Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Jul;75(1):406–412. doi: 10.1016/S0006-3495(98)77525-3

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

L Ujj 1, S Devanathan 1, T E Meyer 1, M A Cusanovich 1, G Tollin 1, G H Atkinson 1
PMCID: PMC1299710  PMID: 9649398

Abstract

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.

Full Text

The Full Text of this article is available as a PDF (111.9 KB).

Selected References

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

  1. Baca M., Borgstahl G. E., Boissinot M., Burke P. M., Williams D. R., Slater K. A., Getzoff E. D. Complete chemical structure of photoactive yellow protein: novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry. Biochemistry. 1994 Dec 6;33(48):14369–14377. doi: 10.1021/bi00252a001. [DOI] [PubMed] [Google Scholar]
  2. Borgstahl G. E., Williams D. R., Getzoff E. D. 1.4 A structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore. Biochemistry. 1995 May 16;34(19):6278–6287. doi: 10.1021/bi00019a004. [DOI] [PubMed] [Google Scholar]
  3. Brack T. L., Delaney J. K., Atkinson G. H., Albeck A., Sheves M., Ottolenghi M. Picosecond time-resolved absorption and fluorescence dynamics in the artificial bacteriorhodopsin pigment BR6.11. Biophys J. 1993 Aug;65(2):964–972. doi: 10.1016/S0006-3495(93)81119-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Genick U. K., Borgstahl G. E., Ng K., Ren Z., Pradervand C., Burke P. M., Srajer V., Teng T. Y., Schildkamp W., McRee D. E. Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science. 1997 Mar 7;275(5305):1471–1475. doi: 10.1126/science.275.5305.1471. [DOI] [PubMed] [Google Scholar]
  5. Genick U. K., Devanathan S., Meyer T. E., Canestrelli I. L., Williams E., Cusanovich M. A., Tollin G., Getzoff E. D. Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein. Biochemistry. 1997 Jan 7;36(1):8–14. doi: 10.1021/bi9622884. [DOI] [PubMed] [Google Scholar]
  6. Henry E. R., Eaton W. A., Hochstrasser R. M. Molecular dynamics simulations of cooling in laser-excited heme proteins. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8982–8986. doi: 10.1073/pnas.83.23.8982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hoff W. D., Düx P., Hård K., Devreese B., Nugteren-Roodzant I. M., Crielaard W., Boelens R., Kaptein R., van Beeumen J., Hellingwerf K. J. Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry. Biochemistry. 1994 Nov 29;33(47):13959–13962. doi: 10.1021/bi00251a001. [DOI] [PubMed] [Google Scholar]
  8. Hoff W. D., van Stokkum I. H., van Ramesdonk H. J., van Brederode M. E., Brouwer A. M., Fitch J. C., Meyer T. E., van Grondelle R., Hellingwerf K. J. Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila. Biophys J. 1994 Oct;67(4):1691–1705. doi: 10.1016/S0006-3495(94)80643-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Imamoto Y., Kataoka M., Tokunaga F. Photoreaction cycle of photoactive yellow protein from Ectothiorhodospira halophila studied by low-temperature spectroscopy. Biochemistry. 1996 Nov 12;35(45):14047–14053. doi: 10.1021/bi961342d. [DOI] [PubMed] [Google Scholar]
  10. Kort R., Vonk H., Xu X., Hoff W. D., Crielaard W., Hellingwerf K. J. Evidence for trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein. FEBS Lett. 1996 Mar 11;382(1-2):73–78. doi: 10.1016/0014-5793(96)00149-4. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Meyer T. E., Cusanovich M. A., Tollin G. Transient proton uptake and release is associated with the photocycle of the photoactive yellow protein from the purple phototrophic bacterium Ectothiorhodospira halophila. Arch Biochem Biophys. 1993 Nov 1;306(2):515–517. doi: 10.1006/abbi.1993.1545. [DOI] [PubMed] [Google Scholar]
  13. Meyer T. E., Fitch J. C., Bartsch R. G., Tollin G., Cusanovich M. A. Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens. Biochim Biophys Acta. 1990 Apr 26;1016(3):364–370. doi: 10.1016/0005-2728(90)90170-9. [DOI] [PubMed] [Google Scholar]
  14. Meyer T. E. Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila. Biochim Biophys Acta. 1985 Jan 23;806(1):175–183. doi: 10.1016/0005-2728(85)90094-5. [DOI] [PubMed] [Google Scholar]
  15. Meyer T. E., Tollin G., Causgrove T. P., Cheng P., Blankenship R. E. Picosecond decay kinetics and quantum yield of fluorescence of the photoactive yellow protein from the halophilic purple phototrophic bacterium, Ectothiorhodospira halophila. Biophys J. 1991 May;59(5):988–991. doi: 10.1016/S0006-3495(91)82313-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Meyer T. E., Tollin G., Hazzard J. H., Cusanovich M. A. Photoactive yellow protein from the purple phototrophic bacterium, Ectothiorhodospira halophila. Quantum yield of photobleaching and effects of temperature, alcohols, glycerol, and sucrose on kinetics of photobleaching and recovery. Biophys J. 1989 Sep;56(3):559–564. doi: 10.1016/S0006-3495(89)82703-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meyer T. E., Yakali E., Cusanovich M. A., Tollin G. Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin. Biochemistry. 1987 Jan 27;26(2):418–423. doi: 10.1021/bi00376a012. [DOI] [PubMed] [Google Scholar]
  18. Schoenlein R. W., Peteanu L. A., Mathies R. A., Shank C. V. The first step in vision: femtosecond isomerization of rhodopsin. Science. 1991 Oct 18;254(5030):412–415. doi: 10.1126/science.1925597. [DOI] [PubMed] [Google Scholar]
  19. Sprenger W. W., Hoff W. D., Armitage J. P., Hellingwerf K. J. The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein. J Bacteriol. 1993 May;175(10):3096–3104. doi: 10.1128/jb.175.10.3096-3104.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wang Q., Schoenlein R. W., Peteanu L. A., Mathies R. A., Shank C. V. Vibrationally coherent photochemistry in the femtosecond primary event of vision. Science. 1994 Oct 21;266(5184):422–424. doi: 10.1126/science.7939680. [DOI] [PubMed] [Google Scholar]

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

RESOURCES