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. 2002 May;82(5):2617–2626. doi: 10.1016/S0006-3495(02)75603-8

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment.

Amir Aharoni 1, Michael Ottolenghi 1, Mordechai Sheves 1
PMCID: PMC1302050  PMID: 11964248

Abstract

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.

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Selected References

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  1. Aharoni A., Hou B., Friedman N., Ottolenghi M., Rousso I., Ruhman S., Sheves M., Ye T., Zhong Q. Non-isomerizable artificial pigments: implications for the primary light-induced events in bacteriorhodopsin. Biochemistry (Mosc) 2001 Nov;66(11):1210–1219. doi: 10.1023/a:1013175000873. [DOI] [PubMed] [Google Scholar]
  2. Aharoni A., Weiner L., Lewis A., Ottolenghi M., Sheves M. Nonisomerizable non-retinal chromophores initiate light-induced conformational alterations in bacterioopsin. J Am Chem Soc. 2001 Jul 11;123(27):6612–6616. doi: 10.1021/ja004035a. [DOI] [PubMed] [Google Scholar]
  3. Aharoni A., Weiner L., Ottolenghi M., Sheves M. Bacteriorhodpsin experiences light-induced conformational alterations in nonisomerizable C(13)=C(14) pigments. A study with EPR. J Biol Chem. 2000 Jul 14;275(28):21010–21016. doi: 10.1074/jbc.M001208200. [DOI] [PubMed] [Google Scholar]
  4. Balashov S. P., Govindjee R., Ebrey T. G. Redshift of the purple membrane absorption band and the deprotonation of tyrosine residues at high pH: Origin of the parallel photocycles of trans-bacteriorhodopsin. Biophys J. 1991 Aug;60(2):475–490. doi: 10.1016/S0006-3495(91)82074-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cao Y., Váró G., Chang M., Ni B. F., Needleman R., Lanyi J. K. Water is required for proton transfer from aspartate-96 to the bacteriorhodopsin Schiff base. Biochemistry. 1991 Nov 12;30(45):10972–10979. doi: 10.1021/bi00109a023. [DOI] [PubMed] [Google Scholar]
  6. Dancsházy Z., Tokaji Z., Dér A. Bleaching of bacteriorhodopsin by continuous light. FEBS Lett. 1999 Apr 30;450(1-2):154–157. doi: 10.1016/s0014-5793(99)00487-1. [DOI] [PubMed] [Google Scholar]
  7. Fischer U. C., Oesterhelt D. Changes in the protonation state of bacterio-opsin during reconstitution of bacteriorhodopsin. Biophys J. 1980 Jul;31(1):139–145. doi: 10.1016/S0006-3495(80)85045-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grigorieff N., Beckmann E., Zemlin F. Lipid location in deoxycholate-treated purple membrane at 2.6 A. J Mol Biol. 1995 Dec 1;254(3):404–415. doi: 10.1006/jmbi.1995.0627. [DOI] [PubMed] [Google Scholar]
  9. Isralewitz B., Izrailev S., Schulten K. Binding pathway of retinal to bacterio-opsin: a prediction by molecular dynamics simulations. Biophys J. 1997 Dec;73(6):2972–2979. doi: 10.1016/S0006-3495(97)78326-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jang D. J., el-Sayed M. A. Deprotonation of lipid-depleted bacteriorhodopsin. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5918–5922. doi: 10.1073/pnas.85.16.5918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lanyi J. K. Bacteriorhodopsin. Int Rev Cytol. 1999;187:161–202. doi: 10.1016/s0074-7696(08)62418-3. [DOI] [PubMed] [Google Scholar]
  12. Lewis A. The molecular mechanism of excitation in visual transduction and bacteriorhodopsin. Proc Natl Acad Sci U S A. 1978 Feb;75(2):549–553. doi: 10.1073/pnas.75.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Losi A., Michler I., Gärtner W., Braslavsky S. E. Time-resolved thermodynamic changes photoinduced in 5,12-trans-locked bacteriorhodopsin. Evidence that retinal isomerization is required for protein activation. Photochem Photobiol. 2000 Nov;72(5):590–597. doi: 10.1562/0031-8655(2000)072<0590:trtcpi>2.0.co;2. [DOI] [PubMed] [Google Scholar]
  14. Luecke H., Schobert B., Richter H. T., Cartailler J. P., Lanyi J. K. Structure of bacteriorhodopsin at 1.55 A resolution. J Mol Biol. 1999 Aug 27;291(4):899–911. doi: 10.1006/jmbi.1999.3027. [DOI] [PubMed] [Google Scholar]
  15. Metz G., Siebert F., Engelhard M. Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation. FEBS Lett. 1992 Jun 1;303(2-3):237–241. doi: 10.1016/0014-5793(92)80528-o. [DOI] [PubMed] [Google Scholar]
  16. Oesterhelt D., Schuhmann L., Gruber H. Light-dependent reaction of bacteriorhodopsin with hydroxylamine in cell suspensions of Halobacterium halobium: demonstration of an apo-membrane. FEBS Lett. 1974 Aug 30;44(3):257–261. doi: 10.1016/0014-5793(74)81152-x. [DOI] [PubMed] [Google Scholar]
  17. Oesterhelt D. The structure and mechanism of the family of retinal proteins from halophilic archaea. Curr Opin Struct Biol. 1998 Aug;8(4):489–500. doi: 10.1016/s0959-440x(98)80128-0. [DOI] [PubMed] [Google Scholar]
  18. Rousso I., Brodsky I., Lewis A., Sheves M. The role of water in retinal complexation to bacterio-opsin. J Biol Chem. 1995 Jun 9;270(23):13860–13868. doi: 10.1074/jbc.270.23.13860. [DOI] [PubMed] [Google Scholar]
  19. Rousso I., Friedman N., Lewis A., Sheves M. Evidence for a controlling role of water in producing the native bacteriorhodopsin structure. Biophys J. 1997 Oct;73(4):2081–2089. doi: 10.1016/S0006-3495(97)78238-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rousso I., Gat Y., Lewis A., Sheves M., Ottolenghi M. Effective light-induced hydroxylamine reactions occur with C13 = C14 nonisomerizable bacteriorhodopsin pigments. Biophys J. 1998 Jul;75(1):413–417. doi: 10.1016/S0006-3495(98)77526-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rousso I., Khachatryan E., Gat Y., Brodsky I., Ottolenghi M., Sheves M., Lewis A. Microsecond atomic force sensing of protein conformational dynamics: implications for the primary light-induced events in bacteriorhodopsin. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):7937–7941. doi: 10.1073/pnas.94.15.7937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schreckenbach T., Walckhoff B., Oesterhelt D. Studies on the retinal-protein interaction in bacteriorhodopsin. Eur J Biochem. 1977 Jun 15;76(2):499–511. doi: 10.1111/j.1432-1033.1977.tb11620.x. [DOI] [PubMed] [Google Scholar]
  23. Schweiger U., Tittor J., Oesterhelt D. Bacteriorhodopsin can function without a covalent linkage between retinal and protein. Biochemistry. 1994 Jan 18;33(2):535–541. doi: 10.1021/bi00168a019. [DOI] [PubMed] [Google Scholar]
  24. Subramaniam S., Lindahl M., Bullough P., Faruqi A. R., Tittor J., Oesterhelt D., Brown L., Lanyi J., Henderson R. Protein conformational changes in the bacteriorhodopsin photocycle. J Mol Biol. 1999 Mar 19;287(1):145–161. doi: 10.1006/jmbi.1999.2589. [DOI] [PubMed] [Google Scholar]
  25. Szundi I., Stoeckenius W. Effect of lipid surface charges on the purple-to-blue transition of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3681–3684. doi: 10.1073/pnas.84.11.3681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thorgeirsson T. E., Xiao W., Brown L. S., Needleman R., Lanyi J. K., Shin Y. K. Transient channel-opening in bacteriorhodopsin: an EPR study. J Mol Biol. 1997 Nov 14;273(5):951–957. doi: 10.1006/jmbi.1997.1362. [DOI] [PubMed] [Google Scholar]
  27. Vonck J. Structure of the bacteriorhodopsin mutant F219L N intermediate revealed by electron crystallography. EMBO J. 2000 May 15;19(10):2152–2160. doi: 10.1093/emboj/19.10.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Xiao W., Brown L. S., Needleman R., Lanyi J. K., Shin Y. K. Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin. J Mol Biol. 2000 Dec 15;304(5):715–721. doi: 10.1006/jmbi.2000.4255. [DOI] [PubMed] [Google Scholar]

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