Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Feb;82(2):1017–1029. doi: 10.1016/S0006-3495(02)75461-1

Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant.

Mary E Hatcher 1, Jingui G Hu 1, Marina Belenky 1, Peter Verdegem 1, Johan Lugtenburg 1, Robert G Griffin 1, Judith Herzfeld 1
PMCID: PMC1301908  PMID: 11806941

Abstract

By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of [zeta-(15)N]lys-, [12-(13)C]retinal-, and [14,15-(13)C]retinal-labeled D85N samples indicate a deprotonated, 13-cis,15-anti chromophore. On the other hand, at neutral pH, the NMR spectra of D85N show a mixture of protonated Schiff base species similar to that seen in the wild-type protein at low pH, and more complex than the two-state mixture of 13-cis,15-syn, and all-trans isomers found in the dark-adapted wild-type protein. These results lead to several conclusions. First, the reversible titration of order in the D85N chromophore indicates that electrostatic interactions have a major influence on events in the active site. More specifically, whereas a straight chromophore is preferred when the Schiff base and residue 85 are oppositely charged, a bent chromophore is found when both the Schiff base and residue 85 are electrically neutral, even in the dark. Thus a "bent" binding pocket is formed without photoisomerization of the chromophore. On the other hand, when photoisomerization from the straight all-trans,15-anti configuration to the bent 13-cis,15-anti does occur, reciprocal thermodynamic linkage dictates that neutralization of the SB and D85 (by proton transfer from the former to the latter) will result. Second, the similarity between the chromophore chemical shifts in D85N at alkaline pH and those found previously in the M(n) intermediate of the wild-type protein indicate that the latter has a thoroughly relaxed chromophore like the subsequent N intermediate. By comparison, indications of L-like distortion are found for the chromophore of the M(o) state. Thus, chromophore strain is released in the M(o)-->M(n) transition, probably coincident with, and perhaps instrumental to, the change in the connectivity of the Schiff base from the extracellular side of the membrane to the cytoplasmic side. Because the nitrogen chemical shifts of the Schiff base indicate interaction with a hydrogen-bond donor in both M states, it is possible that a water molecule travels with the Schiff base as it switches connectivity. If so, the protein is acting as an inward-driven hydroxyl pump (analogous to halorhodopsin) rather than an outward-driven proton pump. Third, the presence of a significant C [double bond] N syn component in D85N at neutral pH suggests that rapid deprotonation of D85 is necessary at the end of the wild-type photocycle to avoid the generation of nonfunctional C [double bond] N syn species.

Full Text

The Full Text of this article is available as a PDF (210.7 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., Kono M., Imasheva E., Lukashev E., Ebrey T. G., Crouch R. K., Menick D. R., Feng Y. Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle. Biochemistry. 1993 Oct 5;32(39):10331–10343. doi: 10.1021/bi00090a008. [DOI] [PubMed] [Google Scholar]
  2. Betancourt F. M., Glaeser R. M. Chemical and physical evidence for multiple functional steps comprising the M state of the bacteriorhodopsin photocycle. Biochim Biophys Acta. 2000 Aug 30;1460(1):106–118. doi: 10.1016/s0005-2728(00)00133-x. [DOI] [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. Brown L. S., Kamikubo H., Zimányi L., Kataoka M., Tokunaga F., Verdegem P., Lugtenburg J., Lanyi J. K. A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5040–5044. doi: 10.1073/pnas.94.10.5040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cao Y., Brown L. S., Sasaki J., Maeda A., Needleman R., Lanyi J. K. Relationship of proton release at the extracellular surface to deprotonation of the schiff base in the bacteriorhodopsin photocycle. Biophys J. 1995 Apr;68(4):1518–1530. doi: 10.1016/S0006-3495(95)80324-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dencher N. A., Dresselhaus D., Zaccai G., Büldt G. Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7876–7879. doi: 10.1073/pnas.86.20.7876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Fodor S. P., Pollard W. T., Gebhard R., van den Berg E. M., Lugtenburg J., Mathies R. A. Bacteriorhodopsin's L550 intermediate contains a C14-C15 s-trans-retinal chromophore. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2156–2160. doi: 10.1073/pnas.85.7.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedman N., Druckmann S., Lanyi J., Needleman R., Lewis A., Ottolenghi M., Sheves M. A covalent link between the chromophore and the protein backbone of bacteriorhodopsin is not required for forming a photochemically active pigment analogous to the wild type. Biochemistry. 1994 Mar 1;33(8):1971–1976. doi: 10.1021/bi00174a001. [DOI] [PubMed] [Google Scholar]
  13. Gochnauer M. B., Kushner D. J. Growth and nutrition of extremely halophilic bacteria. Can J Microbiol. 1969 Oct;15(10):1157–1165. doi: 10.1139/m69-211. [DOI] [PubMed] [Google Scholar]
  14. Govindjee R., Imasheva E. S., Misra S., Balashov S. P., Ebrey T. G., Chen N., Menick D. R., Crouch R. K. Mutation of a surface residue, lysine-129, reverses the order of proton release and uptake in bacteriorhodopsin; guanidine hydrochloride restores it. Biophys J. 1997 Feb;72(2 Pt 1):886–898. doi: 10.1016/s0006-3495(97)78723-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Harbison G. S., Herzfeld J., Griffin R. G. Solid-state nitrogen-15 nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin. Biochemistry. 1983 Jan 4;22(1):1–4. doi: 10.1021/bi00270a600. [DOI] [PubMed] [Google Scholar]
  17. Harbison G. S., Smith S. O., Pardoen J. A., Courtin J. M., Lugtenburg J., Herzfeld J., Mathies R. A., Griffin R. G. Solid-state 13C NMR detection of a perturbed 6-s-trans chromophore in bacteriorhodopsin. Biochemistry. 1985 Nov 19;24(24):6955–6962. doi: 10.1021/bi00345a031. [DOI] [PubMed] [Google Scholar]
  18. Harbison G. S., Smith S. O., Pardoen J. A., Mulder P. P., Lugtenburg J., Herzfeld J., Mathies R., Griffin R. G. Solid-state 13C NMR studies of retinal in bacteriorhodopsin. Biochemistry. 1984 Jun 5;23(12):2662–2667. doi: 10.1021/bi00307a019. [DOI] [PubMed] [Google Scholar]
  19. Harbison G. S., Smith S. O., Pardoen J. A., Winkel C., Lugtenburg J., Herzfeld J., Mathies R., Griffin R. G. Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1706–1709. doi: 10.1073/pnas.81.6.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haupts U., Tittor J., Oesterhelt D. Closing in on bacteriorhodopsin: progress in understanding the molecule. Annu Rev Biophys Biomol Struct. 1999;28:367–399. doi: 10.1146/annurev.biophys.28.1.367. [DOI] [PubMed] [Google Scholar]
  21. Hermone A., Kuczera K. Free-energy simulations of the retinal cis --> trans isomerization in bacteriorhodopsin. Biochemistry. 1998 Mar 3;37(9):2843–2853. doi: 10.1021/bi9717789. [DOI] [PubMed] [Google Scholar]
  22. Herzfeld J., Tounge B. NMR probes of vectoriality in the proton-motive photocycle of bacteriorhodopsin: evidence for an 'electrostatic steering' mechanism. Biochim Biophys Acta. 2000 Aug 30;1460(1):95–105. doi: 10.1016/s0005-2728(00)00132-8. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hu J. G., Sun B. Q., Bizounok M., Hatcher M. E., Lansing J. C., Raap J., Verdegem P. J., Lugtenburg J., Griffin R. G., Herzfeld J. Early and late M intermediates in the bacteriorhodopsin photocycle: a solid-state NMR study. Biochemistry. 1998 Jun 2;37(22):8088–8096. doi: 10.1021/bi973168e. [DOI] [PubMed] [Google Scholar]
  25. Hu J. G., Sun B. Q., Petkova A. T., Griffin R. G., Herzfeld J. The predischarge chromophore in bacteriorhodopsin: a 15N solid-state NMR study of the L photointermediate. Biochemistry. 1997 Aug 5;36(31):9316–9322. doi: 10.1021/bi970416y. [DOI] [PubMed] [Google Scholar]
  26. Hu J., Griffin R. G., Herzfeld J. Synergy in the spectral tuning of retinal pigments: complete accounting of the opsin shift in bacteriorhodopsin. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8880–8884. doi: 10.1073/pnas.91.19.8880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kataoka M., Kamikubo H., Tokunaga F., Brown L. S., Yamazaki Y., Maeda A., Sheves M., Needleman R., Lanyi J. K. Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin. J Mol Biol. 1994 Nov 4;243(4):621–638. doi: 10.1016/0022-2836(94)90037-x. [DOI] [PubMed] [Google Scholar]
  28. Lakshmi K. V., Farrar M. R., Raap J., Lugtenburg J., Griffin R. G., Herzfeld J. Solid state 13C and 15N NMR investigations of the N intermediate of bacteriorhodopsin. Biochemistry. 1994 Aug 2;33(30):8853–8857. doi: 10.1021/bi00196a001. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Luecke H. Atomic resolution structures of bacteriorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this light-driven ion pump. Biochim Biophys Acta. 2000 Aug 30;1460(1):133–156. doi: 10.1016/s0005-2728(00)00135-3. [DOI] [PubMed] [Google Scholar]
  32. Marti T., Rösselet S. J., Otto H., Heyn M. P., Khorana H. G. The retinylidene Schiff base counterion in bacteriorhodopsin. J Biol Chem. 1991 Oct 5;266(28):18674–18683. [PubMed] [Google Scholar]
  33. Mathies R. A., Li X. Y. On modeling the vibrational spectra of 14-s-cis retinal conformers in bacteriorhodopsin. Biophys Chem. 1995 Sep-Oct;56(1-2):47–55. doi: 10.1016/0301-4622(95)00014-o. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Moltke S., Heyn M. P. Photovoltage kinetics of the acid-blue and acid-purple forms of bacteriorhodopsin: evidence for no net charge transfer. Biophys J. 1995 Nov;69(5):2066–2073. doi: 10.1016/S0006-3495(95)80077-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moltke S., Krebs M. P., Mollaaghababa R., Khorana H. G., Heyn M. P. Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions. Biophys J. 1995 Nov;69(5):2074–2083. doi: 10.1016/S0006-3495(95)80078-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. Radionov A. N., Klyachko V. A., Kaulen A. D. Formation of the M(N) (M(open)) intermediate in the wild-type bacteriorhodopsin photocycle is accompanied by an absorption spectrum shift to shorter wavelength, like that in the mutant D96N bacteriorhodopsin photocycle. Biochemistry (Mosc) 1999 Oct;64(10):1210–1214. [PubMed] [Google Scholar]
  41. Rothschild K. J., Braiman M. S., He Y. W., Marti T., Khorana H. G. Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism. J Biol Chem. 1990 Oct 5;265(28):16985–16991. [PubMed] [Google Scholar]
  42. Sass H. J., Schachowa I. W., Rapp G., Koch M. H., Oesterhelt D., Dencher N. A., Büldt G. The tertiary structural changes in bacteriorhodopsin occur between M states: X-ray diffraction and Fourier transform infrared spectroscopy. EMBO J. 1997 Apr 1;16(7):1484–1491. doi: 10.1093/emboj/16.7.1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schulten K., Tavan P. A mechanism for the light-driven proton pump of Halobacterium halobium. Nature. 1978 Mar 2;272(5648):85–86. doi: 10.1038/272085a0. [DOI] [PubMed] [Google Scholar]
  44. Schweiger U., Tittor J., Oesterhelt D. Bacteriorhodopsin can function without a covalent linkage between retinal and protein. Biochemistry. 1994 Jan 18;33(2):535–541. doi: 10.1021/bi00168a019. [DOI] [PubMed] [Google Scholar]
  45. Smith S. O., Lugtenburg J., Mathies R. A. Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy. J Membr Biol. 1985;85(2):95–109. doi: 10.1007/BF01871263. [DOI] [PubMed] [Google Scholar]
  46. Smith S. O., Mathies R. A. Resonance Raman spectra of the acidified and deionized forms of bacteriorhodopsin. Biophys J. 1985 Feb;47(2 Pt 1):251–254. doi: 10.1016/s0006-3495(85)83899-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smith S. O., de Groot H. J., Gebhard R., Courtin J. M., Lugtenburg J., Herzfeld J., Griffin R. G. Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR. Biochemistry. 1989 Oct 31;28(22):8897–8904. doi: 10.1021/bi00448a032. [DOI] [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. Subramaniam S., Lindahl M., Bullough P., Faruqi A. R., Tittor J., Oesterhelt D., Brown L., Lanyi J., Henderson R. Protein conformational changes in the bacteriorhodopsin photocycle. J Mol Biol. 1999 Mar 19;287(1):145–161. doi: 10.1006/jmbi.1999.2589. [DOI] [PubMed] [Google Scholar]
  50. Subramaniam S. The structure of bacteriorhodopsin: an emerging consensus. Curr Opin Struct Biol. 1999 Aug;9(4):462–468. doi: 10.1016/S0959-440X(99)80065-7. [DOI] [PubMed] [Google Scholar]
  51. Turner G. J., Miercke L. J., Thorgeirsson T. E., Kliger D. S., Betlach M. C., Stroud R. M. Bacteriorhodopsin D85N: three spectroscopic species in equilibrium. Biochemistry. 1993 Feb 9;32(5):1332–1337. doi: 10.1021/bi00056a019. [DOI] [PubMed] [Google Scholar]
  52. Vonck J., Han B. G., Burkard F., Perkins G. A., Glaeser R. M. Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin. Biophys J. 1994 Sep;67(3):1173–1178. doi: 10.1016/S0006-3495(94)80585-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoshida M., Ohno K., Takeuchi Y. Altered activity of bacteriorhodopsin in high concentrations of guanidine hydrochloride. J Biochem. 1980 Feb;87(2):491–495. doi: 10.1093/oxfordjournals.jbchem.a132769. [DOI] [PubMed] [Google Scholar]
  54. Yoshida M., Ohno K., Takeuchi Y., Kagawa Y. Prolonged lifetime of the 410-nm intermediate of bacteriorhodopsin in the presence of guanidine hydrochloride. Biochem Biophys Res Commun. 1977 Apr 25;75(4):1111–1116. doi: 10.1016/0006-291x(77)91497-8. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. de Groot H. J., Harbison G. S., Herzfeld J., Griffin R. G. Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: counterion effects on the 15N shift anisotropy. Biochemistry. 1989 Apr 18;28(8):3346–3353. doi: 10.1021/bi00434a033. [DOI] [PubMed] [Google Scholar]
  58. de Groot H. J., Smith S. O., Courtin J., van den Berg E., Winkel C., Lugtenburg J., Griffin R. G., Herzfeld J. Solid-state 13C and 15N NMR study of the low pH forms of bacteriorhodopsin. Biochemistry. 1990 Jul 24;29(29):6873–6883. doi: 10.1021/bi00481a017. [DOI] [PubMed] [Google Scholar]

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

RESOURCES