Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1988 Mar;53(3):367–385. doi: 10.1016/S0006-3495(88)83114-X

The nature of the primary photochemical events in rhodopsin and isorhodopsin.

R R Birge 1, C M Einterz 1, H M Knapp 1, L P Murray 1
PMCID: PMC1330205  PMID: 2964878

Abstract

The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B. (b) The presence of a small activation barrier (~0.2 kcal mol-1) in the 9-cis - 9-trans excited-state surface is associated with the location of the counterion as well as the intrinsic photophysical properties of the 9-cis chromophore. The principal difference between the 1 1-cis -c 1 -transphoto reaction surface and the 9-cis - 9-trans photoreaction surface is the lack of effective electrostatic stabilization of distorted 9 = 10 conformations due to incomplete charge polarization. (c) Hydrogen bonding to the imine proton, ifpresent, does not involve the counterion. We conclude that water in the active site, or secondary interactions with the protein (not involving the CTN), are responsible. (d) All photochemical transformations involve one-bond photoisomerizations.This prediction is based on the observation of a very small excited state barrier for the I -- B photoreaction and a negative barrier for the R - B phototransformation, coupled with the theoretical prediction that all two-bond photoisomerizations have significant S, barriers while one-bond photoisomerizations have small to negative S, barriers.(e) Rhodopsin is energetically stabilized relative to isorhodopsin due to both electrostatic interactions and conformational distortion, both favoring stabilization of R. The INDO-PSDCI calculations suggest that rhodopsin chromophore-CTN electrostatic interactions provide an enhanced stabilization of -2 kcal mol-1 relative to I. Conformational distortion of the 9-cis chromophore-lysine system accounts for -3 kcal mol-1. (f) Energy storage in bathorhodopsin is-60% conformational distortion and 40% charge separation. Our model predicts that the majority of the chromophore protein conformational distortion energy involves interaction of the C,3(-CH3)=CI4--C,5=N-lysine moiety with nearby (unknown) protein residues. (g) Strong interactions between the counterion and the chromophore in R and I will generate weak, but potentially observable charge-transfer bands in the near infrared. The key predictions are the presence of an observable charge-transfer transition at 859 nm (1 1,640 cm- 1) in I and an analogous, but slightly weaker band at 897 nm (11,150 cm-1) in R. Both transitions involve the transfer of an electron from the counterion into low-lying l theta* molecular orbitals.

Full text

PDF
367

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Asato A. E., Denny M., Matsumoto H., Mirzadegan T., Ripka W. C., Crescitelli F., Liu R. S. Study of the shape of the binding site of bovine opsin using 10-substituted retinal isomers. Biochemistry. 1986 Nov 4;25(22):7021–7026. doi: 10.1021/bi00370a039. [DOI] [PubMed] [Google Scholar]
  2. Baasov T., Friedman N., Sheves M. Factors affecting the C = N stretching in protonated retinal Schiff base: a model study for bacteriorhodopsin and visual pigments. Biochemistry. 1987 Jun 2;26(11):3210–3217. doi: 10.1021/bi00385a041. [DOI] [PubMed] [Google Scholar]
  3. Bagley K. A., Balogh-Nair V., Croteau A. A., Dollinger G., Ebrey T. G., Eisenstein L., Hong M. K., Nakanishi K., Vittitow J. Fourier-transform infrared difference spectroscopy of rhodopsin and its photoproducts at low temperature. Biochemistry. 1985 Oct 22;24(22):6055–6071. doi: 10.1021/bi00343a006. [DOI] [PubMed] [Google Scholar]
  4. Barry B., Mathies R. A. Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm. Biochemistry. 1987 Jan 13;26(1):59–64. doi: 10.1021/bi00375a009. [DOI] [PubMed] [Google Scholar]
  5. Birge R. R., Hubbard L. M. Molecular dynamics of trans-cis isomerization in bathorhodopsin. Biophys J. 1981 Jun;34(3):517–534. doi: 10.1016/S0006-3495(81)84865-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birge R. R., Murray L. P., Pierce B. M., Akita H., Balogh-Nair V., Findsen L. A., Nakanishi K. Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4117–4121. doi: 10.1073/pnas.82.12.4117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Birge R. R. Photophysics of light transduction in rhodopsin and bacteriorhodopsin. Annu Rev Biophys Bioeng. 1981;10:315–354. doi: 10.1146/annurev.bb.10.060181.001531. [DOI] [PubMed] [Google Scholar]
  8. Blatz P. E., Mohler J. H., Navangul H. V. Anion-induced wavelength regulation of absorption maxima of Schiff bases of retinal. Biochemistry. 1972 Feb 29;11(5):848–855. doi: 10.1021/bi00755a026. [DOI] [PubMed] [Google Scholar]
  9. Callender R. H., Doukas A., Crouch R., Nakanishi K. Molecular flow resonance Raman effect from retinal and rhodopsin. Biochemistry. 1976 Apr 20;15(8):1621–1629. doi: 10.1021/bi00653a005. [DOI] [PubMed] [Google Scholar]
  10. Cooper A. Energetics of rhodopsin and isorhodopsin. FEBS Lett. 1979 Apr 15;100(2):382–384. doi: 10.1016/0014-5793(79)80375-0. [DOI] [PubMed] [Google Scholar]
  11. Cooper A. Energy uptake in the first step of visual excitation. Nature. 1979 Nov 29;282(5738):531–533. doi: 10.1038/282531a0. [DOI] [PubMed] [Google Scholar]
  12. Crouch R., Purvin V., Nakanishi K., Ebrey T. Isorhodopsin II: artificial photosensitive pigment formed from 9,13-dicis retinal. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1538–1542. doi: 10.1073/pnas.72.4.1538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Einterz C. M., Lewis J. W., Kliger D. S. Spectral and kinetic evidence for the existence of two forms of bathorhodopsin. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3699–3703. doi: 10.1073/pnas.84.11.3699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eyring G., Curry B., Broek A., Lugtenburg J., Mathies R. Assignment and interpretation of hydrogen out-of-plane vibrations in the resonance Raman spectra of rhodopsin and bathorhodopsin. Biochemistry. 1982 Jan 19;21(2):384–393. doi: 10.1021/bi00531a028. [DOI] [PubMed] [Google Scholar]
  15. Eyring G., Mathies R. Resonance Raman studies of bathorhodopsin: evidence for a protonated Schiff base linkage. Proc Natl Acad Sci U S A. 1979 Jan;76(1):33–37. doi: 10.1073/pnas.76.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hargrave P. A., McDowell J. H., Feldmann R. J., Atkinson P. H., Rao J. K., Argos P. Rhodopsin's protein and carbohydrate structure: selected aspects. Vision Res. 1984;24(11):1487–1499. doi: 10.1016/0042-6989(84)90311-0. [DOI] [PubMed] [Google Scholar]
  17. Hong K., Knudsen P. J., Hubbell W. L. Purification of rhodopsin on hydroxyapatite columns, detergent exchange, and recombination with phospholipids. Methods Enzymol. 1982;81:144–150. doi: 10.1016/s0076-6879(82)81024-0. [DOI] [PubMed] [Google Scholar]
  18. Honig B., Ebrey T., Callender R. H., Dinur U., Ottolenghi M. Photoisomerization, energy storage, and charge separation: a model for light energy transduction in visual pigments and bacteriorhodopsin. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2503–2507. doi: 10.1073/pnas.76.6.2503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huppert D., Rentzepis P. M., Kliger D. S. Picosecond and nanosecond isomerization kinetics of protonated 11-cis retinylidene Schiff bases. Photochem Photobiol. 1977 Feb;25(2):193–197. doi: 10.1111/j.1751-1097.1977.tb06897.x. [DOI] [PubMed] [Google Scholar]
  20. Hurley J. B., Ebrey T. G., Honig B., Ottolenghi M. Temperature and wavelength effects on the photochemistry of rhodopsin, isorhodopsin, bacteriorhodopsin and their photoproducts. Nature. 1977 Dec 8;270(5637):540–542. doi: 10.1038/270540a0. [DOI] [PubMed] [Google Scholar]
  21. Kakitani H., Kakitani T., Rodman H., Honig B. On the mechanism of wavelength regulation in visual pigments. Photochem Photobiol. 1985 Apr;41(4):471–479. doi: 10.1111/j.1751-1097.1985.tb03514.x. [DOI] [PubMed] [Google Scholar]
  22. Kliger D. S., Horwitz J. S., Lewis J. W., Einterz C. M. Evidence for a common BATHO-intermediate in the bleaching of rhodopsin and isorhodopsin. Vision Res. 1984;24(11):1465–1470. doi: 10.1016/0042-6989(84)90307-9. [DOI] [PubMed] [Google Scholar]
  23. Leclercq J. M., Sandorfy C. On the possibility of protein-chromophore charge transfer in visual pigments. Photochem Photobiol. 1981 Mar;33(3):361–365. doi: 10.1111/j.1751-1097.1981.tb05430.x. [DOI] [PubMed] [Google Scholar]
  24. Liu R. S., Asato A. E. The primary process of vision and the structure of bathorhodopsin: a mechanism for photoisomerization of polyenes. Proc Natl Acad Sci U S A. 1985 Jan;82(2):259–263. doi: 10.1073/pnas.82.2.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mao B., Ebrey T. G., Crouch R. Bathoproducts of rhodopsin, isorhodopsin I, and isorhodopsin II. Biophys J. 1980 Feb;29(2):247–256. doi: 10.1016/S0006-3495(80)85129-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsumoto H., Yoshizawa T. Recognition of opsin to the longitudinal length of retinal isomers in the formation of rhodopsin. Vision Res. 1978;18(5):607–609. doi: 10.1016/0042-6989(78)90212-2. [DOI] [PubMed] [Google Scholar]
  27. Metzler D. E., Harris C. M. Shapes of spectral bands of visual pigments. Vision Res. 1978;18(10):1417–1420. doi: 10.1016/0042-6989(78)90235-3. [DOI] [PubMed] [Google Scholar]
  28. Monger T. G., Alfano R. R., Callender R. H. Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale. Biophys J. 1979 Jul;27(1):105–115. doi: 10.1016/S0006-3495(79)85205-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Palings I., Pardoen J. A., van den Berg E., Winkel C., Lugtenburg J., Mathies R. A. Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment. Biochemistry. 1987 May 5;26(9):2544–2556. doi: 10.1021/bi00383a021. [DOI] [PubMed] [Google Scholar]
  30. Rafferty C. N., Shichi H. The involvement of water at the retinal binding site in rhodopsin and early light-induced intramolecular proton transfer. Photochem Photobiol. 1981 Feb;33(2):229–234. doi: 10.1111/j.1751-1097.1981.tb05329.x. [DOI] [PubMed] [Google Scholar]
  31. Rothschild K. J., Cantore W. A., Marrero H. Fourier transform infrared difference spectra of intermediates in rhodopsin bleaching. Science. 1983 Mar 18;219(4590):1333–1335. doi: 10.1126/science.6828860. [DOI] [PubMed] [Google Scholar]
  32. Sasaki N., Tokunaga F., Yoshizawa T. The formation of two forms of bathorhodopsin and their optical properties. Photochem Photobiol. 1980 Oct;32(4):433–441. doi: 10.1111/j.1751-1097.1980.tb03785.x. [DOI] [PubMed] [Google Scholar]
  33. Schaffer A. M., Waddell W. H., Becker R. S. Visual pigments. IV. Experimental and theoretical investigations of the absorption spectra of retinal Schiff bases and retinals. J Am Chem Soc. 1974 Apr 3;96(7):2063–2068. doi: 10.1021/ja00814a013. [DOI] [PubMed] [Google Scholar]
  34. Schick G. A., Cooper T. M., Holloway R. A., Murray L. P., Birge R. R. Energy storage in the primary photochemical events of rhodopsin and isorhodopsin. Biochemistry. 1987 May 5;26(9):2556–2562. doi: 10.1021/bi00383a022. [DOI] [PubMed] [Google Scholar]
  35. Smith S. O., Myers A. B., Mathies R. A., Pardoen J. A., Winkel C., van den Berg E. M., Lugtenburg J. Vibrational analysis of the all-trans retinal protonated Schiff base. Biophys J. 1985 May;47(5):653–664. doi: 10.1016/S0006-3495(85)83961-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stryer L. Cyclic GMP cascade of vision. Annu Rev Neurosci. 1986;9:87–119. doi: 10.1146/annurev.ne.09.030186.000511. [DOI] [PubMed] [Google Scholar]
  37. Suzuki T., Callender R. H. Primary photochemistry and photoisomerization of retinal at 77 degrees K in cattle and squid rhodopsins. Biophys J. 1981 May;34(2):261–270. doi: 10.1016/S0006-3495(81)84848-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. WALD G., BROWN P. K. The molar extinction of rhodopsin. J Gen Physiol. 1953 Nov 20;37(2):189–200. doi: 10.1085/jgp.37.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. YOSHIZAWA T., WALD G. Pre-lumirhodopsin and the bleaching of visual pigments. Nature. 1963 Mar 30;197:1279–1286. doi: 10.1038/1971279a0. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES