[51] Bleaching intermediate kinetics of rhodopsin: Picosecond kinetics for squid rhodopsin

368

S P E C T R A L RESPONSES O F V I S U A L P I G M E N T S

[51]

Acknowledgments The authors wish to thank their colleagues George Busch, Wiili Sundstrom, Kevin Peters, Tom Graddis, and WolfgangBaehr for their contributionsto picosecond spectroscopy of visual pigments. The work was supported in part by grants from the National Eye Institute.

[51] B l e a c h i n g I n t e r m e d i a t e K i n e t i c s o f R h o d o p s i n : Picosecond Kinetics for Squid Rhodopsin

By T. KOBAYASHI and S. NAGAKURA Introduction As stated in Article [49] of this volume, the formation of bathorhodopsin was first observed by Yoshizawa and Kito 1 by irradiating cattle rhodopsin at 77 K. The spectra of batho-intermediates of various animals were measured by low-temperature spectrophotometry. 2 Hypsorhodopsin was discovered by irradiating cattle rhodopsin at liquid helium temperature 2,a and has been observed for the cattle, 2 frog, 4 chicken, 5 and squid s rhodopsin systems. Compared with the vertebrate rhodopsins, squid rhodopsin more easily converts into a photosteady state mixture composed of rhodopsin, isorhodopsin, and hypsorhodopsin by irradiation with yellow light (> 480 nm) at liquid helium temperature. Therefore, the squid rhodopsin system is more suitable for detecting hypsorhodopsin in a kinetic study of photobleaching of rhodopsin. As stated in Article [49], squid hypsorhodopsin converts into bathorhodopsin on warming above 35 K in the dark. This indicates that hypsorhodopsin is a precursor to bathorhodopsin. During irradiation with blue light (437 nm) at liquid helium temperature, however, rhodopsin changes to another photosteady state mixture containing bathorhodopsin. This suggests that bathorhodopsin may be produced directly from rhodopsin without any intermediate state. Whether or not hypsorhodopsin is a precursor to bathorhodopsin is a question, therefore, that must be answered by in-

T. Y o s h i z a w a and Y. Kito, Nature (London) 182, 1604 (1958). 2 T. Y o s h i z a w a , in " H a n d b o o k o f S e n s o r y P h y s i o l o g y " (H. J. A. Dartnall, ed.), Vol. 7, Part 1, p. 146. Springer-Vedag, Berlin and N e w York, 1972. T. Y o s h i z a w a and S. Horiuchi, in " B i o c h e m i s t r y and Physiology of Visual P i g m e n t s " (H. Langer, ed.), p. 169. Springer-Verlag, Berlin and N e w York, 1973, 4 S. Horiuchi and T. Y o s h i z a w a , Zool. Mag. 83, 300 (1974). Y. T s u k a m o t o , S. Horiuchi, and T. Yoshizawa, Vision Res. 15, 819 (1975). 6 y . Shichida, F. T o k u n a g a , and T. Yoshizawa, Photochern. Photobiol. 29, 343 (1979).

METHODS IN ENZYMOLOGY, VOL. 81

Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181981-7

[51]

P I C O S E C O N DKINETICS FOR SQUID RHODOPSIN

369

vestigating their formation and decay processes at r o o m t e m p e r a t u r e with the aid o f picosecond time-resolved spectroscopy. The first picosecond s p e c t r o s c o p y study on rhodopsin was carried out by Rentzepis and collaborators for cattle rhodopsin 7 (see Article [50], this volume). T h e y did not, however, consider h y p s o r h o d o p s i n and concluded that bathorhodopsin was formed within 6 psec. Similar e x p e r i m e n t s were made by Sundstrom e t al. 8 and also by Monger e t al. ~ We made picosecond s p e c t r o s c o p y e x p e r i m e n t s for squid rhodopsin at r o o m and liquid nitrogen t e m p e r a t u r e s and succeeded in obtaining the first experimental evidence that hypsorhodopsin is a precursor to bathorhodopsin. 1°'11 Our experimental results will be described in detail and discussed. Experimental Results The picosecond s p e c t r o s c o p y e x p e r i m e n t on squid rhodopsin was made by using a 347-nm light pulse with 20 psec width f r o m a modelocked ruby laser. TM Preparation of the rhodopsin samples solubilized by digitonin is mentioned in the literature) 1 The a b s o r b a n c e change of a sample after excitation was m e a s u r e d at 430 nm, near the absorption m a x i m u m of h y p s o r h o d o p s i n and the result at r o o m t e m p e r a t u r e (21 °) is shown in Fig. 1. We can see from Fig. 1 that the a b s o r b a n c e attains the m a x i m u m value within 19 psec (one step of the echelon used for the generation of a sequence of optical delays for a monitoring beam) and then gradually decreases. It is known that the a b s o r b a n c e of a l l - t r a n s retinal in hexane is increased gradually by irradiation with a 353-nm light pulse because of the a p p e a r a n c e of T-T absorption.13 The rise time of the T-T absorption, however, is 34 psec and is m u c h longer than that for the formation o f hypsorhodopsin. Since both the monitoring and the exciting pulses were 20 psec in width, the time convolution o f the two pulses was calculated at 28 psec. 7 G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, Proc. Natl. Acad. Sci. U.S.A. 69, 2802 (1972). s V. Sundstrom, P. M. Rentzepis, K. Peters, and M. L. Applebury, Nature (London) 267, 645 (1977). 9 T. G. Monger, R. R. Alfano, and R. H. Callender, Biophys. J. 27, 105 (1979). 10 y. Shichida, T. Yoshizawa, T. Kobayashi, H. Ohtani, and S. Nagakura, FEBS Lett. 80, 214 (1977). 11 y. Shichida, T. Kobayashi, H. Ohtani, and T. Yoshizawa, Photochem. Photobiol. 27, 335 (1978). 12T. Kobayashi and S. Nagakura, Chem. Phys. Lett. 43, 429 (1976). 13 R. M. Hochstrasser, D. L. Narva, and A. C. Nelson, Chem. Phys. Lett. 43, 15 (1976).

370

SPECTRAL RESPONSES OF VISUAL PIGMENTS I

I

I

I

[51]

I

0.5

meas.

~

8

~

at

430

nm

0

/

-0.5

i.o

I

I

]

I

~-

I

I

1

I

-*-'-I

c o .£ k)

0.5 meas. o t 5 5 0 n m

./ O

--4-~

]

I

I

0

50

I00

150

I200

Time, psec

FIG. 1. Formation and decay of hypsorhodopsin ( - - 0 - - , top) and formation of bathorhodopsin (---O--, bottom) at room temperature." Squid rhodopsin (2% digitonin extract, pH 10.5) was excited with a 347-nm pulse of 20 psec duration at 2 l°. The absorbances were measured at 430 nm (---O---, top) and 550 nm (----O--, bottom). The points in the figures represent the average of six (top) and ten (bottom) measurements. The error bars show the standard deviation. The relative absorbance changes of 1.0 in the ordinate correspond to an absorbance of 0.23 at 430 nm and 0.31 at 550 nm.

The rise curve o f hypsorhodopsin at room temperature was simulated to the convoluted curve and it was found that the rise time o f hypsorhodopsin was considerably less than 28 psec. The decay time constant o f hypsorhodopsin was estimated at 45 _+ 10 psec by fitting the convoluted curve. The time d e p e n d e n c e o f the absorbance was measured at 550 nm, close to the absorption maximum of (534 nm) of bathorhodopsin and the result is shown in Fig. 1. The absorbance at 550 nm increased after the excitation owing to the formation o f bathorhodopsin. The formation time constant was estimated to be 50 --- 10 psec by a similar method. Since this value agrees with the decay time constant o f hypsorhodopsin within the limitations of experimental error, it is concluded that hypsorhodopsin is a precursor to bathorhodopsin. The experimental result gives the first evidence for the existence of hypsorhodopsin as the precursor at room temperature. In the course of this experiment, particular care was taken to eliminate

[51]

PICOSECOND KINETICS FOR SQUID RHODOPSIN

371

the effect of the photorecovery process, i.e., conversion of alkaline or acid metarhodopsin to rhodopsin, because squid metarhodopsins are rather stable at 21 ° and a small amount of them (< 5%) existed in our preparation after the first excitation. After two further excitations, the sample was replaced. Changes in absorbance at 550 nm (formation of bathorhodopsin) were measured 190 psec after the excitation of rhodopsin and plotted against the laser power (Fig. 2). The straight line with an intercept at zero in the figure indicates that multiphoton processes do not occur under our experimental conditions. Discussion From the experimental results confirming the existence of hypsorhodopsin as a precursor to bathorhodopsin, 1°'11 the photobleaching process for squid rhodopsin can be summarized as shown in Fig. 3. Rhodopsin is converted into hypsorhodopsin by light irradiation, and in the dark hypsorhodopsin converts through several intermediates--bathorhodopsin, lumirhodopsin, and LM-rhodopsin--into acid and alkaline metarhodopsin. Some years ago, Busch e t al. 7 studied the formation and decay of cattle bathorhodopsin at room temperature by measuring the absorbance at 561 nm with LDAO-solubilized rhodopsin excited by a 530-nm light pulse from a mode-locked Nd 3+ laser. Sundstrom e t al. 8 pursued the formation and decay processes of cattle hypsorhodopsin by measuring the absorbance near 430 nm at room temperature after the excitation of rhodopsin with a picosecond laser (6 psec, 530 nm) but they did not succeed.

0,4

m

c

o

0

0.2

0.4

Energy, mJ

FIG. 2. Dependence of the absorbance charge at 550 nm upon the laser power. 11 Squid rhodopsin (2% digitonin extract, pH 10.5) was excited with a 347 nm/psec laser pulse at 21 °.

372

SPECTRAL RESPONSES OF V I S U A L PIGMENTS

[51]

Tp/e( 28 psec Rhodopsin ~ H y p s o r

hodopsin - - v v v ~ ' - I so r hod op si n ( 4 4 6 rim) 472 nm)

(489 nm)

I

"l'i/'"50PSC i >-258" Bathorhodopsm

Lurnirhodopsln

,~¢W~

(515nm) )-65"

LM-Rhodopsin ( 4 8 6 nm)

Acid metorhodopsin (482

rim)

+H _ -H

Alkaline • rnetarhodopsiin (1567 n m )

FIG. 3. Photobleaching process of squid rhodopsin. 11 Photochemical reactions are denoted by wavy lines and thermal (dark) reactions by straight lines. Absorption maxima are shown in parentheses. The transition temperature from one intermediate to the next was measured by low-temperature spectroscopy. 6 ~'l~e were measured at room temperature. 11

After their studies, the picosecond experiment on cattle rhodopsin was carried out very carefully with the use of a Nd/glass laser (6 psec, 530 nm). 14,15 Consequently, it was concluded that the first main intermediate of the bleaching process of cattle rhodopsin is hypsorhodopsin, which is formed with the time constant of 15 _+ 5 psec. The batho-intermediate yields formed directly from excited cattle rhodopsin and formed indirectly through hypsorhodopsin are 7+_~% and 93+1~0~, respectively, in the octyl glucoside buffered solution and 0+_10% and 1007_1°% in the LDAO-buffered solution. Thus the first main intermediate of the photobleaching process is hypsorhodopsin for both squid and cattle rhodopsins. The lifetimes of the triplet state of retinal, '6 unprotonated 17 and protonated retinylidene Schiff bases 18,'a are in the order of 10/xsec. Further14 T. Kobayashi, FEBS Lett. 106, 313 (1979). 15 T. Kobayashi, Photochem. Photobiol. 32, 207 (1980). 16 E. W. Abrahamson, R. G. Adams, and V. J. Wulff, J. Phys. Chem. 63, 441 (1959). 17 T. Rosenfeld, A. Alchalel, and M. Ottolenghi, Photochem. Photobiol. 20, 121 (1974). 18 M. M. Fisher and K. Weiss, Photochem. Photobiol. 20, 423 (1974). 19 A. Alchalel, B. Honig, M. Ottolenghi, and T. Rosenfeld, J. Am. Chem. Soc. 97, 2161 (1975).

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PICOSECOND K I N E T I C S FOR S Q U I D RHODOPSIN

373

more, the quantum yield of isomerization of protonated l l-cis-retinylidene Schiff base is estimated to be less than 0.001 given the assumption that the isomerization occurs only through the triplet state after excitation to the singlet state, and is much smaller than that of rhodopsin. 2° These facts indicate that the isomerization of the retinal chromophore of rhodopsin does not occur through the triplet state but occurs from the excited singlet state. The excited singlet state of twisted 11-c/s-retinal, assumed to be the chromophore of rhodopsin, has a barrierless potential surface for the isomerization at the 11-12 double bond 21 and the isomerization may occur very rapidly. By absorption of a photon, the retinal chromophore is excited to a Franck-Condon state from the ground state, then isomerizes through the barrierless potential surface with accompanying loss of vibrational energy, and finally decays to ground state. According to the theoretical calculation of Suzuki et al., 22 the absorption maximum of retinal Schiff base shifts toward longer wavelengths with increasing protonation. Since hypsorhodopsin has an absorption maximum at 446 nm, which is 88 nm shorter than that of bathorhodopsin (534 nm), the Schiff base of hypsorhodopsin was assumed to be unprotonated. 3 Schaffer et al. 23 calculated the charge density on the nitrogen atom of the Schiff base linkage in the ground state and the lowest excited singlet state as a function of the rotational angle around the 11-12 double bond (0~1-a2). Consequently, they showed that the total charge density on the nitrogen atom in the lowest excited state decreases as 01H2 goes from 180° (cis form) to 100°. This suggests that 11-cis-protonated retinylidene Schiff base may release a proton on the nitrogen atom during the conversion of rhodopsin to hypsorhodopsin. On warming up to 35 K, a small conformational change may occur in protein molecules surrounding the chromophore and the chromophore may again accept a proton from opsin to form bathorhodopsin. This is the proposed mechanism of the hypsorhodopsin formation and decay processes. As a conclusion, bathorhodopsin is mainly formed by the following process: • hv $1 Rhodopsm(S0) ------*

20 H. 21 T. zz H. 2a A.

15 -+ 5 psec (cattle) 50 ± 20 psec (cattle) <28 psec (squid) ~hypsorhodopsin 50 ± 10 psec (squid) ~bathorhodopsin.

J. A. Dartnall, " T h e Visual Pigments." Methuen, London, Wiley, New York, 1957. Kakitani and H. Kakitani, J. Phys. Soc. Jpn. 38, 1455 (1975). Suzuki, T. Komatsu, and T. Kato, J. Phys. Soc. Jpn. 34, 156 (1973). M. Schaffer, T. Yamaoka, R. S. Becker, P h o t o c h e m . Photobiol. 21, 297 t 1975),