J. Phys. Chem. 1996, 100, 6863-6866
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Monodentate vs Bidentate Binding of Lanthanide Cations to PO2- in Bacteriorhodopsin Jennifer A. Griffiths,† Tina M. Masciangioli,† Cecile Roselli,‡ and M. A. El-Sayed*,† School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, and Section de Bioenergetique, DBCM, URA CNRS, CEA Saclay, Gif-Sur-YVette Cedex, France ReceiVed: NoVember 10, 1995; In Final Form: January 22, 1996X
The frequency difference between the symmetric and antisymmetric stretching vibration of PO2- in phosphatidylglycerol phospate (PGP) is used to differentiate between monodentate and bidentate binding of these groups to metal cations in the membrane of bacteriorhodopsin (bR) and phosphatidylglycerol phospate. The binding of Ca2+ to PGP is found to have a frequency difference corresponding to monodentate binding. The symmetric and antisymmetric PO2- bands in bR show similar frequency shifts upon Ca2+ binding, which is independent of pH. This suggests that Ca2+ has a monodentate type binding with the PO2- in bR. In contrast, the PO2- symmetric and antisymmetric frequencies of PGP complexes with trivalent lanthanide cations with higher charge density (Ho3+ and Dy3+) are observed to have smaller separations and to increase their separation with increasing pH toward the value observed for Ca2+ binding. Lanthanide cations (Ho3+, Dy3+, Eu3+, Nd3+, and La3+) binding in bR at pH 4 show small frequency separations that are observed to have similar frequency shifts with pH, the magnitude of which is dependent on the cation. It is proposed that at low pH the lanthanide cations with higher charge density have bidentate binding to bR, while at high pH, complexation with the OH- competes with one of the oxygens of the PO2- for the binding of the lanthanide ion thus changing the bidentate to monodentate type binding.
Introduction Bacteriorhodopsin (bR), which is the only protein found in the purple membrane (PM) of Halobacterium salinarium, is, like the photosynthetic reaction center, a natural photosynthetic system (for reviews see refs 1-3). bR contains as a chromophore an all-trans retinal, which is covalently bound via a Schiff base linkage to the -amino group of Lys216.4-7 Upon absorbing a photon, the protein undergoes a photocycle in which the intermediates shown below have been identified. The final step in the photocycle returns bR to its native structure from which it can begin the cycle again. hν
-H+
+H+
bR570 9 8 J 98 L550 9 8 M412 9 8 N550 f 0.4 ps 625 2 µs 60 µs ms O640 f bR570 This photocycle, which occurs in the absence of oxygen, results in the translocation of protons from the interior to the extracellular side of the purple membrane. The proton gradient created by this mechanism is used by the bacteria for metabolic processes such as ATP synthesis.8 bR has about 4 moles of Ca2+ and Mg2+ bound per mole of bR at ∼pH 6 in the wild type protein.9 Removal of these metal cations changes the purple color of wild type bR (λmax ) 570 nm) to blue (λmax ) 604 nm).9 The consequence of the removal of these cations is that the blue membrane does not undergo a complete photocycle and thus does not pump protons.9 There are two models for the location of the cation-binding sites and the mechanism through which they affect the color and function of the purple membrane. In the model of Szundi and Stoeckenius,10 the metal cations are bound to the membrane surface as free positive charges. The high surface pH generated on the surface by the presence of the cations (the Gouy-Chapman †
Georgia Institute of Technology. CEA Saclay. X Abstract published in AdVance ACS Abstracts, March 15, 1996. ‡
0022-3654/96/20100-6863$12.00/0
effect) is sufficient to keep the counterion (aspartate) deprotonated and to allow the amino acid to act as a proton acceptor during the photocycle. Conversely, several groups11 propose that there are specific binding sites within the protein between specific amino acid residues, suggesting a more direct interaction between the retinal chromophore and the metal cation(s). Multiple spectroscopic studies12,13 have reached the conclusion that there are more than two classes of cation-binding sites in the deionized blue membrane, with different binding affinities and therefore different binding environments. Potentiometric titration13a studies of blue bR with Ca2+ concluded that there are two high-affinity Ca2+ binding sites within the active site of the protein. The results of the potentiometric studies on mutants13b and on bR with the C-terminus cleaved13c suggest the presence of two metal cations within the protein whose binding constants are sensitive to charges on Asp 85 and Asp 212 as well as to other low-affinity binding sites on the surface. The results of FTIR studies of Ca2+-regenerated bR and D85N mutant gave indication of the coupling between Ca2+ and the O- of Asp 85.14 More recent theoretical simulations15 of the two-photon spectrum of the retinal in bR found good agreement with experimental results if the Ca2+ is bound to the O- atoms of Asp 85 and Asp 212. The authors suggest15 that this cation is the second high-affinity cation observed in the potentiometric titrations. It has been observed, however, that not all metal cations interact with bR in the same manner. For example, Hg+ will not regenerate the purple color of bR from the blue deionized bR. Drachev et al.16 found that La3+ has an inhibitory effect on the decay of the M intermediate in the bR photocycle. As the pH is increased from 5.5 to 8.0, this inhibitory effect is increased until at pH 8.0 the M decay is about 103 times slower than in the wild type bR.16 Chemical modification16 of the carboxyl groups in which a positive charge is attached eliminates this inhibitory effect, suggesting that the La3+ may be bound in part to carboxyl groups. In addition, lanthanide cations induce strong aggregation of the deionized purple membrane, presum© 1996 American Chemical Society
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Griffiths et al.
ably because of neutralization of the negatively charged membrane surface. In order to better understand both the mechanism and the differences in the binding mechanism of different cations to bR, we have used infrared spectroscopy to study the deionized purple membrane structure under varying metal cations, cation concentration, and pH. In order to isolate effects of cation binding to the phospholipids, we also study lanthanide complexes with the major charged lipid components of the purple membrane (phosphatidylglycerol and phosphatidylglycerol phosphate). These results are discussed in reference to previous results in our laboratory on the influence of these parameters on the tertiary structure of the protein.17 In addition, the mechanism of lanthanide inhibition of the bR photocycle is discussed. Experimental Section Purple membrane was prepared from the Halobacterium salinarium strain ET1001 as described previously.17 Blue membrane was prepared by passing the purple membrane over a cation exchange column (Bio-Rad). The final pH of the blue membrane was ∼4 with λmax ) 604 nm. All samples were suspended in nanopure water (resistivity 10-18 MΩ cm) and kept in plastic tubes to avoid contamination from other cations. Concentration of the protein was determined using the following extinction coefficients: 570 ) 63 000 cm-1 M-1 and 608 ) 60 000 cm-1 M-1. The purple membrane was regenerated from the deionized blue membrane by addition of solutions of EuCl3 (10mM), DyCl3 (10mM), or HoCl3 (10mM) (Sigma, 99.99% purity). Titrations of the regenerated samples were done using NaOH (10 mM). FTIR samples were prepared by drying a drop of protein solution on an AgCl window in a dessicator for 5 h. Phosphatidylglycerol phosphate and phosphatidylglycerol (Avanti Polar Lipids) dissolved in choloroform (≈10 mg/mL) were equilibrated with the lanthanide solution for 10 h, and samples were prepared as for the bR samples. Spectra were recorded on a Nicolet Magna-IR 550 spectrometer at 2 cm-1 resolution with 100 scans per sample, triangular apodization, and no zero filling. The AgCl window with the sample was placed in a transmission configuration using a universal sample holder. Background spectra were collected using only the AgCl window. All spectra were collected at ambient temperature. Results A. FTIR Studies of Lanthanide Complexes with Phosphatidylglycerol Phosphate (PGP) and Phosphatidylglycerol. Figure 1A shows the FTIR spectra of PGP titrated with Ho3+. There is clearly a shift of the symmetric stretching peak (≈1080 cm-1) to increasingly higher wavenumbers with increasing Ho3+ content. In addition, the antisymmetric stretching peak (≈1200 cm-1) shifts to lower wavenumbers with increasing Ho3+. Figure 1B shows the FTIR spectra of PGP titrated with Ca2+. Addition of Ca2+ results in a shift of the symmetric stretching peak similar to that for Ho3+ (Figure 1). However, the antisymmetric stretching peak of the Ca2+ shifts very slightly to higher wavenumbers, in contrast to the case of Ho3+. These peak shifts are observed to occur continuously as the ratio of cation to lipid increases, i.e., there is no threshold ratio of cation to lipid below which no peak shift is observed. B. FTIR Studies of Lanthanide-Regenerated Deionized bR. Parts a and b of Figure 2 show the IR spectra of Ho3+and Ca2+-regenerated bR in the 900-1400 cm-1 region for both low and high pH samples of each cation. In each part the FTIR spectrum of native bR is shown for comparison. The assignment
Figure 1. (A) FTIR spectra for Ho3+ complexed with phosphatidylglycerol phosphate (PGP) in the phosphate stretching region (1300850 cm-1) with varying Ho3+:lipid ratios: (‚‚‚) 0.5:1; (- - -) 1.0:1; (s); 2:1. (B) FTIR spectra for Ca2+ complexed with PGP in varying ratios: (‚‚‚) 1:1; (- - -) 4:1; (s) 10:1. In parts A and B the arrows indicate the direction of shift of the antisymmetric (≈1200cm-1) and symmetric (≈1080 cm-1) phosphate stretching peaks. In the Ho3+/PGP complex the peaks shift to smaller wavenumber separation. In the Ca2+/PGP complex the two peaks both shift to higher wavenumbers.
of the infrared peaks in this region has been made by Rothschild et al.18 Of predominant interest for our case is the PO2symmetric and antisymmetric stretching bands located at 1095 and 1237 cm-1, respectively in the purple membrane. As observed in our experiments on the titration of PGP with lanthanide cations described above, separation between the two bands is sensitive to the nature and concentration of metal cations bound to the lipid, and thus they could potentially act as an indicator for any changes in lipid environment upon calcium or lanthanide cation binding in bacteriorhodopsin. As seen in Figure 2A, there is a significant change in band shape upon addition of lanthanide cations to deionized bR both in the broad band centered at about 1240 cm-1 and in the 1095 cm-1 region band at low pH. In particular, it appears that there is an increase in the intensity of the absorbance in the 1160-1280 cm-1 region as well as in the 1100 cm-1 region. For Ca2+ regeneration a similar change in band shape is observed (Figure 2B) for the 1100 cm-1 region. However, there is no change in the band shape in the 1160-1280 cm-1 region as observed for the lanthanide regenerated samples. Instead, there is an increase in the peak intensity in the region of the PO2- antisymmetric region at 1237 cm-1. The changes in the FTIR spectra upon lanthanide cation binding are observed to be pH dependent. At higher pH the lanthanide FTIR spectrum in the phospholipid stretching regions
Binding of Lanthanide Cations to PO2-
J. Phys. Chem., Vol. 100, No. 16, 1996 6865
Figure 4. Plot of the ratio of the intensities of the FTIR peaks at 1172 cm-1/1200cm-1 for different lanthanide cations at a ratio of 2:1 ion to bbR. The ratio indicates the relative magnitude of the shift of the antisymmetric PO2- stretching peaks for different lanthanide ions. The influence of lanthanide ions on the phosphate stretching increases with increasing atomic number.
1200cm-1 is plotted for each ion investigated at a ratio of 2:1 ion to bR. As illustrated, at the same cation to bR ratio Ho3+ induces a much stronger change in band shape as compared, for example, to Nd3+. In addition, it is observed that the changes in the peaks described above are continuous with increasing amounts of cation. Discussion
Figure 2. FTIR spectra of blue deionized bR (bbR) regenerated with varying ratios of ions:bbR. For Ho3+-regenerated bbR in part A, the ratios are (‚‚‚) 0.5:1, (- - -) 1.0:1, (s) 2:1. For Ca2+-regenerated bbR in part B, the ratios are (‚‚‚) 1:1, (- - -) 2.0:1, (s) 4:1. For the Ho3+ sample there is an increase in intensity in the 1160-1280 cm-1 region as well as in the 1100 cm-1 region. In contrast, there is no change in the FTIR spectra with increased Ca2+ binding.
Figure 3. pH dependence of the FTIR spectra of Ho3+-regenerated bbR at 2:1 Ho3+ to bbR. At pH 4.5 (- - -) there are large differences in the 1160-1280 and 1100 cm-1 regions compared to wild type bR (s), which is shown for comparison. As the pH is increased, there is a continual shift of the phosphate stretching peaks until at pH 10.4 (‚‚‚) the features of the Ho3+ and wild type bR spectra are similar.
changes to more closely resemble the band shape of native bR. This is clearly shown for the case of Ho3+ in Figure 3. The effect of the lanthanide cations increases with increasing charge density. This is clearly illustrated in Figure 4 in which the ratio of the intensities of the FTIR peaks at 1172 and
Much effort has been put into the investigation of the conformation and dynamics of phospholipids in bilayers. Cations are known to induce strong perturbations on the lipid bilayer (e.g., changes in lipid packing structure and membrane fusion) through binding interaction with the phospholipid head group. We have observed that binding of the lanthanide series cations shifts the antisymmetric PO2- band from ≈1200 to lower frequencies, while the symmetric PO2- band shifts from ≈1080 cm-1 to higher frequencies, resulting in a smaller frequency difference between the symmetric and antisymmetric PO2stretching peaks. The magnitude of this change is dependent on the cation for a fixed mole ratio. This is characteristic of bidentate coordination of the lanthanide cation with oxyanions having equivalent stretching modes.19 For the Ca2+/PGP system it is observed that the symmetric PO2- peak shift from ≈1080 cm-1 to lower frequency, analogous to the shift observed for the lanthanide system. However, the PO2- antisymmetric band shifts from 1200 to a slightly higher frequency, i.e., in the Ca2+/ lipid system the PO2- symmetric and antisymmetric peaks shift in the same direction, making the separation between the peaks larger than in bidentate binding. This suggests that calcium forms a monodentate complex with the lipid. These results are consistent with previous results on the binding of Ca2+ and Tb3+ to the neutral lipid dimysterol phosphatidyl choline.20 The FTIR shifts observed here for the lanthanide- and calcium-regenerated bR samples are consistent with the FTIR shifts observed above for the lipid/cation system. The shifts are similar. This strongly suggests that there is some binding of the lanthanide to a binding site on the phospholipid head group. This is consistent with the 31P NMR experiments by Roux et al.,21 which have shown that the titration of the blue membrane with Nd3+ cations resulted in an increase in the chemical shift anisotropy and paramagnetic line broadening of the phosophorous resonance in the phospholipid phosphate groups. The authors suggest that there is thus a relationship, either spatially or structurally, between the phospholipid head groups and the cation-binding sites in purple membrane. The fact that these effects were observed after the addition of 2:1 or higher Nd3+:bR ratio was pointed out by our group previ-
6866 J. Phys. Chem., Vol. 100, No. 16, 1996 ously22 to support the assignment13 that the low-affinity sites are indeed surface type sites. In addition, the decrease in the frequency separation of the symmetric and antisymmetric bands suggests that the lanthanide cations have a multiple complex binding in bR. The observed change in the FTIR spectrum of the lanthanideregenerated bR to more closely mimic that of native bR at higher pH could be due to the replacement of a phospholipid ligand to the lanthanide with OH-. This would effectively change the binding of the lanthanide cations from bidentate to monodentate, more closely mimicking Ca2+ binding in the system. Previous results have determined that lanthanum cations inhibit the decay of the M412 intermediate.16 Drachev et al.23,24 have also determined that other tri- and divalent cations also have an inhibitory effect on the M412 intermediate. In this work, the authors observe pH effects that depend on the ion and its region of hydrolysis. In general, the lower the region of hydrolysis of the ion, the lower the pH at which significant inhibition occurs. The authors hypothesize that the ions form a mixed coordination compound that includes protein groups (i.e., carboxyl groups) and OH- ions. Whether or not the bidentate binding observed here causes the observed inhibition of the M decay or another type of binding site involving the binding to amino acids (or amino acids and the PO2- groups) cannot be concluded at this time. An X-ray examination25 of the complexation of lanthanide ions with deionized blue bR has shown a change in the hexagonal structure of bR. The native hexagonal structure was regained as the pH increased. The pH at which the conversion took place depended on the charge density of the lanthanide ion. In order to change the bR tertiary structure, the relative positions of the helices need to change. In this work,25 complex binding was proposed to take place between the lanthanide ions and the amino acids (in particular those on different helices) or amino acids and the PO2- groups. A relation is derived25 between the pH at which the conversion is observed and the binding constants of the metal ion to OH- and to O- of the amino acid or the PO2- group of the lipid head groups. Acknowledgment. The authors thank the Department of Energy (Grant No. DEFG03-88ER13828) for support of this work. References and Notes (1) Stoeckenius, W.; Lozier, R.; Bogomolni, R. A. Biochem. Biophys. Acta 1979, 505, 215.
Griffiths et al. (2) Stoeckenius, W. Acc. Chem. Res. 1980, 13, 337. (3) Mathies, R. A.; Lin, S. W.; Ames, J. B.; Pollard, W. T. Annu. ReV. Biophys. Biophys. Chem. 1991, 20, 491. (4) Oesterhelt, D.; Stoeckenius, W. Nature (London) New Biol. 1971, 233, 149. (5) Khorana, H. G. J. Biol. Chem. 1988, 263, 7439. (6) Bridgen, J.; Walker, I. Biochemistry 1976, 15, 792. (7) Heyn, M. P.; Braoun, D.; Dencher, N. A.; Fahr, A.; Holz, M.; Lindau, M.; Seiff, F.; Wallat, I.; Westerhausen, J. Ber. Bunsenges. Phys. Chem. 1988, 92, 1045. (8) (a) Dencher, N.; Wilms, M. Biophys. Struct. Mech. 1975, 1, 259. (b) Belliveau, J. W.; Lanyi, J. K. Arch. Biochem. Biophys. 1977, 178, 308. (9) (a) Chang, C. H.; Chen, J. G.; Govindjee, R.; Ebrey, T. G. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 396. (b) Kimura, Y.; Ikegami, A.; Stoeckenius, W. Photochem. Photobiol. 1984, 40, 641. (c) Kobayashi, T.; Ohtani, H.; Iwai, J.; Ikegami, A.; Uchiki, H. FEBS Lett. 1983, 62, 197. (10) Szundi, I.; Stoeckenius, W. Biophys. J. 1989, 56, 369. (11) (a) Chang, C. H.; Jonas, R.; Melchiore, S.; Govindjee, R.; Ebrey, T. G. Biophys. J. 1986, 49, 731. (b) Ariki, M.; Layni, J. K. J. Biol Chem., 1986, 261, 8167. (c) Corcoran, T. C.; Ismail, K. Z.; El-Sayed, M. A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4094. (12) (a) Dunach, M.; Seigneuret, M.; Rigaud, J. L.; Padros, E. Biochemistry 1987, 26, 1179. (b) Dunach, M.; Seigneuret, M.; Rigaud, J. L.; Padros, E. J. Biol. Chem. 1988, 263, 17378. (c) Dunach, M.; Padros, E.; Seigneuret, M.; Rigaud, J. L. J. Biol. Chem. 1988, 263, 7555. (13) (a) Zhang, Y. N.; Sweetman, L.; Awad, E. S.; El-Sayed, M. A. Biophys. J. 1992, 61, 1201. (b) Zhang, Y. N.; El-Sayed, M. A.; Bonet, M. L.; Lanyi, J. K.; Chang, M.; Ni, B.; Needleman, R. PNAS 1993, 90, 1445. (c) Zhang, Y. N.; M. A. El-Sayed Biochemistry 1993, 32, 14173. (14) Masuda, S.; Nara, M.; Tasumi, M.; El-Sayed, M. A.; Layni, J. K. J. Phys. Chem. 1995, 99, 7776. (15) Stuart, J. A.; Vought, B.; Zhang, C-F; Birge, R. Biospectroscopy 1995, 1, 9. (16) (a) Drachev, A. L.; Drachev, L. A.; Kaulen, A. D.; Khitrina, L. V. Eur. J. Biochem. 1984, 138, 349. (b) Drachev, L. A.; Kaulen, A. D.; Khitrina, L. V. Biochemistry (Moscow) SSR 1988, 53, 581. (17) Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, 31, 667. (18) Rothschild, K. J; Sanches, R.; Clark, N. A. Methods Enzymol. 1982, 88, 696. (19) Nakamoto, K Infrared and Raman Spectra of Inorganic and Coordination Compounds; John Wiley and Sons, Inc.: New York, 1986; 247-249. (20) Petersheim, M.; Halladay, H.; Blodnieks, J. Biophys. J. 1989, 56, 551. (21) Roux, M.; Seigneuret, M.; Rigaud, J. L. Biochemistry 1988, 27, 7009. (22) El-Sayed, M. A; Song, L.; Griffiths, J. A.; Zhang, N. Pure Appl. Chem. 1995, 1, 149. (23) Drachev, L.; Kaulen, A.; Skulachev, Mol. Biol. 1977, 11, 1377. (24) Drachev, L.; Kaulen, A.; Khitrina, L. Biokhimiya (Moscow) 1988, 53, 581. (25) Griffiths, J. A.; El-Sayed, M. A.; Capel, M. In preparation.
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