Thermal denaturation and photochemistry of bacteriorhodopsin from Halobacterium cutirubrum as monitored by resonance raman spectroscopy

Biochimica et Biophysica Acta, 427 (1976) 295-301

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37238 T H E R M A L D E N A T U R A T I O N A N D P H O T O C H E M I S T R Y OF BACTERIOR H O D O P S I N F R O M H A L O B A C T E R I U M C U T I R U B R U M AS M O N I T O R E D BY R E S O N A N C E R A M A N SPECTROSCOPY*

R. MENDELSOHN Division of Chemistry, National Research Council of Canada, Ottawa, Ontario K1A OR6 (Canada)

(Received August 5th, 1975)

SUMMARY Resonance Raman studies of the thermal denaturation of bacteriorhodopsin from Halobacterium cutirubrum show that the N-retinylidenelysine moiety present in the chromophore is N-protonated. This corroborates an earlier suggestion of Lewis et al. ((1974) Proc. Natl. Acad. Sci. U.S., 71, 4462-4466). The widely differing excitation profiles of two - C = C - stretching modes are explained in terms of the light-initiated reaction cycle in the molecule. Glutaraldehyde fixation of bacteriorhodopsin has no effect on the intensity ratio of the two modes, suggesting that no large motion of the protein is necessary for the photoreaction cycle to occur. INTRODUCTION Bacteriorhodopsin is a rhodopsin-like, membrane-bound protein which can be isolated from the cell walls of the extremely halophilic bacteria, Halobacterium halobium and Halobacterium cutirubrum [1-3]. The protein exists in the native membrane as a strongly coloured purple complex absorbing at about 560 nm (the lightadapted molecule absorbs at 570 nm). The chromophore involved is the Schiff base of retinal linked to the e-amino group of a protein-bound lysine residue (retinylidenelysine) [1]. The molecule undergoes a series of light-induced conformational changes during which it functions as a light-driven proton pump [4, 5]. Resonance Raman scattering has been used to probe the structure of the bacteriorhodopsin chromophore [6-8]. The basis of the technique is that intensity enhancement of certain vibrations in chromophoric systems may occur when the Raman spectrum is excited by light of a wavelength that lies within a molecular electronic absorption band. Only certain vibrations coupled to the electronic transition are affected. In the current study, the vibrations of the retinylidenelysine moiety are specifically enhanced to the extent that Raman spectra can be obtained from fairly dilute (10-4-10 -6 M) aqueous solutions, while the non-resonant-enhanced protein modes are lost in the background. Resonance Raman experiments have shown that the electronic structure of the * N.R.C.C. No. 15113.

296 Schiff base in bacteriorhodopsin is considerably perturbed from the free molecule in organic solvent by non-covalent interaction with the protein [6, 8]. In addition, the state of protonation of the chromophore has been studied by Lewis et al. [7], who suggested that the Schiff base nitrogen is protonated. As the Schiff base linkage is possibly directly involved in the proton translocation process [7], further experimental tests concerning this point are indicated. In the current study, additional evidence concerning the state of protonation of the Schiff base nitrogen atom is presented from a resonance Raman study of the thermal denaturation of bacteriorhodopsin. In addition, earlier observations [6, 8] of the variation in Raman intensity with exciting wavelength for two C - - C stretching vibrations of the system are explained in terms of the light-initiated reaction cycle reported by Stoeckenius and Lozier [9]. MATERIALS AND METHODS Bacteriorhodopsin, isolated from H. cutirubrum, as previously described [3], was the generous gift of Professor M. Kates and Dr. S. Kushwaha. The Raman spectrometer consists of a Spex 1400 double monochromator equipped with photomultiplier and photon counting detection. Excitation was provided by a Coherent Radiation Model 52 A Argon ion laser tuned to 4880 A in the experiments reported here. Power levels incident on the sample were about 250 mW. The sample was contained in a melting point capillary and examined with transverse excitation. Temperature was controlled to ± 2 °C by a flow of N2 gas over the sample tube which was placed in an unsilvered vacuum jacketed container. The temperature was monitored with a thermocouple placed near the laser focus. The glycerol, used as solvent for the low temperature experiments, was of spectroscopic grade. Glutaraldehyde fixation was carried out by the method of Brown [10]. RESULTS AND DISCUSSION The resonance Raman spectrum of H. cutirubrum bacteriorhodopsin has been previously reported and tentative assignments for the observed vibrations suggested [8]. Of interest in the current work are modes in the 1500-1700 cm -1 region, which arise primarily from C - - C and C - - N stretching vibrations of the retinylidenelysine.

(i) Thermal denaturation of bacteriorhodopsin The 1450-1700 cm -~ region of the resonance Raman spectrum of bacteriorhodopsin at various temperatures is shown in Fig. 1. The modes at 1529 and 1568 cm-1 arise from - C - - C - stretching vibrations of the Schiff base and will be discussed below. The mode at 1641 cm -a lies in a frequency region expected to contain both the non-resonant-enhanced O-H bending mode of solvent and the resonant-enhanced - C N -+ stretching vibration of a protonated Schiff base [11]. This band was origiI H nally assigned to the O-H bending mode of water on the basis of its disappearance in a 2H20 suspension of bacteriorhodopsin [6]. However, several studies [7, 8, 12] have shown that in 2H20 suspension t h e - C = N -+ vibration resulting from a Schiff base in I 2H

297

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Fig. I. Resonance Raman spectra of 1 • 10-s M bacteriorhodopsin from H. cutirubrum at various temperatures. Excitation source 250 mW of 4880/~ radiation. Spectral slit width, approx. 5 cm-a; time constant, 1 s; scan speed, 1 cm-~/s. which the p r o t o n has been replaced by a deuteron would have a R a m a n frequency also shifted from 1641 cm -1. Hence the original assignment o f the mode based on its disappearance in ZH20 was premature. Further evidence as to the origin of this b a n d was gained f r o m a thermal denaturation experiment, the results o f which are shown in Fig. 1. As the temperature o f the suspension is raised f r o m 23 to 72 °C, the - C = C stretching modes at 1529 and 1568 c m - 1 broaden considerably, indicating an increased mobility o f the c h r o m o p h o r e , while the modes at 1622 and 1641 cm -1 smear out to some extent. A t 85 °C, above the thermal denaturation point, no R a m a n lines are visible. It appears that thermal denaturation shifts the c h r o m o p h o r e absorption band out o f the region where resonance-enhanced R a m a n spectra can be obtained with 4880 A excitation. I f the m o d e at 1641 cm -~ arises from the non-resonant-enhanced O - H bending vibration o f water, it should retain its intensity even u p o n thermal denaturation of the protein. The fact that it disappears (Fig. 1) indicates that the mode is indeed resonant enhanced and therefore arises from the - C = N -+ stretching viI H bration o f an N - p r o t o n a t e d Schiff base. Lewis et al. [7] have arrived at similar conclusions based on low temperature R a m a n experiments with bacteriorhodopsin in

298 HzO and 2HzO suspension. The current study provides an independent line of evidence which corroborates their results. The native bacteriorhodopsin chromophore is therefore a protonated Schiff base.

(ii) Laser-induced photochemistry in bacteriorhodopsin In previous work from this laboratory [8], the variation in Raman intensity with exciting wavelength (excitation profiles) were measured for the two C = C stretching vibrations at 1529 and 1568 cm -1. The measured excitation profiles have widely different shapes [8]. The mode at 1529 cm-a has a maximum in intensity when the exciting wavelength is near 5600 A, and hence is coupled to the pigment electronic transition at 560 nm. The vibration at 1568 cm -1 has very low intensity with excitation at 5600 A, but gains dramatically in intensity as the excitation wavelength is shifted toward the ultraviolet region. The shape of the excitation profile for this vibration indicates that it is coupled to an absorption band in the 4000-4580 A range [8]. Recent flash spectroscopic studies by Stoeckenius and Lozier [9], have shown that bacteriorhodopsin undergoes a fast cyclic photoreaction sequence. The first intermediate in the system is formed by the action of incident light and absorbs at 610 nm. This form then decays rapidly in the dark to other molecular species which absorb successively at 550 and 415 nm [9]. The original (light-adapted) 570 nm form is eventually regenerated from the 415 nm chromophore. It was of interest in the current work to determine whether the mode at 1568 cm-1 in bacteriorhodopsin arises from any of the forms of the molecule which might be induced by the laser beam during the Raman experiment. In Fig. 2A, the resonance Raman spectrum of 1.10 -5 M bacteriorhodopsin in, glycerol/water (3:1, v/v) suspension is shown. The mode at 1472 cm- a arises from a (non-resonance enhanced) C-H deformation of glycerol in the solvent while the modes at 1534 and 1572 cm -1 are the - C = C - stretching vibrations of bacteriorhodopsin shifted from their aqueous suspension values by about 4 cm -a. If the bacteriorhodopsin is cooled to --95 °C in the dark and the Raman spectrum then obtained (Fig. 2B), the mode at 1572 cm-1 is greatly diminished in intensity, while that at 1534 cm -~ is increased significantly (relative to the glycerol band at 1472 cm-1). In addition, a new feature is noted at 1611 cm -1 (Fig. 2B). If the sample is allowed to warm to 23 °C, the Raman spectrum resembles the original (compare Fig. 2A with 2C), showing that the changes on cooling are reversible. If the sample is again cooled to --95 °C, this time while being illuminated with intense (600 roW) 4880 A radiation, the Raman spectrum obtained is shown in Fig. 2D. The mode at 1572 cmis the strongest in the spectrum while the intensity at 1534 cm -~ is greatly reduced from that in Fig. 2B. It is reemphasized that the widely differing Raman spectra shown in Fig. 2B and 2D were obtained under identical experimental conditions, the only difference being the amount of light failing on the sample during the freezing process. The most reasonable explanation for the above observations is that the 1572 cm -~ band arises from one of the species present in the light-initiated reaction cycle which the bacteriorhodopsin molecule undergoes 100-200 times per s at room temperature. Cooling the purple membrane to --95 °C retards formation of the 415 nm form to a large extent while the 610 and 550 nm species still occur [9]. In the light of these results, the most likely candidate for the electronic species giving rise to the 1572

299

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Fig. 2. Resonance Raman spectra of 1'10 -s M bacteriorhodopsin in glycerol/water (3:1, v/v) suspension. A, at 23 °C; B, at --95 °C (cooled in the dark); C, suspension B rewarmed to 23 °C; D, at --95 °C cooled while being irradiated with 600 mW of 4880 A radiation. Spectral parameters as in Fig. 1. The full scale sensitivity was 2000 counts/s in A, C, and D and 5000 counts/s in B.

Raman band is the 415 nm form of the molecule. The Raman data in Fig. 2 can then be explained as follows: At room temperature, the 415 nm form is present (as part of the laser-initiated cycle) and the Raman spectrum reflects the photostationary equilibrium concentration of the native and 415 forms of the molecule. The 1534 cm -1 band arises from the former; the 1572 cm -1 band from the latter. When the sample is cooled to 95 °C in the dark, no photochemistry occurs and none of the 415 nm form is formed in the system during the cooling process. Formation of the 415 nm form is reduced at --95 °C so that little is formed at that temperature by the laser when the Raman spectrum is taken. In this instance (Fig. 2B), the mode at 1572 cm -1 is nearly absent from the Raman spectrum, (consistent with its assignment to the 415 nm form), while that at 1534 cm -~ is more intense than in the room temperature spectrum since no photochemistry has occurred. When, however, the sample is illuminated while being cooled, the frozen suspension shows Raman bands (Fig. 2D) arising from both the 560 and 415 nm forms of the protein. This reflects the fact that a certain amount of the 415 nm form was formed and trapped during the freezing process. In this case the mode at 1534 cm -~ is diminished in intensity (compare Fig. 2D with 2B) with respect to the glycerol band at 1472 c m - ' . cm -1

300 The mode at 1572 cm-~ is expected to be quite strong, as observed. The excitation profile previously reported for the 1572 cm -1 band [8] reflects a balance between the increased activity of the light in causing photochemical reactions as the exciting wavelength is increased towards the 560 nm band of the native molecule [5] and the increased resonance enhancement of the vibration as the exciting wavelength is shifted towards the electronic transition at 415 nm to which it is coupled. Further evidence for the above assignments of the - C = C - stretching modes to particular electronic forms of the protein was gained from the following experiment. The half time of regeneration of the 570 nm form of bacteriorhodopsin from the 415 form is slowed from 10 ms to 13 s in a suspension of the protein in ether-saturated 4 M NaC1 suspension [9]. If the room temperature R a m a n spectrum reflects the photostationary equilibrium concentration of the native and 415 nm forms of the molecule, then the fraction of 415 nm form present should be greater (as measured by an increase in the intensity of the 1568 cm -1 band) in an ether-saturated 4 M NaCI suspension of the pigment. In fact, the intensity of the R a m a n band at 1568 cm-1 is doubled with respect to that at 1529 cm-1 in an ether-saturated 4 M NaC1 suspension at room temperature, compared with its intensity in the absence of ether. As it is apparent that the intensity ratio I (1529)/I (1568) is a measure of the extent of formation of the 415 nm form of the chromophore, it is feasible to use this ratio to investigate situations where the light-initiated cycle may be interrupted. The effect of glutaraldehyde fixation of the protein on the intensity ratio was investigated in order to see whether any large motion of the chromophore occurs during the formation of the 415 nm form. The effect of the fixation process on the purple membrane fragments was readily noted by the formation of rather large aggregates. However, no significant change was noted in the 1 (1529)/• (1568) ratio in the R a m a n spectrum within the error limits of the experiment (-- 7 ~). This suggests that no large motion of the protein is required for formation of the 415 nm form. This result is consistent with other descriptions of molecular rigidity (ref. 13 and Lozier, R. H., Stoeckenius, W., Poo, M., Fein, A. and Cone, R. A., unpublished; quoted in ref. 9) and the fact that the protein spans the entire thickness of the membrane in which it is located [14]. The latter observation illustrates an essential structural feature of the system which enables it to carry out its proton pumping function in the absence of large molecular motion. Several features in the 1500-1700 cm -1 region of the Raman spectra remain unexplained. These include the band at 1603 cm-~ at 23 °C and a new feature observed at 1611 cm -1 at low temperature (Fig. 2). In addition, the mode at 1622 cm -1 has been assigned to the - C ~ N - stretching vibration of an unprotonated Schiff base, and has been used to show that the Schiff base of the 415 nm form is unprotonated [7]. However, the mode must be more complicated than previously described. If the mode arises only from the 415 nm form of the molecule, then it should disappear in situations when the dark reaction leading to this species is prevented. As can be seen in Fig. 2B the mode at 1622 cm-% is still quite intense even though the weakness of the 1572 cm-~ band indicates that very little of the 415 nm form exists under the conditions of the experiment. The mode must therefore have a more complex origin than the - C = N - stretching vibration assigned to it, although the - C = N - stretch certainly contributes to the intensity of the vibration. The utility of resonance Raman spectroscopy for investigation of intermediates

301 in photochemical reaction cycles is clear from the c u r r e n t work. We hope to utilise this technique in both photochemical a n d photobiological systems of interest. REFERENCES 10esterhelt, D. and Stoeckenius, W. (1971) Nat. New Biol. 233, 149-152 2 Blaurock, A. E. and Stoeckenius, W. (1971) Nat. New Biol. 233, 152-154 3 Kushwaha, S. C. and Kates, M. (1973) Biochim. Biophys. Acta 316, 235-243 4 Oesterhelt, D. and Stoeckenius, W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2853-2857 50esterhelt, D. and Hess, B. (1973) Eur. J. Biochem. 37, 316-326 6 Mendelsohn, R. (1973) Nature 243, 22-24 7 Lewis, A., Spoonhower, J., Bogomolni, R., Lozier, R. and Stoeckenius, W. (1974) Proc. Natl. Acad. Sci. U.S. 4462-4466 8 Mendelsohn, R., Verma, A. L., Bernstein, H. J. and Kates, M. (1974) Can. J. Biochem. 52, 774781 9 Stoeckenius, W. and Lozier, R. H. (1974) J. Supramol. Struct. 2, 769-774 10 Brown, P. K. (1972) Nat. New Biol. 236, 35-38 11 Heyde, M. E., Gill, D., Kilponen, R. G. and Rimai, L. (1971) J. Am. Chem. Soc. 93, 6776-6780 12 Oseroff, A. R. and Callender, R. H. (1974) Biochemistry 13, 4243-4248 13 ChigneU, C. F. and Chignell, D. A. (1975) Biochem. Biophys. Res. Commun. 62, 136-143 14 Blaurock, A. E. (1975) J. Mol. Biol. 93, 139-158