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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Aug 1;88(15):6873–6877. doi: 10.1073/pnas.88.15.6873

Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

S Subramaniam 1, D A Greenhalgh 1, P Rath 1, K J Rothschild 1, H G Khorana 1
PMCID: PMC52191  PMID: 1650486

Abstract

We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.

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Selected References

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  1. Ames J. B., Mathies R. A. The role of back-reactions and proton uptake during the N----O transition in bacteriorhodopsin's photocycle: a kinetic resonance Raman study. Biochemistry. 1990 Aug 7;29(31):7181–7190. doi: 10.1021/bi00483a005. [DOI] [PubMed] [Google Scholar]
  2. Aton B., Doukas A. G., Callender R. H., Becher B., Ebrey T. G. Resonance Raman studies of the purple membrane. Biochemistry. 1977 Jun 28;16(13):2995–2999. doi: 10.1021/bi00632a029. [DOI] [PubMed] [Google Scholar]
  3. Braiman M. S., Ahl P. L., Rothschild K. J. Millisecond Fourier-transform infrared difference spectra of bacteriorhodopsin's M412 photoproduct. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5221–5225. doi: 10.1073/pnas.84.15.5221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braiman M. S., Bousché O., Rothschild K. J. Protein dynamics in the bacteriorhodopsin photocycle: submillisecond Fourier transform infrared spectra of the L, M, and N photointermediates. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2388–2392. doi: 10.1073/pnas.88.6.2388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braiman M. S., Mogi T., Marti T., Stern L. J., Khorana H. G., Rothschild K. J. Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212. Biochemistry. 1988 Nov 15;27(23):8516–8520. doi: 10.1021/bi00423a002. [DOI] [PubMed] [Google Scholar]
  6. Butt H. J., Fendler K., Bamberg E., Tittor J., Oesterhelt D. Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump. EMBO J. 1989 Jun;8(6):1657–1663. doi: 10.1002/j.1460-2075.1989.tb03556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duñach M., Berkowitz S., Marti T., He Y. W., Subramaniam S., Khorana H. G., Rothschild K. J. Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants. Evidence for two forms of tyrosine-185----phenylalanine. J Biol Chem. 1990 Oct 5;265(28):16978–16984. [PubMed] [Google Scholar]
  8. Fodor S. P., Ames J. B., Gebhard R., van den Berg E. M., Stoeckenius W., Lugtenburg J., Mathies R. A. Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism. Biochemistry. 1988 Sep 6;27(18):7097–7101. doi: 10.1021/bi00418a064. [DOI] [PubMed] [Google Scholar]
  9. Gerwert K., Hess B., Soppa J., Oesterhelt D. Role of aspartate-96 in proton translocation by bacteriorhodopsin. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4943–4947. doi: 10.1073/pnas.86.13.4943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hauss T., Grzesiek S., Otto H., Westerhausen J., Heyn M. P. Transmembrane location of retinal in bacteriorhodopsin by neutron diffraction. Biochemistry. 1990 May 22;29(20):4904–4913. doi: 10.1021/bi00472a022. [DOI] [PubMed] [Google Scholar]
  11. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  12. Khorana H. G. Bacteriorhodopsin, a membrane protein that uses light to translocate protons. J Biol Chem. 1988 Jun 5;263(16):7439–7442. [PubMed] [Google Scholar]
  13. Koch M. H., Dencher N. A., Oesterhelt D., Plöhn H. J., Rapp G., Büldt G. Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin. EMBO J. 1991 Mar;10(3):521–526. doi: 10.1002/j.1460-2075.1991.tb07978.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kouyama T., Nasuda-Kouyama A., Ikegami A., Mathew M. K., Stoeckenius W. Bacteriorhodopsin photoreaction: identification of a long-lived intermediate N (P,R350) at high pH and its M-like photoproduct. Biochemistry. 1988 Aug 9;27(16):5855–5863. doi: 10.1021/bi00416a006. [DOI] [PubMed] [Google Scholar]
  15. Lin S. W., Mathies R. A. Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism. Biophys J. 1989 Oct;56(4):653–660. doi: 10.1016/S0006-3495(89)82712-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marinetti T., Subramaniam S., Mogi T., Marti T., Khorana H. G. Replacement of aspartic residues 85, 96, 115, or 212 affects the quantum yield and kinetics of proton release and uptake by bacteriorhodopsin. Proc Natl Acad Sci U S A. 1989 Jan;86(2):529–533. doi: 10.1073/pnas.86.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marti T., Otto H., Mogi T., Rösselet S. J., Heyn M. P., Khorana H. G. Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation. J Biol Chem. 1991 Apr 15;266(11):6919–6927. [PubMed] [Google Scholar]
  18. Mogi T., Stern L. J., Marti T., Chao B. H., Khorana H. G. Aspartic acid substitutions affect proton translocation by bacteriorhodopsin. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4148–4152. doi: 10.1073/pnas.85.12.4148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Otto H., Marti T., Holz M., Mogi T., Lindau M., Khorana H. G., Heyn M. P. Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9228–9232. doi: 10.1073/pnas.86.23.9228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Otto H., Marti T., Holz M., Mogi T., Stern L. J., Engel F., Khorana H. G., Heyn M. P. Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1018–1022. doi: 10.1073/pnas.87.3.1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stern L. J., Khorana H. G. Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation. J Biol Chem. 1989 Aug 25;264(24):14202–14208. [PubMed] [Google Scholar]
  22. Stoeckenius W., Lozier R. H., Bogomolni R. A. Bacteriorhodopsin and the purple membrane of halobacteria. Biochim Biophys Acta. 1979 Mar 14;505(3-4):215–278. doi: 10.1016/0304-4173(79)90006-5. [DOI] [PubMed] [Google Scholar]
  23. Subramaniam S., Marti T., Rösselet S. J., Rothschild K. J., Khorana H. G. The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: selective reactivity of different photointermediates. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2583–2587. doi: 10.1073/pnas.88.6.2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Váró G., Lanyi J. K. Kinetic and spectroscopic evidence for an irreversible step between deprotonation and reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Biochemistry. 1991 May 21;30(20):5008–5015. doi: 10.1021/bi00234a024. [DOI] [PubMed] [Google Scholar]

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