6 Discussion. 6.1 Bacterial reaction center X-Ray crystallography of RC mutants

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1 6.1 Bacterial reaction center Proton and electron transfer in bacterial reaction centers were investigated using time-resolved FTIR spectroscopy and X-ray crystallography. The effect of sitespecific mutations was assessed. The implications of these results for the electron and proton transfer mechanism are discussed X-Ray crystallography of RC mutants The structure of the WT RC of Rb. sphaeroides had previously been solved by X-ray crystallography to high resolution [67, 119]. This led to a detailed knowledge of the position of pigments, cofactors, amino acids, water molecules and their relative distances and orientations. With this structural information, it became possible to get a more profound interpretation of spectroscopic data in order to understand the molecular mechanism underlying the function of these proteins. RC mutants provide an excellent tool to investigate the role of specific amino acids in electron and proton transfer processes. Protein crystals from the bacterial RC of Rb. sphaeroides described in this thesis were grown using vapor diffusion as well as lipidic cubic phases. Three kinds of well diffracting crystals (orthorhombic, trigonal and cubic), diffracting to a resolution of 2.5 Å could be obtained. The resolution of the different kinds of crystals does not differ significantly. In comparison to that, the resolution of the recently published high resolution RC WT structure (PDB-ID: 2J8C [67]) reaches 1.9 Å. The procedure for getting RC crystals was not very reproducible. As the purity of the protein needs to be extremely high, the procedure of protein purification was improved. In order to obtain RC crystals with a better resolution, a lot of parameters were optimized [39]. The obtained crystals were frequently thin, poorly ordered and appeared together 92

2 with more or less amorphous precipitate in almost each crystallization experiment. The mechanical stability was low and they tended to break easily during manipulations. Therefore, several attempts were require to get large crystals suitable for X-ray crystallography. In this work, the crystal structure of the L210DN RC mutant was determined for the first time with a resolution of 2.5 Å. The resolution for the L212EA/L213DA RC mutant could be improved compared to the previously published structure determined by Pokkuluri et al. (1K6N, 3.1 Å [100]). The study of the L210DN and L212EA/L213DA RC mutants show no major differences of the general protein structure in comparison to WT structure. The distribution of protein-bound water molecules revealed local differences, which could explain the changed kinetic behavior of the mutant. Several water molecules could be identified to participate in proton transfer. However, the resolution of the structural data has to be improved to obtain a more detailed picture of water molecules in these RC mutants Proton pathway in L210DN mutant RC In WT RC, a proton bound to AspL210 has to be released from the carboxyl group to be transferred to GluL212 (path A in Fig. 6.1). In the L210DN mutant RC (Fig. 5.2), AspL210 is mutated to Asn, and therefore this proton donor to GluL212 is missing [52]. In this case, the proton might instead be donated from a protonated water molecule. The structural data support this view. In WT RCs of Rb. sphaeroides, a chain of conserved water molecules with hydrogen bonding distances can be found between AspL210 and GluL212, including the carboxyl group of GluH122 [67, 119]. The homologous residue AspH125 in Rsp. viridis was also described to be part of a hydrogen bonded cluster of strongly interacting amino acid residues in the proximity of the Q B binding pocket [71, 72]. Recently, GluH122 in Rb. sphaeroides was also suggested to be part of a delocalized proton transport network [57]. Comparison with Rsp. viridis RC structures [70, 71] revealed a similar conserved distribution of water molecules in this part of the structure. In the majority of cases, water molecules that are modeled into protein structures can be assumed to be tightly bound to the protein and are also expected to be present in newly determined structures. In the L210DN mutant, no water molecules between GluH122 and GluL212 can be resolved, as they are not so well ordered. If the same pathway was used in this 93

3 Figure 6.1: Proton transfer pathways in RC. Structural details of the proposed pathways for the first proton in the bacterial RC. Selected amino acid residues of the WT structure (2J8C [67]) are shown as gray sticks. The iron is shown as an orange sphere. Small red spheres depict water molecules. Possible hydrogen bonds are indicated by gray dashed lines. The protein is indicated as a gray surface in the background. The arrows represent the general proton flow from the surface to Q B. (A) Red arrows: AspL210 at the surface of the protein is hydrogen bonded via GluH122, GluH173 and several water molecules to GluL212 and GluM234 in the vicinity of the carbonyl group of Q B. The previously proposed entry point is in the vicinity of HisH126 and HisH128. (B) Blue arrow: ArgH118 and AspM240 at the surface of the protein are hydrogen bonded via GluM236, GluH173 and several water molecules to GluL212 and GluM234 near Q B. An additional, flexible water molecule (shown as black sphere), not resolved in the X-Ray structure, is added to the figure to demonstrate a possible completion of the pathways to its final destination, the carbonyl group of Q B. mutant, protonated water molecules would have to move inside the channel in order to donate the proton to GluL The role of water molecules in proton transfer The distribution of water molecules in WT and L210DN suggests, that water molecules are involved in proton transfer from the cytoplasm to the secondary electron accep- 94

4 tor ubiquinone, Q B. Most likely, the protons are transferred via a Grotthus-like mechanism. Previously, an entry point formed by AspH124, HisH126 and HisH128 was proposed [4, 96, 97]. As visualized in figure 6.1, it was proposed that protons taken up at this entry point would be transferred to Q B via AspL210 [96] and GluL212 [28, 94, 130]. Recently, the existence of another potential proton delivery pathway for the first proton was confirmed by high resolution X-ray structures of WT RC [67]. New water molecules were identified, building a hydrogen bonded network from the protein surface via several amino acids to Q B. The entry point of this alternative proton pathway is distinct from the previously proposed one. It is formed by ArgH118 and AspM240, as visualized in 6.1. The present structural data also demonstrate a possible entry point at AspL210, in the vicinity of HisH126 and HisH128, as it is directly hydrogen bonded to a water molecule at the protein surface. These results support the previously suggested idea [43, 122] that proton pathways to Q B, including their entry points, are not unique. More likely, the proton transfer in RC can be performed via several pathways including different entry points for protons (Fig. 6.1). In agreement with this proposed multiple proton transfer pathway, a single mutation, as in the L210DN mutant, does not lead to the loss of function of the protein. In the case of L210DN, the proton transfer to GluH122 is slowed down, due to the missing proton donor AspL210. The proton might be transferred via an alternative proton pathway. Probably, the proton can be transferred from the entry point at ArgH118 via several amino acid residues and water molecules to Q B. As AspL210 is not directly connected to this proton pathway (pathway B in figure 6.1), its mutation does not significantly influence this alternative proton pathway and a proton can be transferred to Q B. The longer distance of this pathway (B) from the protein surface to Q B in comparison to the shorter pathway (A) from the proposed entry point in the vicinity of HisH126 and His128 might explain the slower time constant of this proton transfer step Structural relevance to PS II Bacterial RCs are thought to be ancestors of PS II. The basic function and structure of bacterial RCs are significantly similar to those of PS II. Three-dimensional structures of PS II have already been determined by X-ray crystallography and the 95

5 Figure 6.2: Possible proton pathway in PS II. Structural details of a possible proton pathway in PS II (2AXT [76]). The protein is indicated as a purple surface in the background. Selected amino acid residues of PS II are shown as purple sticks. The iron is shown as an orange sphere. The cavity between the surface and Q B is indicated by a mesh. The possible proton pathway in PS II begins at Gln D2 239 and Glu D The cavity, big enough for a chain a water molecules, is further framed by the amino acid residues Glu D1 244, Thr D2 243, Tyr D1 246 and Ser D1 268 ending between the bicarbonate and Q B. Illustration by Dr. Christian Burisch (RUB, Bochum, D). spatial arrangement of cofactors and transmembrane helices have also been described to some extent [76]. These structures have shown, together with those of bacterial RCs [67], that RCs of any type possess common structural features. Light induced processes of energy conversion occur in the bacterial RC as well as in PS II, which are both membrane-bound pigment-protein complexes. In both complexes, light absorption results in the transfer of an electron from a primary electron donor by a series of intermediary cofactors to the secondary electron acceptor quinone. After the reduction of the oxidized primary electrn donor, it can be excited again, resulting in the transfer of a second electron. Following, the secondary quinone receives a second electron as well as two protons. Finally it leaves the quinone pocket and is replaced by another quinone [10]. PS II is a significantly larger complex than bacterial RC containing over 20 sub- 96

6 units and 77 cofactors, most of which are chlorophylls [76]. Despite this size difference, both complexes contain a central pair of subunits, L and M in the bacterial RC, and D1 and D2 in PS II. Each one is composed of five helices that form the framework for the organization of the cofactors performing the primary conversion of light energy into a charge-separated state. Although each of the D1 and D2 subunits is larger than its counterpart in the bacterial RC, the differences predominantly lie in the loops and the carboxyl and amino terminus regions, with the transmembrane helices of PS II and bacterial RC being structurally homologous. Both, the protein subunits and the cofactors are related by an approximately twofold symmetry axis, passing from the primary donor to the nonheme iron, which results in the cofactors being organized into two branches. The symmetry of the two branches of cofactors would suggest that electron transfer could proceed equally through either branch. Possible proton pathway in PS II The recently published structure of PS II (PDB-ID: 2AXT [76]) has a resolution of 3.0 Å. No water molecules were determined at this resolution. A cavity analysis of this structure 1 searching for a water channel from the protein surface to Q B and structural comparison with the bacterial RC revealed an analogous proton pathway as in RC. As visualized in figure 6.2, a possible proton pathway in PS II, starting at Gln D2 239 and Glu D2 241 is framed by the amino acid residues Glu D1 244, Thr D2 243, Tyr D1 246 and Ser D1 268 and ends between the bicarbonate and Q B. This pathway corresponds to the described proton pathways in bacterial RCs with the common crosspoint at GluH122. It has to be considered, that PS II does not contain a counterpart to the H-subunit of the bacterial RC. Therefore, no equivalent pathway corresponding to the H-subnit is available in PS II. 1 Cavity analysis was performed by Dr. Christian Burisch (RUB, Bochum, D), data not published. 97

7 6.1.5 FTIR spectroscopic investigation of RC FTIR difference spectroscopy is one of the most promising spectroscopic techniques to study the molecular mechanisms of proteins at the level of individual bonds. It permits to monitor reaction induced changes in vibrational modes of both the protein and the cofactors simultaneously. A large amount of data had previously been obtained on RCs of Rb. sphaeroides by static difference spectroscopy. These studies have led to the precise identification of IR marker bands for specific redox states of cofactors, in particular the primary donor and the two quinone acceptors [23, 22, 14, 84]. Compared to static FTIR difference spectroscopy, time-resolved FTIR difference spectroscopy can monitor the process of a reaction. The fast scan FTIR technique is a well established method to study reactions that can be triggered by light [68]. In specific RC mutants where the first electron transfer reaction is slowed down to the ms time domain, a fast scan FTIR investigation becomes possible. By combining FTIR difference spectroscopy with isotopic labeling, it is possible to detect and assign vibrational modes of single chemical groups in a protein complex as large as RC. In wild type RC, transient signals associated with Q A Q B to Q A Q B electron transfer with time constants in the μs time range could be observed and characterized using step scan FTIR spectroscopy [24, 106]. The first electron transfer from Q A to Q B proceeds within kinetics of about 200 μs at RT [66]. Therefore, it is not accessible to the fast scan FTIR technique. In this work, the fast scan FTIR technique was applied to study the reactions connected with the first electron transfer from Q A to Q B in the L210DN and L210DN/L218DN RC mutants of Rb. sphaeroides. L210DN RC mutant Time-resolved fast scan FTIR measurements of the L210DN RC mutant performed within this thesis [52] lead in combination with earlier published step scan FTIR experiments [105, 52] to a description for the electron transfer between Q A and Q B with three time constants (19 μs, 200 μs and 9 ms) and three recombination time constants (80 ms, 320 ms and 2.6 s). Noticeably, in contrast to the wild type protein, partial P + Q A /PQ A recombination already occurs at 9 ms. In addition, the first time constant for the P + Q B /PQ B recombination is 2.4 times faster than in wild type (320 ms compared to 770 ms) in agreement with UV/VIS measurements [95]. 98

8 Evidently, the changed electrostatic environment in the mutant during the P + Q B state provides less stabilization to the semiquinone Q B, and therefore the lifetime of this state is decreased. L210DN/L218DN RC mutant The L210DN/L218DN RC mutant was investigated in this work for the first time by FTIR spectroscopy. Time-resolved fast scan FTIR measurements reveal one time constant for the electron transfer between Q A and Q B (17 ms) and three recombination time constants (66 ms, 1.6 s and 4.3 s). As in the L210DN mutant, partial P + Q A /PQ A recombination already occurs at an early time constant (17 ms). Additionally, also partial P + Q B /PQ B recombination occurs at this time constant. In contrast to L210DN, the first time constant for the P + Q B /PQ B recombination is 2 times slower than in wild type (1.6 s compared to 770 ms). Evidently, the P + Q B state is more stable in this mutant and therefore the lifetime of this state is increased. Assignment of bands to the intermediary electron donor X In 2003, Remy and Gerwert proposed an intermediary electron donor X in wild type RC [105, 106]. Using step scan FTIR spectroscopy they could show, that the Q B band at 1479 cm 1 appears in wild type RC (200 μs) before the Q A band at 1446 cm 1 disappears (1.1 ms). They suggested that the electron transfer from Q A to Q B involves an intermediary electron donor X. A good candidate for the intermediary electron donor is the iron-histidine complex located between Q A and Q B. Hermes et al. could demonstrate in 2006 by XAS that the iron is not the intermediary electron donor X. The RC mutants L210DN and L210DN/L218DN (investigated in this thesis) exhibit a decelerated rate for the proposed Q A to X + transition. Therefore, this process can be resolved by the fast scan FTIR technique. The analyis of the amplitude spectra allowed the assignment of bands to the intermediary electron donor X (( ) 1533 cm 1, (+) 1373 cm 1, ( ) 1336 cm 1, ( ) 1255 cm 1, (+) 1109 cm 1, ( ) 1100 cm 1, (+) 1094 cm 1 and (+) 1068 cm 1 ). The possible involvement of histidine in the electron transfer process from Q A to Q B was examined by studying 13 C 6, 15 N 3 isotopically labeled histidine isotope effects on FTIR amplitude spectra of L210DN. 99

9 Since the amplitude spectra of RC present a lot of bands, it is difficult to detect the shifts induced by the presence of labeled histidine within the protein. Some of the assigned X-bands are located in the 1100 cm 1 region. As only the side chain of histidine absorbs in the 1100 cm 1 region [5], this region should be the best one to see clear shifts of bands caused by histidine. Histidine bands have already been assigned in this region in protein, e.g. PS II [7, 91]. Thus, this study is focussed on this specific wavenumber scale. Assignment of bands to histidine Histidine bands in isolated histidine: Histidine IR modes have been identified by analyzing the effect of isotopic labeling on isolated histidine and by comparison with published model compound spectra [5]. In the 1100 cm 1 region, three significant bands could be assigned to the change of protonation state in histidine: (+) 1107 cm 1, ( ) 1097 cm 1 and (+) 1088 cm 1. The IR frequency of these bands correspond to modes that have been previously assigned to imidazole ring vibrations based upon comparison with spectra of model compounds [8, 91] and isotopic labeling [12]. The difference in the band position of unlabeled and isotopically labeled histidine is found to be cm 1. Accordingly similar band shifts are expected in isotopically labeled histidine RC protein. However, it has to be considered, that isolated amino acids in solution reveal a different spectroscopic behavior than within a protein molecule. In particular, the environment of the amino acids plays a crucial role, e.g. the amino acids are peptide bonded in protein. In addition, the surrounding amino acids in a protein can be either hydrophobe or hydrophile. Accordingly, slightly different band positions and shifts are expected, by comparing bands observed in spectra of isolated histidine and histidine bands in protein spectra. Histidine bands in RC protein: In this work, histidine marker bands in FTIR amplitude spectra were identified in the L210DN RC mutant from Rb. sphaeroides upon the electron transfer from P + Q A X + to PQ A X using isotopically labeled histidine. The isotope effect on the pure P + Q A X + /PQ A X amplitude spectrum provides IR signatures of the histidine modes that are very comparable to those previously reported to the shift described for histidine labeled PS II by C. Berthomieu [9]. 100

10 Histidines are involved in the reaction described by the 1 st time constant: The assigned histidine bands in the amplitude spectra of L210DN RC mutant during the P + Q A X + to PQ A X transition correspond to histidine bands in isolated histidine. This is in agreement with previously published spectra of model compounds [8, 91] and isotopic labeling in RC and PS II [12, 9]. Consequently, the presented bandshifts during the P + Q A X + to PQ A X transition reflects the protonatio of histidine during the reaction corresponding to the 1 st time constant. Considering that the iron-histidine complex is located between Q A and Q B, the most likely conclusion is that one or more of the included histidines (HisL190, HisL230, HisM219 and HisM266) is / are involved in the Q A to Q B electron transfer. Time-resolved observation of changes in a protonated water cluster Continuumbands in the L210DN and L210DN/L218DN RC mutants: Broad continuum bands in the spectral region of cm 1 are a signature of protonated hydrogen bonded networks within proteins [40, 41]. In br it was shown how the interplay between a strong hydrogen bonded water molecule, a dangling water, and a protonated water complex transfers a proton from the Schiff base, the central proton binding site, to the external medium [41]. The presence of broad positive bands extending between 2900 and 2400 cm 1 has previously been reported in static Q A /Q A and Q B /Q B FTIR difference spectra of Rb. sphaeroides and Rps. viridis [15], as well as in the Q A /Q A spectrum of PS II [91]. However, broad absorbance changes can also be caused by baseline drifts, which often occur during long measuring times to obtain static spectra. Therefore, the time-resolved FTIR measurements were performed to assign clearcut continuum absorbance changes. In this work, we examined the possible involvement of water molecules in the proton transfer processes of the bacterial reaction center. We studied the broad continuum bands in the cm 1 region which have been proposed to contain contributions from protonated water [104, 40]. For the first time, we identified in time-resolved IR measurements in the RC continuum absorbance changes for the Q A to X and the Q B to Q B transition (Fig. 4.5 and 4.4). The different maxima indicate different sizes of the protonated water complex, e.g. an Eigen cluster (H 9 O + 4 ) [35] or a Zundel cluster (H 5 O + 2 ) [132, 50]. The bands in the amplitude spectra indicate a deprotonation of a protonated water cluster during the Q A to Q A transition. Therefore, 101

11 Figure 6.3: Previously proposed mechanism for the electron and proton transfer. Structural details of the WT RC (2J8C [67]). (1) white arrow: Reduction of Q A in 200 ps. (2) yellow arrow: Protonation of AspL210 in 12 μs. (3) orange arrow: Reduction of Q B in 150 μs by an intermediary electron donor X. (4) red arrows: Reduction of Q A by X and protonation of GluL212 by AspL20 in 1.1 ms it is proposed that in a very similar mechanism as in br a proton is transferred in RC from AspL210 to GluL212 via a chain of water molecules. Further experiments have to be performed to obtain such a detailed mechanism as found in br Possible role of water molecules in the electron and proton transfer to Q B in the WT RC Previously proposed mechanism The previously proposed mechanism had 6 steps, as summarized in figure 6.3 (see introduction). (1) The absorption of one photon by the electron donor P induces a very fast electron transfer (200 ps) to the first electron acceptor Q A. (2) The protonation of the amino acid AspL210 occurs in 12 μs. This step is the gate which controls the other following events. (3) Reduction of Q B occurs in 150 μs and takes 102

12 place while Q A is still reduced (Q A ). So the reduction of Q B involves an unknown intermediary electron donor X which gives an electron to Q B. (4) The electron transfer from Q A to X occurs in 1.1 ms. This event is coupled to the proton transfer from AspL210 to GluL212. The proton transfer from AspL210 to GluL212 is proposed to occur not directly between the two amino acids but via a chain of protonated water molecules in a Grotthus-like mechanism. (5) The charge recombination between Q A and P + takes place in 60 ms. (6) The charge recombination between Q B and P + occurs in 770 ms and 3.1 s. Proposition of a new mechanism for the electron and proton transfer The results obtained with the fast scan technique on L210DN mutant containing 13 C 6, 15 N 3 labeled histidine, suggest that in the Q A X + to Q A X transition (step 4) histidines are involved and that histidine(s) is (are) getting protonated during this transition. With this new result, the previous mechanism for the coupled first electron-proton transfer in bacterial photosynthetic reaction center can be modified. The proposed mechanism of electron and proton transfer in the RC is visualized in figure 6.4. (1) The absorption of one photon by the electron donor P induces a very fast electron transfer (200 ps) to the first electron acceptor Q A (blue arrow). (2) As it can be seen in figure 6.4, a hydrogen bonded network (gray dashed lines) exists between Q A and the cytoplasm. This network is connecting Q A, the Fecomplex, Q B, a chain of water molecules (from Q B to the protein surface), different amino acids (including GluL212, GluH173, GluH122 and AspL210) and the cytoplasm. Thanks to this network and long-range electrostatic effects, the reduction of Q A induces a proton uptake from the cytoplasm. This proton is transferred from the cytoplasm to AspL210 in 12μs (yellow arrows). The proton entry point is not totally clear. It has been suggested to be localized in the vicinity of HisH126 and HisH128 [97]. The recently published high resolution structure of RC (PDB-ID 2J8C [67]) reveals a water molecule localized at the protein surface close to AspL210, which could be a good candidate for an entry point of the proton. The residue AspL210 is thought to be the gate which triggers the coupled electron-proton transfer. Indeed, when AspL210 is protonated the gate is open: the protonation of AspL210 disturbs the water chain and it will induce the next electron-proton transfer events. Whereas 103

13 Figure 6.4: Proposed mechanism for the electron and proton transfer. Structural details of the WT RC (2J8C [67]). (1) blue arrow: Reduction of Q A in 200 ps. (2) yellow arrows: Protonation of AspL210 in 12 μs. (3) orange arrow: Shared proton between His and Q B via a protonated water (black sphere). Decreas of the bond order of the C=O bond of Q B in 150 μs. The water represented as a black sphere is not present in the crystal structure. But it is suggested that a flexible water could be present at this place. (4) red arrows: Coupled GluL212 protonation, Q B reduction and Q A oxidation in 1.1 ms. when AspL210 is deprotonated, the gate is closed and no electron-proton event can occur in 1.1 ms. (3) Time-resolved (step scan) FTIR data have shown that a characteristic band of Q B (at 1479 cm 1 ) appears in 150 μs [106]. This result previously led to the conclusion that Q B was reduced in 150 μs and that there should be an intermediary electron donor X between Q A and Q B which gives an electron to Q B while Q A is still reduced. This mechanism has created a lot of controversy when it has been proposed; controversy mainly due to the fact that both quinones were carrying a negative charge at the same time. Now, we propose another assignment for the signal observed at 1479 cm 1 in the 150 μs amplitude spectrum. We propose that 104

14 Figure 6.5: Reaction scheme of the proposed mechanism. Reaction scheme of the proposed electron transfer mechanism in WT involving Q B, a mobile water and a proton from a histidine. this band is not due to a semiquinone C-O (as previously proposed) but to the close interaction between a carbonyl group of Q B and a proton. The proton (positive charge) close to the carbonyl group of Q B attracts the electron density towards the oxygen of the C=O group, resulting in a decrease in bond order of the C=O bond. This is giving rise to an IR signal at 1479 cm 1 that could appear like a Q B band in FTIR data. Our new results on the 13 C 6, 15 N 3 labeled L210DN mutant, involving histidine protonation in step 4 make us suggest that the proton interacting with Q B is given by a histidine (probably HisL190), to the water molecule between Q B and HisL190, as it is illustrated in figure 6.4. We propose that the protonated water which interacts with the C=O group of Q B is the water molecule represented by a black sphere in figure 6.4. This water is not present in the recently published structure 2J8C. But in [67] the authors suggest that there is enough space for a flexible water molecule close to Q B. Such an additional mobile water molecule placed between the Fe-histidine complex and Q B at hydrogen bond distances from amino acids and cofactors would close the missing connection of the hydrogen bonded network between the surface and GluM234 with the oxygen of Q B. The step 3 occuring in 150μs can be summarized this way: A histidine deprotonates and gives its proton to a flexible water (black sphere) located between the histidine and Q B. The protonated water interacts with the C=O group of Q B leading to a decrease in bond order of the C=O bond. (4) The step 4 occurs in 1.1 ms and corresponds to different coupled events (red arrows). The oxidation of Q A is coupled to the proton transfer from AspL210 to GluL212. The proton transfer from AspL210 to GluL212 is proposed to occur not directly between the two amino acids but via a chain of protonated water molecules in a Grotthus-like mechanism. The continuum band observed above 1800 cm 1 in 105

15 the 1.1 ms amplitude spectrum is characteristic of the deprotonation of a protonated water cluster. At the same time Q B is reduced. Due to the initiated protonation of GluL212, there is solvation of Q B by positive charges (protonated Glu212 and the additional mobile protonated water), which allows the transfer of the electron from Q A to Q B. The histidine which initially gives its proton in step 3 is reprotonated in step 4 in 1.1 ms. (5) The charge recombination between Q A and P + takes place in 63 ms. (6) Charge recombination between Q B and P + takes place in 770 ms and 3.1 s. The reactions of this mechanism are summarized in figure Bacteriorhodopsin The photoactive proton pump bacteriorhodopsin is an excellent model system for studying undirectional ion transfer in membrane proteins. This membrane transporter transports actively ions against an electrochemical gradient. Furthermore, it serves as a simple model for seven-transmembrane-helix proteins. Bacteriorhodopsin is one of the most investigated membrane proteins, including both static and time-resolved spectroscopy and X-ray crystallography combined with theoretical studies. However, details of the photocycle are still unknown and have to be investigated. The extracellular region contains a hydrogen bonded network of polar protein residues and seven water molecules between the protonated Schiff base in the protein center and the extracellular surface in the ground state [80, 40]. These water molecules have been identified to participate in the vectorial proton transport [41]. Crystal structures of br mutants, where amino acids that are coordinating water molecules were mutated, revealed changes in the distribution of the water molecules [101, 128]. In contrast to this, the cytoplasmic region contains only three protein bound water molecules in the ground state [80]. No hydrogen bonded network between the cytoplasmic surface and the Schiff base can be observed. A pathway for proton transfer has to be created between the Schiff base and Asp96 over a distance of more than 10 Å in order to reprotonate the Schiff base during the photocycle. It is still unclear how this proton transfer on the cytoplasmic side occurs. Mutating residue 96 from Asp to Glu dramatically slows down the decay of the M state 2. In this work, the X-ray structure of the br mutant D96E was solved and analyzed. 2 Erik Freier (RUB, Bochum, D), private communication 106

16 Structural details are discussed in context of spectroscopic and theoretical data to get a deeper insight of the structure-function relationship Structure solution of br D96E Crystals of the D96E mutant diffracted at the synchrotron up to 2.5 Å. This resolution is comparable to other br mutant structures obtained at the Department of Biophysics [101]. However, it is not as good as the high resoltion structure of wild type br (PDB ID: 1C3W [80]). Crystallization conditions and crystal handling might influence the resolution as discussed in [101]. Twinning: Twinning is a crystal-growth defect that arises when two or more domains within a crystal have different orientations [110]. As a consequence, diffraction spots with different Miller indices are mixed. It has been observed in numerous protein crystal systems, including br [79, 125, 109]. The br crystals analyzed in this thesis were merohedrally twinned. Since the twinning fraction was smaller than 50 % (48.1%) the intensities of the Bragg spots could be corrected [79] using SHELX [115]. Ice rings: (90.5%). Because of deleted reflections due to ice rings the completeness is inferior Structure of br D96E The Asp to Glu mutation of residue 96 does not influence the distribution of the water clusters on the extracellular side of br (Fig. 5.7 and 5.8). The Pentamer and the proton release group have the same arrangement as in the wild type br. The structural changes on the cytoplasmic side are discussed below An additional water molecule on the cytoplasmic side causes structural changes The Asp to Glu mutation in the D96E mutant causes an insertion of a water molecule, which bridges the carboxyl oxygen atom of Glu96 with W502 (6.6). This new water molecule, W666, is additionally hydrogen bonded to the carboxyl oxygen atom of Thr46. An explanation might be the longer side chain of Glu96 in comparison to 107

17 the native Asp96. A 10 ns MD simulation of the D96E structure shows a stable structural motif of this hydrogen bonded chain from Glu96 via water molecules W666 and W502 to Lys216 [127]. As shown in figure 5.5, where the model of D96E is compared with the wild type structure, the position of Thr46 has changed. As a result, the distance between C α atoms of residue 96 and Thr46 increases by over 1 Å. The structure of the D96E mutant reveals that the retinal Schiff base region is influenced by the displacement of Thr46. In the wild type, W501 bridges helices G and F through the hydrogen bonds to the peptide oxygen of Ala215 and the indole nitrogen of Trp182. The hydrogen bond of W501 to Trp182 is essentially unchanged in the D96E mutant (it is 2.7 Å vs 2.8 Å in the wild type). However, the hydrogen bond between W501 and the oxygen atom of Ala215 is weaker, as the interatomic distance increases from 3.0 Å in the wild type to 3.5 Å. This appears to be an indirect consequence of the displacement of helix B at Thr46, as follows. W502 moves with the movement of Thr46 described above because its hydrogen bonds with the peptide oxygen atom of Thr46 and with the peptide oxygen atom of Lys216 are maintained, while a new hydrogen bond with water molecule W666 is created. Ala215 is linked to the cytoplasmic side via the hydrogen bond of Lys216 to W502. Displacement of W502 therefore causes the main chain of helix G to move at Lys216 and Ala215. The side chain of Ala215 moves away from W501. Although the primary main chain and side chain perturbation from the D96E do not reach the retinal region, water molecules in this region are effected. Since these water molecules form hydrogen bonds with functionally important residues and play important roles in the proton transport mechanism [62], their displacement might be important D96E shows an N like distribution of water molecules on the cytoplasmic side Additional water molecules between residue 96 and W502 were reported in the N state of the V49A mutant (PDB ID: 1P8U [112]). The N state structure of V49A contains a continuous hydrogen bonded chain from Asp96 up to the Schiff base built by 2 additional water molecules (W505 and W506), which are coordinated by additional hydrogen bonds to further amino acids (Fig. 6.6.C). The ground state of the D96E mutant shows similarities to the structure of the N 108

18 state of the V49A mutant. Although it would be more relevant to use the N state of the wild type as reference, there is no such stucture available up to date. In order to compare the structural changes in the D96E mutant with N state of wild type structure, molecular dynamic (MD) simulations were performed of a modified N state structure of V49A. An in silico back mutation of Ala49 to Val was introduced to obtain a more wild type like intermediate structure [127, 38]. MD simulations showed as most favorable structure, a stable chain of three water molecules building a pathway from Asp96 to the Schiff base. Figure 6.6 compares the structure models of D96E with with N state of V46A and the simulated N intermediate of the in silico back mutated wild type structure, each superimposed on the WT. In the N intermediate of WT (MD simulation), three water molecules are likely building a proton pathway from Asp96 to the Schiff base [127, 38]. The D96E structure has a similar distribution of 2 water molecules between Glu96 and Lys216. The dramatically decreased time constant of the M to N transition in the D96E mutant can be explained by this configuration of water molecules. D96E seems to have a N-like constellation on the cytoplasmic side of br. The additional water molecule W666 in D96E stabilizes the hydrogen bonded chain from the carboxyl oxygen of Glu96 to the carbonyl oxygen of Lys216. Since the carbonyl oxygen of Lys216 cannot accept a proton, this pathway is a dead end for the proton transfer. In order to build a pathway for proton transfer from Glu96 to the Schiff base, so as to reprotonate the Schiff base, the existing pathway to Lys216 has to be broken by disrupting the hydrogen bonded network. The energy barrier related to this event might be an explanation for the delay of the M to N transition. One additional water molecule connects Asp96 to the carbonyl residue 216 via W502, in what appears to be the beginning of a hydrogen bonded chain that would later extend to the retinal Schiff base. Controlled conformational changes of Asp96 rearrange preordered protein-bound water molecules and influence vectorial proton transfer from the cytoplasmic side to the Schiff base. 109

19 Figure 6.6: Distribution of water molecules on cytoplasmic side. (A) Ground state (BR) of D96E mutant (gray). An additional water molecule (W666) bridges Glu96 with water W502. (B) Ground state (BR) of WT (PDB ID: 1C3W [80]) (green). W502 is located between Asp96 and Thr46. (C) N state of V49A mutant (PDB ID: 1P8U [112]) (yellow). The interhelical space between the Schiff base and Asp96 contains new water molecules, in addition to W502. A continuous, single-file hydrogen bonded chain of W505, W506, W502 and W503 connects the Schiff base nitrogen atom to the carboxyl oxygen of Asp96. (D) Simulated (20 ns) in silico N state of WT [38, 127] (cyan). 110