Structural Basis of the Drastically Increased Initial Electron Transfer Rate in the Reaction Center from a Rhodopseudomonas viridis Mutant Described at 2.00-Å Resolution*

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-Å resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the “special pair” bacteriochlorophyll, DL, is rotated, and its acetyl oxygen is found 1.1 Å closer to the bacteriochlorophyll-Mg2+ of the other special pair bacteriochlorophyll, DM. The rotation of this acetyl group and the increased interaction between the DL ring I acetyl oxygen and the DM-Mg2+ provide the structural basis for the previously observed 80-mV decrease in the D+/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, BA. The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, QB.

Life on earth depends on the ability of photosynthetic organisms to convert solar energy into biochemically amenable energy. A central role in the photosynthetic process is played by the photosynthetic reaction center (RC), 1 an integral mem-brane protein-pigment complex. The RCs from purple bacteria, which catalyze the light-induced reduction of ubiquinone to ubihydroquinone (or ubiquinol) involving the uptake of two protons from the cytoplasm and the oxidation of cytochrome c 2 in the periplasm, are the best characterized membrane protein complexes (see Refs. 1-4 for reviews). The RC of the non-sulfur purple bacterium Rhodopseudomonas (Rp., more recently suggested to be reclassified as Blastochloris (5)) viridis is composed of four polypeptides, namely the L, M, H, and C (a tightly bound tetra-heme cytochrome c) subunits (6) and fourteen cofactors (four heme molecules, four bacteriochlorophyll b, two bacteriopheophytin b, one carotenoid, one non-heme iron, and two quinones, as described previously (7). The four heme molecules are covalently bound by the C subunit, and all other cofactors are non-covalently bound by the L and M subunits. The complex has eleven membrane-spanning helices, five in the L, five in the M, and one in the H subunit. Large parts of the L and M subunits and their associated cofactors are related by a 2-fold rotational symmetry axis perpendicular to the plane of the membrane (7)(8)(9)(10).
Light is absorbed by the bacteriochlorophyll of the B1015 light-harvesting antennae. Excitation energy is then transferred to a dimer of bacteriochlorophyll, the special pair D, thus forming the excited state D*. This decays to D ϩ through electron transfer via the monomeric accessory bacteriochlorophyll B A and the bacteriopheophytin A (11,12) to the primary quinone Q A , which is a menaquinone-9 in the Rp. viridis RC. The electron is then transferred to a secondary quinone, Q B , which is ubiquinone-9 in the Rp. viridis RC. Whereas Q A can accept only one electron, Q B functions as a "two-electron gate" (13), and after a second reduction and the uptake of two protons from the cytoplasm, the ubiquinol leaves its binding site (14,15) to be reoxidized by the cytochrome bc 1 complex (16), which results in the release of protons on the periplasmic side of the membrane. This proton transport produces a transmembrane electrochemical potential that drives ATP synthesis through the ATP synthase (17). The electrons that are released upon quinol re-oxidation are cycled back to the reaction center via a small soluble protein, cytochrome c 2 (18), and ultimately rereduce D ϩ via the tetra-heme C subunit.
For the investigation of the mechanism of the primary electron transfer reactions, RC mutants where the energetics of D* and D ϩ are changed by the removal or addition of hydrogen bonds between the special pair and amino acid side chains have been extensively studied (see Ref. 19 for a review). In both Rhodobacter sphaeroides and Rp. viridis RCs, histidine L168 donates a hydrogen bond to the ring I acetyl group of D L (see Ref. 20 for a review). In a mutant RC from Rp. viridis, where His L168 is replaced by phenylalanine (HL168F), the D/D ϩ redox potential is 80 mV lower than that of the wild-type RC (21). The decay of the excited electronic state D* is accelerated by more than a factor of three. This leads to a remarkable increase in the population of B A Ϫ , thus providing strong support for a stepwise electron transfer mechanism from D* to A with the accessory bacteriochlorophyll B A as a short-lived real electron carrier (21,22).
Here we report the structural characterization of the His L168 3 Phe Rp. viridis RC by crystallographic refinement to a resolution of 2.00 Å. The data collected to resolve the nature of the changes introduced by this mutation turned out to be of sufficient quality to also reliably describe the mode of binding of the triazine inhibitor terbutryn (2-t-butylamino-4-ethylamino-6-methylthio-s-triazine) to the Q B site. Although the terbutryn binding site had been localized by x-ray crystallographic analysis of the RC-terbutryn complex, based on 38,663 unique reflections up to a resolution of 2.9 Å (R-factor ϭ 23.8% (23)), description of the exact nature of protein-terbutryn interactions has had to await the refinement of this higher resolution structure. In addition, possibly biologically relevant modifications of the atomic model also include the crystallographic assignment of a large number of additional tightly bound water molecules, which are of particular relevance to the discussion of proton transfer pathways to the Q B site.

EXPERIMENTAL PROCEDURES
Protein Preparation and Crystallization-The Rp. viridis HL168F mutant was grown, and chromatophores were isolated as described (21). Reaction centers were purified as reported in Refs. 24 and 25, with modifications reported in Ref. 26, and a Q B removal step, which is described in Ref. 27. Crystallization of the RCs was performed as reported (24), except that the pH value was 7.0, and the RCs were crystallized in the presence of 50 M terbutryn (Riedel-de-Haën, Seelze, Germany). Crystals were carefully removed from the depression wells and rinsed with soak buffer (as reported in Ref. 8, except that the pH value was 7.0, and 50 M terbutryn were present). Crystals belong to the tetragonal space group P4 3 2 1 2 and were isomorphous to the original crystals with unit cell dimensions a ϭ b ϭ 223.5 Å, and c ϭ 113.6 Å (8).
Data Acquisition and Processing-Data acquisition was performed in the dark with a temperature of Ϫ10°C at the crystal. Diffraction data were collected using monochromatic synchrotron radiation at a wavelength of 1.07 Å at the GBF/MPG-BW6 beamline (HASYLAB/DESY, Hamburg, Germany) with a MARCCD detector in frames of 0.2°t hrough a continuous angular range of 45°. Diffraction data from just one crystal were processed and scaled using the HKL programs DENZO and SCALEPACK (28) and TRUNCATE from the CCP4 program suite (29) as summarized in Table I.
Initial Model Building and Refinement-Because the crystal used in this study was isomorphous to those of the original (8) and previous work (27,30), difference Fourier and simulated annealing omit map techniques (31) with refined co-ordinates of the structure of the RC complex with a chiral atrazine derivative, PDB entry code 6PRC (30), as a starting structure could be used for the approximation of initial phases and the construction of an initial model.
Crystallographic refinement was performed as described previously (27,30) using iterative cycles of simulated annealing, conventional positional refinement, isotropic overall B-factor refinement, and restrained refinement of individual B-factors with the program X-PLOR (version 3.1; see Ref. 32). Manual inspection and refitting were done with the molecular graphics program O (33). In particular, the assignments of the oxygen and methyl positions in the acetyl groups of the bacteriochlorophyll and bacteriopheophytin molecules were checked as described previously (27), and the same criteria for the assignment of water molecules as described earlier (27,30) were applied. Individual B-factors were refined but restrained to target standard deviations of 1.5 Å 2 for the difference of B-factor values of bonded main-chain atoms (2.0 Å 2 for side-chain atoms) and 2.0 Å 2 for main-chain atoms related by bond angles (2.5 Å 2 for side-chain atoms). The refinement statistics and the quality of the final model are summarized in Table I. Unless stated otherwise, comparisons to the wild-type structure are with respect to the starting structure coordinates 6PRC (30), because these had been refined with the same protocol and parameters as were used here. Figures were prepared with a version of Molscript (34) modified for color ramping (35) and map drawing (36) capabilities.

RESULTS
The structure of the HL168F RC in complex with terbutryn is summarized in Table I. This is the best-defined Rp. viridis reaction center structure to date, and the crystallographic Rfactor and R free T are 19.4% and 21.8%, respectively, for all data between 10.0 and 2.00 Å ( Table I). The position error is estimated to be 0.15 Å. Simulated annealing omit electron density maps (31) were calculated for the site of mutation (Fig. 1) and the terbutryn binding site (Fig. 2). All residues of interest can be detected clearly in the electron density.
As can be seen in Fig. 1A, the Phe side chain of the exchanged residue L168 adopts a conformation very similar to the wild-type His. In the wild-type RC, the His N⑀ atom donates a hydrogen bond to the ring I acetyl oxygen of bacteriochlorophyll D L (23). The position of this oxygen atom in the wild-type RC structure is not covered by the mutant RC electron density (Fig. 1, A and B). In the refined structure of the HL168F RC, this acetyl group has undergone a 20°rotation relative to the wild-type RC, thus shortening the distance between the acetyl oxygen atom and the Mg 2ϩ center of bacteri-   (54). Interestingly, R free T and R were 17.0 and 16.0%, respectively, when calculated with REFMAC and thus significantly lower than those calculated above with X-PLOR. e n obs ϭ number of observed unique reflections used in refinement ϭ 186,255; n par ϭ number of parameters necessary to define the model; this includes three parameters (x, y, and z coordinates) per non-hydrogen atom plus one (isotropic atomic B-factor).
f Based on X-PLOR, version 3.1, parameter files (parhcsdx.pro (55), param19.sol, and param19.ion), RC cofactor parameter files (27), and triazine inhibitor parameter files (30). ochlorophyll D M from 4.1 to 3.0 Å. The distance between the Mg 2ϩ centers of bacteriochlorophylls D M and D L has decreased from 7.7 to 7.4 Å, mainly because of a slight displacement of the D M -Mg 2ϩ toward the D L acetyl oxygen (Fig. 1C). In addition to the acetyl group rotation, an approximately 3°rotation of D L is apparent, leading to a displacement of ring I by between 0.2 (for the NB atom) and 0.4 Å (for the C3B atom), relative to the wild-type RC structure. A slight reduction in the distance between D and B A , as monitored by the distance between the C2B atom of D M and the C2D atom of B A (5.45 Å in the wild-type RC and 5.3 Å in the HL168F RC), is as large as the estimated position error (Table I).
Terbutryn Binding-The pattern of hydrogen bonding geometry for the RC-terbutryn complex (Fig. 2) is very similar to that reported earlier for the binding of atrazine (2-chloro-4ethylamino-6-isopropylamino-s-triazine) and other triazine inhibitors (30). In the following, the respective donor-acceptor distances are given in parentheses. The hydrogen bonding pattern includes, firstly, two tightly bound water molecules, one of which is apparently involved in accepting a hydrogen bond from His L190 N␦ (2.9 Å) and in donating a second hydrogen bond to the N1 of the terbutryn ring (3.0 Å). The other apparently completes the hydrogen-bonding web with hydrogen bonds to the first water molecule (2.7 Å) and to the O⑀ of Glu L212 (2.9 Å). Secondly, the backbone carbonyl oxygen of Tyr L222 is within hydrogen-bonding distance of the terbutryn t-butylamino nitrogen (3.3 Å). In the original RC-terbutryn complex (23), only the two hydrogen bonds from the backbone amide of Ile L224 to the N5 of the terbutryn ring (here, 3.1 Å) and from the aminoethyl nitrogen to Ser L223 O␥ (here, 3.0 Å) had been inferred. In summary, the terbutryn molecule is apparently bound to the protein directly by three hydrogen bonds on the "distal" side and only indirectly via water molecules (and four hydrogen bonds) on the "proximal" side, which is oriented toward the non-heme iron and its ligand His L190. In analogy to the water molecules in the Q B site of the Q Bdepleted RC (3PRC (27)), the two water molecules associated with terbutryn binding are linked by hydrogen bonds to the water molecules of the hydrogen-bonded web (not shown) from the cytoplasmic surface to the Q B site discussed earlier (27). Finally, in Fig. 2, C and D, the refined RC complex with terbutryn is compared with the structure of the RC complex with atrazine (5PRC (30)), where the aromatic ring of Phe L216 is not parallel to the atrazine ring plane at an interplanar angle of 24°. In the RC-terbutryn complex, the Phe L216 ring plane is rotated by 14°to be aligned more parallel to the plane of the terbutryn ring, which itself is tilted by 6°compared with the atrazine ring plane.
Water Molecules-The original (1PRC (10)) structure contained 201 tentative, ordered water molecules, and a Q B -depleted RC structure (3PRC (27)) contained 425. In the new structure presented here, 585 tentative water molecules were included, based on electron density maps and local chemistry. Of these, 328 water molecules (68 more than in 3PRC) are in the periplasmic region of the molecule, three (the same as in 3PRC) are in the transmembrane region, and 254 (68 more than in 3PRC) are in the cytoplasmic region. The latter are of particular interest, because they create new connectivities within the previously described (27) "web" of hydrogen bonds involving amino acid side chains and water molecules and proposed to be relevant for proton uptake from the cytoplasm and proton transfer to the Q B site. A detailed analysis of this further-defined web will be presented elsewhere.

DISCUSSION
Primary Electron Transfer-The mechanism of the initial electron transfer reactions in the RC has been a subject of intense studies (3,37,38). A useful tool in this context are mutant RCs where the energetics of D* and D ϩ are changed by removal or addition of hydrogen bonds between the special pair and amino acid side chains. A hydrogen bond to the bacteriochlorophyll groups of the special pair stabilizes the neutral state of D relative to the oxidized state D ϩ and thus increases the D/D ϩ redox potential (39). In the Rp. viridis HL168F mutant RC studied here, the D/D ϩ redox potential is 440 mV, 80 mV lower than that of the wild-type RC (21). The decay of the excited electronic state D* is accelerated, with the associated time constant being reduced from 3.5 to 1.1 ps. The effects of the His L168 3 Phe exchange on the structure described above, involving the loss of the hydrogen bond to the ring I acetyl group of D L and its 20°rotation, resulting in an increased interaction between the acetyl group oxygen and the Mg 2ϩ of D M , may contribute to the stabilization of the oxidized state D ϩ relative to the neutral special pair and thus at least partially explain the observed lowering of the redox potential and the accelerated rate of initial electron transfer from D* to D ϩ B A Ϫ . Another reason for the increased electron transfer rate may be an increased electronic coupling between D and B A (21). However, the reduction in the distance between D and B A in the HL168F RC discussed above is only on the order of the estimated position error of the structure.
Another previously described spectroscopic feature of the HL168F RC is that the Q y transition of the special pair is shifted by 35 nm to shorter wavelengths (930 nm), indicating perturbations of the electronic structure and/or a changed excitonic coupling of the two bacteriochlorophyll molecules of the special pair (21). In addition to the structural changes of the D L ring I acetyl group, the slight rotation of D L and the reduced Mg-Mg distance between D L and D M may also contribute to this feature. It is expected that the high quality of the determined structure will provide a useful basis for computational studies for a more quantitative description of the observed effects of the His L168 3 Phe mutation.
Terbutryn Binding-The Q B site is a well established site of herbicide action, both in the purple bacterial RC and in the reaction center core of photosystem II, for which the former serves as a model (40 -44). Over 50% of commercially available herbicides function by inhibition of higher plants at the Q B site of the D1 polypeptide of the photosystem II reaction center (45). A commercially very important class of herbicides are the triazines, which were introduced by J. R. Geigy S. A. in the 1950s (46). Prominent examples are atrazine and terbutryn. Interestingly, the binding affinity for the Rp. viridis wild-type RC of terbutryn is 14-fold higher than that of atrazine (47). The reduction in the interplanar angle between the triazine ring and Phe L216 from 24°in the case of atrazine to 17°in the case of terbutryn is probably not sufficient to explain the higher affinity by increasedinteractions. The 6-methylthio group of terbutryn, as opposed to the 2-chloro group of atrazine, apparently fits more snugly to the Q B pocket with the methyl group within van der Waals distance to the C␤ atom and a C␦ atom of Leu L189, as well as to the C␦ atom of Ile L229, thus offering a second explanation for the increased affinity.
Water Molecules-Generally in proteins, internal water not only fills structural cavities, but it is also necessary to stabilize three-dimensional folding (48,49). As has been discussed earlier for the structures 2PRC, 3PRC, and 4PRC (27), some of the additionally determined water molecules make important contributions to crystal packing and are also possibly relevant to the pathways and kinetics of proton transfer to the Q B site (27, 50) and quinol release from the Q B site (27,51). In the specific case of triazine binding, they are also important for optimal inhibitor binding (see Ref. 30 and this work). In particular, we expect that the large number of additionally assigned water molecules will enrich computational simulations of coupled electron-proton transfer to the Q B site.
Concluding Remarks-The present results demonstrate that the structural changes underlying the drastic functional changes associated with replacing His L168 with Phe are limited. In principle, it would appear advantageous for the RC to have a Phe at position L168, because the increased initial electron transfer rate improves the quantum efficiency of photosynthesis (21). However, the associated "blue shift" of the Q y absorption band of the primary donor D also makes energy transfer from the B1015 light harvesting antennae less efficient, whose absorption characteristics are optimized for the natural environment of the Rp. viridis RC. In summary, the presence of His at position L168 demonstrates how nature tunes the RC to function more efficiently with its surrounding antennae, rather than optimize the isolated function of the RC.