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. 2002 Feb;82(2):1004–1016. doi: 10.1016/S0006-3495(02)75460-X

Ultrafast dynamics of phytochrome from the cyanobacterium synechocystis, reconstituted with phycocyanobilin and phycoerythrobilin.

Karsten Heyne 1, Johannes Herbst 1, Dietmar Stehlik 1, Berta Esteban 1, Tilman Lamparter 1, Jon Hughes 1, Rolf Diller 1
PMCID: PMC1301907  PMID: 11806940

Abstract

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.

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Selected References

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  1. Andel F., 3rd, Lagarias J. C., Mathies R. A. Resonance raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome. Biochemistry. 1996 Dec 17;35(50):15997–16008. doi: 10.1021/bi962175k. [DOI] [PubMed] [Google Scholar]
  2. Austin R. H., Beeson K. W., Eisenstein L., Frauenfelder H., Gunsalus I. C. Dynamics of ligand binding to myoglobin. Biochemistry. 1975 Dec 2;14(24):5355–5373. doi: 10.1021/bi00695a021. [DOI] [PubMed] [Google Scholar]
  3. Bischoff M., Hermann G., Rentsch S., Strehlow D. First steps in the phytochrome phototransformation: a comparative femtosecond study on the forward (Pr --> Pfr) and back reaction (Pfr --> Pr). Biochemistry. 2001 Jan 9;40(1):181–186. doi: 10.1021/bi0011734. [DOI] [PubMed] [Google Scholar]
  4. Chory J., Chatterjee M., Cook R. K., Elich T., Fankhauser C., Li J., Nagpal P., Neff M., Pepper A., Poole D. From seed germination to flowering, light controls plant development via the pigment phytochrome. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12066–12071. doi: 10.1073/pnas.93.22.12066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foerstendorf H., Lamparter T., Hughes J., Gärtner W., Siebert F. The photoreactions of recombinant phytochrome from the cyanobacterium Synechocystis: a low-temperature UV-Vis and FT-IR spectroscopic study. Photochem Photobiol. 2000 May;71(5):655–661. doi: 10.1562/0031-8655(2000)071<0655:tporpf>2.0.co;2. [DOI] [PubMed] [Google Scholar]
  6. Foerstendorf H., Mummert E., Schäfer E., Scheer H., Siebert F. Fourier-transform infrared spectroscopy of phytochrome: difference spectra of the intermediates of the photoreactions. Biochemistry. 1996 Aug 20;35(33):10793–10799. doi: 10.1021/bi960960r. [DOI] [PubMed] [Google Scholar]
  7. Frauenfelder H., Wolynes P. G. Rate theories and puzzles of hemeprotein kinetics. Science. 1985 Jul 26;229(4711):337–345. doi: 10.1126/science.4012322. [DOI] [PubMed] [Google Scholar]
  8. Hughes J., Lamparter T., Mittmann F., Hartmann E., Gärtner W., Wilde A., Börner T. A prokaryotic phytochrome. Nature. 1997 Apr 17;386(6626):663–663. doi: 10.1038/386663a0. [DOI] [PubMed] [Google Scholar]
  9. Hughes J, Lamparter T. Prokaryotes and phytochrome. The connection to chromophores and signaling. Plant Physiol. 1999 Dec;121(4):1059–1068. doi: 10.1104/pp.121.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hübschmann T., Börner T., Hartmann E., Lamparter T. Characterization of the Cph1 holo-phytochrome from Synechocystis sp. PCC 6803. Eur J Biochem. 2001 Apr;268(7):2055–2063. doi: 10.1046/j.1432-1327.2001.02083.x. [DOI] [PubMed] [Google Scholar]
  11. Lamparter T., Esteban B., Hughes J. Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure. Eur J Biochem. 2001 Sep;268(17):4720–4730. doi: 10.1046/j.1432-1327.2001.02395.x. [DOI] [PubMed] [Google Scholar]
  12. Lamparter T., Mittmann F., Gärtner W., Börner T., Hartmann E., Hughes J. Characterization of recombinant phytochrome from the cyanobacterium Synechocystis. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11792–11797. doi: 10.1073/pnas.94.22.11792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Li L., Murphy J. T., Lagarias J. C. Continuous fluorescence assay of phytochrome assembly in vitro. Biochemistry. 1995 Jun 20;34(24):7923–7930. doi: 10.1021/bi00024a017. [DOI] [PubMed] [Google Scholar]
  14. Matysik J., Hildebrandt P., Schlamann W., Braslavsky S. E., Schaffner K. Fourier-transform resonance Raman spectroscopy of intermediates of the phytochrome photocycle. Biochemistry. 1995 Aug 22;34(33):10497–10507. doi: 10.1021/bi00033a023. [DOI] [PubMed] [Google Scholar]
  15. Murphy J. T., Lagarias J. C. The phytofluors: a new class of fluorescent protein probes. Curr Biol. 1997 Nov 1;7(11):870–876. doi: 10.1016/s0960-9822(06)00375-7. [DOI] [PubMed] [Google Scholar]
  16. Quail P. H., Boylan M. T., Parks B. M., Short T. W., Xu Y., Wagner D. Phytochromes: photosensory perception and signal transduction. Science. 1995 May 5;268(5211):675–680. doi: 10.1126/science.7732376. [DOI] [PubMed] [Google Scholar]
  17. Remberg A., Lindner I., Lamparter T., Hughes J., Kneip C., Hildebrandt P., Braslavsky S. E., Gärtner W., Schaffner K. Raman spectroscopic and light-induced kinetic characterization of a recombinant phytochrome of the cyanobacterium Synechocystis. Biochemistry. 1997 Oct 28;36(43):13389–13395. doi: 10.1021/bi971563z. [DOI] [PubMed] [Google Scholar]
  18. Rüdiger W., Thümmler F., Cmiel E., Schneider S. Chromophore structure of the physiologically active form (P(fr)) of phytochrome. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6244–6248. doi: 10.1073/pnas.80.20.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Savikhin S., Wells T., Song P. S., Struve W. S. Ultrafast pump-probe spectroscopy of native etiolated oat phytochrome. Biochemistry. 1993 Jul 27;32(29):7512–7518. doi: 10.1021/bi00080a024. [DOI] [PubMed] [Google Scholar]
  20. Schmidt P., Westphal U. H., Worm K., Braslavsky S. E., Gärtner W., Schaffner K. Chromophore-protein interaction controls the complexity of the phytochrome photocycle. J Photochem Photobiol B. 1996 Jun;34(1):73–77. doi: 10.1016/1011-1344(95)07269-1. [DOI] [PubMed] [Google Scholar]
  21. Sineshchekov V., Hughes J., Hartmann E., Lamparter T. Fluorescence and photochemistry of recombinant phytochrome from the cyanobacterium Synechocystis. Photochem Photobiol. 1998 Feb;67(2):263–267. doi: 10.1562/0031-8655(1998)067<0263:faporp>2.3.co;2. [DOI] [PubMed] [Google Scholar]
  22. Smith H. Phytochromes. Tripping the light fantastic. Nature. 1999 Aug 19;400(6746):710-1, 713. doi: 10.1038/23354. [DOI] [PubMed] [Google Scholar]
  23. Song L., El-Sayed M. A., Lanyi J. K. Protein catalysis of the retinal subpicosecond photoisomerization in the primary process of bacteriorhodopsin photosynthesis. Science. 1993 Aug 13;261(5123):891–894. doi: 10.1126/science.261.5123.891. [DOI] [PubMed] [Google Scholar]
  24. Vos M. H., Rischel C., Jones M. R., Martin J. L. Electrochromic detection of a coherent component in the formation of the charge pair P(+)H(L)(-) in bacterial reaction centers. Biochemistry. 2000 Jul 25;39(29):8353–8361. doi: 10.1021/bi000759n. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Yeh K. C., Wu S. H., Murphy J. T., Lagarias J. C. A cyanobacterial phytochrome two-component light sensory system. Science. 1997 Sep 5;277(5331):1505–1508. doi: 10.1126/science.277.5331.1505. [DOI] [PubMed] [Google Scholar]
  27. Zeidler M., Lamparter T., Hughes J., Hartmann E., Remberg A., Braslavsky S., Schaffner K., Gärtner W. Recombinant phytochrome of the moss Ceratodon purpureus: heterologous expression and kinetic analysis of Pr-->Pfr conversion. Photochem Photobiol. 1998 Dec;68(6):857–863. doi: 10.1111/j.1751-1097.1998.tb05296.x. [DOI] [PubMed] [Google Scholar]

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