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. 2002 Apr;82(4):1687–1695. doi: 10.1016/S0006-3495(02)75521-5

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin.

Melinda Lakatos 1, Géza I Groma 1, Constanta Ganea 1, Janos K Lanyi 1, György Váró 1
PMCID: PMC1301968  PMID: 11916830

Abstract

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.

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Selected References

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  1. Bamberg E., Oesterhelt D., Tittor J. Function of halorhodopsin as a light-driven H+ pump. Ren Physiol Biochem. 1994 May-Aug;17(3-4):194–197. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Dancsházy Z., Tokaji Z. Blue light regeneration of bacteriorhodopsin bleached by continuous light. FEBS Lett. 2000 Jul 7;476(3):171–173. doi: 10.1016/s0014-5793(00)01714-2. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Gergely C., Ganea C., Váró G. Combined optical and photoelectric study of the photocycle of 13-cis bacteriorhodopsin. Biophys J. 1994 Aug;67(2):855–861. doi: 10.1016/S0006-3495(94)80545-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Kolbe M., Besir H., Essen L. O., Oesterhelt D. Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution. Science. 2000 May 26;288(5470):1390–1396. doi: 10.1126/science.288.5470.1390. [DOI] [PubMed] [Google Scholar]
  14. Kulcsár A., Groma G. I., Lanyi J. K., Váró G. Characterization of the proton-transporting photocycle of pharaonis halorhodopsin. Biophys J. 2000 Nov;79(5):2705–2713. doi: 10.1016/S0006-3495(00)76508-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Lanyi J. K. Halorhodopsin, a light-driven electrogenic chloride-transport system. Physiol Rev. 1990 Apr;70(2):319–330. doi: 10.1152/physrev.1990.70.2.319. [DOI] [PubMed] [Google Scholar]
  17. Lanyi J. K. Halorhodopsin: a light-driven chloride ion pump. Annu Rev Biophys Biophys Chem. 1986;15:11–28. doi: 10.1146/annurev.bb.15.060186.000303. [DOI] [PubMed] [Google Scholar]
  18. Lanyi J. K. Mechanism of base-catalyzed Schiff base deprotonation in halorhodopsin. Biochemistry. 1986 Oct 21;25(21):6706–6711. doi: 10.1021/bi00369a057. [DOI] [PubMed] [Google Scholar]
  19. Lanyi J. K. Understanding structure and function in the light-driven proton pump bacteriorhodopsin. J Struct Biol. 1998 Dec 15;124(2-3):164–178. doi: 10.1006/jsbi.1998.4044. [DOI] [PubMed] [Google Scholar]
  20. Lindley E. V., MacDonald R. E. A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump. Biochem Biophys Res Commun. 1979 May 28;88(2):491–499. doi: 10.1016/0006-291x(79)92075-8. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Matsuno-Yagi A., Mukohata Y. Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation. Biochem Biophys Res Commun. 1977 Sep 9;78(1):237–243. doi: 10.1016/0006-291x(77)91245-1. [DOI] [PubMed] [Google Scholar]
  24. Oesterhelt D., Hegemann P., Tittor J. The photocycle of the chloride pump halorhodopsin. II: Quantum yields and a kinetic model. EMBO J. 1985 Sep;4(9):2351–2356. doi: 10.1002/j.1460-2075.1985.tb03938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oesterhelt D., Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974;31:667–678. doi: 10.1016/0076-6879(74)31072-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  29. 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]
  30. Schobert B., Lanyi J. K. Halorhodopsin is a light-driven chloride pump. J Biol Chem. 1982 Sep 10;257(17):10306–10313. [PubMed] [Google Scholar]
  31. 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]
  32. Trissl H. W. Photoelectric measurements of purple membranes. Photochem Photobiol. 1990 Jun;51(6):793–818. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  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., 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]

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