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The Photoreactions of Recombinant Phytochrome CphA from the Cyanobacterium Calothrix PCC7601: A Low-Temperature UV-Vis and FTIR Study
Citation key ISI:000262486800029
Author Schwinté, Pascale and G�rtner, Wolfgang and Sharda, Shivani and Mroginski, Maria-Andrea and Hildebrandt, Peter and Siebert, Friedrich
Pages 239-249
Year 2009
ISSN 0031-8655
DOI 10.1111/j.1751-1097.2008.00426.x
Address BIOTECH PARK, 1021 15TH ST, SUITE 9, AUGUSTA, GA 30901-3158 USA
Journal Photochem. Photobiol.
Volume 85
Number 1
Month JAN-FEB
Publisher AMER SOC PHOTOBIOLOGY
Abstract The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV-Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV-Vis and FTIR experiments. Three intermediates have been trapped at -25, -45 and -120 degrees C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at -70 and -140 degrees C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore-protein interactions.
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