Key role of proline L209 in connecting the distant
quinone pockets in the reaction center of
Rhodobacter sphaeroides
J. Tandori†‡, P. Maroti‡, E. Alexov§, P. Sebban†¶, and L. Baciou†

†Centre de Ge´ne´tique Mole´culaire, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France; ‡Department of Biophysics, University ofSzeged, H-6722, Szeged, Hungary; and §Howard Hughes Medical Institute and Biochemistry Department, Columbia University, New York, NY 10032 Edited by Hartmut Michel, Max Planck Institute for Biophysics, Frankfurt, Germany, and approved February 25, 2002 (received for review June 28, 2001) Photosynthetic bacterial reaction centers convert light excitation
side of the complex. The electron is then transferred from QϪA to into chemical free energy. The initial electron transfer leads to the
a secondary quinone QB within 10–100 ␮s (5–7). Both QA and consecutive semireductions of the primary (QA) and secondary (QB)
QB are deeply buried within the reaction center protein. The role quinone acceptors. The Q؊
formations induce proton
of the protein in stabilizing the redox species is essential to uptake from the bulk. Their magnitudes (H؉͞Q؊
and H؉͞QB ,
ensure high forward electron transfer rates and to prevent charge respectively) probe the electrostatic interactions within the com-
recombinations to occur. Although chemically identical, QA and plex. The pH dependence of H؉͞Q؊
A and H؉͞QB were studied in five
QB behave differently. QA, bound to the M subunit in a relatively single mutants modified at the L209 site (L209P3 F,Y,W,E,T). This
hydrophobic pocket, functions as one electron acceptor and is residue is situated at the border of a continuous chain of water
never protonated. At variance, QB, bound to the L subunit, is molecules connecting QB to the bulk. In the wild type (WT), a
surrounded by charged and polar residues and behaves as a proton uptake band is present at high pH in the H؉͞Q؊
A and H؉͞QB
two-electron gate, accepting sequentially two electrons from QA curves and is commonly attributed to a cluster of acidic groups
and two protons from the cytoplasm. In chromatophores, the situated nearby Q
B. In the H؉͞QA curves of the L209 variants, this
semiquinone QB can bind a proton below pH 6.8 (8). However, band is systematically absent but remains in the H؉͞Q؊
B curves.
in isolated RCs, the semiquinones are not directly protonated Moreover, notable increase of H؉͞Q؊
is observed in the L209
but induce the shift of the pKas of ionizable interacting residues, mutants at neutral pH as compared with the WT. The large effects
which results in substoichiometric proton uptake by the protein observed in all L209 mutants are not associated with significant
(9, 10). The proton uptake may occur through a number of water structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L.
molecules and protonatable amino acid residues situated be- & Fritzsch, G. Biochemistry (2001) 40, 4253– 4260). Our data suggest
tween QB and the cytoplasmic surface. Of main interest is that, in the L209 mutants, the QB cluster does not respond to the
to identify the dynamical and structural role of the protein A formation as observed in the WT. We propose that, in the
that contributes to the stability of the QA and QB states and mutants, removal of the rigid proline L209 breaks a necessary
to the energetic and functional connections of their respective hydrogen bonding connection between the quinone sites. These
findings suggest an important role for structural rigidity in ensur-
The partial protonation events that occur on QϪ ing a functional interaction between quinone binding sites.
formations have been studied by spectroscopic techniques by using site-directed mutagenesis (9–22) and by numerical meth- The biological role of bacterial reaction center (RC) mem- ods (23–30). There is a general agreement that the major
brane proteins is to convert light energy into chemical free response of the protein to the QϪB formation is the change of the energy. The sequential absorption of two photons by the system ionization state of acidic residues situated in the QB environ- results into the production of the doubly reduced and doubly ment. These residues (L212Glu, L213Asp, L210Asp, and protonated form of the ultimate electron acceptor of the com- H173Glu) form a strongly interacting cluster, buffering as a plex, a ubiquinone (QB). The formed QBH2 molecule then whole the redox state changes of the quinones. The signature of delivers its reducing power to the cytochrome bc1 complex, this cluster is a notable proton uptake band (Ϸ0.8 Hϩ͞QϪB) at low resulting in the release of protons on the periplasmic side of the pH and at high pH. The high pH proton uptake band system- membrane. The resulting transmembrane proton gradient drives atically disappears in all modified RCs reported so far, where ATP synthesis through the ATP synthase. The reduction of Q L212Glu is absent (11, 14, 21), in both the QB and QA states, coupled to the uptake of protons from the bulk is an important suggesting that the electrostatic effect of this cluster might be step shared by many systems involved either in photosynthesis or extended to the QA environment (12, 13, 15, 18, 31). The long range electrostatic effect between the QA and QB pocket haS The three-dimensional structure of the reaction center from also been proposed on the basis of electrostatic calculations the purple photosynthetic bacterium Rhodobacter (Rb.) spha- (24–26, 30, 32, 33). The existence of electrostatic and͞or eroides is known at atomic resolution (2–4). Three subunits with conformational-mediated interactions between the two quinone a total molecular weight of about 100 kDa compose these RCs.
protein pockets of the bacterial reaction centers have been The transmembrane L and M subunits carry the nine pigments and cofactors: four bacteriochlorophylls, two bacteriopheophyt- ins, two ubiquinones 10, and one non-heme iron atom. The third This paper was submitted directly (Track II) to the PNAS office.
subunit, H, caps the reaction center on the cytoplasmic side of Abbreviations: P, primary electron donor, a noncovalently linked bacteriochlorophyll the membrane. The initial photochemical event induced by the dimer; WT, wild type; L209PY, Pro L209 3 Tyr; L209PF, Pro L209 3 Phe; L209PE, Pro L209 3 absorption of a photon is the creation of the singlet excited state Glu; L209PW, Pro L209 3 Trp; L209PT, Pro L209 3 Thr; QB and QA, primary and secondary of a dimer of bacteriochlorophylls (P3P*), which constitutes the quinone; Rb., Rhodobacter; RC, reaction center; Hϩ͞QA and Hϩ͞QB , proton uptake stoi-chiometries induced by the Q primary electron donor. P* is a strong reducing species that ¶To whom reprint requests should be addressed. E-mail:
initiates the electron transfer reaction through the protein. In about 200 ps, the charge separation occurs between P and the The publication costs of this article were defrayed in part by page charge payment. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
first quinone electron acceptor, QA, situated on the cytoplasmic §1734 solely to indicate this fact.
6702– 6706 ͉ PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10͞cgi͞doi͞10.1073͞pnas.092327799 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 ␮g͞ml) and tetracycline (2 ␮g͞ml). 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 280͞802 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 Rb. sphaeroides RC structure showing the two quinones Q below 10 ␮M. The proton uptake by the RCs was measured on the QB cluster of acidic residues, and the L209P mutation site. Water molecules a home made spectrophotometer by following the absorption connecting QB to the bulk are also represented. Coordinates were taken from 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 unbuf- fered signal. The proton uptake by the RCs (Ϸ2 ␮M) was evoked in experimental works (34–41). The three-dimensional measured at room temperature in the presence of 20 ␮M structure of the protein reveals a large hydrogen bond network bromocresol purple, o-cresol red, or o-cresol-phthaleine, de- in the quinone proteic region involving numerous ionizable, polar residues and water molecules (2, 3, 42, 43). It is therefore The proton uptake stoichiometries were measured in the of particular interest to investigate to what extent this widely spread out hydrogen bond network is involved in balancing the ␮M ferrocene as electron donor to Pϩ and 300 BIOPHYSICS
proton concentration over the key amino acid residues in the two ␮M ferrocyanide. The calibrations were performed by additions of known amounts of HCl (1 M stock; Merck). The proton We report here proton uptake measurements on QϪ uptake stoichiometries because of the formation of QA were formations in RC mutants from Rb. sphaeroides in which measured in the presence of terbutryn (100 ␮M), which prevents L209Pro has been changed by site-directed mutagenesis to threonine (L209PT), tryptophane (L209PW), glutamate The proton uptake by the PQAQB state (⌬HQAQBϪ) is deduced (L209PE), phenylalanine (L209PF), and tyrosine (L209PY).
from the measured value after one flash (⌬Hobs) according to the L209Pro is situated at the border of a chain of hydrogen-bonded water molecules (Fig. 1) that connects QB to the cytoplasmic ⌬H ϩ Ϫ ͓␦ ϩ ␣͑1 Ϫ ␦͔͒⌬H ϩ 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).
⌬HϩϩQAϪ is the proton uptake by the RC in the absence of QB.
The amino acid exchange in the L209PE and L209PT mutants The fraction of RCs without QB activity (␦) and the partition functionally mimics the kinetics of the wild-type (WT) RCs (44).
In the L209PE reaction center, the structure remains unchanged determined from the PϩQϪB 3 PQB charge recombination compared with the WT structure, except the introduced carbox- kinetics monitored at 430 nm. Depending on the strain, ␣ varied ylic side chain of GluL209 located within the water chain (45).
from 0.02 to 0.5 and ␦ from 0.03 to 0.5 as pH is increased from In the L209PW, L209PY, and L209PF variants, the spectroscopic 6 to 10. The occupancy of the QB site was routinely restored by analysis suggested a modification of the network of hydrogen the addition of 60 ␮M ubiquinone-6 (UQ6).
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 The Stoichiometries of Proton Uptake in the Q؊
A State (H؉͞QA ). The
residues (AspL213, ThrL226, and GluH173) and more distantly pH titrations of the Hϩ͞QϪA curves measured in the L209PT, have affected the QB position (45). Despite these different L209PW, L209PE, L209PY, and L209PF mutants in the pH structural changes, the similar proton uptake patterns measured range 6–10.2 are shown in Fig. 2, together with the WT data. The here for all mutants suggest a crucial role for proline L209 in WT Hϩ͞QϪA stoichiometry displays a notable proton uptake connecting both quinone environments.
(Ϸ0.30 Hϩ͞QϪA) at neutral pH and a significant proton uptake band (Ϸ0.45 Hϩ͞QϪA) at high pH, centered at pH 9. In the L209 Materials and Methods
mutants, below pH 8, the Hϩ͞QϪA proton uptake curves are Bacterial Strains and Growth Conditions. The design of the Rb.
superimposable to that of the WT, within the experimental sphaeroides WT or mutant strains harboring pufL mutation on error. However, above pH 8, any of the introduced mutated side PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 6703
pH dependence of the stoichiometries of proton uptake by the PQϪ pH dependence of the stoichiometries of proton uptake by the PQϪ state in RCs of the WT (■), the L209PE (ᮀ), the L209PT (ƒ), the L209PY(E), the state in RCs of the WT (■), the L209PE (ᮀ), the L209PT (ƒ), the L209PY(E), the L209PW (छ), and the L209PF (‚) mutants. Conditions: Ϸ2 ␮M RCs, 0.03% L209PW (छ), and the L209PF (‚) mutants. Same conditions as in Fig. 2, except: Triton X-100, 100 ␮M ferrocene, 300 ␮M ferrocyanide, 50 mM NaCl, 100 ␮M 60 ␮M ubiquinone-6 (UQ6) and no terbutryn present. The error bars reflect the terbutryn, 20 ␮M dye (bromocresol purple, o-cresol red, or o-cresol- respective experimental error of each set of measurements.
phthaleine, depending on the pH). The error bars reflect the respectiveexperimental error of each set of measurements.
reduction of either of the two quinone electron acceptors (QA chains at position L209 cancels the high pH band observed in the and QB) 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 The Stoichiometries of Proton Uptake in the Q؊
B State (H؉͞QB ). Fig.
3 shows the pH titration curves of the Hϩ͞QϪ QA or QB formation. This band disappears concomitantly in the the WT and in the L209PE, L209PF, and L209PY mutants. The Hϩ͞QA and Hϩ͞QB curves in all mutants reported so far in which L212Glu (situated in the QB pocket at more than 15Å from B measurements are focused on the RCs from these three mutants because their three-dimensional structures are available QA) was changed to a non-protonatable residue (11–14, 21). This high pH proton uptake band has been attributed to a change in B stoichiometry is notable (Ϸ0.35– 0.55 the ionization state of L212Glu (11, 14, 18, 21). However, it is B ) at neutral pH. A significant proton uptake band (Ϸ0.80 likely that this band more generally results from the cluster of Hϩ͞QϪB) is observed at high pH, centered around pH 9.7.
strongly interacting acidic groups (L212Glu, L213Asp, L210Asp, In the L209PE mutant, above pH 9, the Hϩ͞QϪB proton uptake curve is superimposable to that of the WT. However, at lower networks (12). Consistently, the AspL2133Asn substitution pH, the Hϩ͞QϪB value is significantly higher than in the WT.
displaces also this band to lower pH (11). The presence of a high Indeed, a value of about 0.60–0.70 Hϩ is measured down to pH pH band is due to the cumulative effects of the strong pair-wise 7, below which the proton uptake increases to about 0.80 Hϩ at interactions within the cluster. Removing any member of the cluster (as observed when one acidic residue is changed to a In the L209PF mutant, above pH 9, the Hϩ͞QϪB value is very non-ionizable residue) shifts to lower pH the highest pKa of both similar, within the experimental error, to that measured in the Hϩ͞QϪ and Hϩ͞QϪ curves, resulting into the apparent disap- WT or in the L209PE mutant. At neutral pH, the Hϩ͞QϪ pearance of the high pH signature of the cluster (P.S., L.B., and stoichiometry is higher than in the L209PE mutant: 0.80 Hϩ͞QϪB are taken up in the pH range 7–9. This value increases up to 1.00 We show here that all L209 mutations specifically suppressed the high pH proton uptake band on Q (Fig. 2) formation but In the L209PY mutant, a similar pattern to the L209PF mutant not on QϪ formation (Fig. 3). Therefore, a complete under- is measured in the pH range 6–9, with a significant high proton standing of the observed effects on both Hϩ͞QϪ uptake (Hϩ͞QϪB Ϸ 0.80–0.90). However, above pH 9, the proton uptake stoichiometries requires a more complex representation.
uptake values drop, Hϩ͞QϪB being equal to Ϸ0.80 at pH 9, but Protonation Events Triggered by the Q؊
A Formation. The high pH
band in the Hϩ͞QϪA stoichiometries is absent in the five mutants Discussion
lacking L209Pro. The crystal structures of the L209PE, L209PY, In the present paper, we have measured the proton uptake and L209PF variants have been determined (45). In the crys- stoichiometries of RC mutants from Rb. sphaeroides in which tallographic structure of the three variants, no changes in the L209Pro has been changed by site-directed mutagenesis to protein backbone were observed, compared with the WT RC threonine, tryptophane, glutamate, phenylalanine, and tyrosine.
structure. The structural models of the variants show some The magnitude of proton uptake induced by the one-electron structural modifications specific to each point-mutation. The 6704 ͉͞cgi͞doi͞10.1073͞pnas.092327799
Scheme representing the response of the protein to the formation of QϪ A and QB in the WT and in the L209P mutants. In the WT, the QA 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 QϪ A and the QB environment. In the QB 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.
structure of the L209PE mutant RC is superimposable to that of the WT except for the introduced glutamate side chain, which regard to the high pH band suggests that, in the L209 mutants, BIOPHYSICS
points toward the hydrogen-bonded water molecules (45) that B cluster to respond to the QA formation is connect QB to the cytoplasmic surface of the RC (2, 3). In the altered (Fig. 4). The rigid side chain of the proline might be of structure of the L209PY and L209PF mutants, both aromatic importance for the conformational coupling between the two side chains are oriented away from the water chain and displace quinone environments. The absence of L209Pro may soften this three surrounding side chains (L213Asp, L226Thr, and coupling, damp the conformational changes, and prevent its H173Glu) by up to 2.6 Å (45). In the structure of the L209PY propagation into the QB site. Therefore, the protein dynamics variant, QB is shifted by Ϸ4 Å and is now located at a position appear to be critical to ensure the connection between the Q similar to that reported for the WT reaction center under illumination (2, 3). In the L209PF variant, the electron density In the L209PY, -F, and -E mutants, the major effect observed map reveals an intermediate QB position between the binding in the QϪ state is an increased proton uptake (0.6–1.0 Hϩ͞QϪ), sites of the WT protein in the dark and that of the L209PY as compared with the WT (0.4 Hϩ͞QϪ) below pH 9. The highest protein. In the L209PE reaction center, the binding site of Q effect is observed in the L209PY and L209PF mutants.
remains unchanged compared with the WT structure (2, 3).
A proposed mechanistic model to explain the amplitude of These different structural effects, but resulting into similar pH proton uptake in the RCs takes into account the movement of lead us to conclude that the absence of ProL209 per se—and not QB from the distal position in its neutral state to the proximal the introduced specific side chain—is responsible for the absence position in the QB state (46, 47). According to this hypothesis, of the high pH band observed in the Hϩ͞QϪ QB in the proximal position is bound via hydrogen bond to GluL212, and stabilizes thereby the protonated form of the Protonation Events Triggered by the Q؊
B in the distal position is likely to favor the B Formation. In the L209PY,
-F, and -E mutants, we do not observe the concomitant drop of 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 the L209PY mutant, we observe a slight acidic shift of the position. However, in the L209PY mutant, where QB is found in the proximal position in its neutral state (45), the same high B high pH band (Fig. 3), as compared with the WT. This shift may be correlated to the observed position of Q amount of proton is taken up as in the L209PF and L209PE structure of this RC variant in its neutral state, which is in the variants for which QB is observed in an intermediate or in a proximal position to the non-heme iron (45). This position has WT-like position, respectively (45). This result does not support been suggested to require GluL212 and AspL213 to be proton- the above proposed mechanistic model. However, in the ated (28). This result would in turn reduce the strength of the L209Tyr mutant, the QB ‘‘proximal’’ position may be tilted by interactions within the cluster in the L209PY mutant, consis- 180° compared with its position in the WT. It could then be that tently with the observed acidic shift of the Hϩ͞QϪB curve.
the terms ‘‘proximal’’ and ‘‘distal’’ should not mean (as it is in the PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 6705
WT) the presence and absence of H-bond between QB and part) by conformational coupling. The current resolutions of the GluL212, respectively, in the special cases of the L209 mutations.
three-dimensional structures of the L209 mutants do not provide It has previously been suggested that, at neutral pH, i.e., in the any structural changes, explaining for the likely modified dy- region where we observe a notable increase of the Hϩ͞QϪB values namics of the protein. Fourier transform infrared spectroscopy in the mutants, protein surface groups are responsible for the that may investigate the global vibration modes of these net- proton binding at the first flash (48, 49). That result would not support the involvement of the ionization state of GluL212 in the works, as well as molecular dynamics calculations, will help to observed phenomenon. In fact, the proton uptake is determined identify the modified interactions in the mutants.
on a time average base of the exposure of the groups to the aqueous solution (40). If this mobility is favored by removal of We thank Marilyn Gunner and Jeroˆme Lavergne for stimulating dis- the proline from the structure, then the exposure on a time- cussions, and Tania Bizouarn for careful reading of the manuscript. E.A.
thanks Barry Honig for the support during this work. This work was supported by the Centre National de la Recherche Scientifique. J.T. was Conclusion
in part supported by a BALATON grant [Hungarian͞French Ministe`re The present paper provides evidence that interactions between des Affaires Etrange`res (No. 00834)], and by a North Atlantic Treaty the QϪA state and the environment of QB is mediated (at least in Organization collaborative research grant (LST.CLG 975754).
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