Chapter 11: Structures of Reaction Centers in Anoxygenic Bacteria

A large proportion of photosynthetically active organisms consists of anoxygenic photosynthetic bacteria. Purple bacteria find their ecological niche in deeper layers of stagnant bodies of water. In all purple bacteria, the photosynthetic pigments and the photosynthetic apparatus are located within a more or less extended system of invaginated intracytoplasmic membranes. Located within these photosynthetic membranes, reaction centers (RCs) [1–8] are defined as the minimal functional units that can catalyze light-induced electron transfer reactions, thus stabilizing the separation of charged species across the membrane. In contrast to the higher plants, algae, and cyanobacteria of oxygenic photosynthesis, which contain the two membrane-bound Photosystems I and II, each of the anoxygenic photosynthetic bacteria has only one type of reaction center. While the iron-sulfur type RCs of heliobacteria and anaerobic green sulfur bacteria resemble that of Photosystem I, the pheophytin-quinone type RCs of purple bacteria are more similar to the RC of Photosystem II. The RC essentially functions as a [reduced soluble electron carrier protein]:quinone photo-oxidoreductase (Figure 1).

The absorption of two photons of light leads to two one-electron oxidations of a soluble electron carrier protein in the periplasm and to the two-electron reduction of a quinone, which is coupled to the uptake of two protons from the cytoplasm. The resulting quinol then leaves its binding site, diffuses in the photosynthetic membrane and is reoxidized by a second membrane protein complex, the cytochrome bc₁ complex, which results in proton release to the periplasm. The electrons are transferred to re-reduce the soluble electron carrier protein in the periplasm. This net proton transport produces a transmembrane electrochemical proton potential that can drive ATP synthesis [9] through a third membrane-spanning complex, the ATP synthase (see Chapter 21 for details).

Unlike Photosystem II, however, the purple bacterial RC is incapable of extracting electrons from water. Instead it must oxidize inorganic or organic molecules available in the environment. According to their electron donor requirements, sulfur and non-sulfur purple bacteria have traditionally been distinguished. In contrast to sulfur purple bacteria (Chromatiaceae, Ectothiorhodospira), non-sulfur purple bacteria (Rhodospirillaceae) do not require inorganic sulfur compounds, such as hydrogen sulfide, but instead use organic electron donors such as malate or succinate as electron donors. Most of what is known today about purple bacterial RCs results from studies on RCs from non-sulfur purple bacteria. These are currently the best characterized membrane protein complexes [1–8].

Most bacterial reaction centers contain four protein subunits (Figure 2), referred to as H, M, L, and C (a tetraheme cytochrome c). Some, however, such as the RCs of Rhodobacter (Rb.) sphaeroides, Rb. capsulatus, and Rhodospirillum (Rs.) rubrum, contain only the H, M, and L subunits. The related RC of the green aerobic thermophilic bacterium Chloroflexus (Cf.) aurantiacus lacks the H subunit. References to representative amino acid sequence information of RC subunits have been compiled [7]. The gene for the H subunit lies on a different operon than those for the other subunits and has been examined less frequently.

Generally, RCs from purple bacteria have been isolated and characterized from Rhodopseudomonas (Rp.) viridis [10], more recently referred to as Blastochloris (Bl.) [11] viridis, Rb. sphaeroides [12], Thermochromatium (Tc.) tepidum [13], Rb. capsulatus and several other purple bacteria [12,14]. Variant RCs have been isolated and characterized from Rb. capsulatus [15–17], Rb. sphaeroides [18,19], and Bl. viridis mutants [20–23]. The methods for isolation (and crystallization) of the RCs from Rb. sphaeroides and Bl. viridis have been reviewed [7,24]. The purification procedures consist of disrupting the bacteria by ultrasonication, isopycnic centrifugation of the chromatophores in a sucrose gradient, and solubilization of the RCs with the detergent N,N-dimethyldodecylamine N-oxide (LDAO) at concentrations of 6% (Bl. viridis) and of 0.5% (Rb. sphaeroides), respectively. The RCs are further purified by a combination of column chromatography steps. In the case of Bl. viridis RCs, molecular sieve chromatography is used exclusively [25]. For the RCs of Rb. sphaeroides, various modifications of a combination of anion exchange chromatography and molecular sieve chromatography [26] have been employed. A procedure for the rapid isolation using Ni²⁺-nitrilotriacetic acid (NTA) affinity chromatography of Rb. sphaeroides RCs with an engineered poly-histidine tag fused to the C terminus of the M subunit has been published [27], and successful crystallization of the isolated material has been reported [28]. A procedure with an engineered His₆-tag fused to the C-terminus of the C subunit of recombinant Bl. viridis RC has yielded material that could be crystallized [23].

The L, M, and H subunits of the Bl. viridis RC contain 273, 323, and 258 amino acid residues (M_r = 30.5, 35.9, 28.3 kDa), respectively [29,30]. The C subunit of Bl. viridis (336 residues, M_r = 40.5 kDa) [31] is a lipoprotein and is anchored in the membrane by a diacylglycerol moiety, which is covalently bound to the N-terminal Cys side-chain via a thioether bond [32]. A recognition site for the covalent attachment of a diglyceride and removal of the signal peptide by signal peptidase II is present in Bl. viridis and Rv. gelatinosus but not in Cf. aurantiacus.

RC preparations have a non-heme iron and four magnesium-containing bacteriochlorophyll cofactors per RC [12], as measured by atomic absorption spectroscopy [33]. In Rb. sphaeroides and Bl. viridis, these are bacteriochlorophyll a and bacteriochlorophyll b, respectively (Figure 3). Those preparations with a tightly bound C subunit have four iron-containing heme groups that are covalently bound to the protein. Apart from these four c-type heme groups, all other cofactors are non-covalently bound by the L and M subunits. In addition to the metal-containing cofactors, these comprise two bacteriopheophytin groups, a carotenoid, and two quinones. In Rb. sphaeroides, these are bacteriopheophytin a, spheroidene, and ubiquinone-10, respectively, whereas Bl. viridis contains bacteriopheophytin b, 1,2-dihydroneurosporene, menaquinone-9 and ubiquinone-9. Similar to the Rb. sphaeroides RC, the Tc. tepidum RC contains four bacteriochlorophyll a and two bacteriopheophytin a groups. Similar to the Bl. viridis RC, the Tc. tepidum RC contains four c-type heme groups, menaquinone-8 and ubiquinone-8. The carotenoid in the Tc. tepidum RC is spirilloxanthin.

Apart from the availability of high resolution crystal structures discussed below, one major reason why, despite its complexity, the purple bacterial RC has become the “hydrogen atom of protein electron transfer” ([34], see also [35,36]) is the richness of its characterization by optical absorption, electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), Fourier-transform infrared (FTIR), resonance Raman (RR), fluorescence, Stark effect, and other types of spectroscopy; comprehensively reviewed in [1–8].

Crystals of the Bl. viridis RC were first grown by vapor diffusion from a protein droplet containing 1.5 m (NH₄)₂SO₄, 0.1% N,N-dimethyldodecylamine N-oxide (lauryl-N,N-dimethyl N-oxide, LDAO) and 3% heptane-1,2,3-triol against a reservoir containing 2–3 m (NH₄)₂SO₄ [25]. They are tetragonal, space group P4₃2₁2 with a = b = 223.5 Å, c = 113.6 Å [37], and one molecule per asymmetric unit (crystal form “A” in Table 1). Using these crystals, the structure of the Bl. viridis RC was solved by multiple isomorphous replacement [37,38] and refined to a crystallographic R-factor of 19.3% up to a resolution of 2.3 Å [39,40]. More recent crystals diffract to at least 1.8 Å resolution (CRD Lancaster, unpublished observations), and the structure has been refined with complete data to 2.0 Å resolution (see Table 2 below) [41].

Three kinds of well-diffracting crystals have been obtained of the Rb. sphaeroides RC (as reviewed by Fritzsch [24]). They are orthorhombic, [42–45] trigonal [46] and tetragonal [47] (crystal forms “B”, “C”, and “D”, respectively, in Table 1). Orthorhombic crystals are grown in the presence of 10–12% poly(ethylene glycol) 4000 (PEG4000), 0.06% LDAO and 3.5–3.9% heptane-1,2,3-triol or 0.8% n-octyl-ß-d-glucopyranoside against a reservoir buffer containing 18–25% PEG4000. The space group is P2₁2₁2₁. The best resolution is 2.8 Å in the direction of the long axis, but worse in the other directions. Using a partially refined coordinate set of the Bl. viridis RC for molecular replacement, three different groups used these orthorhombic crystal forms with slightly different cell dimensions to determine the structure of the Rb. sphaeroides RC. As discussed earlier [48], for all RC structures based on these orthorhombic crystals, the number of observed unique reflections, n_obs, is less than the number of parameters, n_par, required to define the model (cf. Tables 2 and 3 below).

Trigonal crystals can be obtained in the presence of 0.5–1.0 m potassium phosphate, pH 6.5–7.5, 0.06–0.15% LDAO and 1.8–3.0% heptane-1,2,3-triol against a reservoir buffer containing 1.4–1.7 m potassium phosphate. The space group is P3₁21. The best crystals diffract to 1.8 Å [49]. To date, this is the crystal form of the Rb. sphaeroides RC, which has yielded by far the largest number of well-defined structures (cf. Tables 2 and 3 below).

Tetragonal crystals are grown in the presence of 6% PEG4000, 0.85% n-octyl-β-d-glucopyranoside, 2.5% heptane-1,2,3-triol and 0.4% benzamidine hydrochloride against a reservoir solution containing 32% PEG4000 [47]. Crystals belong to the space group P4₃2₁2 with two RCs per asymmetric unit. Data from these crystals have been collected to 2.2 Å resolution [50].

More recently, crystals of the Rb. sphaeroides RC have been obtained using the cubic lipid phase technique [51] of membrane protein crystallization (crystal form “E” in Table 1). Data from these crystals have been collected to 2.35 Å resolution [52].

For the Tc. tepidum RC, orthorhombic crystals have been obtained in the presence of 47% (w/v) PEG4000 as a precipitant in a 15 mm phosphate buffer, pH 7.0, together with 0.36 m NaCl, 0.1% (w/v) NaN₃, and 0.1 mm EDTA (crystal form “H” in Table 1) [53]. Data from these crystals have been collected to 2.2 Å resolution [54].

Tables 2 and 3 list the coordinate sets of those RC structures deposited in the PDB as of 1 August, 2005. Table 3 (below) contains all coordinate sets of variant Rb. sphaeroides RC structures, while Table 2 list all other coordinate sets. Coordinate sets are ordered by their ratio of the number of observed unique reflections, n_obs, to the number of parameters required to define the respective atomic model, n_par. The structures based on the trigonal crystal form satisfy these criteria best, so we shall primarily refer to these when comparing the RC structure from this species to that of Bl. viridis. The structure of the four-subunit Bl. viridis RC is shown schematically in Figures 1 and 2. The RC from Rb. sphaeroides would appear almost identical except for the cytochrome subunit at the top, which would be missing. The Rb. sphaeroides and Bl. viridis RC structures have been compared in detail previously [55,56].

The Bl. viridis RC has an overall length of 130 Å in the direction perpendicular to the membrane. Parallel to the membrane, the maximum width is about 70 Å. The central core of the RC is formed by the L subunit and the M subunit, which possess five membrane-spanning segments each. Both subunits are closely associated and non-covalently bind ten cofactors as detailed above and shown in Figures 1 and 2. Large parts of the L and M subunits and their associated cofactors are related by a two-fold axis of symmetry perpendicular to the plane of the membrane. The H subunit is anchored to the membrane by a single membrane-spanning helix and is attached to the LM core on the cytoplasmic side. On the periplasmic side, the C subunit with its four covalently bound heme groups is attached. The N-terminal diacylglycerol moiety is not visible in the electron density map.

The pigments form two symmetry-related branches, also shown in Figure 2, each consisting of two bacteriochlorophylls, one bacteriopheophytin and one quinone, which both cross the membrane starting from the “special pair” P of two closely associated bacteriochlorophylls near the periplasmic side, followed by the “accessory” bacteriochlorophyll, B, one bacteriopheophytin, H, and a quinone, Q. As indicated in Figure 1, only the branch more closely associated with L subunit is used in the light-driven electron transfer. It is called the A-branch, the inactive one the B-branch. The active branch ends with the primary quinone Q_A, the inactive one with the secondary quinone Q_B. Halfway between both quinones, a non-heme iron is located. The carotenoid, which has a cis double bond at the 15–15′ position in its RC-bound state [57,58], is in van der Waals contact with B_B and disrupts the two-fold symmetry. In both species the crystallographic temperature factors, which are a measure for the rigidity of the structure, are considerably higher along the B-branch than along the A-branch.

Figure 4(a) shows the Cα trace of the L subunit of the Bl. viridis RC. The dominant features are the five long membrane-spanning helices (A–E). They are 21 (helix A), 24 (helices C and E), or 28 (helices B and D) residues long [39]. On the periplasmic side, the connection of transmembrane helices C and D contains a helix (“cd”) of eleven residues and the connection between transmembrane helix E and the C-terminus a helix (“ect”) of nine residues. On the cytoplasmic side, the connection of transmembrane helices D and E contains a helix (“de”) of twelve residues. This region of the structure forms the binding site of the secondary electron acceptor Q_B, which is also included in Figure 4(a). In projection, viewed from the top of the membrane, the transmembrane helices form a semicircular arrangement in the order A, B, C, E, and D [39]. Transmembrane helices A, B, and D are straight, helix E is smoothly curved, and helix C possesses a kink of more than 30°. When the L subunits from Bl. viridis, Tc. tepidum, and Rb. sphaeroides are compared (Figure 4b), an additional eight amino acid residues are found at the C-terminus in the Rb. sphaeroides RC [56].

The M subunit of the Bl. viridis RC is displayed in Figure 5(a). As indicated already by the sequence identity of around 30% between the L and M subunits, the overall protein fold is very similar. The five transmembrane helices of the M subunit are 24 (C), 25 (A,E), 26 (D) or 27 (B) residues long. The connecting helices “cd” (twelve residues) and “ect” (seven residues) on the periplasmic side as well as “de” (14 residues) on the cytoplasmic side, forming part of the Q_A site, are also present. Accompanied by an insertion of seven amino acids (compared with the L subunit), short additional helices are found in the connections of transmembrane helices A and B (helix “ab”, seven residues) on the periplasmic side, and between transmembrane helix D and the connecting helix “de” on the cytoplasmic side (helix “dde”, six residues).

On the cytoplasmic side, the L and M subunits are tightly interwoven. When the L and M subunits are compared, the M subunits are 26 (Bl. viridis) or 25 (Rb. sphaeroides, Figure 5b) residues longer at the N-termini than the L subunits. At the C terminus, the M subunit from Rb. sphaeroides is nine amino acids shorter than the L subunit. The M subunit from Bl. viridis possesses an additional 18 amino acids at the C-terminus, which interact with the C subunit (see also Figures 1 and 2).

The N-terminus of the H subunit (Figure 6) is located on the periplasmic side of the membrane. Residues H12 to H35 form a membrane-spanning helix (Figure 6a), which is an α-helix at its beginning but a π-helix at its very end. The next 70 residues are preferentially in contact with the LM complex. A globular region follows that has been referred to as the “PRC barrel” [59] and contains an extended system of antiparallel and parallel β-sheets. Close to the C-terminus, an α-helix is found.

The structure of the tetraheme cytochrome or C subunit (Figure 7) has been described in detail [40]. It is not related to other known tetraheme protein structures and consists of five segments, an N-terminal segment (C1–C66), the first heme-binding segment (C67–C142), a connecting segment (C143–C225), a second heme-binding segment (C226–C315), and the C-terminal segment(C316–C336). Apart from an α-helix (C25–C34) in the N-terminal segment, the three non-heme-binding segments contain little regular secondary structure. The four hemes and the two heme-binding segments make up the core of the cytochrome subunit. The first heme-binding segment contains the binding sites for heme-1 (c₅₅₄) and heme-2 (c₅₅₆), the second those for heme-3 (c₅₅₉) and heme-4 (c₅₅₂). Each heme-binding site consists of an α-helix that runs parallel to the heme plane, a loop, and the heme attachment site with the sequence Cys-X-Y-Cys-His.

The primary electron donor (“special pair”) is located at the interface of the L and M subunits near the periplasmic side (Figures 1, 2, and 8). It interacts with residues of the transmembrane helices C, D, E and the connections of helices C and D. The special pair bacteriochlorophylls are held in their position by specific interactions with the protein matrix (Figure 8). The first four ligands to the five-coordinated bacteriochlorophyll magnesium are provided by the bacteriochlorin ring nitrogen atoms, and the fifth ligand is provided by the Nε atom of a His side-chain (Figure 8). For the “special pair” bacteriochlorophylls, these His residues (L173 and M200) are close to the N-terminal ends of the L and M subunit transmembrane helices D, respectively. Apart from binding the Mg²⁺ ion, the protein displays several hydrogen bonding interactions with the bacteriochlorophyll molecules, as deduced from the structures [55,60] (Figure 8). The ring I acetyl group (Figure 3a) of P_L is hydrogen bonded to a His residue (His L168 in both the Bl. viridis and Rb. sphaeroides RC, His L176 in the Tc. tepidum RC) in all three RCs. The symmetry-related amino-acid residue near P_M is Phe M197 in Rb. sphaeroides. Thus, no hydrogen bond can be formed. In Bl. viridis and Tc. tepidum, the respective residues are Tyr M195 and Tyr M196, which donate hydrogen bonds to the acetyl carbonyl oxygen of ring I.

Thr L248 donates a hydrogen bond to the ring V keto carbonyl (Figure 3a) of P_L in Bl. viridis, which, in combination with the presence of the bulky Met L127 on the opposite side of the ring, results in ring V being bent towards Thr L248 (Figure 8a). This ring is oriented in the opposite direction in Tc. tepidum and Rb. sphaeroides, where Ala residues are found at the position of the Bl. viridis Met L127, and the Bl. viridis residues Gly L247 and Thr L248 correspond to Cys L247 and Met L248 in Rb. sphaeroides, and to Cys L255 and Ile L256 in Tc. tepidum.

The accessory bacteriochlorophylls B_A and B_B are located between P and the respective bacteriopheophytins H_A and H_B and are in van der Waals contact with both respective neighboring cofactors. The His ligands for the accessory bacteriochlorophylls, L153 (L161 in Tc. tepidum) and M180 (M181 in Tc. tepidum and M182 in Rb. sphaeroides), are situated close to the N-terminal end of the L and M subunit periplasmic helices “cd”, respectively. The average His Nε–Mg distance is 2.1 Å, as is the average distance between the bacteriochlorin N atoms and the respective Mg²⁺. Significant conformational differences between the reaction centers are found only at the ethyl groups – caused by the structural difference between the ethyl group of bacteriochlorophyll a and the ethylidene group of bacteriochlorophyll b (Figure 3a). In all three RCs, the ring V carbonyl oxygen atoms are hydrogen-bonded via a water molecule (“water A”) to His M200 (M201 in Tc. tepidum, M202 in Rb. sphaeroides) and (via “water B”) to His L173, respectively (Figure 8).

Figure 9 shows the location of the bacteriopheophytin H_A between B_A and Q_A for all three reaction centers. At the top, Tyr M208 (M209, M210) appears to be of importance since it is in van der Waals contact with P_M, P_L, and B_A. The symmetry-related residue in the L subunit is Phe L181 (L189, L181). The pattern of hydrogen bonding formed by H_A and H_B with the protein matrix is identical in both species. Trp L100 (L108, L100) and Trp M127 (M128, M129), respectively, donate a hydrogen bond to the ester carbonyls of ring V of H_A and H_B. The carboxyl group of Glu L104 (L112, L104) is calculated [63,77,78] to be protonated and donates a hydrogen bond to the H_A ring V keto group (Figure 9) [60]. This is responsible for the 10 nm redshift of the H_A Q_x band compared with the H_B Q_x band [61], but is not a dominant contributor to the directionality of electron transfer in RCs. The bacteriopheophytin H_A is surrounded by a significant number of Phe residues (Figure 9). Around H_B, these bulky residues are replaced to a large extent by smaller amino acid residues. As seen in Figures 9 and 10, Trp M250 (M251, M252), with its large aromatic side-chain, bridges the gap between H_A and Q_A in all three reaction centers.

Figure 10 shows the binding site of the primary electron acceptor quinone Q_A for all three reaction centers. Q_A is located on the A side where the L subunit dominates (Figure 2), but the quinone ring interacts exclusively with residues of the M subunit. The Q_A-binding site is clearly more hydrophobic than the Q_B-binding site. A major part of the Q_A-binding site is formed by Trp M250 (M251, M252). The ring systems of the tryptophan and Q_A are parallel. Trp M250 (M251, M252) donates a hydrogen bond to Thr M220 (M221, M222). The structural difference between menaquinone as Q_A in Bl. viridis and Tc. tepidum and ubiquinone as Q_A in Rb. sphaeroides causes only minor rearrangements of the Q_A-binding site, which are not shown in Figure 10 for clarity.

The six-coordinate non-heme ferrous iron (Figure 10) is in a distorted octahedron environment, the base plane of which is formed by the three Nε atoms of His L190 (L198, L190), His L230 (L238, L230), and His M217 (M218, M219), and by one carboxyl Oε of Glu M232 (M233, M234). The apices of the octahedron are formed by the Nε atom of His M264 (M265, M266) and the second carboxyl Oε atom of Glu M232 (M233, M234). Such a distorted octahedral coordination had been predicted from Mössbauer and EXAFS results, as reviewed by Feher and Okamura [62]. The average ligand–Fe distances are 2.2 ± 0.2 Å. The four His ligands are located four to eight residues away from the cytoplasmic ends of transmembrane helices D and E of the L and M subunits. The Glu ligand is situated at the N-terminal end of the cytoplasmic helix “dde”, which is only present in the M, but not in the L subunit (see above). The flanking residues Asp M230 (M231, Glu M232), Arg M231 (M232, M233, see also Figure 13 below), and Glu M234 (Asp M235, Glu M236), are important constituents of the “Q_B cluster”, a group of electrostatically strongly interacting, protonatable residues calculated [63] to be important for proton uptake and transfer to the Q_B site coupled to quinone reduction. The His ligands M217 (M218, M219) and L190 (L198, L190) also provide, with their Nδ atoms, the proximal hydrogen bonding partners to the quinones Q_A and Q_B, respectively. The non-heme Fe²⁺ ion can be removed and replaced with Fe²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ in the RC of Rb. sphaeroides [64] and with Zn²⁺ in the RC of Bl. viridis [65]. Apparently, neither Fe²⁺ nor any divalent cation is required for rapid electron transfer from Q_A⁻ to Q_B [64]. However, the presence of a metal ion in the Fe site appears to be necessary to establish the characteristic electron transfer properties of Q_A [64].

The first four ligands to the six-coordinated heme iron are provided by the porphyrin ring nitrogen atoms. The Cys residues of the heme attachment site sequences Cys-X-Y-Cys-His (C87-C91; C132-C136; C244-C248; C305-C309 in Bl. viridis, C85-C89, C130-C134, C225-C229, C285-C289 in Tc. tepidum) form thioether bonds with the heme groups and the His is the fifth ligand to the heme iron. The Met residues C74, C110, and C233 in Bl. viridis (C72, C108, C214 in Tc. tepidum) in the respective parallel helices are the sixth ligands to heme-1, heme-2, and heme-3 (Figure 11a), whereas the sixth ligand to heme-4 is His C124 in Bl. viridis (C122 in Tc. tepidum), which is located in the loop region of the heme-2 binding site (Figure 11b). The average His Nε–Fe distance is 2.0 Å; the average distance between the porphyrin N atoms and the respective Fe²⁺ is 2.05 Å. The average Met Sδ–Fe distance is 2.3 Å.

In the original Bl. viridis RC structure, the Q_B site was poorly defined because it was only partially occupied with the native ubiquinone-9 in the standard RC crystals. However, ubiquinone-2-reconstitution experiments have yielded crystals with full quinone occupancy of the Q_B site [66]. Subsequent X-ray diffraction analysis and refinement has led to a well-defined Q_B-site model (PDB entry 2PRC), with the quinone bound in the “proximal” position, i.e., close to the non-heme iron (hydrogen-bonded to its ligand His L190, see Figure 12a). In the RC structure with a Q_B-depleted Q_B site (3PRC, Figure 12b), refined at 2.4 Å, apparently five, possibly six, water molecules are bound instead of the ubiquinone head group, and a detergent molecule binds in the region of the isoprenoid tail [66]. Using the structures 2PRC and 3PRC as references, the original data set 1PRC [40] was re-examined. While not excluding the presence of a minor fraction of the quinone in the proximal site, this resulted in the suggestion [66] of a “distal” dominant Q_B-binding position for the native ubiquinone-9 (1PRC_new), not hydrogen-bonded to His L190 and further away from the non-heme iron (Figure 12a). A more quantitative analysis [67] of the original data resulted in 20% of the Q_B sites being occupied with quinone in the proximal site, 30% having quinone bound in the distal site, and half of the Q_B sites being empty or having the quinone unaccounted for. A further structure, the RC complex with the inhibitor stigmatellin (4PRC), refined at 2.4 Å, indicates that additional hydrogen bonds stabilize the binding of stigmatellin over that of ubiquinone-2 (Figure 12c). The binding pattern observed for the stigmatellin complex can be viewed as a model for the stabilization of a monoprotonated reduced intermediate (Q_BH or Q_BH⁻) [48,66,68]. This indicates that the Q_B site is not optimized for Q_B binding, but for Q_B reduction to the quinol [68]. In combination with the results of electrostatic calculations, these crystal structures can provide models for intermediates in the reaction cycle of ubiquinone reduction to ubiquinol, as discussed below.

The Q_B site is also a well-established site of herbicide action. Over half [69] of the commercially available herbicides function by inhibition of higher plants at the Q_B site of the D1 polypeptide of the Photosystem II RC (see Section 11.9 for a comparison of the Q_B sites from the bacterial RC and Photosystem II). A commercially very important class of herbicides are the triazines, which were introduced by J.R. Geigy S.A. in the 1950s [70]. A prominent example is atrazine (Figure 3d), first reported in 1957 [71]. According to statistics from 1995, atrazine was used on approximately 67% of all U.S. corn acreage, 65% of sorghum acreage, and 90% of sugarcane acreage. Another well known triazine is terbutryn (2-t-butylamino-4-ethylamino-6-methylthio-s-triazine). X-Ray crystal structures of complexes of the RC with atrazine (PDB entry 5PRC) [58] and terbutryn (PDB entry 1DXR [41]) have been determined at 2.35 and 2.00 Å resolution, respectively (see Table 2). In both cases, three hydrogen bonds bind the distal side of the inhibitors to the protein, and four additional hydrogen bonds, mediated by two tightly-bound water molecules, are apparent on the proximal side, as shown for atrazine in Figure 12(d). In contrast to the proximal binding of stigmatellin (Figure 12c), the triazine inhibitor partially overlaps with both the distal and the proximal ubiquinone binding sites (Figure 12d).

Both Q_A and Q_B sites are buried deep within the photosynthetic reaction center complex, approximately 15 Å from the cytoplasmic surface. Proton transfer to the reduced quinone within the Q_B site could occur by protons moving along a chain of proton donors and acceptors by a “proton wire”, or hydrogen-bonded chain mechanism [72–74]. Possible proton donors and acceptors are protonatable amino acid residues and water molecules (see also Figure 13 below). Several the protonatable residues between the Q_B site and the cytoplasmic surface have been shown to be functionally relevant to the proton transfer process by analysis of site-directed mutations, reviewed in [18,19], and second site revertants [15,75]. The observed effects can be due to the modification of the kinetics and thermodynamics of electron or proton transfer. Electrostatic calculations on the RCs of Rb. sphaeroides [76–78] and Bl. viridis [63,79] led to the identification of residues that can contribute to the changes in equilibrium distributions of protons in the different redox states of the protein, thus helping to determine the role of the functionally important residues.

In combination with the results of electrostatic calculations [63] the crystal structures 3PRC, 1PRC_new, 2PRC, and 4PRC discussed above (cf. Figure 12) can provide models for intermediates in the reaction cycle of ubiquinone reduction to ubiquinol (Figure 12e) [66,68]. The binding of the incoming Q_B to the distal site displaces some of the water molecules present in the “empty” pocket. The quinone ring is flipped around the isoprenoid tail and further water molecules are displaced for the Q_B to occupy the proximal position. This is the position in which neutral Q_B accepts an electron from Q_A⁻. The hydrogen bonds donated to the quinone will automatically lead to a tighter binding of the negatively charged semiquinone Q_B⁻ compared with the neutral Q_B. Additionally, the side-chain of Ser L223 can reorient by rotation of its χ₂ (Cα-Cβ-Oγ-Hγ) torsional angle, thus establishing an additional hydrogen bond to Q_B⁻ [7,66]. Coupled to the transfer of the second electron, the first proton is transferred, possibly via a transiently protonated Ser L223-OH₂⁺ [63], thus forming the monoprotonated, doubly reduced intermediate Q_BH⁻. After transfer of the second proton, movement of the quinol from the proximal to the distal position may be facilitated by increased stacking interactions of the aromatic ring systems with the Phe L216 ring and the diffusion of water molecules back into the pocket. The structures of these intermediates provide explanations for their relative binding affinities, as required for proper enzymatic function of the Q_B site. A rearrangement of hydrogen bonds, most prominently the reorientation of the Ser L223 side-chain for Q_B reduction, as suggested by the scenario in Figure 12, is also calculated to be necessary to make Q_B reduction more favorable than Q_A reduction [78]. These local rearrangements may constitute the conformational changes deduced to be required for function by various experiments [80–83]. A similar mechanism has been proposed for the Rb. sphaeroides RC [84].

The addition of Cd²⁺ or Zn²⁺ decreases the apparent transfer rates of both the first electron from Q_A⁻ to Q_B (k⁽¹⁾_AB Figure 1c) and the second electron from Q_A⁻ to Q_B⁻ (k⁽²⁾_AB, Figure 1c) 10-fold and 20-fold, respectively [83,85–88]. Three mechanisms of inhibition by Cd²⁺ or Zn²⁺ binding have been proposed. These are not mutually exclusive, but highlight the respective dominant effect. First, based on the decrease of k⁽¹⁾_AB upon Zn²⁺ binding, Utschig et al. [83] proposed a gating inhibition mechanism, because the rate-limiting step for k⁽¹⁾_AB was considered to be a conformational gating step, governed by RC dynamics [89]. However, subsequent studies on a Glu L212 → Asn Rb. sphaeroides variant RC [87] indicated that the inhibiting step in Cd²⁺ is proton uptake, leading to the second proposal, namely, His-entry inhibition as the key inhibition mechanism [90,91]. This mechanism is consistent with the location of the Cd²⁺ or Zn²⁺ binding site in the crystal structure (PDB entry 1DV6, Table 2) of the respective complexes of the Rb. sphaeroides RC (cf. Figure 13) [92]. However, the only four-fold decrease in k⁽²⁾_AB in the His H126 → Ala/His H128 → Ala is much less pronounced than the 20-fold decrease observed upon Cd²⁺ binding. Therefore, Gerencsér and Maróti [88] have proposed that proton transfer inhibition by metal binding is caused by induced pK_a shifts of residues along proton transfer pathways.

The electrons used to re-reduce the photo-oxidized special pair P⁺ are provided by soluble electron carrier proteins, such as cytochrome c₂ in the case of Bl. viridis and Rb. sphaeroides, and a high-potential iron-sulfur protein (HiPiP) in the case of Tc. tepidum.

All four hemes of the Bl. viridis RC tetraheme C subunit are located close enough to the surface of the protein to accept electrons from soluble cytochrome c₂. Site-directed mutagenesis in another non-sulfur purple bacterium, Rubrivivax gelatinosus, has led to the identification of a patch of acidic residues immediately surrounding the distal low-potential heme-1 of the tetraheme C subunit that apparently forms an electrostatically favorable binding site for soluble cytochromes. Thus, all four hemes in the C subunit appear to be directly involved in the electron transfer towards the photo-oxidized special pair. Based on these findings, a model was proposed for the transient cytochrome c₂-RC complex for Bl. viridis (Figure 14a). Also, in the case of the Tc. tepidum RC, the surface around heme-1 was found to be the best candidate for a binding site of HiPiP. However, with HiPiP, electrostatic interactions appear to have a negligible influence [93,94] on the binding to the tetraheme cytochrome c subunit. Instead hydrophobic interactions are apparently responsible for docking HiPiP and the C subunit of the RC in Tc. tepidum.

With the Rb. sphaeroides RC, which lacks the C subunit, the photo-oxidized special pair P⁺ is directly re-reduced by cytochrome c₂. The structure of the co-complex of the Rb. sphaeroides RC and cytochrome c₂ has been determined by X-ray crystallography at 2.4 Å resolution (cf. Figure 14b). In these crystals, P⁺ is reduced by cytochrome c₂ at the same rate as measured in solution, indicating that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. The binding interface can be divided into two domains. The first domain contributes to the strength and specificity of cytochrome c₂ binding and is a short-range interaction domain that includes Tyr L162 (cf. Figure 14b), and groups exhibiting non-polar interactions, hydrogen bonding, and a cation–π interaction. The second is a long-range, electrostatic interaction domain that contains complementary charges on the RC and cytochrome c₂. In addition to contributing to the binding, this domain may help steer the unbound proteins into the right orientation.

The distributions within the L and M subunits of the strongly basic amino acid residues, Arg and Lys, and of the strongly acidic residues, Glu and Asp, is shown in Figure 15(a, c, e), for the RCs from Bl. viridis, Tc. tepidum, and Rb. sphaeroides, respectively. Based on electrostatic calculations, only very few of these residues are considered not to be fully ionized, most notably Glu L104 (cf. Figure 9), which is fully protonated, and Glu L212 (cf. Figures 12 and 13), which is predominantly protonated. The residues shown in Figure 15 exhibit an asymmetrical distribution with respect to the cytoplasmic and periplasmic sides. Whereas positively charged residues Arg and Lys predominate on the cytoplasmic, negatively charged residues, Asp and Glu, predominate on the periplasmic side. This observation, as discussed for the Bl. viridis RC [29] is conserved not only in the case of the other RCs (Figure 15), but has since led to the formulation of the “positive-inside” rule [95], which was found to be valid not only for bacterial inner membrane proteins, but also for eukaryotic proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane and across a wide range of organisms [96].

The L and M subunits display a remarkably uneven distribution of Trp side-chains, compared with those of Tyr and Phe side-chains (Figure 15b, d, f). About two-thirds of the Trp residues are found in the hydrophobic surface-to-polar transition zone on the periplasmic side of the complex [39,97]. On the surface of the molecules, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. It has been suggested that Trp residues are involved in the translocation of protein through the membrane and that, following translocation, Trp residues serve as anchors on the periplasmic side of the membrane [97,98].

The original Bl. viridis RC crystal structure (1PRC) contained 201 tentative, ordered water molecules [37]. In more recent structures (cf. Figure 16), between 114 [66] and 384 [41] additional water molecules were assigned, based on electron density maps and local chemistry. Calculation of the water quality factor (QualWat), as introduced by Arnold and Rossmann [99], indicated that the average crystallographic quality of the additionally fitted water molecules was similar to that of the original ones [66]. Generally in proteins, internal water not only fills structural cavities, but it is also necessary to stabilize three-dimensional folding [100,101]. In particular, several water molecules have been described that mediate hydrogen bonding between transmembrane helices in the hydrophobic region of the membrane. Originally, two water molecules were described [39] that mediate hydrogen bonding between transmembrane helices C and E of the L subunit and between transmembrane helix D of the L subunit and transmembrane helix D of the M subunit. The binding of a third water molecule in the transmembrane region was described subsequently [66], mediating hydrogen bonding between transmembrane helices C and E of the M subunit. Recently, a water molecule, referred to as “water A” and located within hydrogen bonding distance of the keto carbonyl of the accessory bacteriochlorophyll B_A (cf. Figure 8), has been shown [102] to have strong effects on the rate of primary electron transfer [103] from the excited primary donor P* (Section 11.8.1). In addition, the participation of water molecules is apparently essential [66,104,105,] for the pathways and kinetics of proton transfer to the Q_B site.

The advent of more refined purification strategies, reviewed earlier [7], in particular involving significantly lower concentrations of detergent used to solubilize the photosynthetic membranes of Rb. sphaeroides and Tc. tepidum, has led to the identification of motionally-restricted phospholipid molecules on the intramembrane surface (Figure 17). Using the standard purification protocol for the Bl. viridis RC with high detergent concentrations [7], no phospholipid is retained (H. Michel, personal communication). However, the binding of two molecules of the detergent LDAO and of a sulfate ion in the Bl. viridis RC (Figure 17a) is clearly analogous to the binding of dipalmitoylphosphatidylethanolamine (DPPE) described for the Tc. tepidum RC (Figure 17b). In turn, the binding site of a β-octyl glucoside molecule in the Tc. tepidum RC (Figure 17b) is occupied by a cardiolipin molecule in the case of the Rb. sphaeroides RC (Figure 17c). The role of Arg M267 (Figure 17c) has been investigated (PDB entry 1UMX, Table 3, Section 11.8.1).

Work on site-directed mutagenesis of photosynthetic reaction centers started with the RC from Rb. capsulatus (see [15,16] for early reviews). This species is genetically very well characterized and able to grow non-photosynthetically under aerobic conditions, as well as under anaerobic conditions using, for example, dimethyl sulfoxide as an electron acceptor. Most importantly, under these latter conditions, the photosynthetic apparatus is fully induced. Unfortunately, the RC from Rb. capsulatus could not be crystallized, thus thwarting proper inspection for structural changes.

The closely related Rb. sphaeroides can be grown under similar non-photosynthetic conditions, so that site-directed mutagenesis is also straightforward. As detailed above, this RC is amenable to inspection by X-ray crystallography for structural changes. Many amino acids that were considered to be of importance for pigment binding, electron transfer [60], in particular the directionality of electron transfer along the A branch, proton transfer to Q_B, phospholipid binding or crystal packing were changed in Rb. sphaeroides RCs, in some cases after the corresponding replacements in the Rb. capsulatus RC had been shown to be important.

When the residues His L173 and His M202 (corresponding to M200 in Bl. viridis, Figure 8) liganded to the special pair bacteriochlorophylls P_L and P_M are replaced by Leu residues, bacteriopheophytins are incorporated as P_L and P_M, respectively. In addition to being analyzed by X-ray crystallography of the orthorhombic crystal form (“B”) at 3 Å resolution (PDB entry 1PST, Table 3), the structure of the “heterodimer” Rb. sphaeroides RC variant His M202 → Leu has subsequently been determined in the trigonal crystal form (“C”) at higher resolution [106] (PDB entry 1KBY). This has allowed a more detailed description of the structural changes associated with this amino acid replacement, which include the loss of water “A”, which, in the wild-type RC, is hydrogen-bonded both to His M202 and the bacteriochlorophyll monomer B_A (Figure 8). Based on the structural (PDB entry 2BOZ) and functional characterization of the Rb. sphaeroides RC variant Gly M203 → Leu, this water molecule “A” has recently been shown to have a strong effect on the rate of light-driven charge separation, in that its removal leads to an approximately eight-fold slowing of the rate of decay of P* [102].

Also remarkable is the replacement of Tyr M210 (Figure 9) with a Phe, taking into account that the symmetry-related residue in the L subunit is Phe L181. In the Tyr M210 → Phe variant RC the rate of initial electron transfer is slowed by a factor of 4–6 [107,108]. X-Ray crystallographic analysis [109] using the orthorhombic crystal form (“B”) did not reveal any significant structural changes except for the absence of the O atom, which appears to be the reason for the decreased observed rate of electron transfer.

Several the Rb. sphaeroides variant RC structures listed in Table 3 involve the replacement of Phe M197, close to P_M (cf. Figure 8c), with Arg. These were combined with the replacement of residues in the carotenoid-binding pocket (Tyr M177, Trp M115) with Phe (PDB entries 1MPS and 1E6D, respectively) [110,111]. The structure of the Phe M197 → Arg/Tyr M177 → Phe variant RC shows an unexpected change in the structure, with a reorientation of the new arginine, the incorporation of a new water molecule into the structure, and the rotation of the P_M ring I acetyl group. The replacement of Phe M197 with Arg was also combined with the replacement of the nearby residue Gly M203 with Asp (PDB entry 1E14). This replacement and the analogous replacement (Gly M201 → Asp) in Rb. capsulatus have been shown to inhibit electron transfer along the A branch. The residue Phe M197 in Rb. sphaeroides corresponds to a Tyr in Bl. viridis and Tc. tepidum (Figure 8). The structure of a Phe M197 → Tyr mutant has also been described [112]. In combination with electrochemically induced FTIR spectra, there is clear evidence for the existence of a newly established hydrogen bond between Tyr M197 and the P_M ring I acetyl group. The residue corresponding to Phe M197 in the L subunit is His L168. Its replacement will be discussed below in the context of Bl. viridis variant RCs.

In the Leu M214 → His variant, corresponding to residue M212 in Bl. viridis (Figure 9) and Rb. capsulatus [113], a bacteriochlorophyll, termed β, is incorporated as H_A instead of a bacteriopheophytin [109]. A very interesting Q_A cofactor exclusion variant RC, involving the replacement of Ala M260 (Figure 10) with a Trp residue, has been structurally described at 2.1 Å resolution (PDB entry 1QOV) [114]. In the Ala M260 → Trp variant, there is no space left in the former Q_A binding pocket for the binding of the native ubiquinone. Instead, in addition to the Trp side-chain, a Cl⁻ ion is found at this position. A recent quintuple variant, combining previously discussed amino acid replacements to inhibit electron transfer via the A-branch (Gly M203 → Asp, Tyr M210 → Phe, Leu M 214 → His), to promote electron transfer via the B-branch (Phe L181 → Tyr), and to exclude the Q_A cofactor (Ala M260 → Trp), has been shown to reduce Q_B via the B-branch and its structure, based on crystal form “C”, has been described at 2.25 Å resolution (PDB entry 1YF6) [115]. A further multiple variant of the Rb. sphaeroides RC containing three replacements (Leu L131 → His/Leu M160 → His/Phe M197 → His) designed to increase the P/P⁺ midpoint potential [116], one replacement (Arg M164 → Tyr) for the incorporation of a Tyr near P, and two replacements (Gly M288 → Asp/Met M168 → Glu), designed to introduce a Mn²⁺ binding-site, has been shown to possess a redox-active Mn²⁺-site, mimicking that of Photosystem II. X-Ray structure analysis of this variant based on crystal form “F” (Table 1) has, however, been limited to 4.5 Å resolution (Table 3). In addition to mutagenesis, cofactors may be removed or replaced chemically with a wide range of similar compounds – reviewed by Gunner [117] in the case of quinones and by Scheer and Struck [118] for bacteriochlorins.

Residues that are either part of or in close proximity to the “Q_B cluster” of interacting residues considered to be relevant for proton transfer to the Q_B site (cf. Figures 12 and 13) have also been replaced by site-directed mutagenesis. Using the orthorhombic crystal form “B”, mutations such as those involving protonatable residues Glu L212 → Gln and Asp L213 → Asn, and a ligand to the non-heme iron His M219 → Cys (corresponding to residue M217 in Bl. viridis, Figure 10), do not lead to detectable structural changes [109]. However, the resolution of the respective data sets was limited to 3.3, 3.0, and 4 Å. Using the trigonal crystal form “C”, the structure of the Asp L213 → Asn variant was more recently shown at 2.1 Å resolution (PDB entry 1RY5, Table 3) [49] to be very similar to that of the wild-type RC, with the exception of the nearby residue Glu H173, which was found in two alternate conformations. Also using the trigonal crystal form, the reaction center double variant Glu L212 → Ala/Asp L213 → Ala has been described at 3.1 Å resolution, revealing unexpected changes in main chain positions (PDB entry 1K6N) [28]. RC variants involving replacement of the nearby residue Pro L209 (Figure 13), with Tyr, Phe or Glu (PDB entries 1F6N, 1FNP, and 1FNQ, respectively) have been determined at 2.6–2.8 Å resolution [119].

In addition, interesting second-site replacements that restore photosynthetic competence have been obtained both for Rb. capsulatus [75,120–122] and Rb. sphaeroides [123,124]. The structures of the Asp L213 → Asn/Arg M233 → Cys and Asp L213 → Asn/Arg H177 → His Rb. sphaeroides RC variants have been determined on the basis of crystal form “C” at 1.8 and 2.75 Å, respectively (PDB entries 1RZH and 1RVJ) [49]. From the structures, alternate proton transfer pathways could be delineated, the main changes occurring near Glu H173 (Figure 13).

The residue Arg M267, found to be involved in cardiolipin binding (Figure 17), has been replaced with a Leu residue and the structure of the corresponding variant RC has been determined at 2.8 Å resolution (PDB entry 1UMX, cf. Table 3) [125]. Instead of cardiolipin, only a phosphate ion was bound by the side-chains of Tyr H30, Trp M271, and His M145. Other than the M267 side-chain, the structure of the protein cofactor system remained unaltered. The mutation did not affect the rate of photosynthetic growth or the functional properties of the RC. However, the thermal stability of the RC was compromised by this amino acid replacement.

The role of crystal contact interactions has been assessed by replacement of amino-acid residues on the surface of the Rb. sphaeroides RC and subsequent crystallization and X-ray structure analysis (PDB entries 1JGW, 1JGX, 1JGY, 1JGZ, 1JH0, cf. Table 3). Depending on the crystal form, significant differences in the resolution limits were associated with the loss of specific interactions between neighboring proteins [126].

Site-directed mutagenesis of the RC from Bl. viridis is possible [127] but more difficult. Bl. viridis can grow only under photosynthetic and, very slowly, under microaerophilic conditions. However, under microaerophilic conditions, the photosynthetic apparatus is not induced and photosynthetic growth conditions exert a selection pressure for revertants and suppressor mutants if the RCs are functionally impaired. In contrast, very interesting herbicide-resistant mutants were obtained by classical selection procedures, with mutations, some of which would not have been made by site-directed mutagenesis [20,128].

Some of these herbicide resistant mutants of the Bl. viridis RC have also been analyzed by X-ray crystallography. In the double mutant Arg L217 → His/Ser L223 → Ala, the side-chain of Asn L213, which is hydrogen-bonded to Ser L223 in the wild type, is rotated towards the cavity that is created by the replacement of Arg L217 by the smaller His [129]. At the same time, Q_B becomes more firmly bound [20]. The mutation Tyr L222 → Phe unexpectedly leads to resistance against the herbicide terbutryn. In the wild type, Tyr L222 forms a hydrogen bond with the peptide carbonyl oxygen of Asp M43. Since this hydrogen bond is now missing, a stretch of the M subunit (M25–50) moves into a new position. The side-chain of Phe L222 rotates by 90° into the herbicide binding site (see above), thereby preventing the binding of terbutryn by steric hindrance [130].

Using site-directed mutagenesis, the highly conserved Tyr L162, positioned halfway between P and the proximal heme-3 (cytochrome c₅₅₉) in the Bl. viridis RC, was exchanged against several amino acids. All mutants grew photosynthetically. The redox potentials of P and c₅₅₉ were changed by the mutations. The structures of two mutants (Tyr L162 → Phe and Tyr L162 → Thr) were determined and found not to differ significantly from the wild-type structure [21]. Analysis of the kinetics of electron transfer led to the conclusion that the tyrosine residue at position L162 is not required for fast electron transfer from c₅₅₉ to P⁺ [21].

Replacement of His L168, which donates a hydrogen bond to the ring I acetyl group of P_L (Figure 8), with Phe leads to a more than three-fold acceleration of the rate of initial electron transfer from the excited primary donor P*. This is associated with the lowering of the oxidation–reduction midpoint potential of P/P⁺ by 80 mV [131]. The structure of the His L168 → Phe variant RC, refined at 2.0 Å resolution (PDB entry 1DXR, cf. Table 2), provides an explanation for these properties. Compared with the wild-type RC, the hydrogen bond to the ring I acetyl group of P_L is removed, the acetyl group is rotated and its acetyl oxygen is found 1.1 Å closer to the bacteriochlorophyll-Mg²⁺ of P_M. Similar findings were subsequently reported for the same amino acid replacement in the Rb. sphaeroides RC [132].

A replacement of Arg C264 by Lys decreases the midpoint potential of heme-3 (cytochrome c₅₅₉) from +380 to +270 mV, i.e., below that of heme-2 (+320 mV, see Figures 11a and 1b) [23]. In the structure of the variant RC at 2.46 Å resolution, no remarkable differences were found apart from the replaced residue itself [23]. The halftime of electron transfer between heme-2 and heme-3 was the same as in the wild-type, indicating that the observed reaction rate is limited by the very uphill electron transfer from heme-2 to heme-4 (Figure 1b) [23].

Previous attempts to determine the structural changes of the RC protein upon illumination of dark-adapted, detergent-grown RC crystals were inconclusive [50,133–135]. Recently, Katona and colleagues [136] grew Rb. sphaeroides RC crystals from a lipidic cubic phase and could describe structural changes at 100 K in the form of a subdomain movement of the H subunit, involving large parts of the PRC barrel, by up to 0.7 Å (Figure 18). The full conformational change is not expected at the low temperature of this experiment. Crystals illuminated at room temperature do not diffract. The observation of this structural change was apparently obscured previously in the case of the other crystal forms due to crystal packing constraints.

Based on the determined structure of the purple bacterial RC, very specific sequence homologies, and azidoatrazine labeling, the RC core of higher plant photosystem (PS) II was proposed to be similar to the LM core of the bacterial RC, with the D1 and D2 proteins corresponding to the L and M subunits, respectively [137–141]. This proposal could be verified experimentally [142]. Similar to the processes discussed in Section 11.6.1 for the bacterial RC, indirect lines of evidence indicate that the reoxidation of Q_A⁻ by Q_B (Q_B⁻) is a conformationally triggered reaction in PS II ([143,144]; for details, see Chapter 16) Suitably designed, modified bacterial RCs mimic tyrosine oxidation [145] and mimic redox-active Mn²⁺ in PS II [146]. Atomic models of the structure of Photosystem II, as determined by X-ray crystallography at 3.5, 3.2, and 3.0 Å resolution, have been presented [147–149]. Each of the D1 and D2 subunits consists of five transmembrane helices organized in a manner almost identical to that of the L and M subunits of the bacterial RC, with a root-mean-square deviation of 1.9 Å for 395 Cα atoms [147]. The Q_B site appears slightly wider in PSII due to the insertion of a residue in the loop connecting the de helix and transmembrane helix E and containing Ser D1–264 (corresponding to Ser L223 of the RC, cf. Figure 19), which may explain the difference in herbicide specificity [150]. Nevertheless, in the case of the inhibitors of both the RC and Photosystem II, such as stigmatellin, atrazine, and terbutryn, the crystal structures of the complexes of these inhibitors with the Bl. viridis RC [41,58,66,130] can serve as useful models of PS II inhibition.

Over 20 years after the publication of the first crystal structure of the Bl. viridis RC, reaction centre crystallography has come of age, providing, in particular, structures of variants and other modified RCs at, generally, higher resolution and quality. These structures, and those of further modified RCs, still to be determined, are prerequisites for an atomic-level understanding of the function and mechanism of action of these fascinating membrane protein complexes. In addition, the improved perspectives for time-resolved X-ray crystallography (see [151] for a recent review) are expected to provide further insight into the dynamical aspects of RC structures.