Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 1993 Dec;65(6):2478–2483. doi: 10.1016/S0006-3495(93)81308-0

Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N.

C Gergely 1, C Ganea 1, G Groma 1, G Váró 1
PMCID: PMC1225989  PMID: 8312486

Abstract

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.

Full text

PDF
2478

Selected References

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

  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. 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]
  3. 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]
  4. 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]
  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. Chernavskii D. S., Chizhov I. V., Lozier R. H., Murina T. M., Prokhorov A. M., Zubov B. V. Kinetic model of bacteriorhodopsin photocycle: pathway from M state to bR. Photochem Photobiol. 1989 May;49(5):649–653. doi: 10.1111/j.1751-1097.1989.tb08437.x. [DOI] [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. 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]
  9. Gerwert K., Souvignier G., Hess B. Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy. Proc Natl Acad Sci U S A. 1990 Dec 15;87(24):9774–9778. doi: 10.1073/pnas.87.24.9774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Groma G. I., Dancshazy Z. How Many M Forms are there in the Bacteriorhodopsin Photocycle? Biophys J. 1986 Aug;50(2):357–366. doi: 10.1016/S0006-3495(86)83469-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hofrichter J., Henry E. R., Lozier R. H. Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy. Biophys J. 1989 Oct;56(4):693–706. doi: 10.1016/S0006-3495(89)82716-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holz M., Drachev L. A., Mogi T., Otto H., Kaulen A. D., Heyn M. P., Skulachev V. P., Khorana H. G. Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2167–2171. doi: 10.1073/pnas.86.7.2167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lanyi J. K. Proton transfer and energy coupling in the bacteriorhodopsin photocycle. J Bioenerg Biomembr. 1992 Apr;24(2):169–179. doi: 10.1007/BF00762675. [DOI] [PubMed] [Google Scholar]
  14. Liu S. Y., Kono M., Ebrey T. G. Effect of pH buffer molecules on the light-induced currents from oriented purple membrane. Biophys J. 1991 Jul;60(1):204–216. doi: 10.1016/S0006-3495(91)82044-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liu S. Y. Light-induced currents from oriented purple membrane: I. Correlation of the microsecond component (B2) with the L-M photocycle transition. Biophys J. 1990 May;57(5):943–950. doi: 10.1016/S0006-3495(90)82614-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lozier R. H., Bogomolni R. A., Stoeckenius W. Bacteriorhodopsin: a light-driven proton pump in Halobacterium Halobium. Biophys J. 1975 Sep;15(9):955–962. doi: 10.1016/S0006-3495(75)85875-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mathies R. A., Brito Cruz C. H., Pollard W. T., Shank C. V. Direct observation of the femtosecond excited-state cis-trans isomerization in bacteriorhodopsin. Science. 1988 May 6;240(4853):777–779. doi: 10.1126/science.3363359. [DOI] [PubMed] [Google Scholar]
  18. Mathies R. A., Lin S. W., Ames J. B., Pollard W. T. From femtoseconds to biology: mechanism of bacteriorhodopsin's light-driven proton pump. Annu Rev Biophys Biophys Chem. 1991;20:491–518. doi: 10.1146/annurev.bb.20.060191.002423. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Needleman R., Chang M., Ni B., Váró G., Fornés J., White S. H., Lanyi J. K. Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base. J Biol Chem. 1991 Jun 25;266(18):11478–11484. [PubMed] [Google Scholar]
  21. 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]
  22. Rothschild K. J. FTIR difference spectroscopy of bacteriorhodopsin: toward a molecular model. J Bioenerg Biomembr. 1992 Apr;24(2):147–167. doi: 10.1007/BF00762674. [DOI] [PubMed] [Google Scholar]
  23. Soppa J., Otomo J., Straub J., Tittor J., Meessen S., Oesterhelt D. Bacteriorhodopsin mutants of Halobacterium sp. GRB. II. Characterization of mutants. J Biol Chem. 1989 Aug 5;264(22):13049–13056. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Trissl H. W. Photoelectric measurements of purple membranes. Photochem Photobiol. 1990 Jun;51(6):793–818. [PubMed] [Google Scholar]
  27. Váró G., Keszthelyi L. Photoelectric signals from dried oriented purple membranes of Halobacterium halobium. Biophys J. 1983 Jul;43(1):47–51. doi: 10.1016/S0006-3495(83)84322-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Váró G., Zimányi L., Chang M., Ni B., Needleman R., Lanyi J. K. A residue substitution near the beta-ionone ring of the retinal affects the M substates of bacteriorhodopsin. Biophys J. 1992 Mar;61(3):820–826. doi: 10.1016/S0006-3495(92)81887-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhou F., Windemuth A., Schulten K. Molecular dynamics study of the proton pump cycle of bacteriorhodopsin. Biochemistry. 1993 Mar 9;32(9):2291–2306. doi: 10.1021/bi00060a022. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]

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

RESOURCES