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. 1998 Dec;75(6):3110–3119. doi: 10.1016/S0006-3495(98)77752-5

Kinetic and thermodynamic study of the bacteriorhodopsin photocycle over a wide pH range.

K Ludmann 1, C Gergely 1, G Váró 1
PMCID: PMC1299982  PMID: 9826631

Abstract

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pKa of the photocycle energetics and the role of several amino acids in the protein.

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Selected References

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  1. Alexiev U., Marti T., Heyn M. P., Khorana H. G., Scherrer P. Surface charge of bacteriorhodopsin detected with covalently bound pH indicators at selected extracellular and cytoplasmic sites. Biochemistry. 1994 Jan 11;33(1):298–306. doi: 10.1021/bi00167a039. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Balashov S. P., Imasheva E. S., Govindjee R., Ebrey T. G. Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release. Biophys J. 1996 Jan;70(1):473–481. doi: 10.1016/S0006-3495(96)79591-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baldwin R. L. Temperature dependence of the hydrophobic interaction in protein folding. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8069–8072. doi: 10.1073/pnas.83.21.8069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bashford D., Gerwert K. Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin. J Mol Biol. 1992 Mar 20;224(2):473–486. doi: 10.1016/0022-2836(92)91009-e. [DOI] [PubMed] [Google Scholar]
  6. Brown L. S., Bonet L., Needleman R., Lanyi J. K. Estimated acid dissociation constants of the Schiff base, Asp-85, and Arg-82 during the bacteriorhodopsin photocycle. Biophys J. 1993 Jul;65(1):124–130. doi: 10.1016/S0006-3495(93)81064-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown L. S., Sasaki J., Kandori H., Maeda A., Needleman R., Lanyi J. K. Glutamic acid 204 is the terminal proton release group at the extracellular surface of bacteriorhodopsin. J Biol Chem. 1995 Nov 10;270(45):27122–27126. doi: 10.1074/jbc.270.45.27122. [DOI] [PubMed] [Google Scholar]
  8. Cao Y., Brown L. S., Needleman R., Lanyi J. K. Relationship of proton uptake on the cytoplasmic surface and reisomerization of the retinal in the bacteriorhodopsin photocycle: an attempt to understand the complex kinetics of the pH changes and the N and O intermediates. Biochemistry. 1993 Sep 28;32(38):10239–10248. doi: 10.1021/bi00089a046. [DOI] [PubMed] [Google Scholar]
  9. Chizhov I., Chernavskii D. S., Engelhard M., Mueller K. H., Zubov B. V., Hess B. Spectrally silent transitions in the bacteriorhodopsin photocycle. Biophys J. 1996 Nov;71(5):2329–2345. doi: 10.1016/S0006-3495(96)79475-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chizhov I., Engelhard M., Chernavskii D. S., Zubov B., Hess B. Temperature and pH sensitivity of the O(640) intermediate of the bacteriorhodopsin photocycle. Biophys J. 1992 Apr;61(4):1001–1006. doi: 10.1016/s0006-3495(92)81907-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dancsházy Z., Govindjee R., Ebrey T. G. Independent photocycles of the spectrally distinct forms of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6358–6361. doi: 10.1073/pnas.85.17.6358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dioumaev A. K. Evaluation of intrinsic chemical kinetics and transient product spectra from time-resolved spectroscopic data. Biophys Chem. 1997 Sep 1;67(1-3):1–25. doi: 10.1016/s0301-4622(96)02268-5. [DOI] [PubMed] [Google Scholar]
  13. Drachev L. A., Kaulen A. D., Komrakov AYu On the two pathways of the M-intermediate formation in the photocycle of bacteriorhodopsin. Biochem Mol Biol Int. 1993 Jul;30(3):461–469. [PubMed] [Google Scholar]
  14. Draheim J. E., Cassim J. Y. Large Scale Global Structural Changes of the Purple Membrane during the Photocycle. Biophys J. 1985 Apr;47(4):497–507. doi: 10.1016/S0006-3495(85)83943-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Eisfeld W., Pusch C., Diller R., Lohrmann R., Stockburger M. Resonance Raman and optical transient studies on the light-induced proton pump of bacteriorhodopsin reveal parallel photocycles. Biochemistry. 1993 Jul 20;32(28):7196–7215. doi: 10.1021/bi00079a017. [DOI] [PubMed] [Google Scholar]
  18. Friedman N., Gat Y., Sheves M., Ottolenghi M. On the heterogeneity of the M population in the photocycle of bacteriorhodopsin. Biochemistry. 1994 Dec 13;33(49):14758–14767. doi: 10.1021/bi00253a014. [DOI] [PubMed] [Google Scholar]
  19. Garty H., Caplan S. R., Cahen D. Photoacoustic photocalorimetry and spectroscopy of Halobacterium halobium purple membranes. Biophys J. 1982 Feb;37(2):405–415. doi: 10.1016/S0006-3495(82)84686-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Grigorieff N., Ceska T. A., Downing K. H., Baldwin J. M., Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Biol. 1996 Jun 14;259(3):393–421. doi: 10.1006/jmbi.1996.0328. [DOI] [PubMed] [Google Scholar]
  23. Han B. G., Vonck J., Glaeser R. M. The bacteriorhodopsin photocycle: direct structural study of two substrates of the M-intermediate. Biophys J. 1994 Sep;67(3):1179–1186. doi: 10.1016/S0006-3495(94)80586-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Hendler R. W., Dancsházy Z., Bose S., Shrager R. I., Tokaji Z. Influence of excitation energy on the bacteriorhodopsin photocycle. Biochemistry. 1994 Apr 19;33(15):4604–4610. doi: 10.1021/bi00181a022. [DOI] [PubMed] [Google Scholar]
  27. Hessling B., Souvignier G., Gerwert K. A model-independent approach to assigning bacteriorhodopsin's intramolecular reactions to photocycle intermediates. Biophys J. 1993 Nov;65(5):1929–1941. doi: 10.1016/S0006-3495(93)81264-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kandori H., Yamazaki Y., Hatanaka M., Needleman R., Brown L. S., Richter H. T., Lanyi J. K., Maeda A. Time-resolved fourier transform infrared study of structural changes in the last steps of the photocycles of Glu-204 and Leu-93 mutants of bacteriorhodopsin. Biochemistry. 1997 Apr 29;36(17):5134–5141. doi: 10.1021/bi9629788. [DOI] [PubMed] [Google Scholar]
  29. Kimura Y., Vassylyev D. G., Miyazawa A., Kidera A., Matsushima M., Mitsuoka K., Murata K., Hirai T., Fujiyoshi Y. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature. 1997 Sep 11;389(6647):206–211. doi: 10.1038/38323. [DOI] [PubMed] [Google Scholar]
  30. Komrakov A. Y., Kaulen A. D. M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH. Biophys Chem. 1995 Sep-Oct;56(1-2):113–119. doi: 10.1016/0301-4622(95)00022-p. [DOI] [PubMed] [Google Scholar]
  31. Krebs M. P., Khorana H. G. Mechanism of light-dependent proton translocation by bacteriorhodopsin. J Bacteriol. 1993 Mar;175(6):1555–1560. doi: 10.1128/jb.175.6.1555-1560.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lozier R. H., Xie A., Hofrichter J., Clore G. M. Reversible steps in the bacteriorhodopsin photocycle. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3610–3614. doi: 10.1073/pnas.89.8.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Luchian T., Tokaji Z., Dancsházy Z. Actinic light density dependence of the O intermediate of the photocycle of bacteriorhodopsin. FEBS Lett. 1996 May 13;386(1):55–59. doi: 10.1016/0014-5793(96)00391-2. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Marque J., Eisenstein L., Gratton E., Sturtevant J. M., Hardy C. J. Thermodynamic properties of purple membrane. Biophys J. 1984 Nov;46(5):567–572. doi: 10.1016/S0006-3495(84)84055-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mowery P. C., Lozier R. H., Chae Q., Tseng Y. W., Taylor M., Stoeckenius W. Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin. Biochemistry. 1979 Sep 18;18(19):4100–4107. doi: 10.1021/bi00586a007. [DOI] [PubMed] [Google Scholar]
  37. Murphy K. P., Privalov P. L., Gill S. J. Common features of protein unfolding and dissolution of hydrophobic compounds. Science. 1990 Feb 2;247(4942):559–561. doi: 10.1126/science.2300815. [DOI] [PubMed] [Google Scholar]
  38. Nagle J. F. Solving complex photocycle kinetics. Theory and direct method. Biophys J. 1991 Feb;59(2):476–487. doi: 10.1016/S0006-3495(91)82241-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Ort D. R., Parson W. W. Enthalpy changes during the photochemical cycle of bacteriorhodopsin. Biophys J. 1979 Feb;25(2 Pt 1):355–364. doi: 10.1016/s0006-3495(79)85297-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pebay-Peyroula E., Rummel G., Rosenbusch J. P., Landau E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science. 1997 Sep 12;277(5332):1676–1681. doi: 10.1126/science.277.5332.1676. [DOI] [PubMed] [Google Scholar]
  42. Popp A., Wolperdinger M., Hampp N., Brüchle C., Oesterhelt D. Photochemical conversion of the O-intermediate to 9-cis-retinal-containing products in bacteriorhodopsin films. Biophys J. 1993 Oct;65(4):1449–1459. doi: 10.1016/S0006-3495(93)81214-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Richter H. T., Brown L. S., Needleman R., Lanyi J. K. A linkage of the pKa's of asp-85 and glu-204 forms part of the reprotonation switch of bacteriorhodopsin. Biochemistry. 1996 Apr 2;35(13):4054–4062. doi: 10.1021/bi952883q. [DOI] [PubMed] [Google Scholar]
  44. Richter H. T., Needleman R., Kandori H., Maeda A., Lanyi J. K. Relationship of retinal configuration and internal proton transfer at the end of the bacteriorhodopsin photocycle. Biochemistry. 1996 Dec 3;35(48):15461–15466. doi: 10.1021/bi9612430. [DOI] [PubMed] [Google Scholar]
  45. Sherman W. V., Caplan S. R. Arrhenius parameters of phototransients in Halobacterium halobium in physiological conditions. Nature. 1975 Dec 25;258(5537):766–768. doi: 10.1038/258766a0. [DOI] [PubMed] [Google Scholar]
  46. Song L., Logunov S. L., Yang D., el-Sayed M. A. The pH dependence of the subpicosecond retinal photoisomerization process in bacteriorhodopsin: evidence for parallel photocycles. Biophys J. 1994 Nov;67(5):2008–2012. doi: 10.1016/S0006-3495(94)80684-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Subramaniam S., Faruqi A. R., Oesterhelt D., Henderson R. Electron diffraction studies of light-induced conformational changes in the Leu-93 --> Ala bacteriorhodopsin mutant. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1767–1772. doi: 10.1073/pnas.94.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Subramaniam S., Gerstein M., Oesterhelt D., Henderson R. Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin. EMBO J. 1993 Jan;12(1):1–8. doi: 10.1002/j.1460-2075.1993.tb05625.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Szundi I., Lewis J. W., Kliger D. S. Deriving reaction mechanisms from kinetic spectroscopy. Application to late rhodopsin intermediates. Biophys J. 1997 Aug;73(2):688–702. doi: 10.1016/S0006-3495(97)78103-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Száraz S., Oesterhelt D., Ormos P. pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy. Biophys J. 1994 Oct;67(4):1706–1712. doi: 10.1016/S0006-3495(94)80644-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tokaji Z. Cooperativity-regulated parallel pathways of the bacteriorhodopsin photocycle. FEBS Lett. 1995 Jan 3;357(2):156–160. doi: 10.1016/0014-5793(94)01344-z. [DOI] [PubMed] [Google Scholar]
  52. Tokaji Z., Dancsházy Z. Kinetics of the N intermediate and the two pathways of recovery of the ground-state of bacteriorhodopsin. FEBS Lett. 1992 Oct 26;311(3):267–270. doi: 10.1016/0014-5793(92)81117-5. [DOI] [PubMed] [Google Scholar]
  53. Tsuda M., Govindjee R., Ebrey T. G. Effects of pressure and temperature on the M412 intermediate of the bacteriorhodopsin photocycle. Implications for the phase transition of the purple membrane. Biophys J. 1983 Nov;44(2):249–254. doi: 10.1016/S0006-3495(83)84296-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Váró G., Duschl A., Lanyi J. K. Interconversions of the M, N, and O intermediates in the bacteriorhodopsin photocycle. Biochemistry. 1990 Apr 17;29(15):3798–3804. doi: 10.1021/bi00467a029. [DOI] [PubMed] [Google Scholar]
  55. Váró G., Keszthelyi L. Arrhenius parameters of the bacteriorhodopsin photocycle in dried oriented samples. Biophys J. 1985 Feb;47(2 Pt 1):243–246. doi: 10.1016/s0006-3495(85)83897-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Váró G., Lanyi J. K. Effects of hydrostatic pressure on the kinetics reveal a volume increase during the bacteriorhodopsin photocycle. Biochemistry. 1995 Sep 26;34(38):12161–12169. doi: 10.1021/bi00038a009. [DOI] [PubMed] [Google Scholar]
  57. Váró G., Lanyi J. K. Effects of the crystalline structure of purple membrane on the kinetics and energetics of the bacteriorhodopsin photocycle. Biochemistry. 1991 Jul 23;30(29):7165–7171. doi: 10.1021/bi00243a018. [DOI] [PubMed] [Google Scholar]
  58. Váró G., Lanyi J. K. Pathways of the rise and decay of the M photointermediate(s) of bacteriorhodopsin. Biochemistry. 1990 Mar 6;29(9):2241–2250. doi: 10.1021/bi00461a006. [DOI] [PubMed] [Google Scholar]
  59. Váró G., Lanyi J. K. Photoreactions of bacteriorhodopsin at acid pH. Biophys J. 1989 Dec;56(6):1143–1151. doi: 10.1016/S0006-3495(89)82761-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Váró G., Lanyi J. K. Protonation and deprotonation of the M, N, and O intermediates during the bacteriorhodopsin photocycle. Biochemistry. 1990 Jul 24;29(29):6858–6865. doi: 10.1021/bi00481a015. [DOI] [PubMed] [Google Scholar]
  61. Váró G., Lanyi J. K. Thermodynamics and energy coupling in the bacteriorhodopsin photocycle. Biochemistry. 1991 May 21;30(20):5016–5022. doi: 10.1021/bi00234a025. [DOI] [PubMed] [Google Scholar]
  62. 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]
  63. Váró G., Needleman R., Lanyi J. K. Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle. Biophys J. 1996 Jan;70(1):461–467. doi: 10.1016/S0006-3495(96)79589-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhang D., Mauzerall D. Volume and enthalpy changes in the early steps of bacteriorhodopsin photocycle studied by time-resolved photoacoustics. Biophys J. 1996 Jul;71(1):381–388. doi: 10.1016/S0006-3495(96)79235-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zimányi L., Cao Y., Needleman R., Ottolenghi M., Lanyi J. K. Pathway of proton uptake in the bacteriorhodopsin photocycle. Biochemistry. 1993 Aug 3;32(30):7669–7678. doi: 10.1021/bi00081a010. [DOI] [PubMed] [Google Scholar]
  66. Zimányi L., Tokaji Z., Dollinger G. Circular dichroic spectrum of the L form and the blue light product of the m form of purple membrane. Biophys J. 1987 Jan;51(1):145–148. doi: 10.1016/S0006-3495(87)83319-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zimányi L., Váró G., Chang M., Ni B., Needleman R., Lanyi J. K. Pathways of proton release in the bacteriorhodopsin photocycle. Biochemistry. 1992 Sep 15;31(36):8535–8543. doi: 10.1021/bi00151a022. [DOI] [PubMed] [Google Scholar]

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