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FTIR Study of the Photoinduced Processes of Plant Phytochrome Phya using Isotope-Labeled Bilins and Density Functional Theory Calculations

Pascale Schwinté ^*, Harald Foerstendorf , Zakir Hussain , Wolfgang Gärtner , Maria-Andrea Mroginski , Peter Hildebrandt  and Friedrich Siebert ^*

^* Sektion Biophysik, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität, Freiburg, Germany; Institut für Radiochemie, Forschungszentrum Dresden-Rossendorf, Dresden, Germany; Max-Planck-Institut für Bioanorganische Chemie, Mülheim a. d. R., Germany; and Institut für Chemie, Technische Universität Berlin, Berlin, Germany

Correspondence: Address reprint requests to F. Siebert, Sektion Biophysik, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität, Hermann-Herderstr. 9, D-79104 Freiburg, Germany. Tel.: 49-761-203-5396; Fax: 49-761-203-5390; E-mail: frisi{at}uni-freiburg.de.

Fourier transform infrared spectroscopy was used to analyze 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-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared 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 (ZZEZZZ), which involves only small protein structural changes.