Natl. Acad. Sci. U.S.A. 78, 2226-2229. Becher BM & Cassim JY (1975) Prep. Biochim. 5, 161-178. Blatz PE, Lane L & Aumiller JC (1975) Photochem. Photobiol.
Bioscience Reports 5, 615-623 (1985) Printed in Great Britain
615
Interaction of tyrosine residues w i t h the chromophore in bacteriorhodopsin
Kazuo TAKEDA, Tatsuo IWASA, Fumio TOKUNAGA, and Lester PACKER Department of Physics, Faculty of Science, Tohoku University, Aobayama, Sendai 980, Japan; and Membrane Bioenergetics Group, Applied Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley, Berkeley, California 94720, U.S.A. (Received 5 July 1985)
We previously reported that the absorption spectrum at low temperatures of iodinated bacteriorhodopsin can be separated into four components with maxima at shorter wavelengths than in native bacteriorhodopsin. In this s t u d y , t h e t i m e c o u r s e of t h e f o r m a t i o n of each s p e c t r a l c o m p o n e n t a f t e r i o d i n a t i o n was analyzed, revealing that these four components correspond to four different iodinated states of tyrosine residues interacting with the retinal chromophore of bacteriorhodopsin. Therefore at least two tyrosine residues interact with the chromophore of bacteriorhodopsin. Bacteriorhodopsin (bR) is a single protein in the purple membrane The chromophore~ retinal, is bound to Lys 216 of bR via a protonated Schiff base (Lemke & Oesterhelt~ 1981a; Bayley et al., 19gl). Upon light absorption, bR undergoes a photochemical reaction cycle which is coupled to proton translocation across the membrane. Light converts bR to a batho-product, K6z~ with the absorption maximum at a longer wavelength than bR. During the photocyle (Iwasa et a l , 1979) retinal isomerizes from the a l l - t r a n s form to 1 3 - c i s and then to a l l - t r a n s (Tsuda et al.~ 19g0)~ and changes occur in the interaction between the chromophore and neighboring amino acid residue% which may be important for the light-dependent proton pump activity. The specific amino acids involved in the photocycle and the m e c h a n i s m of i n t e r a c t i o n with the retinal are as yet unknown. Residues interacting with the chromophore~ however~ appear to play an important role in the proton pumping mechanism (Konishi & Packer, 1978; Hess & Kushmitz, 1979; Scherrer et a l , 1981; Kalisky et al., 1981; Lemke & Oesterhelt~ 1981b; Iwasa et al.~ 1982; Lain & Packer~ 198/4)~ possibly a c t i n g as acceptors or donors of protons for the r e v e r s i b l e deprotonation of the Schiff base at the site of retinal a t t a c h m e n t during the photocycle.
of H a l o b a c t e r i u m halobium.
616
TAKEDA
ET AL.
We p r e v i o u s l y r e p o r t e d that light-.adapted tyrosine-iodinated bR ( T y r - m o d - b R L) consists of four components which have absorption m a x i m a a t s h o r t e r w a v e l e n g t h s t h a n the native pigment. Each component has a c h a r a c t e r i s t i c i nterm edi at e in its photocycle (Iwasa et al., 1992), and arises from one the four d i f f e r e n t iodinated states of tyrosine residues interacting with the retinal. In this investigation, we determined that these four components (comp 1-4) result from d i f f e r e n t degrees of iodination. M a t e r i a l s and M e t h o d s Purple membrane was isolated as described by Becher and Cassim (1975). Iodination of tyrosine residues ol bR was perform ed as in S c h e r r e r e t al. (1991). Tyrosine-iodinated bR (Tyr-mod-bR) was p r e p a r e d with r e a c t i o n t i m e s of 1.0, 1.5~ 2.0, 4.0, and g.0 h. Tyr-mod-bR was suspended in 10 mM phosphate buffer (pH 6.g) and mixed with three volumes of glycerol. The absorption spect ra of these s a m p l e s was measured at low t e m p e r a t u r e s as previously reported (Iwasa et al., 1930). Light adaptation was performed as follows: The sample was irradiated at 500 nm for 60 rain and incubated at 0~ to c o n v e r t M~l 2 i n t e r m e d i a t e to bR. The sample iodinated for t hours is designated as Tyr-mod-bR L (t h). A Xe lamp (Ushio, 500 W) was used as an irradiation light source and the wavelength was sel ect ed with a glass c u t - o f f filter (Toshiba Rt~g, R60, R63, R66, R67, or R69) and/or an i n t e r f e r e n c e filter (Toshiba KLS0 or KL74). Results Since low t e m p e r a t u r e s improve the spectral resolution (Yoshizawa, 1972), we measured absorption s pect ra of iodinated bR at -170~ Fig. I shows absorption spectra of Tyr-mod-bRs, normalized at the absorption maxima. With iodination time, the absorption maximum is blue-shifted from 570 nm to about 540 nm and the spectrum becomes broader. Our previous work (Iwasa et al., I9g2) suggests that the n u m b e r of c o m p o n e n t s i n c r e a s e s with the reaction time. Upon irradiation ol Tyr-mod-bRL (~.5 h) at -iT0~ the absorption spectrum shifts to longer wavelengths than native bR, indicating the formation of batho-products. Eventually, the absorption spectrum reaches the p h o t o s t e a d y - s t a t e mixture which consists oi Tyr-mod-bRs and their batho-products. Since the batho-products r e c o n v e r t to Tyr-mod-bRL in red light like native bR (Iwasa et al., 19g0,g2), we separated the batho-products into components using the reverse photoreaction. The p h o t o s t e a d y - s t a t e m i x t u r e was irradiated at 740 nm and a photoreaction having an isosbestic point at 594 nm was observed (curves 1-6 in Fig. 2a). The di f f er e nc e spectrum of this first component is the same as that of irradiated native trans-bR previously reported (Iwasa et al., / 9 g l ) . Upon f u r t h e r i r r a d i a t i o n at 740 nrn for 30 rain, a d i f f e r e n t photoreaction having an isosbestic point at about 5g6 nm was observed (curves 7-9 in Fig. 2b). The d i f f e r e n c e spectra are the same as that of comp 1 reported previously (Iwasa et al., 19g3). Upon irradiation for an additional 30 rain, the isosbestic point at 596 nm was lost (curve 10). The wavelength of irradiation was then changed from 740
TYROSINE
RESIDUES
IN
BACTERIORHODOPSIN
Visible Absorption Spectra of Iodinated Bacteriorhodopsin I
10
8
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I
o native 9 lhr 1.5 hr 9 2hr
-
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0
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4OO
,
I
,
I
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Fig. i. Relative absorption spectra of lightadapted bR iodinated for 0, 1.0, 1.5, 2.0, 4.0, and 8.0 h. Absorption spectra were measured at -170~ and normalized to 1.0 at the absorption maximum.
nm to longer than 630 nm. Irradiation for 42 min #0 s produced the photoreaction with an isosbestic point at 56g nm (curves 10-20 in Fig. 2c) w i t h curve 20 coinciding with the baseline. The difference spectrum of this component is the same as that of comp 2 reported previously (Iwasa et al., 1992). The results show that Tyr-mod-bRL (t.5 h) consists of native trans-bR, comp I, and comp 2, and that it does not contain comp 3 and comp #, which have an absorption maximum at shorter wavelengths than comp 1 and comp 2. The difference spectrum between each component and its bathoproduct is the same (appropriately normalized) in samples iodinated for d i f f e r e n t lengths of time (data not shown). The difference spectrum b e t w e e n T y r - m o d - b R L and i t s b a t h o - p r o d u c t was reconstituted as described previously (Iwasa et al., 1992). T h e overall d i f f e r e n c e spectrum between the T y r - m o d - b R L (1.5 h) and its photosteady-state mixture (curves 1-20 in Fig. 2a,b,c) reconstituted (Fig. 3b) is identical with the overall difference spectrum within a standard error of 1.5%. Similar experiments were performed with the other samples: Tyr-mod-bRL (l.0 h) and Tyr-mod-bR L (1.5 h) consist of native bRL, comp 1 and comp 2 (Fig. 3a,b); Tyr-mod-bRL (2.0 h) contains additionally comp 3 (Fig. 3c); Tyr-mod-bRL (4.0 h) and
61g
TAKEDA
ET
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Fig. 2. The p h o t o r e v e r s i o n of batho-products generated from Tyr-mod-bR L (1.5 h) at -170~ The base line (0 level) was adjusted to be flat with Tyr-mod-bR L (1.5 h). Curve I: The photosteady state by irradiation at 500 nm for 480 s. (a) Curves 2-7: The products of s u c c e s s i v e irradiations of the photosteady-state mixture with a deep red light (740 nm) for 15, 15, 30, 60, 120, 240 s, respectively. (b) Curve 7: The same curve as curve 7 in (a). Curves 8-10: The products by the further successive irradiations with the light (740 nm) for 480, 960, and 1920 s, respectively. (c) Curve I0: The same curve as curve I0 in (b). Curves 11-20: The products of further irradiations with a red light (> 650 nm) for 15, 15, 30, 60, 120, 240, 480, 960, 1920, and 3840 s, respectively.
AL.
TYROSINE
RESIDUES
IN
BACTERIORHODOPSIN
619
Reconstruction of Difference Spectra Between Tyr--mod--bR L and the Photosteady State Mixture Tyr, mod-bR L (1.0 hr) '
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500
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Overall relative difference spectrum o Reconstituted o Native
g
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tie change in the diffraction pattern, indicating that the color change may be independent of the structural change (Tokunaga et al., 1984). Hence it is more likely that iodinated tyrosines interact directly with the chromophore. Nitration of Tyr 26 in bR shifts the absorption maximum to a shorter wavelength (Lemke & Oesterhelt, 19glb), accelerates formation of M~t z at neutral pH, and slows it at alkaline pH. As a result, the hal/ rise time of M~|~ becomes independent of pH (Rosenbach et al., 1982). Iodination res'uqts in a similar effect; the half rise time of M~I 2 becomes shorter and the half decay time becomes longer (Scherrer et al., 1981). Shortening of the rise time of M~t 2 by iodination corresponds to the appearance of comp l and/or comp 2; and the delay of decay time corresponds to comp 3 and/or comp 4. Iodination of Tyr 26, which a c c e l e r a t e s the rise of Mr corresponds to the formation of comp i or comp 2. Comp 3 or comp tt might be p r o d u ced by iodination of one more tyrosine residue other than Tyr 26, which would a f f e c t the decay process of M4i 2. In any case, our p r e s e n t r e s u l t s require a new model in which at least one more t y r o s i n e r e s i d u e t h a n T y r 26 exists near the chromophore. Our preliminary resuIts using mass s p e c t r o m e t r y show that Tyr 26 was indeed iodinated. Previous fluorescence energy transfer studies on Tyr residues in bacteriorhodopsin have shown t hat certain residues 'see' each other, but have not shown a n y evidence for or against a Tyr residue in proximity to the retinal chromophore. The approches used to address this p r o b l e m h a v e b e e n t h o s e of chemical modification and the substitution of amino acid residues. Model building studies carried out at NASA Ames, using the MOLECULE program, have so far failed to identify Tyr residues which would be candidates for the modification, due to the lack o1 any single definitive experiment that would enable relative configurations of helices to be defined. In the modification described here, t hr e e tyrosines are probably involved, one on the long c h y m o t r y p t i c f r a g m e n t of bR and t h e o t h e r t w o on the short fragment.
TYROSINE RESIDUES IN BACTERIORHODOPSIN
623
Acknowledgements This work was supported by a NASA Ames Research Center/ University of California, Berkeley Interchange agreement and by the O f f i c e of BiologicaI Energy R e s e a r c h , Division of Basic Energy S c i e n c e s , U.S. D e p a r t m e n t of Energy ( C o n t r a c t No. DEACO 3-76SF0009g). The authors are grateful to Drs. Peter Scherrer and Ian Fry for preparation of samples used in this work and to Dr. A. E. Robinson and E. Hrabeta for assistance with the manuscript, and to Drs. R. MacEtroy and A. Pohoril]e of NASA for melecular modelling studies.
References Bayley H, Huang KS, Radhakrishnan R, Ross AR, Takagi Y & Khorana HG (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2226-2229. Becher BM & Cassim JY (1975) Prep. Biochim. 5, 161-178. Blatz PE, Lane L & Aumiller JC (1975) Photochem. Photobiol. 22, 261-263. Hess B & Kuschimtz D (1979) FEBS Lett. I00, 334-340. lwasa T, Tokunaga P & Yoshizawa T (1979) FEBS Lett. I01, 121-124. lwasa T, Tokunaga F & Yoshizawa T (1980) Biophys. Struct. Mech. 6, 253-270. lwasa T, Tokunaga F & Yoshizawa T (1981) Photochem. Photohiol. 33, 539-545. lwasa T, Takeda K, Tokunaga F, Scherrer P & Packer L (1982) Biosci. Rep. 2, 949-958. Kalisky O, Ottolenghi M, Honig B & Korenstein R (1981) Biochemistry 20, 649-655. Konishi T & Packer L (1978) FEBS Lett. 92, 1-4. Lam E & Packer L (1984) International Council of Scientific Unions, Bari, Italy, April 5-7, 1984. Lemke HD & Oesterhelt D (1981a) FEBS Lett. 128, 255-260. Lemke HD & Oesterhelt D (1981b) Eur. J. Biochem. 115, 595-604. Milder ST & Kliger DS (1977) Photochem. Photobiol. 25, 287-291. Rosenbach V, Goldberg R, Gilson C & Ottolenghi M (1982) Photochem. Photobiol. 36, 197-201. Scherrer P, Packer L & Seltzer S (1981) Arch. Biochem. Biophys. 202, 589-601. Tokunaga F, Kataoka M, Sato N, Takeda K, Hisatomi O, Hirai M & Amemiya Y (1982/83) Photon Factory Activity Report, VI-96. Tokunaga F, Kataoka M, Sato N, Takeda K, Hamanaka T & Amemiya Y (1984) Photon Factory Activity Report, in press. Tsuda M, Galccum M, Nelson B & Ebrey TG (1980) Nature 287, 351-353. Yoshizawa T (1972) in: Handbook of Sensory Physiology (Dartnell HJA, ed), pp 146-179, Springer, Berlin.
