Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Nov;79(5):2705–2713. doi: 10.1016/S0006-3495(00)76508-8

Characterization of the proton-transporting photocycle of pharaonis halorhodopsin.

A Kulcsár 1, G I Groma 1, J K Lanyi 1, G Váró 1
PMCID: PMC1301150  PMID: 11053142

Abstract

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.

Full Text

The Full Text of this article is available as a PDF (113.2 KB).

Selected References

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

  1. 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]
  2. Balashov S. P., Govindjee R., Imasheva E. S., Misra S., Ebrey T. G., Feng Y., Crouch R. K., Menick D. R. The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin. Biochemistry. 1995 Jul 11;34(27):8820–8834. doi: 10.1021/bi00027a034. [DOI] [PubMed] [Google Scholar]
  3. Bamberg E., Dencher N. A., Fahr A., Heyn M. P. Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7502–7506. doi: 10.1073/pnas.78.12.7502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bamberg E., Tittor J., Oesterhelt D. Light-driven proton or chloride pumping by halorhodopsin. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):639–643. doi: 10.1073/pnas.90.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bivin D. B., Stoeckenius W. Photoactive retinal pigments in haloalkaliphilic bacteria. J Gen Microbiol. 1986 Aug;132(8):2167–2177. doi: 10.1099/00221287-132-8-2167. [DOI] [PubMed] [Google Scholar]
  6. Brown L. S., Dioumaev A. K., Needleman R., Lanyi J. K. Local-access model for proton transfer in bacteriorhodopsin. Biochemistry. 1998 Mar 17;37(11):3982–3993. doi: 10.1021/bi9728396. [DOI] [PubMed] [Google Scholar]
  7. Dickopf S., Alexiev U., Krebs M. P., Otto H., Mollaaghababa R., Khorana H. G., Heyn M. P. Proton transport by a bacteriorhodopsin mutant, aspartic acid-85-->asparagine, initiated in the unprotonated Schiff base state. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11519–11523. doi: 10.1073/pnas.92.25.11519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duschl A., Lanyi J. K., Zimányi L. Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis. J Biol Chem. 1990 Jan 25;265(3):1261–1267. [PubMed] [Google Scholar]
  9. Dér A., Hargittai P., Simon J. Time-resolved photoelectric and absorption signals from oriented purple membranes immobilized in gel. J Biochem Biophys Methods. 1985 Mar;10(5-6):295–300. doi: 10.1016/0165-022x(85)90063-6. [DOI] [PubMed] [Google Scholar]
  10. Dér A., Oroszi L., Kulcsár A., Zimányi L., Tóth-Boconádi R., Keszthelyi L., Stoeckenius W., Ormos P. Interpretation of the spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2776–2781. doi: 10.1073/pnas.96.6.2776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dér A., Száraz S., Tóth-Boconádi R., Tokaji Z., Keszthelyi L., Stoeckenius W. Alternative translocation of protons and halide ions by bacteriorhodopsin. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4751–4755. doi: 10.1073/pnas.88.11.4751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gergely C., Ganea C., Groma G., Váró G. Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N. Biophys J. 1993 Dec;65(6):2478–2483. doi: 10.1016/S0006-3495(93)81308-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gerscher S., Mylrajan M., Hildebrandt P., Baron M. H., Müller R., Engelhard M. Chromophore-anion interactions in halorhodopsin from Natronobacterium pharaonis probed by time-resolved resonance Raman spectroscopy. Biochemistry. 1997 Sep 9;36(36):11012–11020. doi: 10.1021/bi970722b. [DOI] [PubMed] [Google Scholar]
  14. Haupts U., Tittor J., Bamberg E., Oesterhelt D. General concept for ion translocation by halobacterial retinal proteins: the isomerization/switch/transfer (IST) model. Biochemistry. 1997 Jan 7;36(1):2–7. doi: 10.1021/bi962014g. [DOI] [PubMed] [Google Scholar]
  15. Havelka W. A., Henderson R., Oesterhelt D. Three-dimensional structure of halorhodopsin at 7 A resolution. J Mol Biol. 1995 Apr 7;247(4):726–738. doi: 10.1006/jmbi.1995.0176. [DOI] [PubMed] [Google Scholar]
  16. Hegemann P., Oesterbelt D., Steiner M. The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump. EMBO J. 1985 Sep;4(9):2347–2350. doi: 10.1002/j.1460-2075.1985.tb03937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holz M., Lindau M., Heyn M. P. Distributed kinetics of the charge movements in bacteriorhodopsin: evidence for conformational substates. Biophys J. 1988 Apr;53(4):623–633. doi: 10.1016/S0006-3495(88)83141-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kalaidzidis I. V., Kalaidzidis Y. L., Kaulen A. D. Flash-induced voltage changes in halorhodopsin from Natronobacterium pharaonis. FEBS Lett. 1998 May 1;427(1):59–63. doi: 10.1016/s0014-5793(98)00394-9. [DOI] [PubMed] [Google Scholar]
  19. Keszthelyi L., Ormos P. Displacement current on purple membrane fragments oriented in a suspension. Biophys Chem. 1983 Nov;18(4):397–405. doi: 10.1016/0301-4622(83)80053-2. [DOI] [PubMed] [Google Scholar]
  20. Lanyi J. K., Duschl A., Hatfield G. W., May K., Oesterhelt D. The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins. J Biol Chem. 1990 Jan 25;265(3):1253–1260. [PubMed] [Google Scholar]
  21. Ludmann K., Gergely C., Dér A., Váró G. Electric signals during the bacteriorhodopsin photocycle, determined over a wide pH range. Biophys J. 1998 Dec;75(6):3120–3126. doi: 10.1016/S0006-3495(98)77753-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ludmann K., Gergely C., Váró G. Kinetic and thermodynamic study of the bacteriorhodopsin photocycle over a wide pH range. Biophys J. 1998 Dec;75(6):3110–3119. doi: 10.1016/S0006-3495(98)77752-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ludmann K., Ibron G., Lanyi J. K., Váró G. Charge motions during the photocycle of pharaonis halorhodopsin. Biophys J. 2000 Feb;78(2):959–966. doi: 10.1016/S0006-3495(00)76653-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nagle J. F., Zimanyi L., Lanyi J. K. Testing BR photocycle kinetics. Biophys J. 1995 Apr;68(4):1490–1499. doi: 10.1016/S0006-3495(95)80321-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oesterhelt D., Tittor J., Bamberg E. A unifying concept for ion translocation by retinal proteins. J Bioenerg Biomembr. 1992 Apr;24(2):181–191. doi: 10.1007/BF00762676. [DOI] [PubMed] [Google Scholar]
  26. Oesterhelt D., Tittor J. Two pumps, one principle: light-driven ion transport in halobacteria. Trends Biochem Sci. 1989 Feb;14(2):57–61. doi: 10.1016/0968-0004(89)90044-3. [DOI] [PubMed] [Google Scholar]
  27. Sasaki J., Brown L. S., Chon Y. S., Kandori H., Maeda A., Needleman R., Lanyi J. K. Conversion of bacteriorhodopsin into a chloride ion pump. Science. 1995 Jul 7;269(5220):73–75. doi: 10.1126/science.7604281. [DOI] [PubMed] [Google Scholar]
  28. Scharf B., Engelhard M. Blue halorhodopsin from Natronobacterium pharaonis: wavelength regulation by anions. Biochemistry. 1994 May 31;33(21):6387–6393. doi: 10.1021/bi00187a002. [DOI] [PubMed] [Google Scholar]
  29. Tittor J., Soell C., Oesterhelt D., Butt H. J., Bamberg E. A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide. EMBO J. 1989 Nov;8(11):3477–3482. doi: 10.1002/j.1460-2075.1989.tb08512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Trissl H. W. Photoelectric measurements of purple membranes. Photochem Photobiol. 1990 Jun;51(6):793–818. [PubMed] [Google Scholar]
  31. Váró G. Analogies between halorhodopsin and bacteriorhodopsin. Biochim Biophys Acta. 2000 Aug 30;1460(1):220–229. doi: 10.1016/s0005-2728(00)00141-9. [DOI] [PubMed] [Google Scholar]
  32. Váró G., Brown L. S., Needleman R., Lanyi J. K. Proton transport by halorhodopsin. Biochemistry. 1996 May 28;35(21):6604–6611. doi: 10.1021/bi9601159. [DOI] [PubMed] [Google Scholar]
  33. Váró G., Brown L. S., Sasaki J., Kandori H., Maeda A., Needleman R., Lanyi J. K. Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. 1. The photochemical cycle. Biochemistry. 1995 Nov 7;34(44):14490–14499. doi: 10.1021/bi00044a027. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Váró G., Needleman R., Lanyi J. K. Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. 2. Chloride release and uptake, protein conformation change, and thermodynamics. Biochemistry. 1995 Nov 7;34(44):14500–14507. doi: 10.1021/bi00044a028. [DOI] [PubMed] [Google Scholar]
  36. Váró G., Zimányi L., Fan X., Sun L., Needleman R., Lanyi J. K. Photocycle of halorhodopsin from Halobacterium salinarium. Biophys J. 1995 May;68(5):2062–2072. doi: 10.1016/S0006-3495(95)80385-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Walter T. J., Braiman M. S. Anion-protein interactions during halorhodopsin pumping: halide binding at the protonated Schiff base. Biochemistry. 1994 Feb 22;33(7):1724–1733. doi: 10.1021/bi00173a015. [DOI] [PubMed] [Google Scholar]
  38. Zimányi L., Keszthelyi L., Lanyi J. K. Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 1. Comparison of the photocycles of bacteriorhodopsin and halorhodopsin. Biochemistry. 1989 Jun 13;28(12):5165–5172. doi: 10.1021/bi00438a038. [DOI] [PubMed] [Google Scholar]
  39. Zimányi L., Kulcsár A., Lanyi J. K., Sears D. F., Jr, Saltiel J. Singular value decomposition with self-modeling applied to determine bacteriorhodopsin intermediate spectra: analysis of simulated data. Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4408–4413. doi: 10.1073/pnas.96.8.4408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zimányi L., Lanyi J. K. Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin. The simpler case of the recombinant D96N protein. Biophys J. 1993 Jan;64(1):240–251. doi: 10.1016/S0006-3495(93)81360-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES