Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

Abstract

Photosynthetic bacterial reaction centers convert light excitation into chemical free energy. The initial electron transfer leads to the consecutive semireductions of the primary (Q_A) and secondary (Q_B) quinone acceptors. The Q and Q formations induce proton uptake from the bulk. Their magnitudes (H⁺/Q and H⁺/Q, respectively) probe the electrostatic interactions within the complex. The pH dependence of H⁺/Q and H⁺/Q were studied in five single mutants modified at the L209 site (L209P→F,Y,W,E,T). This residue is situated at the border of a continuous chain of water molecules connecting Q_B to the bulk. In the wild type (WT), a proton uptake band is present at high pH in the H⁺/Q and H⁺/Q curves and is commonly attributed to a cluster of acidic groups situated nearby Q_B. In the H⁺/Q curves of the L209 variants, this band is systematically absent but remains in the H⁺/Q curves. Moreover, notable increase of H⁺/Q is observed in the L209 mutants at neutral pH as compared with the WT. The large effects observed in all L209 mutants are not associated with significant structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L. & Fritzsch, G. Biochemistry (2001) 40, 4253–4260). Our data suggest that, in the L209 mutants, the Q_B cluster does not respond to the Q formation as observed in the WT. We propose that, in the mutants, removal of the rigid proline L209 breaks a necessary hydrogen bonding connection between the quinone sites. These findings suggest an important role for structural rigidity in ensuring a functional interaction between quinone binding sites.

The biological role of bacterial reaction center (RC) membrane proteins is to convert light energy into chemical free energy. The sequential absorption of two photons by the system results into the production of the doubly reduced and doubly protonated form of the ultimate electron acceptor of the complex, a ubiquinone (Q_B). The formed Q_BH₂ molecule then delivers its reducing power to the cytochrome bc1 complex, resulting in the release of protons on the periplasmic side of the membrane. The resulting transmembrane proton gradient drives ATP synthesis through the ATP synthase. The reduction of Q_B coupled to the uptake of protons from the bulk is an important step shared by many systems involved either in photosynthesis or in respiratory chains (1).

The three-dimensional structure of the reaction center from the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is known at atomic resolution (2–4). Three subunits with a total molecular weight of about 100 kDa compose these RCs. The transmembrane L and M subunits carry the nine pigments and cofactors: four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones 10, and one non-heme iron atom. The third subunit, H, caps the reaction center on the cytoplasmic side of the membrane. The initial photochemical event induced by the absorption of a photon is the creation of the singlet excited state of a dimer of bacteriochlorophylls (P→P*), which constitutes the primary electron donor. P* is a strong reducing species that initiates the electron transfer reaction through the protein. In about 200 ps, the charge separation occurs between P and the first quinone electron acceptor, Q_A, situated on the cytoplasmic side of the complex. The electron is then transferred from Q to a secondary quinone Q_B within 10–100 μs (5–7). Both Q_A and Q_B are deeply buried within the reaction center protein. The role of the protein in stabilizing the redox species is essential to ensure high forward electron transfer rates and to prevent charge recombinations to occur. Although chemically identical, Q_A and Q_B behave differently. Q_A, bound to the M subunit in a relatively hydrophobic pocket, functions as one electron acceptor and is never protonated. At variance, Q_B, bound to the L subunit, is surrounded by charged and polar residues and behaves as a two-electron gate, accepting sequentially two electrons from Q_A and two protons from the cytoplasm. In chromatophores, the semiquinone Q can bind a proton below pH 6.8 (8). However, in isolated RCs, the semiquinones are not directly protonated but induce the shift of the pKas of ionizable interacting residues, which results in substoichiometric proton uptake by the protein (9, 10). The proton uptake may occur through a number of water molecules and protonatable amino acid residues situated between Q_B and the cytoplasmic surface. Of main interest is to identify the dynamical and structural role of the protein that contributes to the stability of the Q and Q states and to the energetic and functional connections of their respective environments.

The partial protonation events that occur on Q and Q formations have been studied by spectroscopic techniques by using site-directed mutagenesis (9–22) and by numerical methods (23–30). There is a general agreement that the major response of the protein to the Q formation is the change of the ionization state of acidic residues situated in the Q_B environment. These residues (L212Glu, L213Asp, L210Asp, and H173Glu) form a strongly interacting cluster, buffering as a whole the redox state changes of the quinones. The signature of this cluster is a notable proton uptake band (≈0.8 H⁺/Q) at low pH and at high pH. The high pH proton uptake band systematically disappears in all modified RCs reported so far, where L212Glu is absent (11, 14, 21), in both the Q and Q states, suggesting that the electrostatic effect of this cluster might be extended to the Q_A environment (12, 13, 15, 18, 31). The long range electrostatic effect between the Q_A and Q_B pocket haS also been proposed on the basis of electrostatic calculations (24–26, 30, 32, 33). The existence of electrostatic and/or conformational-mediated interactions between the two quinone protein pockets of the bacterial reaction centers have been evoked in experimental works (34–41). The three-dimensional structure of the protein reveals a large hydrogen bond network in the quinone proteic region involving numerous ionizable, polar residues and water molecules (2, 3, 42, 43). It is therefore of particular interest to investigate to what extent this widely spread out hydrogen bond network is involved in balancing the proton concentration over the key amino acid residues in the two quinone region of the RC protein.

We report here proton uptake measurements on Q and Q formations in RC mutants from Rb. sphaeroides in which L209Pro has been changed by site-directed mutagenesis to threonine (L209PT), tryptophane (L209PW), glutamate (L209PE), phenylalanine (L209PF), and tyrosine (L209PY). L209Pro is situated at the border of a chain of hydrogen-bonded water molecules (Fig. 1) that connects Q_B to the cytoplasmic surface of the RC (2, 3). Our previous reports concerned the characterization of the functional properties of these mutants (35, 44). The x-ray structure of three of these variant proteins (L209PF, L209PY, and L209PE) has also been determined (45). The amino acid exchange in the L209PE and L209PT mutants functionally mimics the kinetics of the wild-type (WT) RCs (44). In the L209PE reaction center, the structure remains unchanged compared with the WT structure, except the introduced carboxylic side chain of GluL209 located within the water chain (45). In the L209PW, L209PY, and L209PF variants, the spectroscopic analysis suggested a modification of the network of hydrogen bonds (35, 44). Consistently, the three-dimensional structures of the L209PF and L209PY mutant RCs show that the mutations have induced local structural changes of three amino acid residues (AspL213, ThrL226, and GluH173) and more distantly have affected the Q_B position (45). Despite these different structural changes, the similar proton uptake patterns measured here for all mutants suggest a crucial role for proline L209 in connecting both quinone environments.

Figure 1.

Rb. sphaeroides RC structure showing the two quinones Q_A and Q_B, the Q_B cluster of acidic residues, and the L209P mutation site. Water molecules connecting Q_B to the bulk are also represented. Coordinates were taken from the PDB entry code 1PCR (3).

Materials and Methods

Bacterial Strains and Growth Conditions.

The design of the Rb. sphaeroides WT or mutant strains harboring pufL mutation on pRK404 were previously described (35). The cells were grown in Erlenmeyer flasks filled to 50% of the total volume with malate yeast medium supplemented with kanamycin (20 μg/ml) and tetracycline (2 μg/ml). The cultures were grown in darkness at 30°C on a gyratory shaker (100 rpm).

Biochemical Techniques.

Cells from Rb. sphaeroides strains (native or harboring the mutation at L209 site) were disrupted by sonication in 20 mM Tris (pH 8) buffer in the presence of DNase and PMSF (1 mM). The intracytoplasmic membranes were purified as described in ref. 35. The membrane solubilization was done first by addition of lauryldimethylamine N-oxide (LDAO; Fluka) to a final concentration of 0.35% in the presence of 100 mM NaCl. The RCs were extracted by a second addition of LDAO to a final concentration of 0.8% in similar conditions. The solubilized RCs were subsequently purified on a DEAE Sepharose CL-6B (Pharmacia) column and eluted at an ionic strength equivalent to 250 mM NaCl. The ratio of absorbance at 280/802 nm was in the range 1.5–1.8 for all RC preparations.

Proton Uptake Measurements.

The RCs were extensively dialyzed against 50 mM NaCl, 0.03% Triton X-100 during 36 h at 4°C. Under these conditions, the Tris buffer concentration was kept below 10 μM. The proton uptake by the RCs was measured on a home made spectrophotometer by following the absorption changes at 585 nm of pH sensitive dyes after one saturating (Yag) laser flash at 532 nm (14). The final proton uptake signal was obtained with subtracting the buffered sample from the unbuffered signal. The proton uptake by the RCs (≈2 μM) was measured at room temperature in the presence of 20 μM bromocresol purple, o-cresol red, or o-cresol-phthaleine, depending on the pH range.

The proton uptake stoichiometries were measured in the presence of 100 μM ferrocene as electron donor to P⁺ and 300 μM ferrocyanide. The calibrations were performed by additions of known amounts of HCl (1 M stock; Merck). The proton uptake stoichiometries because of the formation of Q were measured in the presence of terbutryn (100 μM), which prevents the binding of Q_B.

The proton uptake by the PQ_AQ state (ΔH) is deduced from the measured value after one flash (ΔH) according to the following equation (21):

1

ΔH⁺ is the proton uptake by the RC in the absence of Q_B. The fraction of RCs without Q_B activity (δ) and the partition coefficient between the QQ_B and Q_AQ states (α) were determined from the P⁺Q → PQ_B charge recombination kinetics monitored at 430 nm. Depending on the strain, α varied from 0.02 to 0.5 and δ from 0.03 to 0.5 as pH is increased from 6 to 10. The occupancy of the Q_B site was routinely restored by the addition of 60 μM ubiquinone-6 (UQ₆).

Results

The Stoichiometries of Proton Uptake in the Q State (H⁺/Q).

The pH titrations of the H⁺/Q curves measured in the L209PT, L209PW, L209PE, L209PY, and L209PF mutants in the pH range 6–10.2 are shown in Fig. 2, together with the WT data. The WT H⁺/Q stoichiometry displays a notable proton uptake (≈0.30 H⁺/Q) at neutral pH and a significant proton uptake band (≈0.45 H⁺/Q) at high pH, centered at pH 9. In the L209 mutants, below pH 8, the H⁺/Q proton uptake curves are superimposable to that of the WT, within the experimental error. However, above pH 8, any of the introduced mutated side chains at position L209 cancels the high pH band observed in the WT.

Figure 2.

pH dependence of the stoichiometries of proton uptake by the PQ state in RCs of the WT (■), the L209PE (□), the L209PT (▿), the L209PY(○), the L209PW (⋄), and the L209PF (▵) mutants. Conditions: ≈2 μM RCs, 0.03% Triton X-100, 100 μM ferrocene, 300 μM ferrocyanide, 50 mM NaCl, 100 μM terbutryn, 20 μM dye (bromocresol purple, o-cresol red, or o-cresol-phthaleine, depending on the pH). The error bars reflect the respective experimental error of each set of measurements.

The Stoichiometries of Proton Uptake in the Q State (H⁺/Q).

Fig. 3 shows the pH titration curves of the H⁺/Q proton uptake in the WT and in the L209PE, L209PF, and L209PY mutants. The H⁺/Q measurements are focused on the RCs from these three mutants because their three-dimensional structures are available (45). The WT H⁺/Q stoichiometry is notable (≈0.35–0.55 H⁺/Q) at neutral pH. A significant proton uptake band (≈0.80 H⁺/Q) is observed at high pH, centered around pH 9.7.

Figure 3.

pH dependence of the stoichiometries of proton uptake by the PQ state in RCs of the WT (■), the L209PE (□), the L209PT (▿), the L209PY(○), the L209PW (⋄), and the L209PF (▵) mutants. Same conditions as in Fig. 2, except: 60 μM ubiquinone-6 (UQ₆) and no terbutryn present. The error bars reflect the respective experimental error of each set of measurements.

In the L209PE mutant, above pH 9, the H⁺/Q proton uptake curve is superimposable to that of the WT. However, at lower pH, the H⁺/Q value is significantly higher than in the WT. Indeed, a value of about 0.60–0.70 H⁺ is measured down to pH 7, below which the proton uptake increases to about 0.80 H⁺ at pH 6.

In the L209PF mutant, above pH 9, the H⁺/Q value is very similar, within the experimental error, to that measured in the WT or in the L209PE mutant. At neutral pH, the H⁺/Q stoichiometry is higher than in the L209PE mutant: 0.80 H⁺/Q are taken up in the pH range 7–9. This value increases up to 1.00 H⁺/Q below pH 7.

In the L209PY mutant, a similar pattern to the L209PF mutant is measured in the pH range 6–9, with a significant high proton uptake (H⁺/Q ≈ 0.80–0.90). However, above pH 9, the proton uptake values drop, H⁺/Q being equal to ≈0.80 at pH 9, but only ≈0.30 at pH 10.

Discussion

In the present paper, we have measured the proton uptake stoichiometries of RC mutants from Rb. sphaeroides in which L209Pro has been changed by site-directed mutagenesis to threonine, tryptophane, glutamate, phenylalanine, and tyrosine. The magnitude of proton uptake induced by the one-electron reduction of either of the two quinone electron acceptors (Q_A and Q_B) is an intrinsic observable of the electrostatic interactions associated with the redox function of the RC.

The high pH proton uptake band is commonly observed in the WT RCs from Rb. sphaeroides and Rb. capsulatus either on the Q or Q formation. This band disappears concomitantly in the H⁺/Q and H⁺/Q curves in all mutants reported so far in which L212Glu (situated in the Q_B pocket at more than 15Å from Q_A) was changed to a non-protonatable residue (11–14, 21). This high pH proton uptake band has been attributed to a change in the ionization state of L212Glu (11, 14, 18, 21). However, it is likely that this band more generally results from the cluster of strongly interacting acidic groups (L212Glu, L213Asp, L210Asp, and H173Glu) close to Q_B and also to extended hydrogen bond networks (12). Consistently, the AspL213→Asn substitution displaces also this band to lower pH (11). The presence of a high pH band is due to the cumulative effects of the strong pair-wise interactions within the cluster. Removing any member of the cluster (as observed when one acidic residue is changed to a non-ionizable residue) shifts to lower pH the highest pKa of both H⁺/Q and H⁺/Q curves, resulting into the apparent disappearance of the high pH signature of the cluster (P.S., L.B., and J. Lavergne, unpublished data).

We show here that all L209 mutations specifically suppressed the high pH proton uptake band on Q (Fig. 2) formation but not on Q formation (Fig. 3). Therefore, a complete understanding of the observed effects on both H⁺/Q and H⁺/Q uptake stoichiometries requires a more complex representation.

Protonation Events Triggered by the Q Formation.

The high pH band in the H⁺/Q stoichiometries is absent in the five mutants lacking L209Pro. The crystal structures of the L209PE, L209PY, and L209PF variants have been determined (45). In the crystallographic structure of the three variants, no changes in the protein backbone were observed, compared with the WT RC structure. The structural models of the variants show some structural modifications specific to each point-mutation. The structure of the L209PE mutant RC is superimposable to that of the WT except for the introduced glutamate side chain, which points toward the hydrogen-bonded water molecules (45) that connect Q_B to the cytoplasmic surface of the RC (2, 3). In the structure of the L209PY and L209PF mutants, both aromatic side chains are oriented away from the water chain and displace three surrounding side chains (L213Asp, L226Thr, and H173Glu) by up to 2.6 Å (45). In the structure of the L209PY variant, Q_B is shifted by ≈4 Å and is now located at a position similar to that reported for the WT reaction center under illumination (2, 3). In the L209PF variant, the electron density map reveals an intermediate Q_B position between the binding sites of the WT protein in the dark and that of the L209PY protein. In the L209PE reaction center, the binding site of Q_B remains unchanged compared with the WT structure (2, 3). These different structural effects, but resulting into similar pH dependence of the H⁺/Q stoichiometries in all L209 mutants, lead us to conclude that the absence of ProL209 per se—and not the introduced specific side chain—is responsible for the absence of the high pH band observed in the H⁺/Q proton uptake.

Protonation Events Triggered by the Q Formation.

In the L209PY, -F, and -E mutants, we do not observe the concomitant drop of the high pH band in the H⁺/Q curves, suggesting that no strong rearrangement within the Q_B cluster has occurred. However, in the L209PY mutant, we observe a slight acidic shift of the H⁺/Q high pH band (Fig. 3), as compared with the WT. This shift may be correlated to the observed position of Q_B in the structure of this RC variant in its neutral state, which is in the proximal position to the non-heme iron (45). This position has been suggested to require GluL212 and AspL213 to be protonated (28). This result would in turn reduce the strength of the interactions within the cluster in the L209PY mutant, consistently with the observed acidic shift of the H⁺/Q curve.

The different behavior of the H⁺/Q and H⁺/Q curves as regard to the high pH band suggests that, in the L209 mutants, the capacity of the Q_B cluster to respond to the Q formation is altered (Fig. 4). The rigid side chain of the proline might be of importance for the conformational coupling between the two quinone environments. The absence of L209Pro may soften this coupling, damp the conformational changes, and prevent its propagation into the Q_B site. Therefore, the protein dynamics appear to be critical to ensure the connection between the Q_A and Q_B environments.

Figure 4.

Scheme representing the response of the protein to the formation of Q and Q in the WT and in the L209P mutants. In the WT, the Q state triggers the uptake of substoichiometric protons by the cluster from the outside of the protein. In the case of the L209 mutants, the absence of L209Pro softens the protein, altering the connecting relays between the Q and the Q_B environment. In the Q state, in the mutants, a substantial additional amount of protons is taken from the bulk as a consequence of the disorganization of the hydrogen bond network.

In the L209PY, -F, and -E mutants, the major effect observed in the Q state is an increased proton uptake (0.6–1.0 H⁺/Q), as compared with the WT (0.4 H⁺/Q) below pH 9. The highest effect is observed in the L209PY and L209PF mutants.

A proposed mechanistic model to explain the amplitude of proton uptake in the RCs takes into account the movement of Q_B from the distal position in its neutral state to the proximal position in the Q state (46, 47). According to this hypothesis, Q_B in the proximal position is bound via hydrogen bond to GluL212, and stabilizes thereby the protonated form of the latter, whereas the Q_B in the distal position is likely to favor the anionic form of GluL212 as suggested by the calculations (28). Then the amount of protons that is bound at neutral and alkaline pH is expected to correspond to the fraction of Q_B in the distal position. However, in the L209PY mutant, where Q_B is found in the proximal position in its neutral state (45), the same high amount of proton is taken up as in the L209PF and L209PE variants for which Q_B is observed in an intermediate or in a WT-like position, respectively (45). This result does not support the above proposed mechanistic model. However, in the L209Tyr mutant, the Q_B “proximal” position may be tilted by 180° compared with its position in the WT. It could then be that the terms “proximal” and “distal” should not mean (as it is in the WT) the presence and absence of H-bond between Q_B and GluL212, respectively, in the special cases of the L209 mutations.

It has previously been suggested that, at neutral pH, i.e., in the region where we observe a notable increase of the H⁺/Q values in the mutants, protein surface groups are responsible for the proton binding at the first flash (48, 49). That result would not support the involvement of the ionization state of GluL212 in the observed phenomenon. In fact, the proton uptake is determined on a time average base of the exposure of the groups to the aqueous solution (40). If this mobility is favored by removal of the proline from the structure, then the exposure on a time-averaged base would increase.

Conclusion

The present paper provides evidence that interactions between the Q state and the environment of Q_B is mediated (at least in part) by conformational coupling. The current resolutions of the three-dimensional structures of the L209 mutants do not provide any structural changes, explaining for the likely modified dynamics of the protein. Fourier transform infrared spectroscopy that may investigate the global vibration modes of these networks, as well as molecular dynamics calculations, will help to identify the modified interactions in the mutants.

Abbreviations

P

primary electron donor, a noncovalently linked bacteriochlorophyll dimer

WT

wild type

L209PY

Pro L209 → Tyr

L209PF

Pro L209 → Phe

L209PE

Pro L209 → Glu

L209PW

Pro L209 → Trp

L209PT

Pro L209 → Thr

Q_B and Q_A

primary and secondary quinone

Rb.

Rhodobacter

RC

reaction center

H⁺/Q and H⁺/Q

proton uptake stoichiometries induced by the Q_A/Q or by the Q_B/Q redox transitions, respectively