the haem surface; this residue is without a formal counter ibn. Only the oxygen atoms of the propionic acid groups contribute to the static solvent accessibility of haem 3 (firmly bound water molecules were not included in tlie calculation). Haem 4 has the second largest static solvent-accessible surface of the haems; the propionic acids, and the D pyrrole ring are exposed. Two histidine residues (C124 and C309) are ligands to the haem iron; histidine C124 is in the "first half" of the cytochrome polypeptide chain; the residue analogous to the methionine iron ligands for the other haems is glycine C270. Of the five histidine residues that are iron ligands in the cytochrome, only one (C91) lacks a H-bond from the ND1 atom to the protein; the other four form H-bonds between ND1 and carbonyl oxygen atoms. Subunit interactions Table 9 gives an overview of the accessible surface area buried on each subunit upon formation of the contacts to other subunits, and of the whole RC complex. Each subunit is in contact with each of the others. The sizes of the interfaces range from small (cytochrome-h) to very large (L-M). The L-M interface would appear even bigger if the contributions of the bound cofactors (BChl-b, BPh-b, quinones, non-haem iron) were included in the Table.

14 442 Rps. viridis Reaction Center Subunit M has interfaces considerably larger than L with subunit H and the cytochrome because the 50 residues by which M is larger than L are part of, or are near the contact surfaces (27 at the H-contact, 23 at the C-contact). Apart from numerous van der Waals contacts between the subunits, there exist polar interactions, mostly hydrogen bonds, as listed in Tables 10, 11, and 12. Many of the 201 ordered water molecules are located at or near the subunit interfaces, and play a significant role in subunit interactions (see below). Interactions between subunits L and M The interactions between subunits L and M are distributed very unevenly between the periplasmic and the cytoplasmic side of the membrane. Table 10 lists the H-bonds and salt bridges shorter than 3.5 between subunits L and M. Out of 37 H-bonds, 29 occur near the cytoplasmic membrane surface, one inside the membrane, and only seven near the periplasmic membrane surface. The inter-subunit salt bridges are between Lys L8 and Glu M244, between Arg L217 and Asp M43, and between Asp L218 and Arg M134/Arg M28, all at the cytoplasmic membrane surface. Two of these bridges (L8-M244, and L218-M134) are conserved in the known amino acid sequences of RC L- and M-subunits from purple bacteria (B61anger et al., 1988). As shown in Figure 9, the connections between the transmembrane helices of subunits L and M at the periplasmic side of the membrane are arranged side by side and form the elliptical contact surface with the cytochrome subunit. Most of the polypeptide chain segments in this surface are related by the central local 2-fold symmetry axis. The inter-subunit H-bonds primarily involve residues from the non-symmetric parts of the subunits, notably residues M83 and M86 from one of the sequence insertions in M with respect to L; this may help to prevent formation of homodimers of the L- or M-subunits. Inside the membrane, near the C-contact surface, the subunits are in indirect contact via the interactions between the BChl of the special pair. Observations of self-aggregation of BChl provide experimental evidence for the significance of such interactions (Scherz et al., 1990). The majority of the L-M subunit interactions occur near the cytoplasmic membrane surface and the H-contact site. Here, the subunits L and M interpenetrate. The D and E transmembrane helices and their connection, including the quinone binding site, of each subunit fill a gap between the C and E helices of the other subunit. This leads to intimate interactions between the subunits in this region, including the majority of the polar interactions listed in Table 10. Figure 10 shows tlie polypeptide chains, and interacting side-chains of subunits L and M. Many of the residues forming important polar interactions between subunits L and M are located near the ends of transmembrane helices LB and MB, and in the DE helices of both subunits. Examples are Figure 8. Stereo pair: C~-atoms of the haem-binding segments, side-chains of haem thioether links and iron ligands, and haems, as arranged in the cytochrome subunit's structure. Chain segments deviating significantly from the internal local 2-fold symmetry are indicated by thick bonds.

15 Rps. viridis Reaction Center 443 Table 8 Thioether bridges, iron-protein bonds and polar interactions of haem groups Haem atom Protein atom Distance (A) HE1: Thioether bridges: CAB - SG Cys C CAC - SG Cys C Iron-protein bonds: FE - SD FE - NE2 Met C His C Polar interactions: O1A - N Asn C O2A - N Lys C O1D - N Val C O1D - N Leu C O2D - N Lys C O2D - NH1 Arg C HE2: Thioether bridges: CAB - SG Cys C CAC - SG Cys C Iron-protein bonds: FE - S D Met Cl FE - NE2 His C Polar interactions: HE3:" Thioether bridges: OIA - OH Tyr C OIA - OH Tyr C O1A - NH2 Arg C O2A - NE Arg C CAB - SG Cys C CAC - SG Cys C Iron-protein bonds: FE - SD Met C FE - NE2 His C Polar interactions: OIA - N Val C O1A - N Val C OZA - OH Wat W O2A - OH Wat W OlD - OH Wat W O1D - NH1 Arg C O2D - NE Arg C O2D - NH2 Arg C O2D - NE1 Trp C O2D - OH Wat W HE4: Thioether bridges: CAB - SG Cys C CAC - SG Cys C Iron-protein bonds: FE - NE2 His C FE - NE2 His C Polar interactions: OIA - NE2 Gin C OIA - OH Wat W O2A - OH Wat W OID - OH War W O2D - OG1 Thr C arginines L103 and M130, which occupy equivalent positions in the B helices and which interact with the C-terminal ends of the DE helices of the bpposite subunit. These short helices protrude into the hydrophobic interior of the membrane. Close to the local 2-fold axis, the non-haem iron bindg to glutamic acid M232 and to the four histidine residues L190, L230, M217, and M264, located in the transmembrane helices LD, LE, MD, and ME. These helices are in contact with each other near the iron binding site. Towards the membrane interior, the side-chain of Ser M271 and the carbonyl oxygen atom of L184 form an H-bond; the carbonyl oxygen atoms of L180 and M207, and the side-chain of Asn L183 are H-bOnded to a water molecule near the local 2-fold axis (W301) in the middle of the membrane. Three additional waters with H-bonds to both subunits L and M exist near the periplasmic membrane surface, and four such waters at the cytoplasmic surface.

16 444 Rps. viridis Reaction Center Table 9. Increase of solvent-accessible surface area (,~z) of protein subunits upon removal of one other or all other subunits from the RC complex Context Subunit RC-C RC-L RC-M RC-H Subunit alone C L M H Accessible surface areas (probe size 1.4,~) were calculated for each subunit in the context of the whole RC, in RC models with one subunit removed at a time, and with the subunit model alone. Numbers in rows give increase of accessible surface area of each subunit upon removal of one or all other subunits from the RC model. Residues whose side-chains are involved in polar interactions between subunits L and M are highly conserved in RCs of purple bacteria. Of the 17 side-chains in the L-subunit 14 are identical in the four known amino acid sequences (B61anger et al., Table 10 H-bonds and salt bridges between subunits L and M (H-bond criteria as in Table 5) Near cytoplasmic membrane surface: Glu L6 OE1 - NE1 Trp M252 Lys L8 NZ - OE2 Gin M244 Tyr ) L9 OH - OG1 Thr M241 Tyr L9 OH - OE2 Glu M244 Phe Arg L30 L103 N NH1 - - O O Trp Thr M252 M253 Arg L103 NH2 - O Thr M253 J..eu Llll O - NH1 Arg M245 Gly Ll14 N - O Ala M223 His Ll16 NE2 - OG1 Thr M5 Ser L196 O - N Gly M141 Asn L199 ND2 - OE1 Glu M261 Lys L207 NZ - O Gly M139 His L211 ND1 - O Leu M138 Tyr L215 OH - O Val M131 Arg L217 NH2 - OD2 Asp M43 Asp L218 O - N Tyr M50 Asp Asp L218 L218 OD1 OD1 - - NE NH2 Arg Arg M28 M28 Asp L218 OD2 - NE Arg M28 ASp L218 OD1 - NH1 Arg M134 Val L219 O - NE Arg M130 Val L219 O - NH2 Arg M130 Gly L221 O - N Gly M47 Tyr I.~ OH - O ASp M43 Ile L224 O - N Asp M43 His L230 NE2 - OE2 Glu M232 Arg Arg L231 L231 NE NH2 - - O O Thr Gin M5 M4 Inside membrane: Ala L184 O - OG Ser M271 Near periplasmic membrane surface: Asp L155 OD2 - OH Tyr M196 Asp L155 OD1 - OH Tyr M305 Tyr L169 OH - OD2 Asp M182 Trp L266 NE1 -. O Arg M86 Leu L267 O - NE Arg M86 Leu L267 O - NH2 Arg M86 Tap L272 O - NE2 Gin M ), two are identical in three sequences, and one occurs in two sequences only; in the M-subunit 14 side-chains are involved, of which nine are identical in the four known sequences, one occurs in three, two occur in two, and only two are not conserved at all. Interactions of subunit H with the other subunits The N-terminal residue of subunit H, a formylmethionine, is located at the periplasmic side of the membrane. Here, subunits H and cytochrome have a small contact site, including two H-bonds (see Table 11)in antiparallel ~-sheet style, formed between residues H1 and C212, and H3 and C210. This interaction is extended by a water molecule (W050), bridging between N-H of residue H1 and C------O of residue C212. The transmembrane helix of subunit H follows the surface of the M-subunit, along transmembrane helices ME and MD, and is attached to the M-subunit by polar interactions between the side-chains of Asp Hll and of Trp M295 and His M299. These residues are conserved in the amino acid sequences of purple bacterial RCs (Williams et al., 1986; B61anger et al., 1988). A water molecule (W090), H-bonded to Asp Hll OD2, H12 N, and M288 O may also contribute to these polar interactions. Near the centre of the membrane, Gln H20 forms a H-bond to Trp M199, and possibly also to Ser M277. At the cytoplasmic side of the membrane we find polar interactions of residues Arg H33, Asp H36, and Glu H39 with the M-subunit (see Table 11). Glu H39 forms a salt bridge to Arg M239; both these residues are also conserved in the known RC sequences. The contact site of the transmembrane helix of the H-subunit with helices MD and ME also contains the binding site of the only detergent molecule that is found well ordered in the crystal. This LDAO molecule sits in a cavity formed by.these helices, and the prosthetic groups BA, and QA, as shown in Figure 11. Its interactions with its surroundings are mostly through its hydrophobic tail; its polar head group contacts Asp H56 from a neighbouring RC molecule in the crystal. The refined electron density indicates the binding of a second, partially ordered, detergent molecule in this region. The surface region of the H-subunit (residues H41 to H106) has a large contact surface (778 di 2) with the L-subunit, and a smaller one (113fii 2) with the M-subunit. Residues H46 to H65 do not participate in this contact; part of this section (H47 to H53) is disordered in the crystal. Salt bridges are formed between Lys H66 and Glu M261, and between Glu H84 and Lys L8; all these residues are conserved. The globular domain of the H-subunit sits to a large part "underneath" the L-M. complex. The centre of mass of the domain does not lie on the local 2-fold axis relating subunits L and M; rather> it is shifted to the side opposite to the attachment side of the H transmembrane helix (see Figure 4). Part of its surface pointing towards the membrane is accessible in the crystal; it is likely that in situ this surface is

17 Rps. viridis Reaction Center 445 Figure 9. Stereo pair: Polypeptide chains forming the cytochrome contact surface of subunits L (bonds indicated by double lines) and M (bonds indicated by single lines). The first and last residue of each chain segment is labelled. Also labelled is residue Tyr L162 whose side-chain marks the approximate position of the local 2-fold symmetry axis relating subunits L and M. The side-chains of Tyr L162 and of the residues involved in hydrogen bonds between subunits L and M near the cytoplasmic'membrane surface are drawn with thick bonds. Inter-subunit hydrogen bonds are indicated by broken lines. either in contact with the membrane, or with other membrane proteins, for example light-harvesting complexes. The H-subunit and the light-harvesting proteins can be chemically crosslinked (Peters et al., 1984). Its contact area with subunit M is 966/~2 and includes two salt bridges: Glu H235 and Asp H125 interact with Arg M231, and Arg H181 interacts with Asp M230. These residues are either conserved or conservatively replaced in the known sequences of M- and H-subunits. The contact area of the globular domain of subunit H with subunit L is 541 ~k 2. One salt bridge in this area connects Glu H255 and Arg L12; these residues are not conserved. In addition to the H-bond interactions listed in Table 12, water molecules contribute to polar interactions between subunits (see below). Interactions of the cytochrome with the other subunits The small contact of the cytochrome with subunit H was described in the previous paragraph. The main interactions of the cytochrome in the RC complex are with subunits L and M (see Table 12). Residues from the N-terminal segment of the cytochrome are primarily forming the contacts to the L-subunit, residues from the connecting egment between the haem binding segments contribute most of the interactions with the M-subunit. A smaller contribution to these interactions also comes from the segm4nt binding haems 3 and 4, especially the haem 3 binding region. The following interactions between charged residues are found: Glu C3 with Arg L257, Lys C178 with Asp L268, Arg C202 with Asp M314, Lys C258 and Lys C259 with Asp M304, and Arg C272 with the C terminus of subunit M. None of the residues from subunits L and M mentioned here is conserved in the RC subunits with known sequences (these RCs do not have a bound cytochrome). Bound water molecules also contribute to the interactions between the cytochrome and subunits L and M. ()rdered solvent The majority of the 201 water molecules located during refinement are buried within the RC complex; only 62 have a static accessible surface area of more than 5 ~2. This result simply reflects the higher degree of structural order in the interior of the RC. Subunit interfaces are preferred binding sites for the localized water molecules. Of the 108 water molecules on the periplasmic side of the membrane 39 are in contact only to the cytochrome and the haems, four only to subunit L, and seven only to subunit M. The remaining 58 water molecules have contacts to more than one subunit (16 to cytochrome and L, 30 to cytochrome and M, four to cytochrome, L and M, two to cytochrome and H, one to cytochrome, M and H, and five to L and M). The preference for subunit contacts js even more pronounced on the cytoplasmic side of the membrane, where 20 water molecules are in contact to only one subunit (four to L, three to M, and 13 to H), and 68 waters have contacts to more than one subunit (11 to L and M, 13 to L and H, 27 to M and H, and 17 to L, M, and H). Of the 126 water molecules at subunit interfaces, 41 form H-bonds to more than one subunit: two to subunits L, M, and H, eight to L

18 446 Rps. viridis Reaction Center and M, eight to cytochrome and L, ten to cytochrome and M, four to L and H, and nine to M and H. Thus, waters contribute significantly to subunit interactions in the RC complex. Six of the putative SO~- ions in the refined RC model are located near the cytoplasmic membrane surface (see Figure 4); they could mimic negatively charged lipid head groups, neutralizing the excess of positively charged residues in this region. Four of them (S1, $2, $3, $6) contact one or more positively charged groups from the subunits L, M, and H. One putative sulphate ($4) sits near the N-terminal end of transmembrane helix MA without a formal countercharge in its neighbourhood. This reminds one of ion binding in the bacterial sulphate binding protein (Quiocho et al., 1987). The identity of the remaining two putative sulphate ions is least certain; one of them ($7) sits on a crystallographic 2-fold axis between the H-subunits of adjacent RC molecules, in the crystal. Sulphate $7 has been re-interpreted as an additional detergent molecule in later studies (C. R. D. Lancaster and H. Michel, unpublished). The central cofactors Table 13 lists centre-to-centre distances, and closest distances between atoms in the conjugated ring systems of functional cofactors associated with the subunits L, M, and cytochrome. The angles between ring planes of the various cofactors are given in Table 14. Some of these angles, e.g. the one between Dr. and D,M (Deisenhofer et al., 1984) changed significantly during refinement, mostly due to non-planar deformation of the tetrapyrrole rings: while planes through the ring I atoms of DL and of D,M enclose an angle of only 5.0, the angle between planes defined by the pyrrole nitrogen atoms enclose an angle of 11.3 (Table 14). The degree of non-planarity was determined by measuring the r.m.s, distance of atoms in the tetrapyrrole ring systems from planes that had minimum sums of squared distances from the pyrrole nitrogens. This r.m.s distance is 0.11 A for D, and 0.17 A for DM. Both carbon atoms of ring V in D~ and one of them in DM are more than 0.5 A away from this plane. Figure 10.Stereo pair: polypeptide backbones and selected side-chains of subunits L and M at the H-contact surface. Also shown are QA, Q,, and the non-haem iron. Colours: blue, nitrogen atoms; red, oxygen atoms; blue-green, non-haem iron; light brown, carbon atoms of L-subunit; light blue, carbon atoms of M-subunit; yellow, carbon atoms of quinones. Selected hydrogen bonds are indicated as green broken lines.

19 Rps. viridis Reaction Center 447 T a b l e 11 H-bonds and salt bridges between subunit H and other subunits (H-bond criteria as in Table 5) H-L (24 pairs): Val H44 N - O Leu L2 Val H44 O - N Ala L1 Val H44 O - N Leu L2 Gly H40 O - N Ser L4 Lys H66 NZ - OD1 As^ L199 Val H69 O - N Val L206 Pro H71 O - NZ Lys L205 Arg H82 O - OG Ser L4 Glu H84 OE1 - NZ Lys L8 Glu H84 OE2 - NZ Lys L8 Gly H98 O - N Trp L25 Leu H101 N - O Arg L10 Leu H101 O - N Arg L12 Leu H113 N - O Lys L8 Val H128 N - OE1 Glu L210 Ser H176 OG - OE1 Glu L210 Glu H255 OE1 - NH1 Arg L12 Glu H255 OE1 - NH2 Arg L12 Ala H254 O - N Gly L14 Arg H253 O - NH1 Arg L109 Arg H253 O - NH2 Arg L109 Leu H257 N - O Thr L15 Leu H258 N - O Leu L16 H-M (37 pairs): Asp Hll OD2 - NE1 Trp M295 Asp H11 OD1 - NE2 His M299 Gin H20 OE1 - NE1 Trp M199 Gin H20 NE2 - OG Ser M277 Arg H33 NH1 - O As^ M257 Asp H36 OD2 - OG Ser M262 Asp H36 OD1 - NE1 Trp M266 Asp H36 OD2 - NE1 Trp M266 Glu H39 OE2 - NH1 Arg M239 Lys H66 NZ - OE1 Glu M261 Pro Hl14 O - NH1 Arg M245 Pro H114 O - NH2 Arg M245 Ser Hl16 O - OG1 Thr M241 Ser Hl16 O - NH2 Arg M245 Arg H120 NE - OD1 Asp M238 Arg H120 NH2 - O Asp M238 Ala H121-. N - OD1 Asp M238 Asp H125 OD1 - NH1 Arg M231 Ser H 144 N - O Arg M13 Ser H144 O - N Arg M13 Ala H146 N - O Gin Mll Asp H149 OD2 - N Gin Mll Asp H149 OD1 - OH Tyr M36 Asp H149 OD2 - OH Tyr M36 His H178 NE2 - O Pro M15 Arg H181 NH1 - OD1 Asp M230 Arg H181 NH1 - OD2 Asp M230 Arg H181 NH2 - OD2 Asp M230 Gly H199 O - NE Arg M226 Gly H199 O - NH2 Arg M226 Cys H201 O - NE2 Gln M9 Val Glu H203 H235 N OE1 - - OE1 NH1 Gin Arg M9 M231 Glu H235 OE1 - NH2 Arg M231 Glu H235 OE2 - NH2 Arg M231 Asp H236 OD1 - N Thr M241 H--C (2 pairs): Met H1 O - N Val C212 His H3 N - O Pro C210 The local symmetry in the L-M complex, relating parts of the polypeptide backbones, and especially of the cofactors DM, BB, ~B, QB to DL, BA, CI)^, Q^, respectively (Deisenhofer et al., 1984, 985a), was an important result of the structure determination.of the RC. As subunits L and M, the BChl-b molecules DL and DM of the special pair are related by almost perfect 2-fold symmetry (179.7 rotation). In contrast, B^ and BB are related by a rotation of 175.8, ~^ and ~s by 173.2, and Q^ and QB by These deviations from 2-fold symmetry lead to slightly different distances a~ad angles between cofactors in the A- and B-branch (Deisenhofer & Michel, 1989) as can be seen in Tables 13 and 14. The iron position is about 1.0,~ away from the local 2-fold symmetry axis. Imperfect symmetry is also obvious from the different crystalline order in both branches. The head group atoms of DL, DM, BA, BB, el)a, and Q^ have average B-values between 10.0 ~2 and 12.8/i, 2, the average B-values of the phytyl tail atoms of DL, DM, B^, and ~a are only slightly higher. In contrast, the average B-value of the head group atoms of ~B is 21.1 ~2, and the phytyl taft atoms of BB and ~B are partially disordered (Deisenhofer & Michel, 1989). The QB site is only partially, occupied. Cofactor interactions Table 15 lists the polar interactions of cofactors in the refined RC model. The main interactions between the cofactors and their environment in the RC were correctly described at an intermediate stage of the refinement at 2.9 A resolution (Michel et al., 1986a). Besides more accurate atomic co-ordinates, the main benefit of the refinement at 2.3/~ resolution was the addition of QB, the carotenoid, and of ordered solvent molecules. As can be seen from Table 15, two of the ordered water molecules form H-bonds to the ring V keto carbonyl oxygen atoms of B^ and BB. As shown in Figure 12, these water molecules (W302 and W304) form additional H-bonds with the ND1 atoms of histidines L173 and M200 (tile ligands to the Mg a of the special pair BChl-b), and with backbone carbonyl oxygen atoms of His L168 and Tyr M195 (the side-chains of these residues form H-bonds to the acetyl groups of the special pair BChl-b). Additional water binding sites are between the special pair and the nearest haem of the cytochrome; H-bond networks involve His L168, and Tyr L162 in this region. The interactions of these water molecules must be important for the proper function of the first electron transfer steps in the RC. While the binding pocket of QA is water-free, bound water is a prominent part of file QB binding site, as shown in Figure 13. As QB must be protonated during a functional cycle, the presence of ionizable residues and of Water near its binding site is expected. It is easy to design several possible pathways for protons from the RC surface to the vicinity of QB. Residues in the vicinity of the QB binding site, notably Glu L212, Asn L213, Arg L217, Tyr L222, Ser L223, Ile L229, Asp M43, or their equivalents have been subjects of herbicide-resistant or site-specific mutations in Rb. capsulatus, Rb. sphaeroides, and Rps. viridis; the results of thes"

20 448 Rps. viridis Reaction Center studies have shed some light on the role of individual residues in QB binding and protonation. The emerging view is that Ser L223 is involved in the donation of the first proton, and Glu L212 is the second proton donor (for reviews, see Okamura & Feher, 1992; Leibl et al., 1993). Due to the low occupancy of QB, its exact position cannot be determined with confidence from the crystallographic data. A detailed discussion of QB binding has to await the results of experiments with reconstituted QB, which are currently under wa)~ The carotenoid The carotenoid molecule is located on the M-side of the RC core at about the same distance as the special pair from the cytochrome contact surface. Its middle part is in van der Waals contact with BB. The electron density map at the end of the refinement accounts for a 21-carbon chain, too short for 1,2-dihydroneurosporene; flexibility at both ends of the molecule has to be assumed. The identification of the carotenoid is based on the following observations. The electron density feature is the only one with the right shape for a carotenoid that is located near the special pair. It lies in a plane parallel to the membrane plane; this is in agreement with spectroscopic data on carotenoid orientation (Breton, 1985a,b). Triplet transfer from D to the carotenoid in Rb. sphaeroides requires the presence of an intact BB molecule (Frank & Violette, 1989). Structure analyses of RCs from Rb. sphaeroides indicate that the carotenoid has the same location in these RCs as found in the Rps. viridis RC (Yeates et al., 1988; Arnoux et al., 1989). The carotenoid threads through an opening between transmembrane helices MB and MC; this opening is provided by residues Gly Ml17 (Gly or Ser in the known sequences) and Thr M121 (Thr, Phe, or Ala in the known sequences) on helix MB, Gly M159 (conserved in all four known sequences) and Val M155 (Val, Phe, or Trp in the known sequences) on helix MC, and Val M173 (Val or Pro in the known sequences) in the segment connecting transmembrane helices MC and MD. The corresponding region Figure 11. Stereo pair: the LDAO binding site (labelled LDIH and shown with dot-surface) with transmembrane helices of subunits H and M (helices MD and ME). The course of the polypeptide chains is indicated by ribbons. Also shown are the cofactors BA (labelled BCLAH), ~A (labelled BPLH), and QA. Colours: blue, nitrogen atoms; red, oxygen atoms; blue-green, non-haem iron; pink, carbon atoms of H-subunit; light blue, carbon atoms of M-subunit; yellow, carbon atoms of cofactors and LDAO. Selected hydrogen bonds are indicated as green broken lines.

21 Rps. viridis Reaction Center 449 Table 12 H-bonds and salt bridges between the cytochrome and subunits L and M (H-bond criteria as in Table 5) C-L (21 pairs): Cys C1 O - NE1 Trp L262 Glu C3 N - O Phe L254 Glu C3 OE2 - N Thr L256 Glu C3 OE2 - OG1 Thr L256 Glu C3 OE1 - NE Arg L257 Thr C9 OGI - NE2 His L144 Gin C11 OE1 - N Leu L71 Gin C11 NE2 - OD2 Asp L70 Arg C15 O - ND2 Asn L67 Arg C15 NE - O Pro L68 Arg C15 NH2 - O Asn L67 Leu C17 O - ND2 Ash L159 Ser C18 OG - OE1 Gin L163 Gly C20 O - NE2 Gin L163 Thr C161 OG1 - OT1 Ser L273 Lys C178 NZ - OD2 Asp L268 Tyr C182 OH - OD1 Asp L268 Ala C184 N - OH Tyr L169 Ala C250 N - OD1 Asn L159 Ala C250 O - ND2 Asn L158 Asn C249 OD1 - ND2 Asn L159 C-M (27 pairs): Ser C170 O - NE2 " Gin M87 Tyr C182 O - NE1 Trp M90 Asn C186 O - NZ Lys M97 Asn C186 OD1 - NZ Lys M97 Arg C202 NH1 - OD2 Asp M314 Arg C202 NH2 - OD1 Asp M314 Pro C205 O - ND2 Asn M291 Ala C208 O - N Asp M290 Ala C208 O - N Asn M291 Arg C216 NH1 - O Gly M286 Arg C216 NH2 - O Thr M287 Gly C217 N - O Val M166 Arg C220 O - NE2 Gin M99 Arg C220 NH1 - OH Tyr M191 Leu C223 N - OG Ser M170 Ser C224 OG - O Lys M97 Gin C251 O - ND2 Asn M193 Gin C251 NE2 - OH Tyr M196 Gin C251 NE2 - O Pro M303 Glu C254 OE1 - ND2 Asn M291 Gly C257 O - N Thr M312 Gly C257 " N - O Thr M312 Lys C258 NZ - O Tyr M305 Lys C258 NZ - OD2 Asp M304 Lys C259 NZ - OD1 Asp M304 Arg C272 NE - OT1 Lys M323 Ar~ C272 NH2 - OT2 Lys M323 in the L-subunit does not show a comparable opening. The residues of the L-subunit equivalent to Ml17, M121, M155, M159, and M173 are Thr L90, Leu L94, Phe L128, Gin L132, and Phe L146, respectively Each of these residues in the L-subunit has a side-chain more bulky than that of its equivalent in the M-subunit, thus preventing the opening of a channel between transmembrane helices LB and LC, which could be part of a carotenoid binding site. Furthermore, the tip of the phytyl chain of d)a occupies the same position near pyrrole ring I of BA that is occupied by the carotenoid near BB. This could be one of the causes or an effect of the absence of a carotenoid binding site in the L-subunit. To construct a carotenoid-free RC it may be sufficient to replace Gly Ml17 and/or Gly M159.by residues with medium sized side-chains (Leu, Ile). Because both residues occur in a helix, replacement of Gly would not create problems with backbone conformation. The electron density maps clearly show that the carotenoid molecule is not in an all-trans conformation. Because of the partial disorder, positioning the carotenoid model in its electron density is not straightforward. Only small density features, possibly indicating the position of the methyl groups of the carotenoid, can be used as guidelines. The optimum fit of the model to these features leads to a carotenoid model with the bond between carbon atoms 13 and 14 in cis-conformation, and distortions of the regular geometry nearby Having a cis-bond near the centre of the carotenoid is in agreement with data from Resonance Raman spectroscop)4 however these data indicate that the central bond (15-15') assumes cis-conformation (Lutz et al., 1987; De Groot et al., 1992). The recent finding of a carotenoid in 13-cis conformation in the refined crystal structure of the RC from Rb. sphaeroides wild-type (U. Ermler, G. Fritzsch, S. K. Buchanan & H. Michel, unpublished) is therefore even more surprising. At present we cannot offer an explanation for this discrepancy Crystal packing The RC molecules form crystal contacts almost exclusively with parts of the cytochrome and of the H subunit. A list of residues that come closer than 4.5 A to neighbouring RC molecules in the crystal includes 30 residues from the H subunit, 19 residues and haem I from the cytochrome, the bound LDAO molecule, and only two residues near the N terminus of subunit L and the C-terminal lysine residue of subunit M. Five H-bonds or salt bridges involve residues of the cytochrome; eight H-bonds or salt bridges involve residues from the H subunit, two of these are formed across a crystallographic 2-fold axis by threonines H141 and H167, and their equivalent counterparts. The head group of the bound LDAO molecule interacts with the side-chain of aspartic acid H56 from a neighbouring RC molecule in the crystal. The hydrophobic protein surface in the middle of the RC, which is covered by detergent (Roth et al., 1989), does not contribute to crystal contacts. However, the detergent regions of neighbouring RC molecules may touch each other and thereby contribute to lattice interactions (Roth et al., 1989). Heavy atom binding sites The structure of the RC was solved with the method of multiple isomorphous replacement, using compounds containing mercury or uranium. Between 7 and 11 heavy atom binding sites were found in the derivative crystals suitable for phase determination (Deisenhofer et al., 1984). The mercury

22 Rps. viridis Reaction Center Table 13 Distances between cofactors in RC Upper right: centre to centre Lower left: closest contacts of atoms involved in double-bonds A. Central cofactors: DL DM B^ BB Oh (l)e Fe Q^ QB D~ DM B^ BB O^ FE Q^ Q B. Special pair and haems DL DM HE1 HE2 HE3 HE4 DL DM HE HE HE HE compounds bound mostly to cysteine residues on the surface of the membrane-spanning part of the RC structure. For example, the seven most highly occupied sites of the derivative MDDB (2-methyl-l,4- dichloromercuri-2,3-dihydroxybutane) were associated with cysteines L92, L122, L129, M256, M160, and M296, and with methionine L101. All these residues are inside or at the edge of the surface region covered by detergent (Roth et al., 1989). Thus, in the case of the RC from Rps. viridis, the detergent was no obstacle for heavy atom compounds. On the contrary, because the detergent region does not contribute significantly to crystal packing, heavy atom binding in this region did not cause perceptible non-isomorphism. Uranyl nitrate, the other type of heavy atom compound used, mostly bound to glutamic acid or aspartic acid residues of the H-subunit. One binding site was at the C terminus of this subunit. Only one weakly occupied site was found on the surface of the cytochrome. All these sites are outside the detergent Table 14 Amities between rinl~ planes of cofactors in RC A. Central cofactors D, DM B^ Be ~a OB Q^ QB DL DM B^ - BB - (1)A Q~ B. Haems and special pair Haem 1-haem Haem 2-haem Haem 4-haem Haem 3-DL 37.7 Haem 3-DM region on the polar surface of the RC. Although some of the binding sites of uranyl nitrate are near crystal contacts between neighbouring molecules, the derivatized crystals were nevertheless also highly isomorphous with the native crystals, thus providing an excellent basis for solving the phase problem. Concluding Remarks The structure of the RC from Rps. viridis is an example of a multi-subunit membrane protein complex. Two subunits (L and M) are "true" membrane proteins with multiple membranespanning segments. The other two are "integral" membrane proteins in the operational definition, that they are insoluble in the absence of detergents. This insolubility is caused by their membrane anchors, which are a helix in the case of the H-subunit, and a covalently linked diglyceride in the case of the cytochrome subunit. The diglyceride is disordered, despite the fact that the membrane-spanning parts of the protein show a higher degree of order compared to the extramembraneous parts. The three-dimensional structures of RCs from purple bacteria have helped to understand their function and their mechanism of action. Knowledge of the structures of RCs from purple bacteria has also allowed the drawing of realistic conclusions on the structures of the RCs from other photosynthetic organisms, especially of the photosystem II RC. The atomic model of the RC has been used for theoretical simulations (Chu et al., 1989; Parson et al., 1990; Gunner & Honig, 1991; Treutlein eta!., 1992), and as the basis for investigating both spontaneous and site-specific mutations (Sinning et al., 1989, 1990; Leibl et al., 1993; Laussermair & Oesterhelt, 1992; Wachtveitl et al., 1993; Baciou et al., 1993). Although still not all aspects of the function of the RC are fully understood, it is realistic to expect that at the end of

23 Rps. viridis Reaction Center 451 Table 15 Covalent or polar interactions of central co-factors with the protein environment (bond distances in A) DL: M~' ligand: Polar: MG - NE2 His L OBB - NE2 His L OBD - OG1 Thr L DM: Mg 2 ligand: MG - NE2 His M Polar: OBB - OH Tyr M BA : Mg a ligand: MG - NE2 His L Polar: OBD - OH War W BB: Mg a* ligand: MG - NE2 His MI Polar: OBD - OH Wat W (DA: Polar: OlD - NE1 Trp L OBD - OE1 Glu L (DB: Polar: OlD - NE1 Trp M Fe: Ligands: QA: Polar: FE - NE2 His L FE - NE2 His L FE - NE2 His M FE - OE1 Glu M FE - OE2 Glu M FE - NE2 His M ND1 His M N Ala M QB: (QB position only partially occupied) Polar: 02 - ND1 His L OC Ser L N Gly L anode X-ray generator as described (Deisenhofer et al., 1984). Subsequenfl)4 a second set of intensity data was. collected at DESY using the instrument X31. During this experiment the crystals were kept in an air stream of-4 C. The X-ray wavelength was ~; the crystal to film distance was 75 mm, allowing data collection to 2.3 resolution. The crystals were rotated around the c* axis. To avoid severe overlap of reflections, the rotation interval per film had to be limited to 0.4. In total, seven crystals were needed to completely cover a 45 rotation around the c* axis. In order to find seven crystals that diffracted to 2.3 resolution, about 80 crystals had to be tested. The optical density of the films was measured with an Optronics P1000 scanner with the raster size set to 50 ~tm. Orientation angles, effective mosaicity of the crystals, and crystal to film distance, were determined for each film using the program OSC (Rossmann, 1979; Schmid et d., 1981). Subsequentl)4 reflection intensities were evaluated with the program FILME (Schwager et al., 1975; modifications by W. Steigemann, J. Deisenhofer, and W. S. Bennett). The crystal effective mosaicity of 0.08, in combination with the small rotation intervals, led to a high amount (-80%) of partially recorded reflections. Scaling and merging of the data was done in two steps as described (Deisenhofer et al., 1984): Intensities of fully recorded reflections from each film were used to determine film-to-film scale factors; these scale factors were used during addition of intensities of partially recorded reflections from successive films, and during merging of multiple measurements of reflections for each crystal. Mergeddata from individual crystals were combined into the 2.3 A resolution data set. The R~,se value between the 2.9,~ data and the 2.3 ]~ data is significantly larger (0.12) than the R,~, for each of the data sets alone (-0.09), but a difference-fourier map calculated with the two data sets did not indicate significant changes of the crystal structure. Becauseof the discrepancy between the two data sets, only the 2.9 A data obtained by rotation around a* were used to cover the blind region left during the 2.3,~ data collection. Table 1 lists properties of both data sets used during refinement. the combined efforts of many disciplines we will have a complete and satisfactory concept of the molecules performing the first steps in the photosynthetic light reactions. Progress in understanding other large multi-subunit membrane protein complexes depends critically on structural information. Unfortunately, progress in obtaining such information has been slow; reliable long-term funding of structure analyses is a necessary condition for success. Methods Crystals and data collection RC from Rps. viridis was isolated and crystallized as described (Michel, 1982). The RC crystals,have the symmetry of space group P4~212; the unit cell constants are a = b = A, c = A. With one RC (=145 kda) per asymmetric unit, the Matthews parameter (Matthews, 1968).is 4.9,~S/dalton; disordered solvent, including detergent, occupies about 70% of the crystal volume. The initial structure analysis and the early stages of refinement were done with a set of X-ray reflection intensities to 2.9 A resolution, collected with a rotating Starting model The initial RC model was constructed on interactive graphics display systems (Vector General 3400, Evans & Sutherland PS330) driven by the program FRODO (Jones, 1978). Model building was based on an electron density map at 3 ]~ resolution; the phase information for this map was obtained from m.i.r, experiments, and improved by solvent flattening (Deisenhofer et al., 1984). Complete amino acid sequences for the subunits H, L, and M were available (Michel et al., 1985, 1986b); the amino acid sequence of the cytochrome subunit (Weyer et al., 1987a) was only partially known at the start of refinement. The good quality of the electron density map, and the systematic use of the real-space refinement option in FRODO were prerequisites for the rather low R-value of 0.359, calculated for 46,658 reflections between 7/k and 3 resolution, of the starting model. Refinement Crystallographic refinement of the RC model was done in 16 steps as summarized in Table 2. Each step consisted of cycles of stereochemically restrained least-squares refinement of atomic positions, and, starting at step 4, additional cycles of restrained least-squares refinement of

24 452 Rps. viridis Reaction Center, DL Figure 1:~. Stereo pair: vicinity of the special pair, viewed along the local 2-fold symmetry axis. Shown are the BChl-b molecules De, DM, BA, and BE with the magnesium ligands His L153, His L173, His M180, and His M200. His L168, Tyr M195, Tyr M208, and 3 water molecules (large circles) form hydrogen bonds (shown as broken lines) with the cofactors and among each other. B-values. When these calculations had come near convergence, the model, together with various kinds of maps (see below), was inspected at the display system for error correction, and addition of new details (e.g. side-chains, waters) to the model. Restrained least-squares refinement was done with two different methods. We used the procedure of Jack & Levitt (Jack & Levitt, 1978; Deisenhofer et al., 1985b) during steps I to 13, and the T package (Tronrud et al., 1987) during steps 14 to 16. We turned to T because of its simple and transparent way of defining geometric restraints, a great advantage when the model includes many different types of non-standard groups. Geometry libraries for amino acids, and for bacteriochlorophyll a were obtained from the authors of the T package (Tronrud et al., 1986). Slight modifications were required to construct entries for BChl-b and BPh-b; planarity restraints of the ring systems were redefined with the following planar groups: (CIC, CHC, C4B, NB, C3B, C2B, CIB, CHB, C4A), (CIA, CHA, C4D, ND, C3D, C2D, CID, CHD, C4C), (CID, CHD, C4C, NC, CIC, CHC, C4B), (C1B, CHB, C4A, NA, CIA, CHA, C4D). We designed new entries in the geometry library for formyl methionine, haem, menaquinone-7, ubiquinone-1, 1,2-dihydroneurosporene, LDAO, and (SO4) 2-. Weights for the contributions to the function to be minimized during T refinement were varied empirically during steps 14 to 16; the final set of weights we used is: X-ray term 10-4, bonds 1.5, bond angles 2.5, torsion angles 1.0, trigonal atom planarity 3.0, planes 10, close contacts 10. B-values were refined, starting from step 4, and using reflection data only from outside the 5 ~ resolution sphere. During steps 4 to 13, i.e. while using the Jack-Levitt Figure 13. Stereo pair: environment of Q~, showing residues of subunits L, M, and H, together with the non-haem iron (labelled FE) and water molecules. Water molecules are drawn as large circles, nitrogen atoms as smaller circles, oxygen atoms as double circles. QB is shown with thick bonds, protein side-chains with thin bonds and main chain with single bonds. Hydrogen bonds are drawn as broken lines.

25 Rps. viridis Reaction Center 453 procedure, B-values were averaged separately for backbone and side-chain atoms. Likewise, the B-values of BChl-b, BPh-b, and quinones were averaged separately for head groups, and phytyl tails. In steps 14 to 16 we used the B-value restraint scheme of the T refinement package. This scheme minimizes Z(B~ - Bi) 2 together with Z(IFo~I- IFc,~,l) 2, where B~ and B i are B-values of atoms connected by covalent bonds, and the sum includes all bonded atom pairs. The weights used in T B-value refinement were 3 x 10-3 for the X-ray term, and 1.0 for the B term. Structure factor calculation Throughout the refinement, structure factors were calculated with the two-step FFT procedure (Ten Eyck, 1977). For the work at 2.9 ~ resolution the model electron density maps were calculated with grid spacings of 0.89 ~; the B-values of the atoms were artificially increased by 18 ~2. With these parameters the errors of the F~,~ were kept at less than 2%. For the structure factor calculations at 2.3 ~ resolution we used grid spacings of 0.75 ~, and a B-value addition of 10 ~2. To improve the fit between Fo~ and F,j at low resolution, the contribution of disordered solvent was included routinely as follows. After absolute scaling of Fob, to F~,.c at a resolution higher than 5 ~, the model electron density used in the FFT procedfre was modified such that density values were set to 1.0 where the model density was lower than e/a 3 (i.e. outside the RC molecules) and to zero at all other points. Then a set of structure factors was calculated to 3.5 A resolution from this "solvent map". Subsequentl~ optimum scale factor and B-value for the solvent contribution were calculated with a least squares procedure (program written by M. Schneider and R. Huber). Finally, both contributions to F,l~ were added together; the R-values in Table 2 were obtained with this kind of Fc,~. This procedure was done once per refinement step. Electron density maps and graphics At the end of each refinement step the model was compared with electron density maps at the interactive graphics display system. The m.i.r, map at 3 A resolution (Deisenhofer et al., 1984) was frequently used as an unbiased reference. During steps 1 to 9 phase combination (Hendrickson & Lattman, 1970) with contributions from m.i.r., and from the current model was used to calculate 2Fo~-Fc~c maps. From step 11, with the resolution of the data increased to 2.3 A, we used either Sim-weighted (Sire, 1959) or SIGMAA-weighted (Read, 1986) 2Fo~ - F~lc maps, and SIGMAA-weighted Fob,- F~t maps; especially maps of the latter type often were of supreme quality To save time, inspection of the model during most of the refinement steps was limited to regions of the model with large distortions from ideal geometry, and regions with high positive or negative density in Fot,-F~k maps. Inclusion of ordered solvent Electron density features representing ordered solvent moleckfles could be found in Fo~-F~l maps at 2.3 resolution. Addition of ordered solvent to the model was started in step 12 using a semi-automatic procedure which picked peaks higher than 0.2 e//~ 3 from the Fob, - Fc, jc map, selected the peaks closer than 6/~ to the RC, and listed the closest RC atom. The peak positions found in this way were then inspected at the display, and water. molecules were placed if the electron density was roughly "spherical, and the neighbourhood suitable for hydrogen bonds. Water molecules were removed from the model if their B-values rose above 50 ~2 during the following refinement steps. In seven cases, large density peaks in a suitable polar environment suggested the presence of negatively charged ions; we assumed (SO~)2-t the species most abundant in the crystallization solution, for these sites. Computer programs In addition to computer software already mentioned above, the following programs were used during the structure analysis of the RC. The models of the protein subunits were screened for elements of secondary structure with the procedure of Kabsch & Sander (1983). Lists of hydrogen bonds were computed with the program XPLOR (Brfinger et al., 1989) using parameter lists developed by Treutlein & Schulten (Treutlein et al., 1988, 1992). H-bonds were assigned when the donor-acceptor distance was less than 3.5 A, and the angle (donor-hacceptor) ~vas greater than 120. For donors with undefined hydrogen positions, for example water molecules and the hydroxyl groups of serine, threonine, and tyrosine residues, XPLOR listed only one of the possible H-bonds; additional H-bonds were assigned for these groups by visual inspection. Accessible surface area calculations were performed with the method of Lee & Richards (1971), using a program written by T. J. Richmond. Accessible surface area calculations were also used to analyse the contacts between protein subunits, or between protein and co-factors, e.g. by comparison of accessible surface areas of molecules in isolation, and in a complex (Deisenhofer, 1981). W. Steigemann's PROTEIN package was used for scaling and merging of reflection data, R-value calculations, map calculations, and other routine tasks throughout the refinement. Drawings of molecular models were produced with programs PROJCT (W. Steigemann, unpublished), InsightII (Biosym), and PLUTO (CCP4). The final model was inspected with the program PROCHECK (Laskowski et al., 1993). Nomenclature and data deposition There has been some discussion about a suitable and comprehensible nomenclature, especially for the cofactors in the RC (Hoff, 1988). The nomenclature previously used for the RC from Rps. viridis (Deisenhofer et al., 1985a; Michel et al., 1986a; Deisenhofer & Michel, 1989) was sometimes found too complicated, and suggestions for improvement were made (Hoff, 1988). We therefore use here the following scheme. Residue numbers of protein subunits cytochrome, L, M, and H can be distinguished by the preceding letters C, L, M, and H, respectively The BChl-b of the special pair are numbered DL and DM (formerly BCLP and BCMP) where the subscript indicates attachment to subunit L or M, respectively The accessory BChl-b are called BA and B8 (formerly BCLA and BCMA), the BPh-b are named OA and ~B (formerly BPL and BPM), and the quinones are called Qa and QB; here the subscript indicates the branch. The non-haem iron and the carotenoid are numbered FE1 and NSl, respectively The cytochrome's haem groups were given the numbers HE1 to HE4, according to the order in which they are attached to the polypeptide chain.

26 454 Rps. viridis Reaction Center Refined atomic co-ordinates of the Rps. viridis RC are available under code 1PRC from the Brookhaven Protein Data Bank (Bernstein et al., 1977). Under the same code the Data Bank also holds a data set with h, k, I, Fo~, Fore, and ~=~ betweer/20 A and 2.3 A resolution (qb~ is the calculated phase angle). Acknow/edgements We thank Dr. U. Errnler and C. R. D. Lancaster for reading the manuscript, Dorothee Staber for assistance with preparation of the manuscript, and Diana Diggs for help with Figure 4. This work was supported by the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft (SFB 143), and the Fonds der Chemischen Industrie. References " Alegria, G. & Dutton, P. L. (1987). Construction and charact4rization of monolayer films of the reaction centre cytochrome c protein from Rhodopseudomonas viridis. 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