FTIR Study of the Photoinduced Processes of Plant Phytochrome phyA using isotope-labeled Bilins and DFT Calculations
Pascale Schwinté 1, Harald Foerstendorf 2, Zakir Hussain 3, Wolfgang Gärtner 3, Maria-Andrea Mroginski 4, Peter Hildebrandt 4 and Friedrich Siebert 1*
1 Universität Freiburg
2 Forschungszentrum Dresden-Rossendorf
3 Max-Planck-Institut für Bioanorganische Chemie
4 Technische Universität Berlin
* To whom correspondence should be addressed. E-mail: frisi{at}biophysik.uni-freiburg.de.
Submitted on February 14, 2008
Revised on March 10, 2008
Accepted on 25 March 2008
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Abstract |
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FTIR spectroscopy was employed to analyse the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global 13C-labelling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the FTIR difference spectra. All IR difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE 
ZZZ) which involves only small protein structural changes.
Key Words:
chromophore-protein interaction, conformational change, isotope-labeling, photoisomerization, resonance Raman, vibrational spectroscopy
